2015 ada posters 1930-2373 - home | diabetes...objective: animal studies suggest that...

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INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

1926-PHow to Transform a Metabolic Syndrome Score in an Insulin Sen-sitivity Value?MICHEL P. HERMANS, EVARISTE BOUENIZABILA, K. DANIEL AMOUSSOU-GUENOU, SYLVIE A. AHN, MICHEL F. ROUSSEAU, Brussels, Belgium, Brazzaville, Congo, Republic of the, Cotonou, Benin

The metabolic syndrome (MetS) predicts cardiovascular risk and incident type 2 diabetes mellitus (T2DM). The presence of a MetS is defi ned by the clustering of ≥3 out of 5 cardiometabolic criteria (hyperglycemia; hyperten-sion; enlarged waist; low HDL-cholesterol; and hypertriglyceridemia), each of which is connected with insulin resistance (IR). It is not known whether the severity of MetS, ranked from the sextet of scores’ range [0/5 to 5/5], is linearly related to reduced insulin sensitivity (IS) and/or lesser hyperbolic product across the glycemic spectrum.

We analyzed 839 adults (54 normoglycemic; 785 with abnormal glucose homeostasis, among whom 711 T2DM), whose IS was assessed together with their cardiometabolic phenotype.

There was a signifi cant gradient according to interval-scale MetS score in insulinemia; BMI; (visceral) fat; hepatic steatosis; and macroangiopathy. There was an inverse linear relationship between increasing MetS scores and decreased IS, allowing to defi ne an IR-predicting linear equation: IS (%) = [-15.1*MetS score] + 109.4 (R2 = 0.221). For each MetS category, mean IS values did not signifi cantly differ between groups of patients across the gly-cemic spectrum. The hyperbolic product (β-cell function x IS) and/or its loss rate were inversely related to MetS severity.

In conclusion, IS is linearly and inversely related to MetS severity across the 6 scores. This novel way to exploit information intrinsic to the MetS cri-teria provides an easy and costless means to quantify IS across the glycemic spectrum. Moreover, MetS severity is associated with a lesser hyperbolic product, and a higher loss of the latter.

1927-PPossible Involvement of Undercarboxylated Osteocalcin upon Reg-ulating Insulin Secretion in Patients with Type 2 DiabetesYUICHI TAKASHI, YOKO MATSUZAWA, JUN SAITO, MASAO OMURA, TETSUO NISHIKAWA, Yokohama, Japan

We examined the effect of undercarboxylated osteocalcin (ucOC) on the ability of insulin secretion, evaluated by glucagon loading test (GLT) and meal tolerance test (MTT). UcOC has been shown to regulate insulin secretion in rodents. However, data on the correlation between ucOC and glucose metabolism in humans is limited and controversial. We recruited 50 patients (41 men and 9 postmenopausal women) with type 2 diabetes under written informed consent, and performed both GLT and MTT, and analyzed the relationship between ucOC and C-peptide response (CPR). All patients were free of insulin therapy and drugs known to infl uence bone metabolism, such as vitamin D, bisphosphonate. Number of patients who were treated with DPP-4 inhibitor, sulfonylurea, glinide, metformin, thiazolidinedione and alpha-glucosidase inhibitor were 17, 19, 15, 19, 4 and 12 patients. The av-erage of age, duration of diabetes, BMI, and HbA1c of the subjects were 59.2 y.o., 7.8 years, 26.2 kg/m2 and 9.4%, respectively. UcOC was shown to correlate positively with CPR in GLT and CPR after meal taking (r = 0.32, p = 0.025; r = 0.29, p = 0.047). Furthermore, we focused on the patients with HbA1c less than 8.0% because we thought they had little infl uence of glucose toxicity. UcOC was shown to correlate positively more strongly with CPR after glucagon loading and CPR in GLT (r = 0.60, p = 0.0088; r = 0.67, p = 0.0025) than with CPR after meal taking (r = 0.51, p = 0.035). In both groups, ucOC was not shown to correlate with CPR before glucagon loading or meal taking. It is suggested that ucOC correlate with not basal but bolus insulin secretion and may directly induce insulin secretion from β-cells without in-volving gut-related insulin stimulation in patients with type 2 diabetes. UcOC is suggested to be a new marker to evaluate the reserve capacity of β-cell function, such as the bolus insulin secretion ability.

1928-P20/(Fasting C-Peptide × Fasting Plasma Glucose) Is a Novel Index of Insulin Resistance with Small Effect of Hepatic Insulin ClearanceTSUYOSHI OKURA, YOUHEI FUJIOKA, RISA NAKANISHI, HIDEKI SHIOCHI, KEISUKE SUMI, KYOKO SHOJI, AYUMI MURAWAKI, KAZUHIKO MATSUZAWA, SCHOICHIRO IZAWA, CHIEKO SAKAI, MASAHIKO KATO, SHIN-ICHI TANIGUCHI, KAZUHIRO YAMAMOTO, Yonago, Japan

We developed a simple and new insulin resistance index derived from a glucose clamp and a meal tolerance test (MTT) in Japanese patients with type 2 diabetes mellitus.

Twenty patients [mean fasting plasma glucose (FPG) 7.3 mmol/L, HbA1c 7.5%, BMI 28.4 kg/m2] underwent a MTT and a glucose clamp. Participants were given a test meal (460 kcal). Plasma glucose, insulin, and C-peptide im-munoreactivity (CPR) were measured at 0, 30, 60, and 120 min. HOMA-IR and Hepatic Insulin Clearance (HIC: AUC-Insulin/AUC-CPR ratio) were calculated from the MTT results. The glucose disposal rate (GDR) was measured during hyperinsulinemic-euglycemic glucose clamps.

The mean GDR in all patients was 5.08 mg·kg-1·min-1. The index 20/(F-CPR × FPG) was correlated strongly with GDR (R=0.72), better than HOMA-IR (R=−0.53). 20/(F-CPR × FPG) was able to estimate GDR, we would like to name this index “CPR-IR.” The median value of HIC in all patients was 6.0. In the patients with low hepatic insulin clearance (HIC<6.0), HOMA-IR and CPR-IR were correlated equally with GDR (R=-0.68 and 0.69). In the patients with high hepatic insulin clearance (HIC>6.0), whereas HOMA-IR did not cor-relate with GDR (R=-0.21), CPR-IR correlated with GDR (R=0.64).

CPR-IR is a simple and effective index of insulin resistance in the patients with T2DM, and performs better than HOMA-IR with small effect of hepatic insulin clearance.

Table. The Correlation between GDR and CPR-IR, HOMA-IR.CPR-IR HOMA-IR

All patients R=0.72 R=-0.53Low hepatic insulin clearance (HIC<6.0) R=0.69 R=-0.68High hepatic insulin clearance (HIC>6.0) R=0.64 R=-0.21


Guided Audio Tour: Insulin Secretion In Vivo (Posters: 1929-P to 1934-P), see page 13.

& 1929-PLimitations of Standardized C-Peptide Kinetics for Estimation of β-Cell ResponsivityRON T. VARGHESE, FRANCESCA PICCININI, MEERA SHAH, CHIARA DALLA MAN, ROBERT A. RIZZA, CLAUDIO COBELLI, ADRIAN VELLA, Rochester, MN, Padova, Italy

Standard kinetics of C-Peptide clearance enable deconvolution of insu-lin secretion from changing plasma C-Peptide concentrations. However, it is uncertain if standard parameters apply to all conditions under which β-cell function is measured. As part of a series of experiments examining the mechanisms underlying prediabetes, we studied 60 nondiabetic individuals (38.7±2.2 years, BMI 28.3±0.8 Kg/M2) on 2 occasions in random order using an oral glucose challenge. On one occasion, free fatty acid (FFA) elevation to cause insulin resistance, was achieved by infusion of intralipid + heparin. On the other study day subjects received the same amount of glycerol pres-ent in the intralipid. On a 3rd study day, after a euglycemic clamp where somatostatin inhibited endogenous insulin secretion, subjects received a C-Peptide bolus to calculate individual kinetic for clearance. β-cell responsivity (ϕTotal) in the presence or absence of FFA elevation was estimated using the oral C-Peptide minimal model using either standard or individual C-Peptide kinetics. ϕTotal estimated using individual kinetics correlated well with those estimated using standard kinetics in the presence (46±4 vs. 46±4 10-9 min-1, R2 =0.59, p<0.01) or absence (56±4 vs. 53±4 10-9 min-1, R2 =0.66, p<0.01) of FFA elevation. Analysis of the components of ϕTotal reveals generally worse correlation for ϕDynamic in the presence (R2 =0.42, p<0.01) compared to the absence (R2 =0.68, p<0.01) of FFA elevation. Similarly, ϕStatic in the presence (R2 =0.50, p<0.01) and absence (R2 =0.76, p<0.01) of FFA elevation. The great-est discrepancies arise in basal β-cell responsivity during FFA elevation (ϕBasal - R2<0.01, p=0.71) compared to glycerol infusion (R2 =0.79, p<0.01). We conclude that results obtained with standard C-Peptide kinetics correlate well with those obtained using individual kinetics. However individual pa-rameters are required for optimal measurement of ϕBasal at least in response to acute decreases in insulin action.

Supported By: National Institutes of Health (R01DK078646)

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& 1930-PExposition to Glucocorticoids during Fetal Life Is Associated with Insulin Secretion Defect at Adult AgeJEAN-PIERRE RIVELINE, JEAN-LOUIS NGUEWA, BAZ BAZ, PHILIPPE BOUDOU, BERTRAND BLONDEAU, BERNADETTE BRÉANT, YVES MOREL, JEAN-FRANCOIS GAUTIER, Paris, France, Lyon, France

Objective: Animal studies suggest that glucocorticoids (GCs) inhibit the development of beta cells during fetal life. In order to address if such inhibi-tion may occurs in humans, we investigated in a case-control study beta-cell function in healthy subjects who have been exposed to GCs during early fetal life, at a period of beta cells development.

Patients and Methods: Women carrying congenital adrenal hyperplasia (CAH) mutation are treated by dexamethasone (DXM) during pregnancy to avoid sexual ambiguity in affected fetus. DXM is stopped around the 18th week as soon as the mutation is excluded in the fetus. We investigated such healthy adults (exposed group, n=12) compared to a age, BMI, gender matched control group (n=12). We measured insulin secretion using oral glucose tolerance test (OGTT) and insulin action by a hyperinsulinemic euglycemic clamp (80 mU/m2/minute).

Results: Age (23.8±3.4 vs. 23,8±2,9 years), BMI (23.4±3.3 vs. 22,99±2,88 kg/m2) and anthropometric characteristics (waist circumference, fat mass, sys-tolic blood pressures and diastolic blood and fat) were similar between the two groups. At T0, glycemia and insulinemia were increased in the exposed group in comparison to the control group (Glycemia: 4.83±0.43 vs. 4.55±0.30 mM, p = 0,05 and Insulinemia: 7.67±2.78 vs. 5.16±1.06 mUI/l, p=0.006). During OGTT, at T120, glycemia and insulinemia were similar while at T30, glycemia was increased in exposed group (7.72±1.27 vs. 6.36±1.20 mM, p=0.009) but insulinemia was similar (50.18±15.67vs 41.79±18.71 mUI/l, NS). Insulinogenic index was decreased in exposed group (15.87±5.12 vs. 22.13 ± 13.33, p=0.05) while Insulin sensitivity (Mvalue) was similar in both groups.

Conclusion: These results suggest that early fetal exposure to glucocor-ticoids is associated with a decreased early insulin secretion in response to oral glucose at adult age.

& 1931-PEffects of Anesthesia on Metabolic Responses to Mixed Meal Tolerance Test in Cynomolgus Monkeys with Naturally Occurring DiabetesXIAOLI WANG, YONGQIANG LIU, BINGDI WANG, GUOFENG SUN, KEEFE CHNG, YONG-FU XIAO, YI-XIN (JIM) WANG, Taicang, China, Kannapolis, NC

Mixed meal tolerance test (MMTT) is commonly used in diabetic research in experimental animal models and humans to evaluate glucose metabolic regula-tion by entero-insular axis. Since anesthesia can affect gastric intestinal physiol-ogy, it could in turn affect the MMTT responses. The aim of the present study is to evaluate the impact of sedation on MMTT responses in normal (n=7, Control), pre-diabetic (n=6, Pre-DM), and diabetic (n=7) cynomolgus macaques based on their metabolic profi les including fasting glucose and insulin as well as ivGTT responses. Following oral administration of a standard liquid mixed meal, both the hyperglycemic and insulinogenic responses were greater in the conscious than sedated monkeys in the Control, but not in DM group, while in Pre-DM group, only the hyperglycemic, but not insulinogenic response was higher in con-scious than sedated condition (Fig. 1). Thus, the present data demonstrated for the fi rst time in nonhuman primates (NHP) that anesthesia can blunt the normal physiological responses to MMTT, which may be attributed by attenuation of entero-insular axis with slow gut movement and reduced nutritional absorption, hence inhibition of incretin hormone secretion. In addition, the diminished MMTT responses in DM NHPs may also indicate an attenuated entero-insular axis.

& 1932-PsnoRNA Loss-of-Function Potentiates Glucose-stimulated Insulin SecretionJIYEON LEE, MARIA S. REMEDI, JANA MAHADEVAN, FUMIHIKO URANO, JEAN E. SCHAFFER, St. Louis, MO

Reactive oxygen species (ROS) participate in signaling cascades that regulate insulin secretion. Recent studies implicate small nucleolar RNAs (snoRNAs) from the rpL13a locus in regulation of ROS responses. To examine the physiological role of these non-coding RNAs, we generated a knockin mouse model with loss-of-function of the 4 intronic snoRNAs from this locus (snoless). Fasting plasma glucose and insulin levels were indistinguishable between wild type (WT) and snoless mice; however, intraperitoneal glucose tolerance testing (GTT) demonstrated improvement of glucose tolerance in snoless mice (area under curve for WT vs. snoless: 7733 477 vs. 5293 558 mg/dL-min, p < 0.01). Serum insulin levels were higher in snoless mice 30 minutes following glucose challenge, suggesting enhanced insulin release in response to glucose (WT vs. snoless: 418 29.96 vs. 531 36.29 pg/ml, p < 0.05). While -cell mass was similar in the two genotypes, islets isolated from snoless mice showed lower basal ROS tone, resistance to ROS stimuli, and enhanced glucose-stimulated insulin secretion (WT vs. snoless: 596.05 105.35 vs. 957.11 32.69 pg/ml, p < 0.05). Hence, our studies reveal an impor-tant and unpredicted role for snoRNAs in the regulation of insulin secretion and suggest that these non-coding RNAs could be novel targets for improv-ing glucose homeostasis.

& 1933-PGlucagon Is a Key Factor in Clock Gene Regulation of Glucose MetabolismSIRI MALMGREN, BO AHRÉN, Lund, Sweden

Metabolic regulation of glucose homeostasis is partly controlled by os-cillations in the transcription of concurrent CLOCK genes. Their main syn-chronizer is light; however, they are also affected by nutritional intake and hormones. Since dysregulation of glucagon accompanies disrupted insulin secretion in type 2 diabetes, we asked how glucagon regulation is affected by circadian rhythm and if regulation of clock genes by glucagon is crucial for normal glucose homeostasis.

We therefore performed oral meal challenges in wt and glucagon recep-tor knock-out mice (GCGR-/-) and measured glucagon, glucose and insulin in

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plasma at zeitgeber times (ZT) 3 (3h after start of 12h/12h light/dark cycle; light phase) and ZT15 (dark phase). Upcoming experiments include analysis of CLOCK gene expression in liver and islets, liver glycogen content analysis and hyperinsulinemic hypoglycemic clamp.

We found a marked difference in basal glucose (4.2±0.2 vs. 7.4±0.3 mmol/l, p<0.0001), peak glucose (8.0±0.4 vs. 13.6±0.6 mmol/l, p<0.0001), and glucose-stimulated insulin (675±176 vs. 2249±536 pmol/l, p<0.0001) in GCGR-/- compared with wt animals at ZT3. In contrast, there was no signifi -cant difference in glucose or insulin levels between groups at ZT15. When applying the quantitative insulin sensitivity check index (QUICKI), we found that GCGR-/- differ greatly from wt in insulin sensitivity at ZT3 (0.97±0.11 vs. 0.47±0.02, p<0.0001) but not at ZT15 (0.70±0.02 vs. 0.65±0.03, p=0.81). The glucagon response 5 minutes after meal ingestion was signifi cantly higher at ZT3 than at ZT15 (5.2±1.0 vs. 4.2±1.0, p=0.04) in wt animals.

We conclude that glucagon signaling is of important for the circadian glu-cose rhythm since there is a more pronounced glucagon response during the light than during the dark phase. This is further highlighted by the fact that GCGR-/- mice differs in glucose dynamics from wt animals only during the light phase. Upcoming experiments will reveal mechanisms of this glucagon regulation of circadian rhythm and CLOCK genes.

Supported By: Kungliga Fysiografen i Lund

& 1934-PAldosterone Excess State Causes a Chronic Infl ammation in the Pancreatic IsletRIEKO GOTO, TATSUYA KONDO, SAYAKA KITANO, KAORU ONO, RINA MAT-SUYAMA, MOTOYUKI IGATA, JUNJI KAWASHIMA, TAKESHI MATSUMURA, HIROYUKI MOTOSHIMA, SEIYA SHIMODA, EIICHI ARAKI, Kumamoto, Japan

Primary aldosteronism (PA), often accompanies impaired glucose toler-ance (IGT) or diabetes. We previously reported at 73rd ADA that IGT or dia-betes were apparent in 76% of 58 patients with PA. This study was designed to understand the detailed underlying mechanism of IGT caused by excess state of aldosterone (Ald).

For this study, SD rats were given 1% NaCl in drinking water and randomly placed in one of the following 28 day treatments: 1) C: vehicle (n=6), 2) Ald: 0.75 µg/hr Ald (n=6), 3) Ep: Ald infusion with 100 mg/kg/day oral eplerenone (Ep) (n=6). Physical, biochemical and histological analysis were performed. Gene and protein expression in islets were measured by qRT-PCR and West-ern blotting, respectively.

In the experiments, insulinogenic index was signifi cantly lower in Ald or Ep compared to C. Adjustment of serum potassium by low potassium fed for C did not alter the result, suggested that low insulin secretion was not induced by reduced potassium level. Islet/pancreas area ratio was reduced in Ald as compared to C, which was completely restored in Ep. Pro-infl ammatory M1 Macrophages (MΦ) were accumulated to the islets in Ald. Since IL-6 was reported to enhance insulin secretion by increasing GLP-1 secretion from α-cells, we examined IL-6 and GLP-1 in α-cells. IL-6 mRNA and protein ex-pression were signifi cantly increased in Ep treated α-cells. Furthermore in Ep, GLP-1 was expressed in α-cells, and plasma glucagon concentration was suppressed. In addition, ER stress related genes in islets were signifi cantly reduced in Ep.

Taken together, a state of excess aldosterone may cause islet chronic in-fl ammation with accumulation of M1 MΦ, and induce apoptosis of β-cells. Ep may protect β-cells in part through the decrease of glucagon secretion by inducing IL-6 in α-cells.

1935-PImpaired Islet Mitochondrial Function and Biogenesis in the Cohen Diabetic RatAVIRAM KOGOT-LEVIN, ITAMAR RAZ, ANN SAADA, SARAH WEKSLER-ZANGEN, Jerusalem, Israel

Mitochondria plays a pivotal role in glucose stimulated insulin secretion (GSIS) by coupling glucose oxidation to ATP production. We demonstrated blunted GSIS associated with impaired mitochondrial morphology in hyperg-lycemic-Cohen diabetic sensitive rat (CDs) fed a diabetogenic diet (HSD). We examine islet-mitochondrial function and biogenesis as well as the differ-ential expression of mitochondrial biogenesis genes in relation to diabetes development. Normoglycemic-CDs fed regular-diet (RD) exhibited reduced mitochondrial complex IV activity and ATP content that decreased further-more in hyperglycemic-CDs (Fig. 1A). We found marked reduction in the ex-pression of genes involved in mitochondrial biogenesis in islets of Diabetic-CDs (Fig. 1B) while reduced mtDNA content and PGC-1α protein levels were observed on both diets (Fig. 1C&D). Thus, the genetic susceptibility of CDs partially explains the marked reduction in mitochondrial function and GSIS

observed in the diabetic-CDs fed HSD. These fi ndings emphasizes the role of both genetic susceptibility and harmful environment in the development of overt diabetes in Cohen rats.

Supported By: Joint Lower Saxony-Israeli Research Projects

1936-PRedundant Effect of GIP and GLP-1 on Insulin Secretion in Normal MiceGIOVANNI PACINI, BO AHRÉN, Padova, Italy, Lund, Sweden

Glucagon like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) augment glucose-stimulated insulin secretion. Since they are both released after meal ingestion, they might cooperate with a synergistic mechanism for stimulating a stronger incretin effect. The aim of this study was therefore to evaluate whether a combination of these two peptides elicits an augmented effect on glucose mediated insulin release.

Female C57BL/6 mice were injected intravenously with glucose alone (35mg/mouse; n=83), with glucose+GIP (n=27), with glucose+GLP-1 (n=35), or with glucose+GIP+GLP-1 (n=17). Dose of incretins was 3 nmol/kg. Beta cell function (BCF) was assessed as the ratio of the incremental 50 min area under the curve (ΔAUC) of insulin to that of glucose.

Insulin levels reached after glucose alone (AUC=12±0.4 nmol/l min) were potentiated by adding both GIP (20±2; P<0.0001) and GLP-1 (13±1; P=0.017). BCF was 21±1 nmol/mol after glucose alone and rose to 40±3 nmol/mol with GIP (P<0.0001) and to 26±1 nmol/mol with GLP-1 (P=0.0007). When both peptides were given together with glucose, insulin ΔAUC was signifi cantly higher than after glucose alone (7.4±1.8 vs. 4.0±0.5 nmol/l min; P=0.014), but not signifi cantly different from insulin ΔAUC after GLP-1 (6.2±0.8 nmol/l min) or GIP (10.9±1.4 nmol/l min) alone. Again, glucose tolerance was increased compared to glucose alone (P=0.002) and BCF was higher after the combina-tion than after glucose alone (33±3 versus 21±1 nmol/mol; P<0.0001) but not signifi cantly different from GLP-1 or GIP alone.

We conclude that both GIP and GLP-1 do not cooperate in their respective incretin effects after combined administration. This suggests that the incre-tin effect is not dependent on cooperative action of the incretin hormones and thus that the release of the two incretin hormones is redundant for the regulation of islet function.

1937-PCirculating MicroRNA Species and Role in Human Insulin Resist-anceELIZABETH MA, MINSUNG KANG, XIANGQIN CUI, WEI ZHANG, YUCHANG FU, W. TIMOTHY GARVEY, Birmingham, AL

Levels of microRNA species have been shown to be altered in tissues and plasma in metabolic disease models, although minimal data are avail-able regarding insulin resistance in humans. To assess this, plasma samples were obtained from non-diabetics classifi ed as insulin sensitive (IS) or insulin resistant (IR) based on hyperinsulinemic clamp. miRNA expression arrays were used to screen 3 different pooled samples of 5 from each group. 12 candidate small and miRNAs were identifi ed and further analyzed for as-sociations with markers of insulin resistance in 15 IS and 15 IR subjects. Certain species were correlated with clamp measures and/or fasting insu-lin, including miR-133a (clamp; r=0.38, p=0.04), snRNA U6 (r=0.31, p=0.09), miR-133a (fasting insulin; r=-0.47, p=0.01), miR-34a (r=-0.37, p=0.05), miR-150 (R=-0.46, p=0.01), and SNORD61 (r=-0.51, p<0.01). Other miRNAs were

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correlated with triglycerides, including miR-107 (r=0.46, p=0.01), miR-222 (r=0.39, p=0.03), miR-33 (r=0.39, p=0.04), miR-140 (r=0.35, p=0.06), and miR-199a (r=0.43, p=0.02). Furthermore, relative levels of mi/snRNAs associated with IR were correlated with one another (U6, 133a, 150, and SNORD61), while miRNAs associated with elevated triglycerides were also inter-related (107, 222, 33, 140, and 199a).

In 1) Different circulating mi/snRNA species are correlated with measures of insulin sensitivity and triglycerides in humans; 2) miRNAs associated with IR and elevated triglycerides are separately inter-correlated. The data sug-gest that different subsets of miRNAs are involved in the development of insulin resistance versus dyslipidemia in cardiometabolic disease, and point to novel markers and potential therapeutic targets.

Supported By: U.S. Department of Veterans Affairs; National Institutes of Health (DK-038765, DK-083562); University of Alabama at Birmingham Diabetes Research Center (P30DK079626)

1938-PIntravenous and Oral Glucose Tolerance Tests-Derived Measures in Nondiabetic IndividualsSTEVEN M. HAFFNER, CARLOS LORENZO, MARKKU LAAKSO, San Antonio, TX, Kuopio, Finland

Insulin secretion is already compromised prior to the onset of diabetes. We hypothesized that onset of secretory decline is infl uenced by the as-sessment of both insulin secretion and its relationship with plasma glucose. We examined this issue in the EUGENE2 Kuopio cohort (263 non-diabetic Finnish offspring of type 2 diabetic individuals aged 24-50 years). Insulin sensitivity was directly measured by the hyperinsulinemic clamp. Insulin secretion was estimated from the fasting state (homeostasis model assess-ment [HOMA]-β cell) and oral (insulinogenic index) and intravenous glucose tolerance tests (IVGTT early and late phases). HOMA-β cell and IVGTT late phase were directly related to 2h glucose (p <0.001 for both), whereas IVGTT early phase increased within the normal range of 2h glucose (p=0.025) and decreased afterwards (p=0.050). After the adjustment for insulin sensitivity, IVGTT early phase was inversely related to 2h glucose (p=0.002). However, unadjusted and adjusted insulinogenic index had a strong and inverse rela-tionship with 2h glucose (p=0.002 and <0.001, respectively). In summary, the insulinogenic index may detect an early secretory defect within the normal 2h glucose range.

Supported By: Academy of Finland (124243)

1939-PExtrahepatic Cholestasis Is Associated with Reduced Beta-Cell Function in Nondiabetic SubjectsTERESA MEZZA, VINSIN ALICE SUN, SIMONA MOFFA, GIAN PIO SORICE, CA-TERINA CONTE, CHIARA MARIA ASSUNTA CEFALO, ANDREA MARI, ANDREA GIACCARI, Rome, Italy, Padova, Italy

Several studies have shown that cholestasis is associated with altered glucose tolerance. However, the relationship between cholestasis and β-cell function has not been fully evaluated in the clinical setting. To investigate whether cholestasis, as evidenced by hyperbilirubinemia, affects β-cell function and insulin secretory response, we performed OGTT and hyperg-lycemic clamps (HC) followed by arginine stimulation in 44 patients (27 F/17 M, 51±15 yrs) scheduled for pancreatoduodenectomy for periampullary dis-eases, all without known history of type 2 diabetes (T2D). Based on bilirubin levels (BL), subjects were divided into 2 groups: with resolved cholestasis and/or normal BL (NChol, n=21, BL: 0.29±0.03 mg/dl) and with active extra-hepatic cholestasis (Chol, n=23, BL: 4.30±0.69 mg/dl). Amylasemia was simi-lar between the 2 groups (76.6±15.8 vs. 86.9±10.3UI/L). To evaluate β-cell function, β-cell glucose sensitivity (GS) during HC was calculated as the ratio of insulin secretion (IS) and glucose increments. Chol group displayed a signifi cantly lower Insulinogenic index (II) (0.46±0.06 vs. 0.76±0.11, p=0.01),

while no differences were detected in the Matsuda index (5.89±0.88 vs. 4.66±0.67, p=NS). The incremental 1st phase (p=0.01), 2nd phase IS (p=0.02) and GS (57.5±10.5 vs. 92.3±11.3 pmol·min-1·m-2·mM-1, p=0.03) were signifi -cantly lower in Chol group. Analysis of the entire group revealed an inverse correlation between BL and II (r=-0.40; p<0.05), and Arginine-stimulated IS (AIS) (r=-0.51; p<0.01). Our data indicate that cholestasis associates with im-paired β-cell function and possibly reduced mass (as estimated by AIS). We speculate that, in subjects with extrahepatic cholestasis, impaired GS could represent a major determinant of the observed insulin secretory defect in response to both glucose and arginine stimulus. Further investigation of this mechanism might improve understanding of the pathogenic events leading to altered insulin secretion in T2D.

1940-PImpact of 1-Hour Postchallenge Glucose on the Relationship be-tween Insulin Sensitivity and SecretionYUKA SATO, RIE OKA, YASUTO NAKASONE, KEISHI YAMAUCHI, TORU AIZAWA, Matsumoto, Japan, Toyama, Japan

The impact of postchallenge glucose on the hyperbolic function between insulin sensitivity (SI) and secretion (β) is unknown. Subjects with NGT (n=1,623), nondiabetic hyperglycemia (NDH) (n=555), or DM (n=86) were ana-lyzed using OGTT indices of SI (ISIMatsuda, 1/HOMA-IR and 1/fasting IRI) and β (insulinogenic index, Stumvoll-1 (ST-1) and -2). Of the nine SI-β pairs, the 1/HOMA-IR - ST-1 combination best recapitulated the hyperbolic SI-β relation-ship, with a fi tted line slope exactly -1.00 (log-linear standardized major axis regression, SMATR ver 2.0); this combination was subsequently utilized. Di-viding the NGT subjects according to 1hPG quartiles (Q) disrupted the hyper-bola, and the fi tted line for the SI-β correlation steepened progressively for Q1-Q4: the slopes (95% CI) were -0.66 (-0.73 to -0.61), -0.68 (-0.75 to -0.62), -0.85 (-0.92 to -0.78), and -1.26 (-1.37 to -1.16) (p trend <0.05). For NDH and DM, the slopes were -1.55 and -1.92, respectively. The median SI and β were also progressively lower from NGTQ1 to Q4 and from NDH to DM, with a predominant attenuation of β. In summary, an elevated 1hPG was associated with steepening and downward shifting of the SI-β regression line, indicat-ing an impaired beta cell response to an attenuated SI among subjects with an elevated 1hPG. This may represent primary beta cell dysfunction, beta cell insult from a low level of hyperglycemia, or a combination thereof.

Supported By: Japan Ministry of Health, Labour and Welfare

1941-PDefi ning the Pathophysiology of Low Body Mass Index DiabetesAKANKASHA TIWARI, RIDDHI DAS GUPTA, ANNEKA WICKRAMANAYAKE, MICHELLE CAREY, PADMANABAN V, MOHAN JAMBUGULAM, CHAITHANYA KOCHERLAKOTA, HARSHA JAYATILLAKE, DANIEL T. STEIN, NIHAL J. THOMAS, MEREDITH HAWKINS, New York, NY, Vellore, India, Bronx, NY

Millions of individuals with low body mass index (BMI) globally have diabe-tes of unclear etiology. These include patients with Fibrocalculous Pancreatic Diabetes (FCPD) and Lean Diabetes (LD), defi ned by the presence or absence of pancreatic calcifi cations on ultrasound. We present the fi rst studies using gold-standard methodologies to assess insulin secretion in FCPD and LD, com-pared to type 1 (T1D), type 2 diabetes (T2D) and nondiabetic (ND) subjects.

Mixed-meal tolerance tests (MMTT) with C-peptide deconvolution were performed in n=22 Indian males with FCPD (age 30±2 y, BMI 19.7±0.6 kg/m2, HbA1c 9.0±0.3%) and n=6 with LD (age 36±4 y, BMI 18.3±0.1 kg/m2, HbA1c 11.6±1.3%), and compared with n=12 age, BMI matched ND, n=16 T1D (HbA1c 9.1±0.3%) and n=12 T2D subjects (age 36±2 y, BMI 26.0±0.3 kg/m2, HbA1c 9.7±0.6%). Therapeutic regimens were intensifi ed for two weeks to correct glucose toxicity and match fructosamine levels.

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Glucose and C-peptide responses to MMTT suggest subjects with FCPD (14.5± 2.2 pmol/kg/min) and LD (15.0 ± 2.9 pmol/kg/min) have markedly im-paired insulin secretion relative to both ND and T2D (p<0.001), and not sta-tistically different from T1D (Fig. 1). Thus, both in the presence and absence of pancreatic calcifi cations, patients with low BMI diabetes have compara-bly decreased insulin secretion, consistent with nutritional effects on beta cell development or function. This has signifi cance for millions of patients worldwide.

1942-PEffects of Dietary-induced Weight Loss on Cardiovascular Risk Fac-tors and HDL Functionality in Modestly Obese African American and White American Women with PrediabetesTRUDY GAILLARD, KWAME OSEI, Cincinnati, OH, Columbus, OH

In the current study, we investigated the effects of 6 month-dietary-induced weight loss (DIWL) on cardiovascular risk factors, glucose homeo-stasis and HDL functionality (paraoxonase1 {PON1}) in modestly obese Af-rican American (AA) and White American (WA) women with prediabetes. One hundred and eight modestly obese women were recruited (67 AA and 41 WA; mean BMI 37.8±6.3kg/m2) with prediabetes. We performed meta-bolic studies at 0, 3 and 6 months. Corresponding cardiovascular risk factors (lipids/lipoproteins, infl ammatory marker {c-reactive protein}, oxidized-LDL, interlukin-6, adiponectin) and PON1 were measured. Insulin sensitivity index (Si), glucose effectiveness (Sg), acute insulin response to glucose (AIRg) and disposition index (DI) were obtained (Bergman’s Minmod). The weight loss program consisted of a hypo-caloric formula (~1200 kcal/day) and lifestyle modifi cation education. The mean BMI was greater in AA than WA (38±8 vs. 34±8kg/m2). DIWL resulted in 12% and 22% (-5.5kg vs. -9.6kg) weight loss, respectively. Mean fasting and 2hr serum glucose, insulin and c-peptide lev-els decreased signifi cantly at 3 and 6 months vs. 0 month in AA and WA. Mean Si, Sg, AIRg, and DI did not improve in either group. Despite identical Si, serum triglycerides were signifi cantly lower in AA than WA at baseline and during DIWL. However, the modest weight loss had no signifi cant ef-fects on lipids and lipoproteins and cardiovascular risk factors in AA and WA. In conclusion, we found ethnic differences in the magnitude of weight loss in AA and WA. Modest weight loss had only minimal impact on glucose homeostasis, cardiovascular risk factors and HDL functionality in modestly obese AA and WA women with prediabetes.

Supported By: American Diabetes Association (1-11-CT-39 to K.O.)

1943-PDecreased Expression of Genes Regulating Glucose Stimulated In-sulin Secretion in the Cohen Diabetic RatSARAH WEKSLER-ZANGEN, ITAMAR RAZ, DEVORAH SOIFERMAN, AVIRAM KOGOT-LEVIN, Jerusalem, Israel

The Cohen-diabetic sensitive rat (CDs) fed a diabetogenic diet (HSD) ex-hibit diabetes due to a selective impairment in glucose stimulated insulin

INTEGRATED PHYSIOLOGY—LIVER

secretion (GSIS). Recently we detected a 50% reduction in GSIS of CDs fed a normal diet (RD), prior to diabetes, suggesting a genetic impairment in β cell function. This study aims to defi ne the expression of genes that regulate the GSIS pathway in CDs-islet. We found >50% decrease in the expression of PDX1, Insulin, GLUT-2 and Glucokinase genes in islets of normoglycemic-CDs fed RD (Fig. 1). Following HSD feeding, upon diabetes onset, only low residual expression levels remained. Similarly, expression of K-ATP channel subunits ABCC8 and KCNJ11 decreased in CDs fed RD, further reducing on HSD (Fig. 1). Thus, expression of genes regulating glucose-sensing and secretion-coupling are both reduced in normoglycemic-CDs. Exposure to the diabetogenic diet prominently diminish the expression of these genes and GSIS resulting in hyperglycemia. The Cohen rat model demonstrate the role of genetic susceptibility and harmful environment in the development of overt diabetes.

Supported By: Joint Lower Saxony-Israeli Research Projects


Guided Audio Tour: Liver Metabolism In Vivo (Posters: 1944-P to 1951-P), see page 13.

& 1944-PA Quantitative, Mass Spectrometry-based Platform for Phenotyp-ing Mouse Liver Metabolic Flux In VivoCLINTON M. HASENOUR, MARTHA L. WALL, D. EMERSON RIDLEY, CURTIS C. HUGHEY, FREYJA D. JAMES, DAVID H. WASSERMAN, JAMEY D. YOUNG, Nash-ville, TN

Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, we developed a scalable method to quantitatively assess hepatic glucose and intermediary metabolism in con-scious, unrestrained mice. 13C and 2H isotopic tracers were delivered intra-venously in chronically catheterized, short (9 hr) and long-term (19 hr) fasted C57BL/6J mice. GC-MS, mass isotopomer distribution (MID), and metabolic fl ux analysis (MFA) were performed on three, 40 µL plasma glucose samples obtained from arterial circulation of individual mice during the euglycemic, isotopic steady state. As anticipated, glucose-6-phosphate (G6P) derived from glycogen diminished and endogenous glucose production (EndoRa) was exclusively gluconeogenic in prolonged fasting. Gluconeogenic fl ux from phosphoenolpyruvate (PEP) remained stable while that from glycerol expe-rienced a slight increment in the transition from a short to long-term fast. Citric acid cycle (CAC) fl ux (i.e., citrate synthase (CS)) reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast dura-tion; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were several fold higher than CS fl ux in long-term fasted mice. The method presented here capitalizes on state-of-the-art in vivo methodology and fl ux modeling to quantify the effect of a basic physiological stress on hepatic glucose and intermediary fl uxes in mice. The small plasma require-ments limit stress, decrease sampling error, and affi rm steady state glucose kinetics within each study. The versatile modeling approach utilized here also serves as a predictive and diagnostic tool for optimizing future in vivo inquiry.

Supported By: National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases (DK59637, DK50277, DK076169, DK07563)

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& 1945-PThe Mitochondrial Pyruvate Carrier Regulates Hepatic Gluconeo-genesisKYLE S. MCCOMMIS, ZHOUJI CHEN, XIAORONG FU, WILLIAM G. MCDONALD, JERRY R. COLCA, ROLF F. KLETZIEN, SHAWN C. BURGESS, BRIAN N. FINCK, St. Louis, MO, Dallas, TX, Kalamazoo, MI

Pyruvate is a major substrate for mitochondrial oxidative metabolism and for hepatic gluconeogenesis. Gluconeogenesis from cytosolic pyruvate re-quires pyruvate transport across the inner mitochondrial membrane into the matrix. Mitochondrial pyruvate carrier 1 and 2 (MPC1 and MPC2) proteins me-diate mitochondrial pyruvate import and have emerged as targets for insulin sensitizing drugs. To evaluate the effects of MPC2 defi ciency on gluconeo-genesis, mice harboring a conditional allele of Mpc2 were generated. Liver-specifi c MPC2 knockout (LS-MPC2-/-) mice were viable and outwardly normal. Consistent with the concept that gluconeogenesis from pyruvate requires mi-tochondrial pyruvate import, LS-MPC2-/- mice exhibited lower blood glucose concentrations during pyruvate tolerance tests, after prolonged fasting, or following streptozotocin-induced insulin defi ciency. Isolated hepatocytes from LS-MPC2-/- mice produced less glucose and had lower incorporation of 13C-labeled pyruvate into TCA cycle intermediates compared to WT hepatocytes. An MPC inhibitor (UK5099) and an insulin-sensitizing drug known to bind the MPC (MSDC-0602) attenuated glucose production by isolated hepatocytes in an MPC2-dependent manner. Residual pyruvate metabolism in LS-MPC2-/- hepatocytes or after MPC inhibitor was mediated by pyruvate conversion to alanine, which can be transported into the mitochondrial matrix independently of the MPC and then transaminated back to pyruvate. Indeed, alanine-stimu-lated gluconeogenesis was normal in LS-MPC2-/- mice. Inhibitors of alanine aminotransferase (ALT), which interconverts pyruvate and alanine, markedly attenuated glucose production and 13C-labeled pyruvate fl ux in LS-MPC2-/-, but not WT mice. Finally, adenovirus-mediated suppression of ALT2 by shRNA further reduced blood glucose concentrations in LS-MPC2-/- mice after bolus pyruvate injection. Altogether, these data are consistent with an important role for the MPC in regulating hepatic gluconeogenesis.

& 1946-PPriming Effect of a Morning Meal on Glucose DisposalMARY C. MOORE, MARTA SMITH, GUILLAUME KRAFT, MASAKAZU SHIOTA, BEN FARMER, DOSS NEAL, PHIL WILLIAMS, DALE S. EDGERTON, ALAN D. CHER-RINGTON, Nashville, TN

Meal tolerance has been found to improve over the course of the day in some but not all studies. Given the importance of the liver in meal tolerance, such a fi nding would suggest that a morning meal might prime the liver to extract more glucose during subsequent same-day meals. We used hepatic balance and tracer (3H-glucose) techniques to examine this in chronically catheterized conscious dogs. After an overnight fast, the dogs received a 4h duodenal infusion of glucose (10 mg.kg-1.min-1; CHO group, n=6) or normal saline (SAL group; n=6), followed by a 2h post-infusion fasting period and then a 4h clamp period with infusions of somatostatin; intraportal insulin (4xbasal), glucagon (basal), and glucose (4 mg.kg-1.min-1); and peripheral venous glucose to maintain arterial glucose at 200 mg/dL, thus increasing the hepatic glucose load to 2xbasal. Deep venous plasma insulin (26±8 vs. 6±1 µU/mL) and glucose (147±6 vs. 117±3 mg/dL) concentrations were higher (P<0.05) during duodenal infusion of CHO vs. SAL, respectively. During the clamp, arterial plasma glucose was 199±2 mg/dL in both groups. Net hepatic glucose uptake during the clamp was 80% higher in CHO vs. SAL (5.8±0.7 vs. 3.2±0.3 mg.kg-1.min-1; P<0.05) and unidirectional (tracer-determined) hepatic glucose uptake was 70% greater (4.6±0.7 vs. 2.7±0.2 mg.kg-1.min-1; P<0.05). In terminal liver biopsies, glycogen concentrations were 40% greater (P<0.05), glycogen synthase activity tended to be higher (0.04±0.01 vs. 0.02±0.01 µmol/g liver/min; P=0.11), and glycogen phosphorylase activity was lower (0.18±0.05 vs. 0.37±0.10 µmol/g liver/min, P<0.05) in CHO vs. SAL. Thus morning glucose administration markedly facilitated hepatic glucose uptake and storage later in the same day. It remains to be determined whether the priming effect on the liver is dependent on prior hyperglycemia, hyperinsu-linemia, or both.

Supported By: National Institutes of Health (DK018243)

& 1947-PIn Vivo Identifi cation of Phospho-Serine/Threonine (pS/T) Motifs on IRS1 Upregulated by Hepatic mTOR Complex 1 (mTORC1) ActivityKYLE D. COPPS, NANCY HANCER, MORRIS F. WHITE, Boston, MA

Insulin activates S/T kinase pathways that, in cultured cells, can “feed back” to promote or impair signaling through their upstream regulators, the

insulin receptor substrates IRS1 and IRS2. While S/T-phosphorylation of the IRS presents an attractive mechanism for regulating insulin sensitivity, the large number of S/T residues in each protein has complicated assessment of their function in tissues. We used a panel of 25 mono-specifi c antibodies to identify insulin-stimulated pS/T common to Irs1 in liver, skeletal muscle, and cardiac muscle of i.v. insulin-stimulated mice. These include pS265 and pS302 (motif: RxRxxpS/T) and pS307 (pS/TP), each of which can be phosphorylated by the mTORC1 S6 kinase (S6K) pathway. We activated mTORC1 in liver of Irs1 A302 (AA) and control S302 (SS) knock-in mice using Cre adenovirus-me-diated deletion of Tsc1. Consistent with earlier studies, Tsc1 deletion in these mixed-background mice (B6x129) caused liver enlargement—though not hy-perinsulinemia that would indicate compensation for insulin resistance. Glu-cose tolerance was also unimpaired by transduction with Cre (versus control lacZ) adenovirus in AA and SS mice, despite moderate impairment of hepatic Akt activation by insulin in both. Loss of Tsc1, and consequent elevated basal S6k activity, did not upregulate putative S6k (RxRxxpS/T) sites in either AA or SS liver, but did stimulate pS307 and other pS/TP sites. In both AA and SS primary hepatocytes, partial Tsc1 loss activated mTORC1, and reduced the insulin-stimulated binding of PI3 kinase (p110) to IRS1. This result is compat-ible with—but does not formally prove—a net negative effect of mTORC1-induced IRS1 phosphorylation at sites other than S302. We conclude that, in the hepatic compartment, normal to elevated mTORC1 activity in itself has little ability to dysregulate systemic glucose homeostasis, despite demon-strable negative effects upon signaling through IRS1 and Akt.

& 1948-PEffects of Glucagon-Like Peptide-1 on Glucagon Secretion, Endog-enous Glucose Production, and Whole Body Lipolysis in Patients with Nonalcoholic Fatty Liver DiseaseANDERS E. JUNKER, LISE L. GLUUD, GERRIT V. HALL, JENS J. HOLST, FILIP K. KNOP, TINA VILSBØLL, Hellerup, Denmark, Hvidovre, Denmark, Copenhagen, Den-mark

We evaluated the glucagon-suppressive effect of glucagon-like peptide-1 (GLP-1) and its potential effects on endogenous glucose production and whole body lipolysis in non-diabetic patients with non-alcoholic fatty liver disease (NAFLD).

Ten non-diabetic patients with biopsy-verifi ed NAFLD (NAFLD activity score 2.5±1.0) and 10 matched controls underwent a 2-hour intravenous GLP-1 (0.8 pmol × kg-1 × min-1) and placebo infusion on separate days. Since GLP-1-mediated glucagon suppression has been shown to be glucose-dependent, plasma glucose was clamped at fasting level during the fi rst hour, then raised and clamped at “postprandial level” (fasting plasma glucose level plus 3 mmol/L) for the remaining hour. We evaluated relative plasma levels of glucagon, endogenous glucose production and whole body lipolysis rates with stable isotopes and respiratory quotient using indirect calorimety.

Compared to controls, patients with NAFLD were insulin resistant (ho-meostatic model assessment (HOMAIR): 3.8±2.2 vs. 1.6±1.5, P=0.003) and had fasting hyperglucagonemia (7.5±5.3 vs. 5.8±1.5 mmol/L, P=0.045). Simi-lar relative glucagon suppression was seen in both groups during GLP-1 infu-sion at fasting (-97±75 vs. -93±41 pmol/L × min-1 P=0.566) and “postprandial” plasma glucose levels (-108±101 vs. 97±53 pmol/L × min-1, P=0.196). Patients with NAFLD had impaired GLP-1-induced suppression of endogenous glu-cose production at fasting and “postprandial” glucose levels and impaired elevation of respiratory quotient during “postprandial” glucose levels.

Patients with NAFLD exhibited fasting hyperglucagonemia, but intact GLP-1-mediated glucagon suppression independently of plasma glucose concentrations. Preserved glucagonostatic effect of GLP-1 in patients with NAFLD may be important to sustain normoglycemia.

& 1949-PA Fasting-induced LncRNA Regulates Hepatic and Systemic Glu-cose MetabolismXIANGBO RUAN, PING LI, LING YANG, ANDREW CANGELOSI, HAIMING CAO, Bethesda, MD

Long non-coding RNAs constitute a signifi cant portion of the mamma-lian transcriptome and have been implicated in diverse processes in cells. However, their physiological signifi cance remains poorly understood. Here we report a fasting-induced lncRNA (lncLGR) that regulates the activities of glucose kinase (GCK), a known diabetic gene and a key regulator of he-patic glucose fl ux. We demonstrate that lncLGR expression is suppressed by insulin in primary hepatocytes and in diabetic mice. Hepatic depletion of lncLGR in mice induces substantial accumulation of glycogen in the liver. Gene expression analysis in glycogen synthesis pathway shows that lncLGR depletion enhances hepatic GCK expression and activities, which is mani-

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fested by increased glucose-6-phosphate (G6P) content in liver. Consisently, lncLGR over-expression suppresses GCK expression and leads to reduced contents of glycogen and G6P in the liver. Mechanism study shows that lncL-GR specifi cally binds to hnRNPL, a RNA binding protein (RNP) with no known role in regulating glucose metabolism. We further demonstrate that hnRNPL directly binds to GCK promoter and suppresses its activity. Furthermore, HnRNPL knockdown induces GCK expression, and this effect is completely blocked by lncLGR over-expression arguing that their synergistic actions are required for regulating GCK transcription in vivo. Taken together, our data reveals a novel regulatory mechanism of hepatic glucose fl ux where a fast-ing-induced lncRNA suppresses GCK expression through a RNP, providing the fi rst evidence that a lncRNA-RNP complex critically regulates systemic glucose metabolism.

Supported By: National Institutes of Health (HL006103-02)

& 1950-PRenal Sympathetic Denervation Normalizes Hepatic Insulin Sensi-tivity in Obese Insulin Resistant CaninesMALINI S. IYER, ORISON O. WOOLCOTT, JEREMY E. KORMAN, VIORICA IONUT, MORVARID KABIR, RICHARD N. BERGMAN, CATHRYN KOLKA, Los Angeles, CA

Activation of the sympathetic nervous system is associated with obesity and insulin resistance. Renal Sympathetic Denervation (RDN) is a clinical procedure that has been used to treat drug-resistant hypertensive patients. Clinical RDN has also been suggested to improve glucose metabolism and insulin sensitivity (SI), although the mechanism is unknown. To determine whether RDN improves insulin sensitivity, and to study the mechanism of improvement we performed surgical bilateral RDN in obese normotensive insulin resistant dogs.

Dogs were fed high fat diet for 6 weeks (HFD, n=4) to induce insulin re-sistance. SI was measured using euglycemic hyperinsulinemic clamp before (w0) and after HFD. As expected, HFD reduced whole body SI (8.9±0.6 * 10-4 dLkg-1min-1pM-1 at w0 to 6.3±0.8 * 10-4 dLkg-1min-1pM-1 at HFD, P<0.05) as well as hepatic SI (-1.4±0.4 * 10-4 dLkg-1min-1pM-1 at w0 to 0.2±0.4 * 10-4 dLkg-1min-1pM-1 at HFD, P<0.05). Bilateral surgical RDN was performed at the end of the HFD by cutting all visible nerves along the renal artery and painting the renal artery with 10% phenol in ethanol solution. Animals were allowed to recover for 10 days; denervation was confi rmed by measuring noradrenaline in the renal cortex (right renal cortex: 97.4±56 ng/g in den-ervated vs. 279.9±68.3 ng/g in sham, left renal cortex: 67.4±6 ng/g in den-ervated vs. 278.9±115.6 ng/g in sham). No changes in blood pressure were observed with HFD and RDN. However, despite continued HFD, hepatic insu-lin sensitivity was totally normalized by RDN (-1.3±0.2 * 10-4 dLkg-1min-1pM-1 at RDN, P<0.05 vs. HFD, P=ns vs. w0). Bilateral Renal Denervation improves sensitivity to insulin, independent of changes in the blood pressure, primar-ily by reversing diet-induced hepatic insulin resistance. These data support crosstalk between renal sympathetic nerves and hepatic glucose regulation. Pathways involved remain to be established.

Supported By: R01DK029867, 5R37DK027619

& 1951-PInsulin Pattern during the FSIGT Accurately Refl ects Liver Insulin DegradationISAAC ASARE BEDIAKO, REBECCA L. PASZKIEWICZ, MIGUEL BURCH, RICHARD N. BERGMAN, STELLA P. KIM, Los Angeles, CA

Hyperinsulinemia is a critical factor in maintaining glucose tolerance dur-ing insulin resistance. While increased insulin secretion can play a role in hyperinsulinemia, evidence continually supports dynamic changes in hepatic insulin clearance as a major factor in compensatory plasma insulin. Thus it is of paramount importance to establish an accurate method to measure insulin clearance. Liver insulin clearance is often indirectly assessed via the hyperinsulinemic euglycemic clamp (EGC) or the frequently sampled intra-venous glucose tolerance test (FSIGT). To evaluate these methods, we com-pared them in the canine model to a direct method which requires access to the hepatic portal vein. Using the dog model, which allows for portal vein access, we measured insulin clearance directly, and from the EGC vs. FSIGT. Direct method: Paired portal/peripheral insulin infusion protocol (PPII) - in-sulin is infused at 3 rates into either the portal (one day) or peripheral vein (other day). Insulin clearance is accurately calculated as (1-mpo/mpe); m is the ratio of the slopes of insulin infusion vs. concentration for the 2 delivery routes. Indirect methods: 1) EGC: metabolic clearance rate (MCR) - ratio of the insulin infusion rate to steady-state plasma insulin level; 2) FSIGT: frac-tional disappearance rate of insulin (FCR) - determined by the rate of decline of plasma insulin after insulin injection. In a population of healthy dogs (n=4; insulin sensitivity (SI) range= 5.5-10.6 dL/kg/min/pM), FCR measured from

FSIGT had a strong correlation with the direct PPII method (r=0.64). Surpris-ingly, MCR (n=10; SI range=1.8-10.6) during the EGC was not signifi cantly correlated with hepatic extraction (r= 0.082). Thus, FCR from the FSIGT is an excellent measure of liver insulin clearance. However, MCR from the EGC may not be an acceptable surrogate measure of hepatic insulin clearance. Insulin clearance measured from the EGC may refl ect insulin disappearance from nonhepatic tissues, such as the kidneys and skeletal muscle.

1952-PDiscovery of In Vivo Regulators of Glucagon ActionWEI SONG, DAOJUN CHENG, SHANGYU HONG, PAVLOS PISSIOS, NORBERT PERRIMON, Boston, MA

In addition to insulin resistance, enhanced glucagon action leading to ex-cess hepatic glucose production is also associated to hyperglycemia and type 2 diabetes. Characterizing physiological regulators of glucagon signal-ing will provide novel insights of diabetes pathogenesis. We fi rst generated a glucagon-like-induced hyperglycemia fl y model that overexpresses AKH, glucagon homolog, and performed a large-scale in vivo RNAi screen of ki-nases and phosphatases according to circulating sugar level change. 46 novel kinases and phosphatases were identifi ed as physiological regulators of glucagon-like signaling. One of the top hits, BABO, the homolog of activin type I receptor ALK4/5 in mammal, was found to directly enhance AKH sig-naling via SMAD-dependent transcription of the AKH receptor in fat body, functionally equivalent to liver. Furthermore, we confi rmed that this regula-tion is conserved in the mammalian system. In mouse primary hepatocytes, ALK/SMAD signaling, activated by activin A or TGF-beta1, increases the glucagon receptor transcription, glucagon sensitivity and glucagon-induced hepatic glucose production. Particularly, this enhanced glucagon action can be potently abolished by ALK4/5 inhibitors. Taken together, we established a conserved hyperglycemia model to explore in vivo regulators of glucagon action and open up therapeutic opportunities for diabetes treatment.

Supported By: Howard Hughes Medical Institute

1953-PHepatic ATP Content Is Lower and Relates to Glycemic Control in Type 1 DiabetesSOFIYA GANCHEVA, ALESSANDRA BIERWAGEN, PAUL BEGOVATZ, JAN OLOF JESPER LUNDBOM, SABINE KAHL, PETER NOWOTNY, GUIDO GIANI, BARBARA HOFFMANN, JULIA SZENDROEDI, MICHAEL RODEN, Düsseldorf, Germany

Increased lipid availability and hyperglycemia associate with lower he-patic energy metabolism in insulin resistant patients with type 2 diabetes. Here, we examined the role of insulin resistance and hyperglycemia for he-patic energy metabolism in patients with type 1 diabetes (T1D). Forty-seven newly diagnosed T1D patients (age: 33.5±8.9 years; body mass index (BMI): 24.8±3.8 kg/m²) with known diabetes duration of less than one year under-went euglycemic-hyperinsulinemic clamps to assess whole-body (M-value) and hepatic insulin sensitivity (insulin-mediated suppression of endogenous glucose production). Hepatic fat content (HCL), ɣ-adenosine triphosphate (ɣATP) and inorganic phosphate (Pi) concentrations were quantifi ed by multinuclei magnetic resonance spectroscopy (31P/1H-MRS). Healthy hu-mans with comparable age and BMI served as control group (CON; n=53, age: 33.3±9.9 years; BMI: 24.1±2.6 kg/m²). The T1D patients featured near-normal glycemia (haemoglobin A1c; A1c: 6.2±1.0%) and whole-body insulin sensitivity (9.0±3.7 mg*kg-1*min-1), but had moderate hepatic insulin resis-tance (74±20%). T1D and CON showed comparable hepatic Pi (1.89±0.53 vs. 2.02±0.58 mmol/l) and HCL levels (1.4±2.8% vs. 1.3±2.2%). However, hepatic ɣATP concentrations were 15% lower in T1D (2.26±0.55 vs. CON: 2.65±0.55 mmol/l, P<0.001). In the entire study population, hepatic ɣATP related nega-tively to A1c (r=-0.23, P=0.03). Within the T1D group, neither hepatic ɣATP, nor Pi displayed correlations with whole-body and hepatic insulin sensitivity, whereas Pi related negatively to waist circumference (r=-0.33, P=0.02). In conclusion, T1D patients with short-duration of disease and good metabolic control already exhibit hepatic insulin resistance and lower hepatic energy metabolism. Interestingly, these abnormalities coexist in the absence of ste-atosis, but are not necessarily related.

Supported By: German Center for Diabetes Research; German Federal Ministry of Education and Research; German Federal Ministry of Health, Ministry of Innova-tion, Science and Research of the State of North Rhine-Westphalia

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1954-PPaternal Hyperglycermia Induces Transgenerational Inheritance of Susceptibility to Hepatic Steatosis Involving Altered Liver MethylomeXIAOQIU XIAO, YUYAO ZHANG, CHUAN PENG, SHUIQIN CHAI, YANG YU, XIAOP-ING WEI, HENG WANG, HONGYING WANG, QIFU LI, JIBIN LI, Chongqing, China

The prevalence of nonalcoholic fatty liver disease (NAFLD) rises dramati-cally recently. Previous studies showed that offspring of obese or diabetic dams developed abnormal liver lipid metabolism. It is unknown whether paternal metabolic abnormalities will affect offspring. Present study in-vestigated consequences of offspring born from hyperglycermic fathers by employing a single dose of streptozotocin (STZ). Male SD rats were injected ip with STZ, with citrate buffer (CB) as control. Blood glucose levels higher than 16 mM were selected to breed with normal female rats. Offspring from STZ or CB treated fathers (STZ-O and CB-O) were maintained in the same lit-ter size and weaned to standard chow diet. Body weight, glucose and insulin tolerance tests (GTT and ITT) were performed; hepatic histology, blood bio-chemical index and hormones were assayed; expression of factors involved in lipid metabolism was performed; MeDIP-sequencing was used to identify differential DNA methylation profi le in the liver of CB-O and STZ-O. STZ treatment induced a signifi cant hyperglycermia; beginning from 12-weeks of age, body weight of STZ-O was signifi cantly higher than CB-O; progres-sive impairment of GTT was observed in STZ-O compared with CB-O; ITT results showed decreased insulin sensitivity in STZ-O; histological obser-vation showed a higher degree of lipid accumulation and steatosis scoring in STZ-O, accompanied by upregulation of key components in lipogenesis, without altering lipid transport and oxidation pathways. MeDIP-sequencing assay demonstrated paternal hyperglycermia altered overall methylome patterns, with a large portion of differentially methylated genes focus on bile secretion, lipid and ketone metabolism, macromolecular localization by Gene Ontology and KEGG pathways analysis. Our study revealed that pa-ternal hyperglycermia predisposes offspring to developing NAFLD, which is possibly associated with altered liver methylome.

Supported By: National Basic Research Program of China (2012CB517505); National Natural Science Foundation of China (81270947)

1955-PGlucagon Regulation of Liver Adaptation to Nutritional Support Is PPARα-DependentYOLANDA F. OTERO, TAMMY M. BARNES, TERI D. STEVENSON, ALICIA D. LOCKE, CARLO MALABANAN, OWEN P. MCGUINNESS, Nashville, TN

Nutritional support (NS) helps meet metabolic demands of patients. Un-fortunately, hyperglycemia can develop in critically ill patients receiving NS, negatively impacting outcomes. We found NS increases FGF21 and enhanc-es the body’s glucose uptake capacity. However, elevated glucagon (GCG), observed in severely ill patients, reverses the enhanced glucose uptake. As PPARα regulates FGF21 and is a GCG target, we sought to determine if it is needed for the NS-induced increase in FGF21 and the ability of GCG to blunt the response to NS. Hyperinsulinemic-euglycemic clamps were performed in WT and PPARα knock-out (KO) mice that received NS (3 days), with or without chronic GCG infusion. The clamp glucose infusion rate (GIR) was lower in KO. Chronic GCG decreased GIR in WT but not in KO. The decreased GIR was due to persistent liver glucose production. In WT, GCG increased liver expression of a gluconeogenic gene (G6pc) and an inhibitor of pyruvate oxidation (Pdk4). In KO, G6pc was increased and was not amplifi ed by GCG. Further, GCG did not increase PDK4 in KO. Liver FGF21 was markedly sup-pressed in KO, and was not increased by GCG. In white adipose tissue (WAT), FGF21 and adiponectin were unaffected. No changes in peripheral glucose uptake were observed. Thus, the NS-adaptive response and the normal in-duction of FGF21 are PPARα-dependent. Moreover, PPARα contributes to GCG regulation of liver glucose metabolism during NS.

Table. Glucose Infusion Rate and Gene Expression.WT WT+GCG KO KO+GCG

Glucose Infusion Rate (mg/kgmin) 27±3 13±1 * 9±2 12±4Liver Fgf21 (Rq) 1.0±0.3 1.6±0.2 0.02±0.0 0.2±0.1†Liver G6pc (Rq) 0.6±0.1 11±2.4* 24.5±16 17.5±14Liver Pdk4 (Rq) 0.7±0.1 2.7±0.3* 0.6±0.3 0.4±0.05†WAT Fgf21 (Rq) 1±0.2 1.1±0.1 1.3±0.4 0.8±0.2WAT AdipoQ (Rq) 0.9±0.1 0.7±0.04 0.9±0.1 1±0.04†

*p †p

Supported By: DK43748, DK78188, DK059637

1956-PFructose Decreases Mitochondrial Fatty Acid Oxidation in Liver through Regulation of Protein Acetylation and SuccinylationSAMIR SOFTIC, HANS LAURITZEN, BRIAN O’NEILL, MENGYAO LI, JEREMIE BOUCHER, OLGA ILKAYEVA, MATTHEW RARDIN, ERIC M. VERDIN, BRADFORD W. GIBSON, CHRISTOPHER B. NEWGARD, C. RONALD KAHN, Boston, MA, Dur-ham, NC, Novato, CA, San Francisco, CA

Non-alcoholic fatty liver disease (NAFLD) is a frequent complication of obesity and metabolic syndrome, and is highly correlated with the level of in-sulin resistance. In the present study we assessed the effects of high carbo-hydrate diet and high fat diet (HFD) on obesity, insulin resistance and NAFLD development in C57Bl6/J mice using 6 different diets. Three groups were on normal chow, one with regular drinking water (Chow + H2O), the second with 30% fructose in drinking water (Chow + Fruct), and the third group with 30% glucose in drinking water (Chow + Gluc). The next three groups were all on high-fat diet and again on regular water (HFD + H2O), fructose (HFD + Fruct), or glucose (HFD + Gluc).

The HFD + Fruct group gained the most weight over 10 week study period, developed the most severe insulin resistance and had more advanced liver disease than all other groups. The HFD + Gluc group appeared to be protected from metabolic derangements as compared to the HFD +H2O group. The HFD + Fruct group also exhibited profound changes in mitochondrial morphology. This was associated with an increase in the acetylation and succinylation of the majority of mitochondrial proteins involved in fatty acid metabolism as assessed by mass spectrometry without any changes in levels of Sirt3 or Sirt5, the major mitochondrial deacetylase and desuccinylase. Succinylation of long chain acyl-CoA dehydrogenase at lysine 279 and succinylation of trifunctional enzyme subunit alpha at lysine 359 were increased by 16- and 6.2-fold, respectively, in HFD + Fruct group, while they were decreased by 50 and 35 percent in HFD + Gluc group, as compared to Chow. Thus, dietary fructose, but not glucose, alters post-translational modifi cation of enzymes involved in mitochondrial fatty acid metabolism, such that when mice are challenged with high-fat diet they develop accelerated obesity and a more severe form of fatty liver disease and insulin resistance.

Supported By: K12-HD000850

1957-PSaroglitazar Shows Therapeutic Benefi ts in Mouse Model of Nonal-coholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepa-titis (NASH)MUKUL R. JAIN, SURESH R. GIRI, BIBHUTI BHOI, CHITRANG TRIVEDI, RAM-CHANDRA RANVIR, SHEKHAR KADAM, PRABODHA SWAIN, PANKAJ R. PATEL, Ahmedabad, India

NAFLD and NASH are common clinico-pathological conditions affecting millions of patients worldwide. Although numbers of therapeutic options have been explored for management of NAFLD/NASH, no pharmacologi-cal treatment is yet approved. Saroglitazar is a novel PPARα/γ agonist that shows anti-hyperlipidemic, anti-hyperglycemic and insulin sensitizing effects. C57BL/6 mice fed with choline-defi cient amino acid-defi ned, high-fat diet (CDAHFD) containing 60 kcal% fat,is known to develop a condition similar to human NASH. Following eight-weeks of CDAHFD feeding, animals were treated with saroglitazar (1 and 3 mg/kg/day) or fenofi brate (300 mg/kg/day) for 8 weeks and maintained on CDAHFD. Saroglitazar (1 and 3 mg/kg) showed dose-dependent and signifi cant (p<0.001) reduction in serum ALT (38 and 57%), AST (33 and 56%) and MCP-1 (41 and 42%) levels when com-pared with untreated (CDAHFD-fed) disease control animals. Liver lipid accu-mulation was also signifi cantly attenuated (60-70%, P<0.001) by saroglitazar treatment.Fenofi brate (300 mg/kg) also showed reduction in serum ALT (44%), AST (51%) and MCP-1 (37%), however, the elevated liver lipids were not affected by fenofi brate treatment. The expression of pro-infl ammatory genes such as MMP-9, TNF-α and pro-fi brotic marker genes such as α-SMA were also suppressed in saroglitazar-treated animals. Histological investi-gation of liver revealed reduction of steatosis, ballooning, infl ammation and fi brosis in animals treated with saroglitazar. The NASH Score of saroglitazar (3mg/kg) group was 4.7 vs. 17.8 for untreated control animals and 16.4 for fenofi brate-treated animals. The results indicate that saroglitazar appears to be a promising drug for the management of NAFLD/NASH.

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1958-PInhibition of the Hypothalamic Supramammillary Nucleus (SuMN) Increases Insulin Resistance and Gluconeogenic and Proinfl amma-tory Status in the LiverYAHONG ZHANG, SHUQIN LUO, MICHAEL EZROKHI, YELENA TRUBITSYNA, AN-THONY H. CINCOTTA, Tiverton, RI

The SuMN dopamine (DA) neurons project to the area of the biological clock, suprachiasmatic nucleus (SCN) where the peak in circadian DA ac-tivity is of crucial importance in maintaining peripheral insulin sensitivity. High fat diet (HFD) feeding that induces insulin resistance (IR) is coupled to loss of the coincident daily circadian peaks in DA release at the SCN area and DA neuronal activity at the SuMN. Supportively, we found that inhibi-tion of the SuMN neuronal activity (including its dopamine neurons) by local infusion of a GABAa agonist/AMPA antagonist cocktail induces systemic IR in HFD-resistant animals as assessed by glucose tolerance test (69% decrease in Belfi ore insulin sensitivity index, P=0.002). We further exam-ined the potential molecular mechanisms in liver and muscle underlying the insulin resistance induced by such SuMN inhibition in rats. HFD-resistant rats were maintained on HFD and divided into two groups wherein SuMN was locally infused through an implanted osmotic pump for 3 weeks with ei-ther vehicle or GABAa agonist/AMPA antagonist cocktail (muscimol/CNQX: 3.6/1.8 nmol/day to inhibit SuMN activity (SuMNi). Relative to vehicle con-trols, such SuMNi signifi cantly increased the hepatic protein expression levels of the gluconeogenic enzyme PEPCK (25%, p=0.009) and the key pro-infl ammatory factors SOCS3 (31%, p=0.048), JNK (24%, p=0.031) and p-JNK (28%, p=0.044) known to markedly potentiate hepatic IR. In the muscle, such treatment increased total mTOR expression (43%, p=0.046, one-tailed t-test) which can facilitate muscle IR. Thus, inhibition of SuMN neuronal activity in-duces a high gluconeogenic/proinfl ammatory status in the liver and increas-es mTOR abundance in the muscle that can potentiate hepatic and muscle insulin resistance, respectively. These fi ndings implicate an important role for SuMN activity in the regulation of peripheral metabolism.

1959-PEvaluation of the Effect of Enteral Lipid Sensing on Endogenous Glu-cose Production in HumansCHANGTING XIAO, SATYA DASH, CECILIA MORGANTINI, KHAJAG KOULAJIAN, GARY F. LEWIS, Toronto, ON, Canada

Administration of lipids into the upper intestine of rats acutely decreases endogenous glucose production in the pre-absorptive state, likely through a gut-brain-liver axis involving accumulation of long-chain fatty acyl CoA, release of cholecystokinin and subsequent neuronal signaling. It remains unknown, however, whether a similar gut-brain-liver axis is operative in humans. Here we examined in humans endogenous glucose production in response to either 20% Intralipid (a synthetic lipid emulsion composed of primarily polyunsaturated long-chain fatty acids) or normal saline directly infused into the duodenum. Following an overnight fast, nine healthy male volunteers were infused either Intralipid or normal saline, in random order, 4 to 6 weeks apart, through an intraduodenal tube at the rate of 30 ml per hour for 90 min. Endogenous glucose production was assessed under pancreatic clamp conditions with stable isotope enrichment techniques. Under these experimental conditions, plasma glucose concentration or endogenous glu-cose production were not affected by intraduodenal infusion of Intralipid, compared with saline infusion. We conclude that Intralipid infusion into the duodenum at this rate does not elicit detectable effects on glucose levels or endogenous glucose production in healthy men, which may refl ect important interspecies differences between rodents and humans with respect to the putative gut-brain-liver axis in regulation of glucose homeostasis.

Supported By: Canadian Diabetes Association

1960-PThe Unfolded Protein Response Regulates Hepatic Fatty Acid Me-tabolism and Hepatic Steatosis through Peroxisome Proliferator-Activated Receptor AlphaXUQING CHEN, FEIFEI ZHANG, QI GONG, ZHIMIN HU, YAMEI HAN, JING GAO, ZHONGNAN YANG, YU LI, Shanghai, China

Aberrant fatty acid metabolism plays important roles in the develop-ment of hepatic steatosis. Hepatic steatosis is increased in defective fatty acid metabolism, refl ecting chronic increases in endoplasmic reticulum (ER) stress. However, whether the unfolded protein response (UPR), induced as an adaptive response by ER stress, also modulates fatty acid metabolism is unclear. Here we show that the nuclear receptor PPARα couples the UPR and hepatic fatty acid metabolism. Pharmacological or genetic activation of the UPR using tunicamycin and thapsigargin, or by overexpression of active

N-terminal domain of ATF6, the UPR transducer, respectively triggered acti-vation of PPARα downstream targets, such as CPT1α and MCAD, the marker of mitochondrial fatty acid oxidation in HepG2 cells. Conversely, attenua-tion of the UPR using an adenovirus-delivered dominant negative form of ATF6 (DN-ATF6) inhibited PPARα agonist Wy14643- or thapsigargin-induced fatty acid oxidation gene expression in hepatocytes. Interestingly, GST pull-down assays showed that ATF6 physically interacted with PPARα and its transcriptional coactivator PGC1α. Overexpression of DN-ATF6 decreased the transcriptional activity of 3XPPRE-driven reporter induced by Wy14643 or PPARα/RXR complex, and further inhibited oxygen consumption rates in HepG2 cells. These data indicate that ATF6 is necessary for transcriptional activity of PPARα and hepatic mitochondria fatty acid oxidation. Because knockdown of ATF6 potently attenuated the UPR gene expression, such as GRP78 and CHOP, and exacerbated diet-induced hepatic steatosis, our re-sults demonstrate 1) how cross-talk between the UPR and nuclear receptor coordinates at the transcriptional level, and contributes to hepatic lipid ho-meostasis; 2) modulation of the UPR through ATF6 represents an alternative avenue to improve liver function and treat hepatic steatosis in obesity.

Supported By: National Natural Science Foundation of China (81270930, 31471129)

1961-PPotential Usefulness of Intrahepatic Lipid Accumulation and Liver Function Tests to Identify Insulin Resistance Phenotype in Non-obese Type 2 DiabetesYASUHIKO FURUKAWA, YOSIFUMI TAMURA, KAGEUMI TAKENO, TAKASI FU-NAYAMA, HIDEYOSI KAGA, TAKAHIRO WATANABE, SAORI KAKEHI, YOSIO FU-JITANI, RYUZO KAWAMORI, HIROTAKA WATADA, Tokyo, Japan

Despite low body mass index (BMI), Asian people often develop type 2 diabetes. In addition to reduced insulin secretion, etiological difference of insulin resistance (IR) between Caucasian and Asian might be involved in this phenomenon. Previous data demonstrated that non-obese Asians eas-ily develop non-alcoholic fatty liver disease (NAFLD) which is considered as cause and result of IR. These data suggested that intrahepatic lipid (IHL) ac-cumulation and liver dysfunction could be markers of IR in non-obese type 2 diabetes. To test this hypothesis, we recruited 16 non-obese (BMI<25kg/m2) type 2 diabetes (BMI 21.9±2.0 kg/m2 HbA1C 6.8±0.5%). Using a hyperinsu-linemic euglycemic clamp with glucose tracer, we evaluated insulin sensitiv-ity (IS) in muscle and liver, respectively. We also measured IHL by 1H-MRS and serum liver function tests, such as AST, ALT and γ-GTP. Based on the up-per limit of normal IHL level (4%) in general non-obese Japanese cohort, we divided the subjects into low IHL group (n=11;1.3 (0.46-2.39)%) and high IHL group (n=5;10.3 (6.26-12.7)%). Compared with low IHL group, high IHL group showed lower muscle IS (6.79 (5.48-7.54) mg/kg/min vs. 3.87 (3.84-5.66) mg/kg/min, P=0.06) and higher γ-GTP (25.3±9.1 IU/l vs. 41.2±17.4 IU/l, P<0.05), while hepatic IS was comparable between the groups. Correlation analysis in all subjects revealed that IHL was not signifi cantly correlated to IS in muscle and liver, however, all liver function tests are signifi cantly correlated to both hepatic and muscle IS, respectively. Among them, ALT was a best predictor for IS both in muscle (r=-0.84, P<0.01) and liver (r=-0.60, P<0.05), whereas ~90% of ALT values in this study subjects were within nor-mal limit (<40 IU/l). These data suggested that small elevation of ALT even within the normal range, rather than IHL accumulation, is a useful marker to identify IR phenotype in non-obese type 2 diabetes.

1962-PHepatic SIRT1 Stimulates Hepatic Autophagic Initiator ULK1 as well as Elicits FGF21 Production and Its Role in Inducing Beige Adi-pocytes in Insulin-resistant MiceXIANLIANG RUI, YU LI, JONG WOO LEE, TING LU, ALLISON NOCON, MENGWEI ZANG, Boston, MA

While our recent studies indicate that the NAD-dependent deacetylase SIRT1 induces hepatic FGF21 production and secretion and protects against hepatic steatosis, the underlying mechanisms remain mysterious. Because improved hepatic steatosis and browning of adipose tissue are attributed to the induction of autophagy and a newly identifi ed hormone FGF21, we hypothesize that hepatic SIRT1 might be functionally linked to the regulation of autophagy and FGF21 endocrine effects in insulin-resistant states. Liver-specifi c SIRT1 knockout mice (SIRT1 LKO) and their wild-type littermates (WT) were placed on a high fat, high sucrose (HFHS) diet and a HFHS diet supplemented with resveratrol (130 mg/kg/day). Our results showed that autophagy activity, as refl ected by LC3-II accumulation and p62 degrada-tion, was impaired in livers of HFHS-fed WT mice. Autophagy defects were exacerbated in SIRT1 LKO mice with more severe hepatic steatosis, insulin

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resistance, and obesity. Strikingly, autophagy markers, phosphorylation of ULK1, an initiate kinase of autophagy, and FGF21 production were elevated in resveratrol-treated WT mice and the response to resveratrol was dimin-ished in SIRT1 LKO mice. Adenoviral overexpression of FGF21 acted on white adipose cells in vivo and in culture to induce interferon regulatory factor 4 (IRF4), a critical thermogenic regulator, and a brown-fat-like program. More-over, FGF21 promoted the development of smooth muscle cells, precursors of beige adipocytes, into thermogenic adipocytes in vitro, by increasing expres-sion of IRF4 and thermogenic genes (UCP1, DIO2, and Cidea). Collectively, ULK1-stimulated hepatic autophagy and FGF21-induced beige adipocytes may represent a novel mechanism for benefi cial effects of hepatic SIRT1 on metabolic disorders.

Supported By: National Institutes of Health

1963-PMetabolic Reprogramming in Male Offspring in a Nondietary Model of Liver Insulin ResistanceDARIO F. DE JESUS, ERCUMENT DIRICE, ABDELFATTAH EL OUAAMARI, CARMEN JERÓNIMO, AMÉLIA M. SILVA, ROHIT N. KULKARNI, Boston, MA, Porto, Portugal, Vila Real, Portugal

While several studies have focused on investigating the effects of early nutritional insults that increase the likelihood of developing type 2 diabetes in the offspring, virtually none include non-dietary models manifesting hy-perglycemia and hyperinsulinemia. We aimed to determine the effects of paternal versus maternal insulin resistance on developmental programming in the offspring of the liver-specifi c insulin receptor knockout (LIRKO) mice. At 8 weeks of age LIRKO males and females showed insulin resistance and glucose intolerance. We examined male control offspring of LIRKO males (LM), LIRKO females (LF), and control mothers and fathers (C) weaned either on a chow (21% fat) or high-fat diet (HFD, 60% fat). On a chow diet, LM and LF exhibited lower body weights at 4 weeks of age and compensated by gaining weight with age. After 3 weeks on HFD, LF but not LM gained more weight compared to C. LM and LF exhibited insulin resistance compared with C on both chow (LM vs. C: area under the curve (AUC) p<0.01 and LF vs. C: (AUC)p<0.01; n=5-8) and HFD (LM vs. C: (AUC) p<0.05 and LF vs. C: (AUC) p<0.01, n=4-5) cohorts at 9 weeks of age. LM and LF maintained insulin resistance with age and after 12 weeks on HFD became hyperglycemic (LM, 110.2±6.2; LF, 107.2±4.1; C, 78.3±6.9 mg/dL; LM vs. C and LF vs. C: p<0.01, n=4) while LF showed hyperinsulinemia (LF, 2.63±0.66; LM, 1.62±0.67; C, 0.52±0.11 ng/ml; LF vs. C: p<0.05, n=4) during fasting. The livers of LF on chow diet were light-er and became heavier on HFD compared with controls. Analysis of genes involved in regulation of liver fatty acid metabolism revealed downregula-tion of CD36, PPARα and PPARγ genes in LM and LF mice on chow diet while the same groups on HFD showed upregulation of CD36, PPARα and PPARγ compared with C. Together these data suggest that prenatal hyperinsuline-mia and hyperglycemia have detrimental effects on the transcriptional regu-lation of hepatic metabolism in the offspring that may contribute to their impaired growth and metabolic response to dietary challenges.

Supported By: R01DK67536

1964-PDifferences in Experimental Design Explain the Divergent Effects of Brain Insulin in Controlling hGP in Dogs and RatsANDY SHIN, NIKA FILATOVA, ELAINE DELLINGER, CHRISTOPH BUETTNER, New York, NY

There is controversy whether insulin regulates hepatic glucose production (hGP) via the brain. Clamp studies performed in rats, originally described by Rossetti et al., suggest that brain insulin action is critical in mediating the abil-ity of insulin to suppress hGP by altering autonomic outfl ow. To the contrary, in clamp studies performed in dogs, centrally infused insulin fails to suppress hGP. Here we examine the hypothesis that differences in the experimental design, such as the timing of the somatostatin infusion during the clamp, ex-plain these variances in brain insulin action between rodents and dogs and not a species-specifi c difference per se. Of note, somatostatin has been used to treat autonomic failure in humans indicating that somatostatin can alter autonomic outfl ow. In the rat studies, brain insulin is infused for 4 hrs before a clamp, including the infusion of IV somatostatin, is performed. To the contrary, in dogs a clamp with IV somatostatin infusion is induced for 2 hrs before the brain insulin infusion is started. We fi nd that when somatostatin is omitted in the rat studies and a clamp is performed by infusing insulin at replacement levels (which does not lead to measurable hyperinsulinemia) intracerebroven-tricular (ICV) insulin fails to suppress hGP. Vice versa, the ICV co-infusion of insulin with somatoststatin prevents brain insulin to suppress hGP, even when somatostatin is infused peripherally during the last two hr clamp period. These

results indicate that in rats 1. brain insulin suppresses hGP only when IV soma-tostatin is used during the clamps, and that 2. somatostatin can interfere with the ability of brain insulin to suppress hGP potentially by altering the electro-physiological effects of insulin. Thus, the ability of brain insulin to suppress hGP in rats is a somatostatin dependent phenomenon and, hence, experimen-tal and not species differences likely account for the divergent effects of brain insulin in controlling hGP in dogs and rats.

Supported By: American Diabetes Association (7-11-CD-02 to C.B.)

1965-PStatin Treatment Induces Dysglycemia by Enhancing Hepatic Glu-coneogenesis via Autophagy InductionEUN SEOK KANG, HYE JIN WANG, EUN YEONG CHOE, HYE-SUN PARK, GYURI KIM, KYU YEON HUR, MYUNG-SHIK LEE, HYNAGKYU LEE, CHUL HOON KIM, YOUNG-BUM KIM, Seoul, Republic of Korea, Incheon, Republic of Korea, Boston, MA

Statins are widely used to lower blood cholesterol levels but have been shown to increase the risk of type 2 diabetes mellitus. However, the mo-lecular mechanism underlying diabetogenic effects remains to be eluci-dated. Here we show that statins signifi cantly increase the expression of key gluconeogenic enzymes (e.g., glucose 6-phosphatase (G6Pase) and phos-phoenolpyruvate carboxykinase 1) in vitro and in vivo and promote glucose output by hepatocytes. Defi ciency of autophagy-related 7 protein and beclin 1 blocked this effect, suggesting that autophagy plays a role in the diabe-togenic effects of statins. In high-fat diet-fed mice treated with statins, autophagic vacuoles and gluconeogenic gene expression in the liver were increased, leading to elevated hepatic glucose production, hyperglycemia, and insulin resistance. Together, these data demonstrate that chronic statin treatment leads to insulin resistance through the activation of hepatic gluco-neogenesis, which is tightly coupled to hepatic autophagy.

1966-PHypothalamic Orexin as a Chronotherapeutic Target for Type 2 DiabetesHIROSHI TSUNEKI, TAKASHI NAGATA, KANTA KON, TSUTOMU WADA, TOSHI-YASU SASAOKA, Toyama, Japan

Synchronization between daily rhythm of glucose metabolism and sleep/wake cycle is required for maintaining glucose homeostasis. However, the chronotherapy of type 2 diabetes has not been developed. Hypothalamic orexin neurons are daily activated for stabilizing wakefulness. We have also found that endogenous orexin regulates daily rhythm of glucose metabolism, according to sleep/wake cycle, for preventing insulin resistance during ag-ing. In the present study, we investigated whether timely activation or in-activation of orexin system improves glucose metabolism in type 2 diabetic mice. First, to clarify the infl uence of orexinergic activation, we examined the effect of nicotine possessing the ability to stimulate orexin neurons. Oral nicotine consumption mainly at night in nocturnal wild-type (WT) mice for three weeks suppressed hepatic gluconeogenesis in pyruvate tolerance test at daytime resting period, without affecting body weight, food and water intake, and serum insulin and leptin levels. Orexin knockout (KO) and hepatic parasympathectomy abolished the nicotine effects. Under high fat-fed con-dition, the nighttime nicotine consumption reduced daytime blood glucose in WT but not orexin KO mice. In type 2 diabetic db/db mice, nighttime but not daytime intraperitoneal injection of nicotine for two weeks reduced daytime blood glucose via suppression of hepatic gluconeogenesis. This time-of-day-dependent effect was comparable to the effect of centrally-administered orexin A. Next, to clarify the infl uence of orexinergic inactivation, we ex-amined the effect of an orexin receptor antagonist in db/db mice. The day-time but not nighttime treatment of the antagonist for two weeks improved glucose tolerance. Taken together, amplifi cation of the daily orexinergic ac-tion by timely administration of orexin agonist or antagonist appears to be a novel chronotherapeutic strategy for type 2 diabetes.

1967-PRosiglitazone Ameliorates Iron Dysregulation in Livers from ZDF RatsJORDAN M. JOHNSON, DREW H. SMITH, DAVID C. WRIGHT, CHAD R. HANCOCK, RICHARD WATT, Provo, UT, Guelph, ON, Canada

Type 2 diabetes (T2D) has been recently linked to elevated circulating and tissue iron levels. Elevated iron increases oxidative stress leading to β-cell failure and insulin resistance. Although the adverse effects of elevated iron are well-established, it is not clear if this is a result of the diabetic condition or if elevated iron status contributes to the development of insulin resis-tance and T2D. We investigated the total and labile hepatic iron content from Zucker Diabetic Fat (ZDF) rats compared with ZDF rats that received

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treatment with the insulin sensitizer Rosiglitazone (ZDF ROSI). We hypothe-sized that livers from ZDF rats would exhibit iron dysregulation via increases in total and labile iron, and that treatment with Rosiglitazone (ROSI) would prevent elevated iron levels. Six week old ZDF rats were fed ad libitum either a chow diet or the same chow diet supplemented with 100mg/kg ROSI for six weeks. An increase in total iron was observed in livers from ZDF rats (37.5% ±7% SEM) when compared with lean controls (LC) (p= 0.003), while ROSI treatment prevented this increase. Labile iron concentration was increased similarly by 35.5% ± 7% in ZDF livers (p=0.01), and again restored to control levels with ROSI treatment. Although hepatic ferritin levels were not differ-ent between LC and ZDF rats, ROSI treatment reduced hepatic ferritin by 37% ± 12% (p=0.003). Interestingly, ZDF livers exhibited a nearly 2-fold in-crease (83% ±12% p=0.03) in the iron exporter ferroportin. Livers from ROSI treated rats tended to express lower levels of ferroportin, although this dif-ference was not signifi cant (p=0.08). These results provide novel evidence that T2D induces high iron levels that are reversible by treatment with an insulin sensitizer. Reduction in liver iron by ROSI treatment and not by iron reduction therapy implicates insulin resistance as a cause of iron dysregula-tion possibly by increased iron absorption.

1968-PEndogenous Glucose Production during Hypoglycemia in Patients with Newly Diagnosed and Long-Standing Type 1 DiabetesSABINE ZENZ, WERNER REGITTNIG, MARTINA URSCHITZ, MARTINA BRUNNER, STEFAN KORSATKO, REINGARD RAML, SOPHIE NARATH, CHRISTOPH MAGNES, BERND TSCHAPELLER, THOMAS AUGUSTIN, THOMAS R. PIEBER, Graz, Austria

Long-standing diabetes duration is a major risk factor for severe hypo-glycemia in type 1 diabetes. Spontaneous recovery from hypoglycemia is driven by endogenous glucose production (EGP). This study compared EGP response during hypoglycemia in 7 patients with newly diagnosed type 1 diabetes (NDD, mean duration 2±1 years) and 7 patients with long-stand-ing type 1 diabetes (LSD, mean duration 27±11 years, p<0.001 vs. NDD). Glucose was normalized during an overnight fast by intravenously regu-lar insulin. After steady state all subjects underwent a hyperinsulinemic, stepwise hypoglycemic clamp with the plateaus of 5.5, 3.5, 2.5 mmol/L and a recovery phase from hypoglycemia. Insulin infusion was stopped 15 minutes after achieving the nadir glucose plateau. EGP was determined by a stable isotope tracer ([6, 6-2H2]-glucose) technique. A stable enrichment was achieved throughout the experiments. Before hypoglycemic induc-tion, mean basal EGP was comparable in both groups (NDD: 2.1±0.2 vs. LSD: 2.0±0.2 mg/kg/min, p< 0.902, mean ± SD). Hyperinsulinemia induced a comparable suppression of EGP in both groups during euglycemia (5.5 mmol/L, 0.4±0.3 vs. 0.2 ±0.1 mg/kg/min, p<0.383), and at the fi rst hypogly-cemic plateau (3.5mmol/L, 0.6±0.3 and 0.5±0.2 mg/kg/min, p<0.535). At nadir plateau (2.5 mmol/L) a trend to a higher EGP at NDD compared with LSD (0.8±0.5 vs. 0.4±0.1 mg/kg/min, p<0.097) was observed. However, during recovery from hypoglycemia NDD subjects had signifi cantly higher EGP compared to LSD subjects (1.2±0.5 vs. 0.7±0.3mg/kg/min, p<0.017). This study demonstrates that in subjects with short diabetes duration EGP is higher during hypoglycemia than in subjects with LSD, thus likely con-tributing to the differences in the risk of hypoglycemia.

1969-PLiver Circadian Factor BMAL1 Regulates the Stability of mTORC2 Complex and Promotes Insulin-dependent De Novo LipogenesisLEI YIN, DEQIANG ZHANG, XIN TONG, BLAKE ARTHURS, Ann Arbor, MI

The clock protein BMAL1 has been shown to participate in circadian regu-lation of lipid metabolism. However, its contribution to insulin-regulated he-patic lipid synthesis remains unclear. Both Bmal1-/- and acute liver-specifi c Bmal1-depleted mice exhibit reduced levels of lipogenic gene expression in the liver upon food intake, suggesting that BMAL1 is required for refeeding-induced lipogenesis. Moreover, Bmal1-/- primary mouse hepatocytes dis-played greatly reduced rate of de novo lipogenesis and lowered expression of lipogenic enzymes, supporting the notion that that BMAL1 regulates lipid biosynthesis in hepatocytes in a cell-autonomous manner. Further studies showed that restoring AKT activity by a constitutively active mutant of AKT nearly normalized the rate of de novo lipogenesis in Bmal1-/- hepatocytes. Thus, our study uncovered a novel metabolic function of hepatic BMAL1, which promotes de novo lipogenesis through insulin-mTORC2-AKT signal-ing during refeeding (1). How BMAL1 regulates RICTOR expression and the mTORC2 complex activity is currently unknown. Our preliminary results show that Bmal1 defi ciency accelerated degradation of RICTOR and enhanced ubiquitination of RICTOR, indicating that BMAL1 protects RICTOR from ubiq-uitination-dependent proteasome degradation likely via its transcriptional

targets. Identifi cation of such BMAL1 targets will provide a direct link that connects the molecular clock and nutrient sensing pathways in liver cells.

Reference:1. D. Zhang, et al Liver clock protein BMAL1 promotes de novo lipogenesis

through insulin-mTORC2-AKT signaling J. Biol Chem 2014 289 (37): 25925-35. Supported By: DK099593

1970-PGastric Bypass Surgery Modestly Improves Hepatic Insulin Sensi-tivity of Fat-Fed Osborne-Mendel RatsDANIEL F. VATNER, RASMUS RABØL, SARA A. BEDDOW, SACHIN K. MAJUM-DAR, DONGYAN ZHANG, MARIO KAHN, NICHOLAS STYLOPOULOS, LEE M. KA-PLAN, GERALD I. SHULMAN, VARMAN T. SAMUEL, New Haven, CT, Boston, MA

The mechanisms responsible for the amelioration of diabetes after Roux en Y gastric bypass surgery (RYGB) remain debated. Changes in incretin responses and improvements in islet function are well-documented, while improvements in insulin sensitivity have been observed, though to varying degrees in differ-ent models. Here we assessed insulin action by hyperinsulinemic-euglycemic clamps in chronically fat-fed Osborne-Mendel rats 10 weeks after RYGB com-pared to sham-operated, ad-lib fed animals (SHAM), and a food restricted, weight matched group (WM). RYGB and WM both resulted in a ~30% decrease in body weight. RYGB did not alter fasting plasma glucose or basal rates of glu-cose production, but did reduce fasting plasma insulin concentrations by ~30% [P <0.05]. While RYGB did not affect whole body insulin stimulated glucose dispos-al, insulin suppression of hepatic glucose production improved by almost 3-fold [SHAM: 17±9%; WM: -12±19%; RYGB: 49±8%]. Surprisingly, hepatic triglyceride or diacylglycerol content was not altered 10 weeks after RYGB in these fat-fed rats. Hepatocellular insulin signaling was also unchanged as assessed by IRS2 tyrosine phosphorylation and Akt2 phosphorylation. Increased circulating bile acids are associated with improved insulin action, and we found plasma bile ac-ids were increased by 2.6-fold by RYGB [P < 0.01]. Though bile acids can increase deiodinase expression and thyroid hormone activity, the hepatic expression of thyroid hormone responsive genes were unchanged by RYGB. Bile acids may also promote secretion of FGF15/19, which impacts insulin sensitivity. Consistent with this hypothesis, protein expression of the FGF15/19 target PGC1α was sup-pressed, as were two downstream proteins, PEPCK and G6Pase.

Conclusion: RYGB surgery modestly improves hepatic insulin sensitivity in chronically fat-fed Osborne-Mendel rats without affecting insulin signaling, an effect that may be mediated in part by changes in bile acid circulation and FGF15/19.

1971-PIntegrin-linked Kinase Impairs Hepatic Insulin Action in High-Fat Fed MiceASHLEY S. WILLIAMS, LOUISE LANTIER, DEANNA P. BRACY, FREYJA D. JAMES, AMBRA POZZI, ROY ZENT, DAVID H. WASSERMAN, Nashville, TN

Integrin-linked kinase (ILK) is a downstream integrin signaling protein that facilitates signal transduction from the extracellular matrix to the inside of the cell. Recent studies from our laboratory show that selective deletion of hepatic ILK in vivo results in the complete restoration of hepatic insulin sensi-tivity in high fat (HF) fed C57BL/6J mice. In this study, we sought to determine the mechanisms whereby this occurs. Mice with a hepatocyte specifi c dele-tion of ILK (ILKlox/loxAlbcre) and their wild-type littermates (ILKlox/lox) were fed a chow or HF diet (60%) for 16 wks. After a 5h fast, mice were sacrifi ced or underwent a 4 mU·kg-1·min-1 hyperinsulinemic, euglycemic (insulin) clamp (IC). There was no difference in the glucose infusion rate (GIR) or endoRa in chow mice during the IC. As reported previously, the GIR was increased in HF ILKlox/loxAlbcre mice and endoRa was fully suppressed. Insulin signaling was assessed in livers from HF mice after the IC using an antibody microarray and western blot analysis. Key signaling proteins were upregulated in HF ILKlox/loxAlbcre mice including Akt2, TBK1, and GSK3a as well as several proteins in the MAPK cascade such as Raf1, MAP2K7, MAP2K5 and MAPK9. Consistent with glucose fl ux measurements, phosphorylation of Akt at S473 and FoxO1 at S256 were increased and gluconeogenic gene expression was suppressed in HF ILKlox/loxAlbcre mice. There was no difference in glycogen synthesis between genotypes during the insulin clamp. Mitochondrial function was as-sessed in isolated mitochondria from livers of 5h fasted mice. Consistent with improved insulin action, glutamate/malate stimulated state 3 respiration was increased in HF ILKlox/loxAlbcre mice. In summary, hepatic ILK deletion has no effect on insulin action in lean mice, but sensitizes the liver to insulin during the challenge of HF feeding. This effect corresponds to changes in the expres-sion and activation of key signaling pathways as well as a greater capacity for hepatic mitochondrial glucose oxidation.

Supported By: DK050277, DK20593

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1972-PExogenous Administration of DLK1-Fc Ameliorates Hepatic Steato-sis and Suppresses Gluconeogenesis via Activation of AMPKEUGENE HAN, YONG-HO LEE, SO RA KIM, MI RA YUN, HYUN MIN KIM, HYE-SUN PARK, HYE-JIN YOON, BYUNG-WAN LEE, EUN SEOK KANG, HYUN CHUL LEE, BONG SOO CHA, Seoul, Republic of Korea

Activation of hepatic Notch signaling pathologically enhances lipogenesis and gluconeogenesis in liver associated with increased insulin resistance. Delta-like 1 homolog (DLK1) is an imprinted gene that inhibits adipogenesis and is associated with muscle development. Recent evidence indicates that DLK1 interacts with Notch1 and functions as an inhibitory regulator of Notch signaling. Therefore, we aimed to investigate the metabolic effect of DLK1 in vitro and in vivo.

Male db/db mice were randomly assigned to two groups: (1) vehicle-treat-ed, and (2) DLK1-Fc-treated groups. DLK1-Fc (25mg/kg) was intraperitoneally injected twice a week for 4 weeks.

Hepatic triglyceride content and lipid droplets in liver tissues as well as serum levels of liver enzymes were markedly decreased in DLK1-Fc-treated db/db mice. Furthermore, DLK1-Fc-treated mice showed signifi cantly lower levels of fasting and random glucose with improved glucose and insulin tol-erance, compared to the vehicle-treated group. In the liver of db/db mice and HepG2 cells, DLK1-Fc treatment induced phosphorylation of AMPK and ACC, indicating activation of fatty acid oxidation pathway. Moreover, DLK1-Fc suppressed glucose production from hepatocytes, which was blocked after co-treatment of compound C. DLK1-Fc-treated hepatocytes and liver tissues showed decreased expression of PEPCK and G6Pase. DLK1-Fc triggered the phosphorylation of AKT, followed by cytosolic translocation of FOX01 from nucleus in hepatocytes. DLK1-Fc-treated mice also exhibited decreased amount of macrophage content in epididymal fat without affecting adipo-cyte morphology, compared to the vehicle-treated db/db mice.

In conclusion, our study demonstrated that exogenous administration of DLK1-Fc reduced hepatic steatosis and inhibited gluconeogenesis via AMPK activation. This implicates that DLK1 may provide a novel therapeutic ap-proach for the treatment of non-alcoholic fatty liver disease and diabetes.

Supported By: National Research Foundation of Korea (2012R1A1A2043812)

1973-PFlavin Monooxygenase 3 Is Required for FoxO1 Expression and De-velopment of the Diabetic PhenotypeJI MIAO, Boston, MA

Despite the long-standing and well-documented association between insulin resistance and cardiovascular disease, the key targets of insulin relevant to the development of cardiovascular disease are not known. Non-biased profi ling of the livers of male Liver Insulin Receptor Knockout (LIRKO) mice revealed the enzyme fl avin monooxygenase 3 (FMO3) to be the sec-ond most highly induced transcript and its product, trimethylamine N-oxide (TMAO), to be the most highly induced metabolite. TMAO alters cholesterol metabolism and increased plasma levels of TMAO are closely associated with atherosclerosis in mice and humans. Knockdown of FMO3 using anti-sense oligonucleotides suppressed FoxO1, a central metabolic regulator. Thus, knockdown of FMO3 entirely prevented the development of hypergly-cemia, hyperlipidemia and atherosclerosis in male LIRKO mice. FMO3 is also highly elevated in livers of streptozotocin-treated and ob/ob mouse models of type 1 and 2 diabetes, respectively. Moreover, FMO3 knockdown in ob/ob mice also suppressed FoxO1 and had benefi cial metabolic effects. However, the effects of insulin resistance on FMO3 expression were much more mod-est in females as FMO3 expression in Mus musculus is sexually dimorphic. Taken together, these data indicate that FMO3 is required for FoxO1 expres-sion and the development of metabolic dysfunction, and suggest that treat-ments to suppress hepatic FMO3 may prevent hyperglycemia, dyslipidemia and cardiovascular disease in diabetic patients.

Supported By: HL109650, DK100539

1974-PNeuronal Signals from the Hepatic Amino Acid/mTOR/S6K Pathway Regulates Systemic Lipid MetabolismKENJI UNO, HIDEKI KATAGIRI, Sendai, Japan

Metabolism in different tissues/organs is systemically under coordinated regulation, to allow adaptation to a variety of environmental conditions. Neuronal signals as well as humoral factors have attracted attention in this inter-tissue metabolic network. In obesity, serum amino acids (AAs) are el-evated and the downstream target of AA signaling, the mTOR/S6K pathway, is activated in organs such as the liver. Herein, we focused on the roles of hepatic AA signaling in systemic regulation of metabolism. Adenoviral

expression of an AA transporter (SNAT2) in the liver increased hepatic AA uptake and activated the hepatic mTOR/S6K pathway. Under this condition, serum triglycerides (TGs) in SNAT2-mice were markedly elevated, especially in postprandial states. The hypertriglyceridemia is derived from decreased TG-hydrolysis with the down-regulation of adipose lipoprotein lipase (LPL) expression. Hepatic expression of Rheb, an activator of mTOR signaling, also induced postprandial hypertriglyceridemia, and hepatic expression of dom-inant-negative form of S6K inhibited hypertriglyceridemia in SNAT2-mice, indicating hepatic mTOR/S6K involvement. Furthermore, surgical denerva-tion, pharmacological deafferentation of hepatic vagus and β-adrenergic blocker administration inhibited the postprandial hypertriglyceridemia and suppressed adipose LPL down-regulation in Rheb-mice. These results indi-cate that this novel inter-tissue mechanism is mediated by a neuronal relay originating from the hepatic AA/mTOR/S6K signaling and is responsible for the regulation of systemic lipid metabolism. Furthermore, blockade of this pathway suppressed hypertriglyceridemia in obese mice. Thus, this neuronal mechanism produces inter-nutrient coordination at the whole-body level and makes an important contribution to the development of obesity-related hy-pertriglyceridemia.

1975-PImpacts of Heat Shock Protein 72 in Hepatic Glucose Metabolism in Model Mice of Type 2 DiabetesTATSUYA KONDO, RINA MATSUYAMA, SAYAKA KITANO, RIEKO GOTO, KAORU ONO, JUNJI KAWASHIMA, HIROYUKI MOTOSHIMA, TAKESHI MATSUMURA, HIROFUMI KAI, EIICHI ARAKI, Kumamoto, Japan

Cell stress, such as endoplasmic reticulum stress or oxidative stress is one of the key factors in pathophysiology of type 2 diabetes. Molecular chaper-one, which modulate protein folding and/or assembly and protect cells from those stresses, may be a favorable target for diabetic treatment.

Heat shock protein (HSP) 72 is a major inducible heat shock protein against heat, ultraviolet or infection, and works as a molecular chaperone to pro-tect cells from infl ammatory stresses. Induction of HSP72 by pharmacologic agent or mild electrical stimulation with heat shock improves glucose intol-erance in diabetic model mice. In this study, we investigated glucose me-tabolism in whole body HSP72 defi cient (KO) mice to explore the impacts of HSP72 in diabetes.

Male HSP72 KO mice or control (C) mice were subjected to a high-fat diet (HFD) regimen for 16 weeks. Several metabolic parameters and cellular stress markers were evaluated.

KO mice showed signifi cantly higher body weight after 10 weeks of HFD (KO: 36.9 g vs. C: 33.4 g). Fasting blood glucose was signifi cantly increased after 11 weeks of HFD (KO: 143.3 mg/dL vs. C: 115.5 mg/dL). Random fed blood glucose and food intake were comparable. Upon glucose challenge, blood glucose levels at any time points measured were higher in KO mice. Upon insulin tolerance test, KO mice exhibited insulin resistant phenotype. Epididymal, mesenteric and retroperitoneal fat masses were all increased in KO. Hepatic steatosis was obvious in KO liver. Upon insulin stimulation from inferior vena cava, phosphorylation of Akt was decreased by approximately 50% in KO liver extracts, with increased activation of c-jun N-terminal ki-nase. Hepatic gluconeogenesis was not suppressed in KO mice.

In summary, defi ciency of HSP72 leads to increased visceral adiposity, in-sulin resistance, glucose intolerance and fatty liver. As induction of HSP72 is benefi cial to treat diabetes, our observations strongly indicate the abun-dance of HSP72 is critical in diabetic pathophysiology.

Supported By: Japan Ministry of Education, Culture, Sports, Science and Tech-nology

1976-PThe Role of ChREBP in the Development of High-Fat Diet-induced Fatty LiverKATSUMI IIZUKA, HIROYUKI NIWA, TAKEHIRO KATO, WU WUDELEHU, HIROMI TSUCHIDA, YUKIO HORIKAWA, JUN TAKEDA, Gifu, Japan

Carbohydrate Response Element Binding Protein (ChREBP) regulates de novo lipogenesis and switches from glycogenosis to triglyceride synthesis. In ChREBP knockout (KO) mice, de novo lipogenesis and hepatic acyl-CoA oxidation are suppressed. We tested whether high-fat diet (HFD) causes fatty liver in ChREBP KO mice. Male wild type (WT) and KO mice were fed a HFD from 8 weeks of age. Oral glucose tolerance, olive oil load + tyloxapol injection (intestinal lipid absorption test), and tyloxapol injection tests (VLDL secretion test) on WT and KO mice were performed. In HFD fed KO mice, body and fat weight were much lower than those in WT mice, because of decreased food intake, especially during the dark. Glucose clearance in KO mice was slightly improved compared with WT mice. Liver weight of HFD

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fed KO mice was much higher than that of HFD fed WT mice. Livers of WT mice had histologically small fat droplets near the central vein and large fat droplets near the portal vein. In contrast, livers of KO mice had various sizes of lipid droplets around all the hepatic lobules. Liver cholesterol content in KO mice was similar to that in WT mice, and liver triglyceride content in KO mice was only slightly lower than that in WT mice. Fasted plasma free fatty acid, beta-hydroxybutylate and fi broblast growth factor-21 (FGF21) levels, refl ecting beta oxidation of fatty acyl-CoA in liver, were much lower in KO mice than in WT mice. Consistently, the expression of hepatic peroxisome proliferator-activated receptor alpha and FGF21 mRNA in KO mice was lower than that in WT, whereas lipogenic genes (Fasn) mRNA in liver of KO mice were suppressed. In addition to the decreased beta oxidation of acyl-CoA, increased intestinal lipid absorption and decreased VLDL secretion in KO mice suggest that KO mice have properties that cause hepatic lipid accumu-lation. In conclusion, ChREBP gene deletion failed to alleviate HFD-induced fatty liver, because of decreased beta oxidation of acyl-CoA, increased in-testinal lipid absorption, and decreased VLDL secretion.

1977-PAdiponectin-SOGA Dissociation in Type 1 Diabetes: A Marker of Adiponectin Resistance?TERRY COMBS, JANET K. SNELL-BERGEON, DAVID M. MAAHS, BRYAN C. BERGMAN, MARIE LAMARCHE, LAURA IBERKLEID, ROLAND TISCH, PHILIPP E. SCHERER, ERROL B. MARLISS, Chapel Hill, NC, Aurora, CO, Montreal, QC, Canada, Dallas, TX

Adiponectin plays an important role in glucoregulation. Circulating levels are elevated in type 1 diabetes (T1D) patients and nonobese diabetic (NOD) mice, consistent with tissue resistance to adiponectin that could affect gly-cemia. It stimulates liver cell production of the Suppressor Of Glucose from Autophagy (SOGA), a protein that inhibits glucose release. We hypothesize: 1, that elevated adiponectin in T1D fails to increase SOGA and its glucoregulatory effects; 2, the circulating C-terminal fragment of SOGA is a surrogate marker for liver SOGA. Adiponectin and SOGA were measured by Western blots in serum from adult T1D and control participants undergoing a 3-stage hyperin-sulinemic clamp for the Coronary Artery Calcifi cation in T1D study (n=10 per group). SOGA was also measured in liver and plasma from diabetic and control NOD mice (n=6 per group). The 13-29% decrease of SOGA in the liver of dia-betic NOD mice, was associated with a 30-37% reduction of circulating SOGA (P<0.05) and the two were correlated (r=0.826, p=0.001). SOGA correlated negatively with glucose Ra (measured by 2H-glucose) at stages 1 and 2 of the clamp (r=-0.84 and -0.65, P<0.022). In T1D patients, fasting serum adiponec-tin was elevated 50-60% whereas SOGA was decreased 30-50% (P<0.05). During the high insulin dose (40 mu/m2), at stage 3, T1D participants required lower glucose infusion rates than control participants (4.1 + 0.9 vs. 7.6 + 1.1 mg/kg/min; p<0.05).Thus: the reductions of liver SOGA in diabetic NOD mice implies hepatic resistance to adiponectin signaling, and their correlation of SOGA between liver and circulation supports the utility of circulating SOGA as a surrogate marker of its hepatic regulation. The dissociation between serum adiponectin and serum SOGA in T1D, its negative association with Ra, and the reductions of liver and circulating SOGA in NOD mice, may provide a targeted pathway for restoring the glucoregulatory effects of circulating adiponectin.

Supported By: National Institutes of Health (DK075573 to T.C.) (HL061753 to J.K.S-B.) (DK075360 to D.M.M.) (DK056350 to R.T.) (DK055758 to P.E.S.); Canadian Institutes of Health Research (MOP-62889 to E.B.M.)

1978-PEffect of Vildagliptin and Metformin Combination Therapy on Liver Fat Content in Type 2 Diabetes MellitusZAOPING CHEN, JUN LIU, XINMEI HANG, Shanghai, China

The aim of this study to investigate the effect of vildagliptin and metformin combination therapy on liver fat content in type 2 diabetes mellitus. A total of 70 patients with low dose of metformin monotherapy (metformin 500mg two times per day) were randomly divided into two groups: The combined treat-ment group (CTG) and high dose of metformin group (HDMG). The CTG was giv-en with Vildagliptin 50mg two times per day, the HDMG was given metformin 1000mg two times per day. Fasting blood glucose, HbA1c, insulin, blood lipid, liver function, BMI and liver fat content were detected before treatment and after 24 weeks of treatment. The liver fat content was determined by CT. The liver fat content of CTG and HDMG had no obvious difference before treat-ment, and both decreased signifi cantly after treatment (CTG 4.23±5.03 vs. 2.66±4.39, P<0.05 in before treatment vs. after treatment, HDMG 2.71±3.06 vs. 1.31±3.16, P<0.05 in before treatment vs. after treatment). But there have no signifi cant differences after treatment between two groups. Correlation analysis revealed that the extent of reduction of liver fat content had positive

corrections with alanine aminotransferase (ALT) decreased (r=0.630, P<0.05) after treatment, and had inverse corrections with baseline glycosylated he-moglobin (r=-0.227, P<0.05). Multiple stepwise regression analysis revealed that the degree of ALT decreasd was the independent corrected to the extent of reduction of liver fat content (P<0.05). The results suggest that vildagliptin combined with low dose of metformin treatment can reduce the content of liver fat in patients with type 2 diabetes, which has the similar effect of high dose of metformin treatment.

1979-PLiver-derived ApoJ Regulates Insulin Signaling via LRP2 in Insulin-Target TissuesJI A. SEO, MIN-CHEOL KANG, LEANDRO MAURA, MICHELLE CHUNG, JEONG HO KIM, SOO HYUN HONG, INES S. LIMA, YOUNG-BUM KIM, Seoul, Republic of Korea, Boston, MA

Our previous data demonstrate that liver-selective deletion of apolipo-proteinJ (L-ApoJ) resulted in insulin resistance and lack of ApoJ in sera (Diabetes 2014; 63; A000). To further determine the mechanism by which hepatic ApoJ regulates insulin action, L-ApoJ defi cient mice were injected with insulin intraperitoneally (10 U/kg, 10 min). Insulin’s ability to increase in-sulin receptor phosphorylation was notably reduced by ~40-65% in muscle, adipose tissue, and liver in L-ApoJ defi cient mice compared to control mice. Concurrently, downstream signaling components of insulin, including IRS1/2, Akt, AS160, GSK3, and GS, were also markedly decreased in the absence of hepatic ApoJ. These effects were independent of adiposity. Surprisingly, ApoJ protein was absent in muscle, adipose tissue, heart, pancreas, and kidney in L-ApoJ defi cient mice. To test the possibility that ApoJ in these tissues is delivered from the liver, adenovirus encoding ApoJ was expressed in liver of L-ApoJ defi cient mice. Adenovirus-mediated ApoJ expression in liver led to a signifi cant increase in ApoJ level in sera and in aforementioned metabolic organs of L-ApoJ defi cient mice, suggesting liver-produced ApoJ can be released in the circulation and transported to metabolic tissues. Moreover, we found that ApoJ protein in muscle and in sera was normal despite the fact that ApoJ gene was selectively deleted in muscle. To further determine whether circulating ApoJ is transported into muscle via ApoJ’s potential receptor LRP2, mice lacking LRP2 in muscle were studied. ApoJ level in muscle was decreased by ~50% in muscle-selective ApoJ-defi cient mice compared to control mice, leading to an increase of circulating ApoJ. However, normal ApoJ level was detected in liver, adipose tissue, heart, and brain. These data demonstrate that liver-derived ApoJ plays a key role in regulating insulin signaling in an autocrine or a paracrine manner via LRP2. Thus, hepatic ApoJ could be a therapeutic option for the treatment of type 2 diabetes.

Supported By: American Diabetes Association (7-12-BS-094 to Y-B.K.)

1980-PThe Role of Starvation-induced Autophagy in Gluconeogenesis and KetogenesisMOTOYUKI KONDO, AYANO TAKAGI, SHINJI KUME, SHIN-ICHI ARAKI, TAKASHI UZU, HIROSHI MAEGAWA, Otsu, Japan

Starvation is one of the strongest stimuli to activate autophagy. However, the exact role of starvation-induced autophagy remains unclear. In this study, we examined the physiological role of autophagy in metabolic organ which contribute to maintain systemic energy homeostasis during starvation.

We examined a role of autophagy in gluconeogenesis and ketogenesis using liver specifi c Atg5 knockout mice (L-Atg5-/-), skeletal muscle specifi c Atg5 knockout mice (M-Atg5-/-), kidney proximal tubular epithelial cells spe-cifi c Atg5 knockout mice (K-Atg5-/-), and control mice (Atg5fl ox/fl ox).

There were no signifi cant differences in metabolic phenotypes under ad-libitum feeding among 4 groups. After a 36-h fast, fasting-induced increase in plasma ketone levels was signifi cantly lower in L-Atg5-/- mice compared to other groups. But, the impairment of ketogenesis in L-Atg5-/- mice was incomplete, suggesting that some other organs compensated for loss of he-patic ketone production.

We fi rst focused on skeletal muscle because it is an important organ to provide amino acids. There were no signifi cant differences in both plasma and tissues ketogenic-amino-acid levels between M-Atg5-/- and Atg5fl ox/fl ox at 36-hour fasting.

Interestingly, starvation-induced formation of lipid droplets and elevation of expression level of HMG-CoA synthase were observed not only in the liver but also in the kidney. We then hypothesized that the kidney compen-sated for the loss of ketone generation in L-Atg5-/- mice. We generated the mice lacking Atg5 in both liver and kidney (LK-Atg5-/- mice). LK-Atg5-/- mice showed further decline in plasma ketone concentration during a 36-h fast,

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compared with L-Atg5-/- mice. These results suggest that autophagy main-tains energy homeostasis during starvation via regulating ketogenesis, and that the kidney potentially has a function to generate ketone bodies.

Supported By: Japan Ministry of Education, Culture, Sports, Science and Tech-nology

1981-PHigh-Fat Diet Causes an Increase in Pyruvate Carboxylase in LiverARIJEET K. GATTU, DANIEL F. VATNER, SARA A. BEDDOW, LAUREN PAOLELLA, VARMAN T. SAMUEL, West Haven, CT, New Haven, CT

An increase in hepatic pyruvate carboxylase (PC) protein expression (but not mRNA expression) is strongly associated with HbA1c in humans and in rodents fed a high-fat fed (HFF) and without changes in other gluconeogenic enzymes. We hypothesized increased fatty acids promote post-translational modifi cation of PC that increases PC content. We studied male, Sprague-Dawley rats that were either regular chow (RC) fed or HFF for 3day, 2 weeks and 4 weeks. Hepatic steatosis was evident in all HFF rats. PC mRNA expres-sion was not altered at any time point. PC protein or activity was not altered after 3day HFF compared to RC. In contrast, PC protein expression increased ~ 45% (P<0.04) and ~100% (P<0.03), and activity increased ~40 % and ~65% (P<0.001), after 2 and 4 weeks HFF, respectively. This was associated with a ~2-fold increase in PC lysine acetylation (K-Ac) in HFF rat livers compared to RC (P<0.001). HFF did not alter PC sumoylation or lysine-succinylation. The increase in K-Ac was associated with a decrease in PC ubiquitination in HFF rats. To further test the role of K-Ac, primary hepatocytes from RC and HFF rats cultured with 500 µM linoleic acid (LA) or BSA alone for 16-18 hours. PC protein was only increased in hepatocytes isolated from HFF rats supple-mented with LA (HFF/LA). Primary hepatocytes were treated with 10ug/ml cyclohexemide to inhibit tranlsation. While PC protein expression decreased by ~50% (P<0.02) after 12H in RC/BSA hepatocytes, PC protein content was stable in HFF/LA hepatocytes. The hepatic mRNA expression of key acetyl-transferases, deacetylases or acyl dehydrogenases were unchanged by HFF. Prior studies have demonstrated that HFF can increase hepatic acetyl CoA concentration. To assess whether K-Ac could be driven primarily by substrate, primary hepatocytes from RC rats were incubated for 16 hours with 50 mM sodium acetate. This resulted in a ~70% increase in PC protein compared to control.

Conclusion: High-fat feeding promotes substrate mediated K-Ac of PC, which increases PC stability, content and activity.

Supported By: I01BX000901

1982-PRegulation of Liver Carnitine Palmitoyltransferase 1a in Streptozo-tocin-induced DiabetesJANOS KERNER, NANA OWASU, CHIEH A. LEE, MARIA K. STOLL, TIMOTHY KERN, CHARLES L. HOPPEL, Cleveland, OH

Carnitine palmitoyltransferase 1a (CPT1a) by virtue of inhibition by malonyl-CoA represents the rate-controlling step of mitochondrial fatty acid oxidation. The purpose of this study was to assess whether posttranslational modifi ca-tion of CPT1a is the underlying mechanism of the known decreased malonyl-CoA sensitivity and thus increased hepatic fatty acid oxidation in diabetes.

Diabetes was induced by intraperitoneal injection of streptozotocin and the rats were treated with insulin to avoid hypercatabolism (Diabetic group). Insulin was withheld in half of the diabetic animals four days before eu-thanasia to induce ketoacidosis (Diabetic ketoacidotic group). Untreated rats were used as controls. Kinetic properties (CPT1a activity, malonyl-CoA sensitivity) and posttranslational modifi cation of CPT1a were determined on Percoll-purifi ed liver mitochondria. In addition, we measured hepatic acyl-carnitine profi les.

Diabetes and diabetic ketoacidosis resulted in signifi cantly decreased sen-sitivity of CPT1a to malonyl-CoA inhibition (Ki values) and a slight increase in catalytic activity. This decreased malonyl-CoA sensitivity was associated with increased tyrosine phosphorylation and increased lysine acetylation. No apparent differences were observed in serine and threonine phosphory-lation. Furthermore, in liver mitochondria from diabetic animals we observed a novel, hitherto not described protein, which strongly cross-reacted with CPT1a antibodies. As judged by its electrophoretic mobility (SDS-PAGE) this protein is smaller in size and was absent in mitochondria from control rats.

In addition, diabetes was associated with an increase in liver carnitine con-tent characterized by increased long-chain acylcarnitines and acetylcarnitine.

These data are consistent with increased mitochondrial fatty acid oxi-dation caused by decreased malonyl-CoA sensitivity of CPT1a and suggest posttranslational modifi cation of CPT1 as the underlying mechanism.

Supported By: American Diabetes Association (7-12-BS-078 to C.L.H.)

1983-PEffects of PPARα Agonists on In Vivo Whole Body and Tissue Spe-cifi c Free Fatty Acid (FFA) Metabolism in the RatKRISTINA WALLENIUS, NIGEL TURNER, ANN KJELLSTEDT, THERESE HAGSTEDT, NICHOLAS D. OAKES, Mölndal, Sweden, Sydney, Australia

We studied the effects of PPARα agonists (either AZα 1 µmol/kg/day or WY-14,643; 30 µmol/kg/day for 3-4 wk) on lipid metabolism in male Sprague Dawley rats fed a high fat diet starting 1 week before treatment. Indices of local FFA utilization (Rf

*) and storage (Rfs) were calculated using the partially metabolizable [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP) and [U-14C]-palmi-tate (14C-P) tracers. Whole body FFA oxidation rate (Rox) was assessed us-ing 3H2O production from 3H-palmitate. As expected AZα decreased plasma TG (3h fasted state) and increased liver weight compared to vehicle treated rats. During the tracer studies, in the 10 h fasting state AZα modestly in-creased plasma glucose and did not affect FFA levels. In liver, a major sink for whole body FFA disposal, AZα did not alter either Rf

* or Rfs (per unit tis-sue weight) implying no change in hepatic tissue uptake or metabolic fate of plasma FFA. In skeletal muscle, the sciatic nerve stimulated contraction induced increase in calf muscle Rf

* was actually dampened by AZα, with no effect on Rfs, indicating a treatment induced reduction in local FFA oxidation. To further explore the potential of PPARα agonism to enhance FFA oxidation under conditions of increased energy demand, acute effects of the uncou-pling agent 2,4-dinitrophenol (DNP, 15 mg/kg i.v.) were studied in WY-14,643 and vehicle treated groups. DNP stimulated Rox to a similar extent in both vehicle (2.3±0.3x basal) and WY-14,643 (2.7±0.5X basal) groups. This was despite the fact that, compared to vehicle, WY-14,643 induced a marked increase in hepatic FFA oxidation capacity (assessed ex vivo). In conclusion, our results suggest that PPARα agonism failed to enhance tissue specifi c FFA oxidation in either skeletal muscle or liver under conditions of basal or elevated energy demand. These results show that pharmacological ap-proaches aimed at increasing the machinery for oxidation of lipid may not succeed in enhancing FFA oxidation.

1984-PAdipose Tissue Dysfunction and Hepatic Fibrosis in Diabetic Pa-tientsEMILIA RUSU, GEORGIANA ENACHE, MARIANA JINGA, FLORIN RUSU, CRISTINA PARPALA, RAMONA DRAGUT, RALUCA NAN, HORATIU CRISTIAN POPESCU VAL-CEANU, MIHAELA POSEA, ANDREEA DIANA DRAGOMIR, MARILENA STOIAN, ILEANA TEODORU, ADRIAN COSTACHE, GABRIELA RADULIAN, Bucharest, Roma-nia, Calarasi, Romania, Câmpulung, Romania

The aim of this study was to investigate the interaction between adipose tissue hormones and hepatic fi brosis (HF) in diabetic patients. We conducted a cross-sectional multicenter study which included a total of 144 patients with type 2 diabetes, 74 women (51.4%). There were followed the anthro-pometric indicators (weight, height, waist circumference, body mass index (BMI). The biochemical parameters followed were fasting plasma glucose (FPG), lipid profi le, liver function tests, blood count. Serum concentrations of adiponectin, leptin, resistin, insulin, TNF-alpha, IL-6 were measured with ELISA method. Insulin resistance (IR) was estimated by the homeostasis model assessment (HOMA). Liver fi brosis was non-invasively assessed using the Forns index; a value >6.9 was a predictor for signifi cant fi brosis. Multi-variate analysis based on backward logistic regression was used to evaluate the association between hepatic fi brosis and metabolic factors. Median age was 53.2 years. The prevalence of fi brosis evaluated by Forns Index was 24.35% (n=35). Median serum concentrations of adiponectin in patients with HF were signifi cantly lower (p=0.042). Median serum levels of TNF-alpha, IL-6, resistin in patients with HF were signifi cantly higher than in controls (all p<0.05). In univariate analysis the following parameters were signifi cantly related to fi brosis: age, BMI, triglycerides, ALT, GGT, FPI, HOMA-IR, leptin, resistin, IL-6, TNF-alpha levels. In multivariate analysis, independent predic-tors of fi brosis were age (OR: 1.13, 95% CI: 1.02-1.34), BMI (OR: 2.11, 95% CI: 1.01-3.46), insulin resistance (OR: 2.32, 95% CI: 1.48-3.66), and TNF-alpha (OR: 1.2, 95% CI: 1.1-1.74).

This study showed an elevated prevalence of hepatic fi brosis in diabetic patients, especially in patients aged over 54 years with obesity and IR. In the present study, the predictive factors for hepatic fi brosis in diabetic patients were: age, body mass index, HOMA-IR over 4, and TNF-alpha.

Supported By: European Social Fund/Romanian Government (POSDRU/159/1.5/S/137390)

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INTEGRATED PHYSIOLOGY—MACRONUTRIENT METABOLISM AND FOOD INTAKE

1985-PExtrahepatic Effects and Metabolic Dysregulation in Irs1/Irs2 Liver Knockout MiceOLIVER STOHR, AMANDA PLESSMANN, KYLE COPPS, MORRIS F. WHITE, Boston, MA

LDKO (hepatic Irs1/Irs2 double knockout) mice are hyperglycemic, insulin resistant and hyperinsulinemic. At 10 months, LDKO mice display nodules characteristic of transformed tissue. LDKO liver exhibits increased phospho-STAT3 (Signal transducer and activator of transcription 3) levels linked to mitochondrial respiration in other studies. Elevated pSTAT3 (Ser727) was as-sociated with increased expression of mitochondrial complex I and complex II in LDKO livers and isolated crude mitochondria, whereas the expression levels of the other complexes were unchanged. Respiratory capacity of complex I increased slightly whereas complex II displayed a strong increase. Regardless, basal and maximal respiration capacity decreased indicating hepatic mitochondrial dysfunction in LDKO mice. JNK (c-Jun N-terminal kinase) phosphorylation increased in LDKO liver suggesting that elevated hepatic infl ammation might be involved. A state of enhanced infl ammation might lead to an increased cytokine production and secretion from the livers of these mice, contributing to systemic insulin resistance. However, serum levels of pro-infl ammatory cytokines_including TNFα, IL-6, IL-9, IL-1α and MCP-1 were normal. We investigated skeletal muscle in LDKO mice to assay for insulin resistance stemming from hepatic Irs knockout. Skeletal muscle displayed normal insulin signaling; however, [14C]2-DOG uptake by type I and type II fi bers of gastrocnemius muscle of LDKO mice was reduced and only weakly increased by insulin. pJNK in muscle was unchanged in LDKO versus control mice. Other muscles showed no difference in response to insulin in [14C]2-DOG uptake. These results indicate that the genetic hepatic insulin resistance of LDKO mice has little effect to promote systemic infl ammation, and only minor effects upon glucose uptake by muscle, whereas hepatic mitochondrial dysfunction might contribute to metabolic dysregulation and hepatic infl ammation.

Supported By: American Diabetes Association (7-12-MN-82 to M.F.W.)

1986-P


Guided Audio Tour: Macronutrient Metabolism and Food Intake (Posters: 1987-P to 1993-P), see page 15.

& 1987-PPKC-θ-induced Phosphorylation of AMPK on Serine491 Causes Leptin Resistance in Diet-induced ObesityYOSSI DAGON, WEIXIA JIAN, ISMAIL SYED, ODILE D. PERONI, BARBARA B. KAHN, Boston, MA

Resistance to the anorexigenic action of leptin plays a major role in the development of obesity. AMPK is a key regulator of energy balance and food intake through actions in the hypothalamus. Inhibition of AMPKα2 cata-lytic subunit (α2AMPK) kinase activity in the hypothalamus is necessary for leptin’s anorexigenic effects. Leptin inhibits α2AMPK activity by phos-phorylating α2AMPK on serine491. In diet-induced obesity (DIO), leptin fails to modulate hypothalamic α2AMPK activity and to decrease food intake. We aimed to identify the mechanism by which DIO impairs leptin-induced α2AMPK regulation.

High fat (55%) diet (HFD) for 15 weeks increased hypothalamic α2AMPK-serine491 phosphorylation by 46% and suppressed α2AMPK activity (9.6±0.8 nmol/g per min versus CHOW diet - 12.3±1.1, p<0.05). This was associated with activation of hypothalamic PKC-θ, a known inducer of leptin resistance in DIO mice.

In GT1-7 neurons, both PKC-θ overexpression and activation by Phorbol-12-Myristate-13-Acetate stimulated α2AMPK-serine491 phosphorylation and suppressed α2AMPK activity. Conversely, PKC-θ inhibition with Ro 31-8220 suppressed α2AMPK-serine491 phosphorylation and increased α2AMPK ac-tivity. PKC-θ-induced α2AMPK-serine491 phosphorylation impaired the abil-ity of leptin to phosphorylate α2AMPK on serine491, resulting in failure of leptin to regulate α2AMPK activity.

Hypothalamic PKC-θ inhibition with Ro 31-8220 administration i.c.v in DIO mice restored leptin’s (20 ng i.c.v) regulation of hypothalamic α2AMPK and anorexigenic action (Ro 31-8220 3.8g/24h vs. Ro 31-8220 + leptin 2.7g/24h; p<0.05).

In sum, PKC-θ inhibits α2AMPK activity by phosphorylating it on serine491. HFD induced PKC-θ activation impairs leptin’s effect on α2AMPK serine491

phosphorylation, resulting in failure of leptin to modulate α2AMPK activity and to decrease feeding. In conclusion, the mechanism for PKC-θ induced leptin resistance in obesity involves phosphorylation of α2AMPK on ser-ine491.

Supported By: JPB Foundation

& 1988-PmTOR Phosphorylates HSF-1 to Control Protein Folding Fidelity in Parallel with Protein BiogenesisJIA YOU, JU HO YOUN, GIULIO ROMEO, MARK JEDRYCHOWSKI, STEVEN GYGI, BRENDAN D. MANNING, JONGSOON LEE, STEVEN E. SHOELSON, Boston, MA

Feeding and nutrients induce the activity of hepatic mTORC1, most nota-bly a burst in protein synthesis. We have found that transcriptional activity of heat shock factor 1 (HSF-1) in liver parallels mTORC1 activity during fast-ing/refeeding in mice. HSF-1 controls the heat shock response (HSR) through synthesis of protein folding chaperones, including heat shock proteins in the HSP40, HSP70, HSP90 families. mTOR activation by TSC2 deletion and inhibi-tion e.g. with Torin1 upregulates or inhibits HSF-1 activation, respectively. Mass spectrometry showed that in parallel with promoting protein synthesis through phosphorylation of S6 kinase and 4E-BP1, nutrient-activated mTORC1 phosphorylates Ser 303, 307 and 363 of HSF-1 to signal nuclear entry and transcriptional activation. Phosphorylated HSF-1 Ser307 was detected in the nucleus only. RNAseq analyses of livers from wt and hepatocyte-specifi c HSF-1 knockout mice (Hsf1/fl ox x Alb-Cre) were conducted following fasting and refeeding. In addition to the induction of many chaperones, refeeding in wt mice upregulated expression of selected Xbp1 targets and other genes relevant to protein glycosylation and degradation - all of which were dimin-ished in livers of Hsf1-deleted mice, despite lean and obese Hsf1-deleted mice having similar metabolic phenotypes compared to wt. These fi ndings demonstrate that in parallel with promoting protein biogenesis, the mTOR/HSF-1 axis directly upregulates the cellular machinery required for proper folding and synthetic fi delity of nascent proteins, i.e. proteostasis, includ-ing elements of both classical heat shock and unfolded protein responses (UPR). These fi ndings therefore suggest that nutrients and obesity, acute and chronic activators of mTOR, respectively, co-translationally activate

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HSR and elements of the UPR, and that enhanced proteostasis associated with obesity is a normal physiological response to nutrient-mediated mTOR activation, as opposed to a cause of insulin resistance.

Supported By: American Diabetes Association (1-12-CT-71, 7-12-MN-77 to S.E.S.); National Institute of Diabetes and Digestive and Kidney Diseases

& 1989-PEicosapentaenoic Acid but Not Docosahexaenoic Acid Promotes a Brite Phenotype in Primary Human AdipocytesMANUELA ELSEN, DANIELA DINNIES, TOMAS JELENIK, MICHAEL RODEN, TANIA ROMACHO, JUERGEN ECKEL, Düsseldorf, Germany

Promoting the induction of UCP1-expressing brown-like (brite/beige) adi-pocytes within white adipose tissue (WAT) represents a strategy to counter-act obesity. This so-called browning process may be triggered by polyunsat-urated fatty acids (PUFAs). Indeed, intake of the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) prevents the development of obesity and increases WAT oxidative metabolism in rodents. Therefore, we hypothesize on a particular role for n-3 PUFAs in WAT browning in humans.

Human adipose-derived stem cells (hASCs) isolated from the subcutane-ous depot were exposed to 20 µM of the n-3 PUFAs EPA and DHA, arachidon-ic acid (ARA, n-6) and oleic acid (OA, n-9) during adipocyte differentiation (12 days). Treatment of hASCs with EPA, DHA, and ARA increased lipid accumu-lation and HSL protein levels, indicating enhanced adipogenesis. Adiponec-tin protein levels were higher in hASCs exposed to EPA and DHA. Expression of the brown marker genes UCP1 and CPT1B were solely upregulated by EPA, while ARA signifi cantly enhanced mRNA levels of the WAT-specifi c marker TCF21. The benefi cial effects of EPA on UCP1 and CPT1B expression were abrogated when EPA was combined with ARA. Protein levels of OXPHOS complexes in total, an indicator of mitochondrial density, were not altered by any of the fatty acids. However, citrate synthase activity was increased in EPA treated hASCs. Moreover, spare respiratory capacity was reduced in hASCs challenged with ARA. Importantly, EPA was also able to upregulate UCP1 and CPT1B expression in hASCs when only added from day 8 to 12 of differentiation.

In conclusion, solely the n-3 PUFA EPA but not DHA promotes a brite gene expression pattern and improves mitochondrial function, providing a poten-tial mechanism for the benefi cial anti-obesity effects of dietary n-3 PUFAs. Remarkably, EPA can even trigger a brite phenotypic shift in the late stage of differentiation.

Supported By: European Commission (ADDIO-PIEF-2012-328793 to T.R.)

& 1990-PImpaired Cholesterol Production by Glia Results in Protection from Obesity and Increased Respiratory Exchange Ratio in MiceHEATHER FERRIS, FRED DAVIS, C. RONALD KAHN, Boston, MA

We have previously shown impaired insulin-mediated cholesterol synthe-sis in the brain in mouse models of both type 1 and type 2 diabetes. It is unknown if this contributes to the metabolic effects or increased cognitive decline seen in some patients with diabetes. Almost all brain cholesterol is synthesized in the brain, as it does not readily cross the blood brain barrier. Studies of neurons and glia in culture suggest that glia are the major source of cholesterol synthesis in the brain and are required for healthy neuronal fi ring. To test this hypothesis in vivo we made a conditional knock-out of SREBP2, a key transcriptional regulator of the cholesterol synthetic path-way, in glia using a GFAP-Cre. The resulting SREBP2 GFAP-/- mice were vi-able but had a 30% decrease in brain volume. SREBP2 GFAP-/- mice were protected against age and high fat diet induced obesity with no change in linear growth. Despite reduced body mass, these mice ate more per gram of body weight than their littermate controls, indicating alterations in energy expenditure. Metabolic cage assessment revealed that the respiratory ex-change ratio (RER) for these mice was dramatically increased in the fed state, but not in the fasting, suggesting preferential burning of carbohydrates but the preserved ability to switch to fat as a fuel source. Parameters normally associated with alterations in RER and energy expenditure, such as activ-ity, leptin, thyroid hormone and catecholamines were not changed in the SREBP2 GFAP-/- mice. While total food intake was similar in the two groups of mice, there was a shift in feeding patterns, suggesting an alteration in circadian rhythms. Using wheel-running cages in constant darkness it was clear that SREBP2 GFAP-/- mice had a longer circadian period. In conclusion, mice with impaired cholesterol synthesis in glia, as occurs in uncontrolled diabetes, display alterations in bodyweight, substrate utilization, and circa-dian rhythm which may contribute to diabetes pathology.

Supported By: 5K08DK097293-03

& 1991-PDistinct Roles of Circulating Oleate and Palmitate in Energy Homeo-stasisJANG YOUN, YOUNG TAEK OH, Los Angeles, CA

We recently showed that a fall in plasma FFA level activates the hypotha-lamic-pituitary-adrenal (HPA) axis to increase plasma ACTH and corticoster-one in rats. Because these hormones increase lipolysis in adipocytes, these responses may serve as a mechanism for feedback control of plasma FFA levels. A follow-up study showed that this regulation was mainly mediated by palmitate (C16:0), but not oleate (C18:1) or linoleate (C18:2), apparently in-consistent with earlier fi ndings that when injected into the brain, oleate, but not palmitate, was sensed to reduce food intake. In the present study, we tested if food intake is regulated by circulating oleate. Rats received an i.v. infusion of olive, saffl ower, or coconut oil (100 mg/h; n=6 for each group), to-gether with heparin, to raise circulating oleate, linoleate, or saturated fatty acids, respectively. Food intake decreased similarly (~30%) with saffl ower and coconut oil infusions, which might be responses to the energy intake due to the oils infused. In contrast, olive oil infusion had a more profound ef-fect to reduce food intake (~70%; P < 0.01 vs. other oils), consistent with the concept that food intake is regulated by circulating oleate. To further sup-port this, oleate infusion (0.5 mg/h; n=6) decreased overnight food intake by 26% (P < 0.001), but no signifi cant effect was seen with linoleate, octanoate, or vehicle (0.1% albumin) infusion. These data, taken together, suggest the intriguing possibility that circulating oleate and palmitate may be involved in different regulations; oleate, which originates largely from food, may signal food intake for the brain to subsequently reduce food intake. In contrast, palmitate, which can be produced from other major fuels, such as glucose and amino acids, may signal the amount of available fuels for the brain to regulate fat storage or mobilization via the HPA axis. Thus, there may be a “division of labor” between oleate- and palmitate-sensing mechanisms for separate regulation of energy balance and storage/mobilization.

Supported By: American Diabetes Association (7-12-BS-214 to J.Y.)

& 1992-PPotentiation of Insulin Response and Reduction of Postprandial Gly-cemia, Free Fatty Acids, and Glucagon after Lunch and Dinner by Eating vs. Skipping Breakfast in Type 2 Diabetic PatientsDANIELA JAKUBOWICZ, JULIO WAINSTEIN, BO AHRÉN, ZOHAR LANDAU, YOSEFA BAR-DAYAN, MAAYAN BARNEA, OREN FROY, Holon, Israel, Lund, Swe-den, Rehovot, Israel

Reduction of postprandial hyperglycemia (PPHG) is major target in treat-ment of type 2 diabetic patients (T2D). Skipping breakfast (B) has been as-sociated with higher HbA1c and overall PPHG in T2D. We aimed to explore the effect of eating vs. skipping B on PPHG after subsequent isocaloric lunch (L) and dinner (D). In crossover design 22 T2D, aged 57±1 y; BMI 28.2±0.6 kg/m2; HbA1c 7.7±0.08%, were randomly assigned to 2 meal test days: one day (YesB) with B, L and D, another day (NoB) the B was omitted. Postprandial plasma glucose, insulin, free fatty acids (FFA), glucagon and intact gluca-gon-like peptide-1 (iGLP-1) after isocaloric (700 kcals; % of CH: protein: fat: 47:31:22) L and D were assessed.

AUC180 after Lunch in YesB vs. NoB, was lower for glucose response (30566±19 vs. 41804±31 mg/dl*min p<0.0001 by - 37%), higher for insulin (6213±12 vs. 5156±86 µU/ml*min p<0.0001 by 17%) and for iGLP-1 by 19% p<0.0001 while plasma FFA and glucagon were more suppressed by -41.3% and -14.8% p<0.0001 respectively. Insulin peaked higher and earlier at 30 min after L in YesB vs. at 60 min after L in NoB.

AUC180 after Dinner in YesB vs. NoB, was less for glycemic response (37368±247 vs. 47310±88 mg/dl*min p<0.0001 by - 26.6%), enhanced insulin (5593±15 vs. 5150±99 µU/ml*min p<0.0001 by 7.9%.) and iGLP-1 by 16.5% p<0.0001. More suppressed FFA by -29.6% p<0.0001 and reduced glucagon by -11.5% p<0.0001. Insulin peaked higher at 60 min after D in YesB vs. at 90 min in NoB condition.

AUC for glycemic response correlated positively with FFA (R2=0.78 p< 0.0001) and with glucagon (R2=0.73 p< 0.0001) and negatively with iGLP-1 (R2=- 0.79 p< 0.0001) AUC responses after L and D.

Conclusion: Eating vs. skipping B led to reduced PPHG after Lunch and Din-ner. This was accompanied in YesB, by higher iGLP-1, higher and faster pran-dial insulin release and more suppressed plasma FFA and glucagon. Avoiding breakfast omission should be a strategy toward reduction of PPHG in T2D.

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& 1993-PLong-Term Exposure to a High-Fat Diet Results in Low Plasma Branched-chain Amino Acids in Mice Despite Type 2 DiabetesYUSUKE ADACHI, KENJI NAGAO, HIROKO JINZU, ERI IKEGAMI, MAIKO MORI, YASUSHI NOGUCHI, Kawasaki, Japan

Branched-chain amino acids (BCAA) are important nutrient signals that affect metabolism. Many studies have demonstrated that the supplementa-tion of BCAA has positive effects on obesity and hyperglycemia. Despite its salutary infl uence on metabolic disorders, based on the frequently observed increase of circulating BCAA in human and animal T2DM, BCAA may in fact trigger type 2 diabetes (T2DM). The hypothesized mechanism linking the in-creased plasma BCAA levels and T2DM involves BCAA-mediated activation of mTOR, which results in inhibiting insulin signaling. However, we found a T2DM model exposed to a long-term (16 weeks) high fat diet (LHFD) which shows decreased rather than increased plasma BCAA levels. To understand the physiological roles of BCAA in T2DM, we analyzed metabolic pheno-types and amino acid profi les of LHFD mice. LHFD mice showed obesity, hyperglycemia, hyperinsulinemia and hypoadiponectinemia, confi rming that LHFD mice have typical T2DM phenotypes. Still, LHFD mice showed a siz-able reduction of BCAA (leucine, isoleucine and valine) in plasma (14-22% decrease, P < 0.001) and liver (40-47% decrease, P < 0.001) but a slight re-duction in skeletal muscle (5-14% decrease, P < 0.05). A marked increase of BCAA was observed in adipose tissue (33-50% increase, P < 0.05). There was also a strong correlation between plasma and hepatic BCAA levels (r=0.91, P < 0.0001), suggesting that perturbation in hepatic BCAA metabolism pre-dominantly contributes to the reduced plasma BCAA in LHFD. Despite the reduced BCAA, the hepatic mTOR pathway was signifi cantly stimulated (67% increase in phospho-4EBP1, P < 0.005), indicating that the activation of mTOR is independent of BCAA. More importantly, short-term exposure to high fat diets (6 weeks) induced T2DM but did not alter BCAA metabolism. Taken together, these results would support a hypothesis that BCAA has benefi cial effects rather than negative effects in metabolic disorders.

1994-PEffect of Endogenous GLP-1 on Food Intake Before and After RYGB in Patients with Type 2 DiabetesMARIA S. SVANE, NILS B. JØRGENSEN, CARSTEN DIRKSEN, KIRSTINE N. BOJS-EN-MØLLER, SIGNE TORÄNG, VIGGO B. KRISTIANSEN, JENS J. HOLST, STEN MADSBAD, Hvidovre, Denmark, Copenhagen, Denmark

Roux-en-Y Gastric Bypass (RYGB) produces a large, sustainable weight loss probably by reducing energy intake. Exaggerated meal-induced secre-tion of the appetite-regulating gut hormone GLP-1 has been suggested as an important mechanism.

We investigated ad libitum (ad lib) energy intake and meal-induced gut hormone secretion before (pre) and 3 months (3mo) after RYGB in patients with type 2 diabetes (T2D) and evaluated the effect of GLP-1 receptor an-tagonism.

Nine patients with T2D (age: 50±3 years, gender (f/m): 3/6, BMI: 39.2±2.4 kg/m2, diabetes duration: 5.7±1.3 years, HbA1c: 48±4 mmol/mol) underwent two standard liquid meal tests on separate days at two visits (pre and 3mo after RYGB) with infusion of either saline or the GLP-1 receptor (GLP-1R) antagonist, exendin 9-39. Patients met fasting, basal blood samples were drawn, the liquid meal was given and blood was sampled frequently for 4 hours. Subsequently patients were served an ad lib meal of pasta Bolognese (energy content 533 kcal/100 g).

Ad lib energy intake was reduced by ~40% after RYGB compared to before (pre: 2009±181kJ vs. 3mo: 1151±107 kJ, p<0.01). GLP-1R blockade increased ad lib intake before surgery (+16±5%, p=0.039), whereas no effect was seen postoperatively.

Meal-induced secretion of GLP-1 (AUC pre: 2.7±0.4 nmol x min/L; 3mo: 5.5±0.5, p<0.01) and PYY (AUC pre: 4.6±0.3; 3mo: 5.8±0.5, p<0.01) increased markedly after RYGB. Before RYGB GLP-1R blockade increased GLP-1 secre-tion (+29±10%, p=0.02), whereas PYY secretion was unaffected (+7±8%, p=0.6). After surgery GLP-1R antagonism increased not only GLP-1 secretion (+57±12%, p<0.01) but also PYY secretion signifi cantly (+53±10%, p<0.01).

In conclusion, RYGB reduces ad lib energy intake and this is associated with increased GLP-1 and PYY secretion. In patients with T2D GLP-1R block-ade increases food intake before RYGB, but surprisingly not after, which may be explained by a concurrent hypersecretion of PYY. Thus GLP-1 and PYY may act in concert to inhibit appetite after RYGB.

Supported By: University of Copenhagen

1995-PObesity without Glucose Intolerance in Mice Fed a High Starch DietAMANDA E. BRANDON, TUONG-VI NGUYEN, EURWIN SURYANA, LEWIN SMALL, EDWARD W. KRAEGEN, GREGORY J. COONEY, Darlinghurst, Australia, Sydney, Australia

A high fat diet in mice leads to the accumulation of fat in adipose tissue and non adipose tissues (liver, muscle) and is linked to the development of glucose intolerance and insulin resistance. A diet high in starch can also lead to the accrual of adipose tissue but the effects on glucose metabolism are less clear. We housed 3 groups of C57BL6 mice for 30 weeks at a thermoneutral tem-perature of 29oC and fed one group a standard chow diet (20% protein, 11% fat 69% carbohydrate), another a high starch (Hi-ST) diet (14% protein, 28% fat, 58% carbohydrate) and another a high fat diet (Hi-F) (14% protein, 58% fat, 28% carbohydrate). Body weight, food intake, body composition (DEXA), glucose tolerance and insulin tolerance were assessed over the feeding period and tissue sampled for analysis at the end of the feeding period. Both Hi-ST and Hi-F diet mice gained signifi cantly more body weight than chow-fed mice (42.8±1.5g; 38.4±1.7g; 29.8±0.6g, respectively) and had signifi cantly more fat mass by DEXA scanning (Hi-ST 8.5±0.7g; Hi-F 8.5±0.7g; chow 4.4±0.2g). Energy intake was higher in the Hi-ST and Hi-F groups compared to chow. Despite a similar degree of body fatness, Hi-ST mice had signifi cantly better glucose tolerance than Hi-F mice (incremental Area Under the Curve 535±56 versus 888±59; P<0.001) and similar glucose tolerance to lean, chow-fed mice (iAUC 528±53). Insulin levels during the GTT were highest in Hi-ST mice but both Hi-ST and Hi-F mice had increased levels of triglycerides in liver compared to chow-fed mice (Hi-ST 20.3±0.9µmol/g; Hi-F 23.6±1.9µmol/g; chow 10.3±0.9µmol/g). Immunoblotting showed decreases in fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC) protein in adipose tissue and steroyl CoA desaturase (SCD1) and ACC in the liver of Hi-F mice. These results indicate that mice that become obese on a Hi-ST diet do not develop glucose intolerance as do similarly obese mice fed a Hi-F diet suggesting these models may be useful for determining mechanistic links between obesity and reduced insulin action.

1996-PEffi cacy of High Protein vs. High Carbohydrate Diet on Remission of Impaired Glucose Tolerance (IGT) to Normal Glucose Tolerance (NGT) and Physiological Parameters in Obese AdultsFRANKIE B. STENTZ, CHANNING GARBER, ABBAS KITABCHI, Memphis, TN

The purpose of this study is to determine if a High Protein (HP) or High Car-bohydrate (HC) diet is more effective in conversion of patients with impaired glucose tolerance (IGT) to normal glucose tolerance (NGT), physiological pa-rameter that are affected and to identify if HP or HC diet contributes to DNA methylation changes that can be linked to conversion from IGT to NGT.

18 obese, pre-diabetic adults were randomized to a HP or HC diet for 6 months(mo) with all food provided. The HP diet consisted of 30% protein, 30% fat, 40% carbohydrates while the HC diet consisted of 15% protein, 30% fat, 55% carbohydrates distributed by percentage of daily kcals derived for each subject. An Oral Glucose Tolerance Test (OGTT) was performed at Baseline (BL) and 6 mo to determine IGT/NGT status. A 2 hr glucose between 140 to 199 mg/dl was considered IGT. Food pick up and weight checks were weekly.

The HP diet had a 100% (9/9) conversion rate to NGT while the HC diet had a 44.4% (4/9) conversion rate. Both diet groups had weight loss and improve-ment in insulin sensitivity determined by Matsuda Index [HP(BL 2.0 ± 0.4; 6 mo 6.6 ±0.5)], [HC(2.1±0.5; 6 mo 3.7±0.4)] and decrease in HbA1c [HP(BL 5.99±.05; 6 mo 5.53±.02)], [HC(5.9±.04; 6 mo 5.69±.06)] and TNFa [HP(BL 11.2±0.3; 6 mo 5.4±0.2)], [HC(11.0±0.3; 6 mo 7.8±0.2)]. Baseline total genome methylation (TGM) was 5.8 ±0.5% for HP group and 5.7±0.4% for the HC group. The HP group had a 6 mo TGM of 3.9±0.4% while the HC group was 4.6±0.4% in the remission patients and 5.3±0.5% in the non-converting pa-tients. Both diets resulted in improvement in glucose tolerance and insulin sensitivity but the HP diet was most effective. The decrease in DNA methy-lation from 0 to 6 mo may correlate with reduced blood glucose. Studies to determine specifi c genes involved in DNA methylation may be important.

Supported By: American Diabetes Association (7-12-CT-41 to A.K.)

1997-PPharmacological Activation of AMPK Suppresses Infl ammation through a Novel AMPK/mTOR/NF-κB AxisJU HO YOUN, GIULIO ROMEO, JIA YOU, JONGSOON LEE, STEVEN E. SHOELSON, Boston, MA

AMPK is a major cellular sensor for energy depletion. Although its role in metformin’s metabolic actions is debated, AMPK is a viable therapeutic target in T2D - validated by results of the TINSAL trials showing substantial glucose and lipid lowering by salicylate (SA) in patients with T2D. The anti-

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infl ammatory drug SA inhibits NF-κB in addition to activating AMPK. Genetic ablation and siRNA knockdown of AMPK both activate mTOR and NF-κB, and prevent SA from inhibiting NF-κB. Our results also show for the fi rst time that NF-κB inhibition and AMPK activation, two seemingly indepen-dent events, are linked through mTOR. SA and AICAR bind AMPK to directly enhance phosphorylation of AMPK itself and Ser487 in the tumor suppressor TSC2. This promotes TSC1/TSC2 GAP activity to inhibit Rheb, the small GT-Pase responsible for mTOR activation. We further show this inhibits mTORC1, but not mTORC2, which suppresses the potential for infl ammatory stimuli to active IKKβ, the kinase responsible for canonical NF-κB activation. Loss of mTOR activity suppresses TRAF6-mediated ubiquitination of IKKγ, the scaf-folding component of the IKK signalsome. IKKγ and TRAF6 ubiquitination are upregulated in AMPK-defi cient cells, leading to NF-κB activation. By con-trast, either pharmacological (Torin1) or genetic (Rheb siRNA knockdown) inhibition of mTOR signifi cantly reduce TRAF6 and IKKγ ubiquitination and TNF-α induced IKKβ phosphorylation, IκBα turnover, and NF-κB activation. Three major signaling nodes are thus affected by AMPK activation: AMPK itself, the mTORC1 complex, and NF-κB. AMPK-phosphorylated TSC complex and Rheb link AMPK to inhibited mTOR, whereas TRAF6 and IKKγ ubiquitina-tion link mTOR to IKKβ and NF-κB activation. In this setting NF-κB inhibition is thus inextricably linked to AMPK activation. This suggests that clinical AMPK activators will suppress infl ammation and impact cardiovascular dis-ease, which accounts for >85% of deaths in patients with T2D.

Supported By: American Diabetes Association (1-12-CT-71, 7-12-MN-77 to S.E.S.); National Institute of Diabetes and Digestive and Kidney Diseases

1998-PSupraMammillary Nucleus (SuMN) Dopaminergic Communication with the Biological Clock (Suprachiasmatic Nucleus; SCN) Area Regulates Peripheral Insulin SensitivityMICHAEL EZROKHI, SHUQIN LUO, YAHONG ZHANG, YANG LI, YELENA TRUBIT-SYNA, ANTHONY H. CINCOTTA, Tiverton, RI

SuMN dopamine (DA) neurons provide a primary dopaminergic input to the peri-SCN area, where circadian DA activity is critical in maintaining periph-eral insulin sensitivity. Silencing of SuMN neurons with GABA stimulation and NMDA inhibition (SuMNi) in normal rats results in insulin resistance. It is not known however if or to what extent this SuMNi effect on insulin resistance derives from the reduction in SuMN - SCN DA communication. This study therefore investigated whether SCN circadian-timed DA infusion could reverse insulin resistance induced by SuMNi. Sprague-Dawley rats re-sistant to the weight gain effects of a high fat diet (HFD) were maintained on such HFD and randomized to one of three treatment groups (SuMNi [GABAa receptor agonist/AMPA receptor antagonist cocktail {muscimol/CNQX: 3.6/1.8 nm/day} infusion via osmotic pumps directly to SuMN], SuMNi plus SCN circadian timed DA infusion [4 nm in 0.2 µl artifi cial CSF at the right side SCN for one minute daily at onset of daily locomotor activity], or con-trol [vehicle SuMNi and SCN infusions]; N=10/each group). After 2 wks of treatment all animals were subjected to a glucose tolerance test (GTT) (3 g glucose/kg) followed in 48 hr by a pyruvate tolerance test (PTT) prior to sacrifi ce 48 hr later for analyses of body fat and liver lipid levels. Relative to vehicle, SuMNi increased the GTT glucose AUC by 25% (P<0.005), the PTT glucose AUC by 26% (P<0.0001) and insulin resistance (Belfi ore insulin sen-sitivity index) by 100% (P<0.005), liver lipid level by 50% (P<0.03), and total body fat by 96% (P<0.01). The SCN DA treatment reversed the dysglycemia, insulin resistance induced by SuMNi (P<0.02), reduced the SuMNi effect on liver lipids (by 50%, P=0.05) and did not alter the SuMNi effect on body fat. These results suggest that SuMN circadian DA communication with the SCN regulates peripheral insulin sensitivity independent of other SuMN effects on body fat.

1999-PSR-135, a Peroxynitrite Decomposing Catalyst, Ameliorates Beta-Cell Defects and Insulin Resistance in B6D2F1 Mice Fed a High-Fat DietMICHAEL JOHNS, ROBERT FYALKA, JENNIFER A. SHEA, WILLIAM L. NEUMANN, SMITA RAUSARIA, ELIWAZA MSENGI, MARYAM IMANI-NEJAD, HARRY ZOL-LARS, TIMOTHY MCPHERSON, JOSEPH SCHOBER, JOSHUA WOOTEN, GUIM KWON, Edwardsville, IL

Peroxynitrite has been implicated in β-cell dysfunction and insulin resis-tance in obesity. Chemical catalysts that destroy peroxynitrite, therefore, may have therapeutic value for treating type 2 diabetes. To this end, we have recently demonstrated that Mn (III) bis (hydroxyphenyl) dipyrromethene complexes, SR-135 and its analogues, can effectively catalyze the decompo-sition of peroxynitrite in vitro and in vivo through a 2-electron mechanism. To

study the effects of SR-135 on glucose homeostasis in obesity, B6D2F1 mice were fed with a high fat-diet (HFD) for 12 weeks and treated with vehicle, SR-135 (5 mg/kg), or a control drug SRB for 2 weeks. SR-135 signifi cantly reduced fasting blood glucose and insulin levels, and enhanced glucose tol-erance as compared to HFD control, vehicle or SRB. Islets isolated from mice treated with SR-135 showed signifi cantly greater glucose-stimulated insulin secretion and elevated insulin content than control animals. Moreover, SR-135 restored islet architecture, decreased islet size, and reduced tyrosine nitration and apoptosis. These results suggest that a peroxynitrite decom-posing catalyst enhances insulin sensitivity and β-cell function and survival under nutrient overload.


2000-PEndocannabinoids and Food Intake: What If It All Came from the Periphery?JENNIFER VINERA, FILIPE DE VADDER, AUDE BARATAUD, CARINE ZITOUN, ADE-LINE DUCHAMPT, MAUD SOTY-ROCA, GILLES MITHIEUX, Lyon, France

Food intake is a key physiological process with constant crosstalk be-tween the brain and the peripheral organs. The endocannabinoid system through its CB1 receptors (CB1Rs) is a key system controlling energy homeo-stasis and feeding behavior. CB1R pharmacological antagonists like Rimona-bant (SR141716) are potent hypophagic agents but their site and mechanisms of action remain elusive.

Initially, Rimonabant-induced hypophagia was considered as a central mechanism since CB1Rs control the activity of many neurons regulating food intake. Surprisingly, several studies reported that CB1Rs might also modu-late feeding from peripheral sites. Indeed, a subset of CB1Rs is located on gut nerve terminals, which are known to mediate satiety signals. One key question is to elucidate how peripheral CB1Rs could modulate feeding. A peripheral function known for its satiety effect is intestinal gluconeogenesis (IGN). Glucose produced from IGN can be detected by a sensor connected to the portal neural system eventually curbing hunger at a central level. Hence, we hypothesized that peripheral CB1Rs could modulate food intake by induc-ing IGN.

Here we showed that CB1R blockade with either Rimonabant (acting both centrally and peripherally) or AM6545 (restricted to the periphery) led to a reduction in food intake, suggesting that peripheral CB1Rs play a crucial role in the regulation of food intake. We also observed a concomitant induction of the intestinal glucose-6-phosphatase (G6Pase) activity. Consistently, we found that Rimonabant induced neuronal activation in satiety centers and in central relays of both spinal and vagal pathways. Interestingly, these ef-fects were abolished by specifi c denervation of portal sensitive nerves with capsaicin-treatment. Strikingly, the hypophagic effect of Rimonabant or AM6545 was abolished in mice lacking GP6ase specifi cally in the intestine.

Altogether, our data suggest that the Rimonabant-induced hypophagia is mediated by peripheral CB1Rs and depends on IGN.

2001-PGreater Insulin Resistance and Paradoxically Lower Postprandial Peak Glucose in Black Compared with White Women: The Federal Women StudyPAOLA C. ALDANA, CAROLINE K. THORESON, MICHELLE T. DUONG, MADIA RICKS, LILIAN S. MABUNDO, ANNE E. SUMNER, STEPHANIE T. CHUNG, Bethes-da, MD

Black women have greater hyperinsulinemia to oral and intravenous (IV) glucose compared to whites. It is unknown if these differences persist dur-ing a normal meal. We performed a mixed meal test (MMT), OGTT and FSIGT in 28 non-diabetic female federal employees (15 African American, 6 African, 7 White; age 46±8y (mean±SD); BMI 28±5kg/m2). Area under the curve (AUC) for insulin and C-peptide levels were used to quantify insulin response and hepatic insulin extraction (HIE) during the OGTT and MMT. Acute insulin re-sponse to IV glucose (AIRg) and insulin sensitivity index (SI) were calculated from the FSIGT. Black women had lower SI (3.1±0.8 vs. 5.5±2.2 mU/L-1.min-1, P<0.01) and greater insulin response to glucose independent of mode of ad-ministration (insulin AUCOGTT 274 ± 173 vs. 143 ± 53, P=0.03; insulin AUCMMT 5249±3333 vs. 2698±1545, P=0.03; AIRg 625±354 vs.399±216, P=0.1). Peak meal glucose was lower in black women (Figure) but glucose did not differ by race during the OGTT or FSIGT. During the OGTT and MMT, black women had lower HIE (76±9 vs. 84±5% and 75±8 vs. 85±4%, respectively, P<0.05). Black women were more insulin resistant, yet had lower peak glucose dur-ing a meal. This paradoxical postprandial glucose response may be partially explained by greater hyperinsulinemia. Racial differences in postprandial factors such as gastric emptying should also be considered.

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Supported By: National Institute of Diabetes and Digestive and Kidney Diseases

2002-PPlasma Acylcarnitines Are Increased in the Model of Acute Satu-rated Fatty Acid Diet-induced Insulin ResistanceMARLIES K. OZIAS, JURAJ KOSKA, DAWN C. SCHWENKE, PETER D. REAVEN, Phoenix, AZ

Excess dietary saturated fat is an important risk factor in the development of insulin resistance (IR). Perturbations to the fatty acid oxidation result in elevated acylcarnitines that have been proposed to interfere with insulin ac-tion. Using our acute model of dietary saturated fatty acid (SFA)-induced IR we examined relationships of plasma and muscle acylcarnitine species to IR.

In a series of crossover studies, 21 subjects with normal or impaired glu-cose tolerance consumed a SFA-rich, high-calorie diet and a standard Ameri-can Heart Association (AHA) diet, each for 24 hours (with 2-week washout). IR was determined by steady state plasma glucose (SSPG) by insulin sup-pression test. Carnitine species were measured by mass spectrometry in plasma and skeletal muscle 4-hr after the fi nal breakfast.

SSPG was increased (i.e. increased IR) with the SFA diet compared with AHA diet (median 237 vs. 140 mg/dl, p<0.0001). Total acylcarnitines were high-er while free carnitine was lower after SFA diet compared with AHA diet (14 vs. 7 and 39 vs. 50 micromol/l, respectively, p<0.0001 both). Elevated plasma acylcarnitine species included acetyl-, 4:0-, 6:0-, 8:0-, 10:0-, 12:0-, 14:0-, 16:0-, and 18:0-carnitine. On the SFA diet, SSPG positively correlated with acyl-carnitines (r=0.64, p=0.002), including acetyl- (r=0.61, p=0.003), 4:0- (r=0.44, p=0.046), 12:0- (r=0.47, p=0.03), 16:0- (r=0.65, p=0.002) and 18:0-carnitine (r=0.45, p=0.04). Only 18:0-carnitine was higher in the skeletal muscle with the SFA diet (3.2 vs. 1.8 nmol/g, p=0.02) but it was not associated with SSPG.

Plasma, but not muscle, acylcarnitines were elevated after 24 hours of a SFA diet and were positively correlated with SSPG. These data support a role for plasma acylcarnitines in acute dietary induced IR in humans. In this model, changes in skeletal muscle acylcarnitines do not appear to be associated with IR.

Supported By: American Diabetes Association (1-14-MERCK-07 to P.D.R.)

2003-PProtective Effects of Curcumin in Mice Given a High-Fat/High-Sug-ar DietMICHAEL ROUSE, JENNIFER L. FIORI, SUSAN WALKER, ISABEL GONZALEZ MARISCAL, SARA SANTA-CRUZ CALVO, JOSEPHINE M. EGAN, Baltimore, MD

Curcumin, a natural polyphenol and primary component of turmeric, is re-ported to serve as a therapeutic agent in metabolic diseases, including type 2 diabetes (T2DM). Certain factors, such as obesity, chronic infl ammation, fatty liver, and islet dysfunction, all contribute to the development of T2DM. Due to the putative therapeutic properties of curcumin, we investigated if 6 months of daily curcumin added to diet was protective of the metabolic consequences induced by a high fat/high sugar (HFS) diet in mice. Initially, mice on HFS + curcumin had reduced body weight compared to mice on HFS alone (44.8±0.9 vs. 47.3±0.4 g BW, p < 0.05), despite the fact that these mice actually ate more food per week (28.1±0.4 vs. 24.1±0.5 g food). HFS + curcumin mice had lower fasting blood glucose (179±6 vs. 202±6 mg/dL) and insulin (0.9±0.2 vs. 0.5±0.1 ng/mL) compared to HFS mice, suggesting cur-cumin supplementation leads to improved insulin sensitivity. Additionally,

we found that HFS+curcumin mice had smaller sized islets (~71% reduction compared to HFS) in keeping with reduced insulin requirement. By oil red o staining of liver, HFS mice had neutral triglycerides and lipids increase by 57.6% compared to mice on a standard chow diet. Furthermore, triglyceride and lipid staining was signifi cantly reduced by 8.8% in HFS+curcumin mice compared to mice on HFS alone. In plasma obtained at the end of the study, we detected signifi cantly elevated levels of GM-CSF, MIP-1α, MIP-1β, IL-10, and IL-12 (p40) in HFS+curcumin mice compared to HFS mice, suggesting an enhanced activated immune system. In addition, we found that the expres-sion of TXNIP mRNA, a pro-oxidant and pro-apoptotic protein is signifi cantly elevated 2.3-fold in islets cultured under high fat/high glucose conditions, whereas curcumin treatment prevented the subsequent increase of TXNIP mRNA. In conclusion, the fi ndings of our study suggest that curcumin is a potent, natural therapeutic agent that probably works in a complex, multi-faceted manner to protect against insulin resistance.

2004-PCold Induced Brown and White Adipose Tissue Lipidomic DynamicsMATTHEW D. LYNES, MICHAEL A. KIEBISH, YU-HUA TSENG, Boston, MA, Fram-ing ham, MA

Brown adipose tissue activation leads to increased energy expenditure and enhanced glucose metabolism. Although cold exposure is an effective way of activating brown fat, therapeutic cold challenge has proven uncomfort-able for humans, making identifi cation of circulating factors that mediate the thermogenic effect an attractive therapeutic target to ameliorate metabolic abnormalities. Recently, circulating lipid species that activate cell surface receptor mediated signaling cascades have been identifi ed, suggesting lipids may act as hormones in inter-organ communication. In this study, we sought to investigate the lipidomic profi le of brown fat, white fat and serum from mice that were cold challenged in order to prospectively identify potential lipidomic mediators of thermogenesis. Using mice housed in thermoneutral and cold conditions to quantify the lipid content of different adipose tissues and serum, we defi ned the dynamics of the lipidomic profi le in response to cold challenge. Several classes of lipids form a profi le of brown adipose tissue that is distinct from white adipose. Further, specifi c lipid classes are cold responsive in differ-ent adipose tissue depots. By taking a bioinformatic approach, we have begun to identify target lipid species that may signal activation of thermogenesis in brown fat. We have identifi ed a serum lipid which is able to activate thermo-genesis in mice presumably via promoting fatty acid utilization in brown fat, thereby protecting animals from the decreased body temperature normally seen in response to an acute cold challenge. Taken together, these fi ndings highlight a diverse set of lipids that defi ne tissue identity, are dynamic in the cold, demonstrate the ability of a circulating lipid to activate brown adipose tissue thermogenesis, and may have translational potential in treating human obesity, diabetes and other related metabolic disorders.

Supported By: American Diabetes Association (7-12-BS-191 to Y-H.T.)

2005-PLimonenediol Ameliorates Lipid Profi les by Upregulation of Sirt1 Activity and Expression in Cultured Cells and High-Fat Diet-fed MiceLICHT MIYAMOTO, HARUNA AIHARA, WENTING XU, MEINA JIN, YOSUKE TOMI-DA, TOMOMI YAMAOKA, NAONOBU TANAKA, YASUMASA IKEDA, AKIRA SHI-GENAGA, AKIRA OTAKA, TOSHIAKI TAMAKI, YOSHIKI KASHIWADA, KOICHIRO TSUCHIYA, Tokushima, Japan

Sudachi (Citrus Sudachi) is a small sour citrus and grows exclusively in Tokushima prefecture in Japan. Recently our collaborators reported that ad-ministration of freeze-dried peel of Sudachi decreases serum triglyceride (TG) levels in obese human subjects. Thus we aimed to determine the active com-ponents from Sudachi peel in the current study. Here we reports limonenediol as an active ingredient to ameliorate lipid profi le via sirt1 pathway.

Administration of Sudachi peel improved serum TG levels and extended lifespan of Zucker fatty rats. Sudachi peel also reduced TG levels in cultured C2C12 myotubes. Thus, we assumed that sirt1 might be involved and con-ducted a cell-based screening using C2C12 cells by an index of sirt1 activity.

Two compounds out of 35 molecules purifi ed from Sudachi peel increased sirt1 activities, one of which found to be limonenediol. Limonenediol also in-creased activities and expression levels of sirt1 in hepatic Fao cells in dose- and time-responsive manners. Limonenediol reduced intracellular TG levels, which was sensitive to a sirt1 inhibitor, nicotinamide. In high fat diet-fed mice, repetitive administration of limonenediol for 10 days did not improve glucose tolerance but reduced fatty liver, serum TG and cholesterol to the same levels as those of healthy mice with increase in sirt1 activities in the gastrocnemius muscle and liver.

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Therefore, limonenediol will be one of the active components of Sudachi peel regulating lipid metabolism, which should be mediated by sirt1 activa-tion. The sirt1 activation is supposed principally due to the upregulation of its expressions.

In conclusion, we found that limonenediol ameliorates lipid metabolism in cultured cells and in vivo with increase in the activities and expression levels of sirt1 which should be involved in the metabolic action. Limonenediol will be a novel lead compound regulating lipid homeostasis by modulating sirt1 expression levels.

2006-PNo Difference in Ad Libitum Energy Intake in Healthy Men and Woman Consuming Beverages Sweetened with Fructose, Glucose, or High-Fructose Corn SyrupJESSICA N. KUZMA, GAIL CROMER, DEREK K. HAGMAN, CHRISTIAN L. ROTH, KAREN FOSTER-SCHUBERT, EMILY WHITE, SARAH E. HOLTE, D. SCOTT WEIGLE, MARIO KRATZ, Seattle, WA

The intake of sugar-sweetened beverages (SSBs) is associated with weight gain and obesity. These beverages typically contain a combination of fructose and glucose, in the form of high fructose corn syrup (HFCS), or sucrose. There are two prominent hypotheses for why SSBs promote excess energy intake: one, the human body may not fully compensate for liquid calo-ries; alternatively, the high fructose content of SSBs may fail to trigger re-sponses in energy homeostasis-regulating hormones in the same manner as glucose. To address whether the relative amounts of fructose vs. glucose in sweetened beverages modifi es their effect on ad libitum energy intake, we conducted a randomized, controlled, double-blind cross-over dietary inter-vention trial in 24 healthy adults (12 normal weight, 12 overweight or obese). A standardized diet was provided at 125% of estimated energy needs and consumed ad libitum for three separate 8-day dietary periods. Solid foods provided were identical in all three dietary periods. In addition, subjects consumed four servings per day of beverages sweetened with fructose, glu-cose, or HFCS (55% fructose, 45% glucose), constituting 25% of estimated calorie requirements (mandatory consumption). Overall energy intake was determined by weigh-back of all uneaten foods. Mean ± standard deviation energy intakes were 2,970 ± 482, 2,950 ± 535, and 2,940 ± 460 kcal per day during the fructose, HFCS, and glucose phases, respectively, which did not differ from each other signifi cantly (p=0.880), and yet on average, were ~16% higher than the participant’s estimated total energy requirements. These data demonstrate that the type of sugar used to sweeten SSBs does not differentially affect total energy intake. Rather, our research suggests that the overconsumption of energy in individuals consuming SSBs is due to the fact that sugar is consumed in liquid form.

Supported By: 5R21HL108257, P30DK017047, P30CA015704, R25CA094880

2007-P5-HT2C Receptors in the Brainstem Regulate Energy Balance and Glucose HomeostasisTIEMIN LIU, KEVIN W. WILLIAMS, ERIC BERGLUND, CHEN LIU, YONG GAO, TING YAO, JOEL K. ELMQUIST, Dallas, TX

Serotonin (5-HT) acts via 5-HT2C receptors (5-HT2CRs) in the brain to regulate feeding behavior and glucose homeostasis. Drugs that activate 5-HT2CRs including d-fenfl uramine (d-Fen) and Lorcaserin are effective ap-petite suppressants. However, it is still unclear which 5-HT2CR-expressing neurons are required to mediate these effects. To address this question, we generated mice lacking 5-HT2CRs in brainstem neurons including the dorsal motor nuclues of the vagus nerve (DMV) and the nucleus of the solitary tract (NTS) in the caudal brainstem. In chow-fed male mice defi cient for 5-HT-2CRs failed to infl uence body weight or composition compared to controls. In contrast, high-fat diet (HFD)-fed male mice lacking 5-HT2CRs in both the DMV and NTS displayed increased body weight and adiposity. Interestingly, we found that independent of changes in body weight deletion in brainstem neurons impaired insulin tolerance. Collectively, these results suggest that 1) 5-HT2CRs signaling in brainstem neurons including those in the NTS are required to regulate feeding behavior and energy balance; 2) 5-HT2CRs sig-naling in parasympathetic preganglionic neurons of the DMV are required to regulate glucose homeostasis.

Supported By: American Diabetes Association (7-11-MN-16 to J.K.E.); American Heart Association (14SDG20370016)

2008-PVytorin Inhibits the Pro-infl ammatory Effects of Cream in Type 2 Diabetic PatientsPARESH DANDONA, HUSAM GHANIM, KELLY GREEN, SANAA ABUAYSHEH, AN-TOINE MAKDISSI, AJAY CHAUDHURI, Buffalo, NY

Our previous work has shown that ingestion of cream or fast food meal induces an increase in oxidative stress and infl ammation in peripheral blood mononuclear cells (MNC). Since vytorin (combination of simvastatin and ezetamibe) lowers plasma concentrations of LDLc, which exerts a pro-infl ammatory effect, we hypothesized that vytorin intake in type 2 diabetics may exert an anti-infl ammatory effect and also inhibit the pro-infl ammatory effect of cream. Twenty type 2 diabetic patients were randomized to either vytorin or placebo treatment for 6 weeks. Patients in both groups ingested 33g of cream (about 70% saturated fat) and fasting and post-cream chal-lenge blood samples were obtained before and after treatment. The two groups had comparable age, BMI, HbA1c and gender distribution. Total cho-lesterol and LDLc concentrations were lowered at 6 weeks following vytorin (P<0.05). Cream induced signifi cant increases in MNC expression of IL-1β (by 105±18%), TNFα (by 97±12%), CD68 (by 48±8%), PECAM (by 66±10%), TLR-4 (by 84±11%) and TLR-2 (by 67±9%) at 0 week. Fasting MNC expression of IL-1β and CD68 fell by 21±7 and 24±10, respectively, (P<0.05) following vytorin. The increase in expression of IL-1β, CD68, TNFα and PECAM following cream was suppressed signifi cantly by 74±15%, 68±13%, 67±14% and 45±9%, re-spectively, compared to 0 week) in the vytorin group. In addition, the expres-sion of TLR-2 and TLR-4 were paradoxically suppressed following cream at 6 weeks by 21±8% and 18±7%, respectively, vs. 0hr in vytorin group. Vytorin treatment also suppressed fasting and cream induced increase in LPS con-centrations in plasma (by 24% and 26%, P<0.05, vs. baseline at 0 week). Fasting concentrations of CRP, FFA and IL-18 also fell signifi cantly by 32±11%, 19±8% and 15±4%, respectively, following vytorin. We conclude that vytorin reduces fasting and cream-induced expression of pro-infl ammatory media-tors. Further studies are needed to investigate whether the observed effects are due to simvastatin or ezetamibe or both.

2009-PA Diet Enriched with Omega-3 Fatty Acids Improves Insulin Sensi-tivity in Adipose Tissue in Morbidly Obese SubjectsPIM W. GILIJAMSE, ARVID SCHIGT, BART A. VAN WAGENSVELD, MARIETTE T. ACKERMANS, MAX NIEUWDORP, J.A. ROMIJN, MIREILLE J. SERLIE, Amsterdam, Netherlands

Omega-3 (ω-3) fatty acids (FA) have demonstrated potential to improve insulin sensitivity in rodents.

We studied the effect of oral ω-3 FA supplementation (4g EPA and 1.7g DHA/day) on insulin sensitivity in morbidly obese subjects. Subjects were randomized to a eucaloric diet intervention consisting of a 50% ad libitum diet supplemented with liquid meals with (FO) or without fi sh oil (controls) to meet caloric requirements. Participants consumed the diets for 4 weeks prior to bariatric surgery. Before and after the dietary interventions a two-step hyperinsulinemic euglycemic clamp was performed to assess insulin sensitivity. Liver and abdominal subcutaneous and visceral (VAT) adipose tissue biopsies were taken during surgery.

We included 20 patients (n=10 per group, 45.3 ± 10.4 yrs; BMI 43.8 ± 6.7 kg/m2). BMI did not differ at baseline and did not change during the diet in-tervention. The FO diet increased EPA concentrations in liver and adipose tis-sue (expressed as % of total FA, 3.8 ± 1.2% and 0.11 ± 0.03% in the FO group vs. 1.0 ± 0.4% and 0.06 ± 0.02% in the control group, respectively (p<0.001)). The FO diet improved insulin-mediated suppression of FFA levels, represent-ing insulin sensitivity of adipose tissue (p=0.007). There were no signifi cant differences in hepatic or peripheral insulin sensitivity of glucose metabolism before and after treatment between and within the diet groups. The FO diet decreased Protein kinase A (PKA) expression in VAT by 1.5 fold (=0.02). There were no signifi cant differences in the expression of other key glucose or fatty acid metabolism associated genes in liver or adipose tissue.

In morbidly obese patients, a 4 week high omega-3-fatty acids enriched eucaloric diet improves insulin-mediated suppression of lipolysis and lowers expression of PKA in visceral adipose tissue.

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2010-PEffect of Dietary Phosphate on Glucose Metabolism by 18F-FDG PET/CT in RatsMAERJIANGHAN ABUDULI, HIROKAZU OHMINAMI, TAMAKI OTANI, HITOSHI KUBO, HARUKA UEDA, EIJI TAKEDA, YUTAKA TAKETANI, Tokushima, Japan, Fu-kushima, Japan

Recent studies have suggested that excess intake of phosphate would be a risk factor for progression of chronic kidney disease and cardio-vascular complication. Our previous studies also suggest that endothelial dysfunction mediated by high phosphate diet-induced hyperphosphatemia may contrib-ute to increase the risk for cardiovascular mordidity and mortality. However, the effect of dietary phosphate intake on the development of metabolic disorder such as obesity and type 2 diabetes is still under investigation. In this study, we investigated the effect of dietary phosphate loading on glucose metabolism in healthy rats. Male, Sprague-Dawley rats were di-vided into three groups and fed experimental diets including 0.2% (LP), 0.6% (CP) or 1.2% (HP) phosphate, respectively for 4 weeks. We observed high phosphate diet suppressed body weight gain and visceral fat accumulation, also decreased respiratory quotient. However, mRNA expression related to fat oxidation did not increase in the WAT and the liver of HP group. We speculated that high phosphate diet may stimulate the brown adipose tis-sue (BAT) activity. High phosphate intake signifi cantly increased UCP-1and PGC1a expression in BAT. We thus measured of BAT activity using 18F-FDG PET/CT imaging in rats. Unexpectedly, dietary phosphate didn’t signifi cantly increase 18F-FDG uptake in BAT. Interestingly, kinetic evaluation showed 1.6-fold decrease in hexokinase activity and BAT infl ux rate constant for the glucose in HP group. CT Hounsfi eld units (HUs) of BAT were decreased in HP group than LP group, BAT volume may also contribute to the fall in HUs of BAT. Considering that the HP group rats may inhibited glucose transport and hexiokinase activity in the BAT, resulting that the BAT in the HP group mainly consume the stored lipid for energy rather than glucose. Our data indicates that HP diet can negatively regulate lipid synthesis in liver and increased lipid oxidation through upregulated UCP1 expression in the BAT, thereby pre-venting visceral fat accumulation.

2011-PEffect of Gpr120-Selective Agonism on Glucose Metabolism in Zucker Diabetic Fatty RatsTONYA L. MARTIN, JIANYING LIU, MATTHEW RANKIN, THOMAS KIRCHNER, SIMON HINKE, GEORGE HO, MEGHAN TOWERS, S. PAUL LEE, YUANPING WANG, MICHAEL P. WINTERS, MARK J. WALL, MARK MACIELAG, ALESSANDRO POCAI, Spring House, PA

Gpr120 is a G-protein-coupled receptor activated by long chain fatty acids expressed predominantly in adipose tissue and colon. Chronic activation of GPR120 has been shown to improve insulin sensitivity in diet-induced obese mice. Here, we have identifi ed three structurally distinct series of selective orally bioavailable Gpr120 agonists (A, B and C). These compounds activate rat Gpr120 receptors with EC50 values of 57 nM, 196 nM and 16 nM, re-spectively and have no activity toward the other polyunsaturated fatty acid receptor, Gpr40 (EC50[Ca 2+] values > 5µΜ) Compounds A, B and C were dosed in Zucker Diabetic Fatty (ZDF) rats, a model of insulin resistance and diabetes to evaluate the effect of chronic Gpr120 activation on insulin sensi-tivity and glucose tolerance.

Male ZDF rats were treated once daily for 17 days with vehicle or com-pounds A, B or C at 30 mg/kg; rosiglitazone at 10 mg/kg was used as a positive control. Food intake and body weight were monitored during the study and an insulin tolerance test (ITT) and an oral glucose tolerance test (OGTT) were performed on day 10 and 15, respectively. At the end of the study plasma samples were collected to measure the corresponding plasma exposures to ensure target coverage.

Chronic Gpr120 agonism did not affect food intake or body weight. On day 10, compound A and compound C signifi cantly improved insulin sensitivity during the ITT. No signifi cant improvement was observed with compound B. On day 15, treatment with compound C resulted in a modest improvement in glucose tolerance during the OGTT while compounds A and B had no effect.

These data demonstrate that selective Gpr120 agonism leads to a modest improvement in insulin sensitivity and minor effect on glucose tolerance in ZDF rats.

2012-PBlunted Neutrophil CD11b Expression after High-Fat Feeding in Obese Children despite Elevated Neutrophil CountsABRAHAM S. CHIU, GOUTHAM GANESAN, FRANK ZALDIVAR, PETER HORVATH, SHLOMIT RADOM-AIZIK, PIETRO GALASSETTI, Irvine, CA

Obesity-related cardiovascular risk is linked to altered infl ammation: chronic infl ammation, including altered neutrophil (PMN) function; and acute, repeated exacerbations from stimuli such as post-prandial hyperlipi-demia. Underlying mechanisms are unclear, especially in children, despite the recent alarming increase in pediatric obesity. We therefore studied PMN responses to high-fat or placebo meals (1.5g fat/kg or no-calorie meals) in 6 healthy (HC, BMI < 85%), 7 overweight (OW, BMI 85-95%), 9 obese (Ob, BMI >95%) children. CBC analysis and fl ow-cytometry assessment of PMN surface markers (CD66, CD11b) were performed at baseline and at 1-h & 2-h post meal. In HC, high fat increased PMN CD11b expression (MFI) already at 1-h post (37568±5045), which was severely blunted in Ob (21143±2667, p<.02) and OW (23052±5442, p=.06). This occurred despite PMN counts that were 15% greater in Ob vs. HC through the study, and triglyceride levels (TG, mg/dl) that were higher in Ob vs. HC at baseline (89±16 vs. 60±8), and in-creased proportionally by 2-h post (Ob, 195±41; HC 124±17). Placebo caused no measurable changes. Our data indicates that the infl ammatory changes in pediatric obesity may include a paradoxical dichotomy between increased chronic infl ammation (leukocytosis, possibly from sustained hyperlipidemia) and a compensatory blunting of immune responses to acute pro-infl amma-tory stimuli.

Supported By: National Institutes of Health (P01HD048721-061, K24DK085223-01A1, UL1TR000153); Newkirk Center for Science and Society

2013-PEffect of Sugar vs. Mixed Breakfast on Plasma Ghrelin and P-300 Brain PotentialsLIVIO LUZI, STEFANO PAINI, ANDREA CAUMO, LUCA ANDREONI, ILEANA TER-RUZZI, MICHELE STERLICCHIO, Milan, Italy, San Donato Milanese, Italy

Breakfast consumption is associated with nutritional benefi ts. It promotes the ghrelin regulation of satiety and may increase the cognitive performance. We investigated the effects of glucose and different commercial breakfasts on 1) glucose increment and ghrelin suppression; 2) cognitive processing of sensory information assessed by frontal P 300 evoked potentials.

Twelve healthy subjects (8M/12F; BMI 20.8±0.51; 31±0.7 y) were studied. All participants completed 2 trials (Study A and Studies B) in a randomised-crossover fashion. All subjects underwent Study A: OGTT 50 g glucose and 3 commercial breakfast packs as test-meal breakfasts Study B1: milk (125 ml) and cereals (30 g); B2: milk (220 ml), apple (200 g) and cream choco-late fi lled sponge cake (30 g); B3: milk (125 ml), bread (50 g), apple (150 g)

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INTEGRATED PHYSIOLOGY—MUSCLE

and hazelnut cream chocolate (15 g). Plasma glucose and ghrelin excursions were assessed. Frontal P 300 evoked potentials were measured before and after (180 min) each study. Data (expressed as mean±SEM) showed normal distributions.The comparison among studies was performed using within-subjects ANOVA (with Greenhouse-Geisser correction) followed by Dun-nett’s post-hoc test (Study A=control). P<0.05 was considered statistically signifi cant.

The peak value of glycemia occurring 30 min after consumption (mg/dl) was signifi cantly lower during B2 (86.5±4.6) and B3 (92.2±5.1), but not during B1 (109.8±4.2), as compared to A (123.8±6.5). Ghrelin after 120 min (pg/ml) attained the lowest level in study B3 (332.1±30.2), but the decrement with respect to A (420.6±44.2) did not achieve statistical signifi cance. The incre-mental (post-pre) frontal P 300 potential (ms) showed a signifi cant anticipa-tion only in B3 (-33.6±5.9) with respect to A (9.7±6.6).

Our results indicate that cereal-based B1 elicited the highest glucose re-sponse, whereas mixed breakfast B3 elicited the highest inhibition of ghrelin and the most pronounced effect on the cognitive performance.

Supported By: Soremartec Italia S.r.l.

2014-PAcute Meal Challenge, Metabolic Flexibility, and Glucostatic Pa-rameters in Lean, Insulin-Sensitive and Obese, Insulin-Resistant ChineseEHSAN PARVARESH RIZI, TZE PING LOH, SUE-ANNE TOH, CHIN MENG KHOO, Singapore, Singapore

Acute meal challenge is used to examine the modulation in postprandial metabolic response. Here, we studied the metabolic effects of different test meals in healthy subjects with different metabolic phenotype. We recruited 9 lean (22.0±0.2 kg.m-2), insulin-sensitive (LIS) and 9 obese (30.1±0.7 kg.m-2), insulin-resistant (OIR) normoglycemic Chinese males. LIS and OIR subjects were 23.2±0.2 and 28.6±1.4 years old, respectively. Following a 10-h over-night fast, all subjects were randomised to undergo a 600-kcal meal chal-lenge of high carbohydrate (HC, 57% carbohydrate), high fat (HF, 52% fat) or high protein (HP, 55% protein), with 7 days intervals. Fasting and post meal (up to 360 min) blood samples were collected for plasma glucose, serum insulin, triglyceride, and non-esterifi ed fatty acids (NEFA) measurements. Fasting and 6-h post meal indirect calorimetry was performed to measure respiratory quotient (RQ). LIS subjects showed greater RQ increment after meal than OIR subjects. The RQ increment was greater with HC than HF or HP meal. HC meal produced higher plasma glucose, whereas HP meal produced higher serum insulin in both LIS and OIR individuals. Postprandial NEFA responses were similar between LIS and OIR subjects, and were less suppressed with HF meal. Serum triglyceride levels increased with all meals, but highest seen with HF meal. Insulinogenic index was higher among OIR than LIS subjects, and HP meal induced greatest response compared with HC or HF meal. The Matsuda index was higher among LIS than OIR subjects, and highest values seen with HF meal. We found out that LIS subjects are more metabolic fl exible than OIR ones. Different test meal induces different metabolic responses, i.e. HC meal greater glucose exposure, HP meal great-er insulin response, HF meal greater triglyceride exposure and less NEFA suppression. Hence, it is important to match the test meal to experimental hypothesis.

2015-PDoes High-Fat Meal Infl uence Fasting Glycemia in Patients with Type 2 Diabetes?EMMANUELLE LECORNET-SOKOL, AGNÈS HARTEMANN, SOPHIE JACQUEMI-NET, MARINE HALBRON, Paris, France

Patients with type 2 diabetes often report the infl uence of the composi-tion of the previous meal on fasting glycemia, especially in the upper range of HbA1c. The aim of the study is to assess the infl uence of a high fat meal on fasting glycemia.

Sixteen type 2 diabetic patients were included in a prospective random-ized cross-over study. Inclusion criteria were: age >18 years old, body mass index >26kg/m², creatinin clearance > 60ml/min, stable oral treatment, HbA1c between 7 and 9.5%, patients reporting variation of their fasting glycemia. Exclusion criteria were: type 1 diabetes, renal or hepatic failure, treatment with insulin or agonist GLP-1. A common standard meal (containing 30g of lipids: 10g of butter and 20g of oil) was compared to a high fat meal (stan-dard meal enriched in fat, 60g of lipids: 30g of butter and 30g of oil).

The characteristics of the population were: BMI=30.1 ± 4.3kg/m2, HbA1c=7.7 ± 0.5%, duration of diabetes=152.5 ± 98.5 months, retinopathy: 35%. The mean fasting glycemia twelve hours after standard meal and high fat meal was respectively 7.51 mmol/l (3.50-9.58) and 7.12 mmol/l (4.78-

11.18), p=0.91. Aera under the curve of glycemia and triglycerids were not different after the two meals: (99.04 vs. 93.90mmol/l; p=0.71, and 25.18 vs. 25.06mmol/l; p=0.60). No difference was observed either regarding fasting insulinemia (11.55 vs. 8.79mU/l; p=0.31), CRPus (2,03 vs. 2,56mg/l; p=0.70), Interleukin-6 (4.21 vs. 4.02 pg/ml; p=0.88) or GLP-1 (5.08 vs. 5.75 pmol/l; p=0.64). There was no correlation between AUC of glycemia and AUC of triglycerids (-0.22, p=0.41), adiponectin (0.15, p=0.59), leptin (0.07, p=0.77), CRPus (0.29, p=0.22), IL6 (0.33, p=0.14), fasting glycemia (0.44, p=0.10). Nev-ertheless a correlation was observed between AUC glycemia and breakfast post-prandial glycemia (0.70, P=0.01).

Using meal with a real life composition, a unique high fat enriched meal does not infl uence the following fasting glycemia in patients with a long history of type 2 diabetes and under oral treatment.

Supported By: Institut Benjamin Delessert


Guided Audio Tour: Muscle Metabolism In Vivo (Posters: 2016-P to 2021-P), see page 17.

& 2016-PAJS1669, a Novel Small-Molecule Muscle Glycogen Synthase Acti-vator, Improves Glucose Metabolism with Reducing Body Fat Mass in MiceKAZUHIRO NAKANO, Kawasaki, Japan

Glycogen, an important component of whole-body glucose metabolism, is synthesized primarily in the liver and skeletal muscle. Impaired glycogen synthesis and turnover are a common defect in insulin-resistance and type 2 diabetes. Since glycogen synthase (GS) is a key enzyme involved in the synthetic process, it presents a promising therapeutic target for treatment of type 2 diabetes. In this study, we identifi ed a novel, potent, and orally available GS activator AJS1669 (sodium 2-[[5-[[4-(4,5-difl uoro-2-methyl-sulfanyl-phenyl) phenoxy] methyl] furan-2-carbonyl]-(2-furylmethyl) amino] acetate). In vitro, AJS1669 activated GS subtype 1 (GYS1) in a concentration-dependent manner, with GYS1 stimulation further potentiated in the pres-ence of glucose-6-phosphate (G6P), an allosteric activator of GS. The action of AJS1669 in the skeletal muscle was elicited at lower concentrations than in the liver. Additionally, AJS1669 dose-dependently increased glycogen level and improved glycogen metabolism in human skeletal muscle cells. Repeated administration of AJS1669 over 4 weeks lowered blood glucose and HbA1c level in ob/ob mice. AJS1669 also improved glucose tolerance in a dose-dependent manner, and decreased body fat mass. These effects were not associated with excessive body weight gain or any adverse ef-fects on liver function. The mRNA levels of genes involved in mitochondrial fatty acid oxidation and mitochondrial biogenesis were elevated in skeletal muscle tissue following AJS1669 treatment. In contrast to ob/ob mice, nor-mal animals, AJS1669 administration elicited no alterations in body weight or glucose-lowering effects. These results demonstrate the possibility that pharmacological agents that activate GYS1, the main GS subtype found in the skeletal muscle, have potential for use as diabetes treatments of a new type, aimed at improving glucose metabolism in the skeletal muscle.

Supported By: Ajinomoto Pharmaceuticals Co., Ltd.

& 2017-PEpigenetic Regulation of Skeletal Muscle Fiber Composition through MicroRNAsCHARLES A. STUART, MARY E. HOWELL, MARIANNE F. BRANNON, MICHAEL H. STONE, WILLIAM L. STONE, Johnson City, TN

Slow-twitch (type I) fi ber content of skeletal muscle correlates with insulin responsiveness and inversely with risk for diabetes. Exercise training causes muscle fi ber shifts either to type I or away from type I, depending on whether the training is endurance or resistance and on the pre-training fi tness sta-tus of the subject. Laser capture microdissection (LCM) samples of human muscle fi bers have shown a mixture of myosin heavy chain genes expressed in each specifi c fi ber type, quantifi ed by RNAseq and by protein identifi cation with mass spectroscopy. A microRNA (miR-23) was identifi ed by sequence homology that targets the 3’UTRs of mRNA for three myosin heavy chains that characterize fast-twitch (type IIx) fi bers (genes MYH1, MYH2, and MYH4). Additional microRNAs were identifi ed targeting the mRNAs of the myosin heavy chains that predominate in slow-twitch fi bers (genes MYH6 and MYH7). In normal control subjects, miR-23a was expressed 1.5-fold higher in type I fi bers than in type IIx fi bers, quantifi ed by real-time PCR of

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LCM samples. Serum concentrations of miR-23 in 12 control subjects aver-aged 66% higher than in 12 metabolic syndrome subjects (50% vs. 39% type I fi bers, respectively). Quantifi cation of microRNAs by miRarrays in muscle from three recreational runners (mean 72% type I fi bers) compared to three metabolic syndrome subjects (mean 20% type I fi bers) showed a range of 1.8 to 3.1-fold higher content of components of the miR-23/27/24 cluster. We conclude that miR-23 may be a major factor in the regulation of skeletal muscle fi ber composition. This microRNA is likely to act by suppression of MYH1, MYH2, and MYH4 myosin heavy chain gene expression, favoring the slow-twitch phenotype. Additional microRNAs have been identifi ed that tar-get the 3’UTRs of genes that code for other parts of the muscle contractile apparatus whose expression is changed by exercise-related muscle fi ber shifts, suggesting that several additional microRNAs may have regulatory roles in these changes.

& 2018-PGlobal Kinome Interactome in Human Skeletal Muscle Revealed by ATP Affi nity Probes and ProteomicsYUE QI, ABDULLAH MALLISHO, DANJUN MA, XIANGMIN ZHANG, MICHAEL CARUSO, DIVYASRI DAMACHARLA, RODNEY BERRY, NISHIT SHAH, MAJED AB-DULLAH ALHARBI, BERHANE SEYOUM, ZHENGPING YI, Detroit, MI

Protein kinases are core modulators in cell signaling (such as insulin signal-ing) and their functions are regulated by their protein interaction partners. However, currently, no large scale analysis have been reported for kinase interaction partners in human skeletal muscle, a key tissue in the pathogen-esis of insulin resistance and type 2 diabetes (T2D). In the present study, lysate proteins from muscle biopsies from 4 lean healthy subjects were labeled with an ATP probe, while equal amount of lysate proteins without probe labeling were served as nonspecifi c controls. Co-Immunoprecipitations were performed to pull down the labeled proteins as well as their interaction partners, followed by in-solution tryptic digestion. The resulting tryptic pep-tides were analyzed by HPLC-ESI-MS/MS using an Orbitrap Elite, followed by bioinformatics. We identifi ed 542 distinct human proteins with at least 2 unique peptides and with an enrichment ratio greater than 10 fold compared to the nonspecifi c control. Of the 542 identifi ed proteins, 58 are kinases, the rest proteins were considered interaction partners of those kinases. This is the largest kinome interactome in human tissue to date. Among the 58 kinas-es, 14 kinases are involved in the insulin signaling pathway, 7 participate in glucose metabolic process, and 7 contribute to pathway related to diabetes, such as MAPK, PKA, ILK, PFKM, PGK1 and HK1. In order to fully understand the signaling pathways of these kinases, we also mapped interaction part-ners of kinases into pathways. Eighty of these kinase interaction partners are involved in pathways related to diabetes, 22 in glucose disposal, 11 in lipid oxidation, 5 in protein degradation, 5 in protein synthesis and 4 in infl amma-tion. These results indicated the establishment of an ATP probe based pro-teomics platform to profi le the kinome interactome in human skeletal muscle, which may be useful to provide novel insights into the molecular mechanism of skeletal muscle insulin resistance and T2D.

Supported By: National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases (R01DK081750 to Z.Y.)

& 2019-PSuccinate-Supported Skeletal Muscle Mitochondrial Respiration and ATP Production in a Mouse Model of Type 2 DiabetesFAN BAI, BRIAN D. FINK, LIPING YU, WILLIAM I. SIVITZ, Iowa City, IA

Mitochondrial respiration on the complex II substrate, succinate, is gen-erally assessed in the presence of rotenone (rot) to block reverse electron transport (RET) to complex I and prevent oxaloacetate (OA) accumulation, which would otherwise inhibit succinate dehydrogenase. Prior studies in our laboratory showed that skeletal muscle (SkM) mitochondrial ATP production during respiration on succinate alone (without rot) is highly dependent on [ADP]. Respiration actually peaked at low [ADP], but then dropped off mark-edly as [ADP] was increased; an effect likely due to OA inhibition. Since mi-tochondrial function and complex I activity is impaired in diabetes, we tested the hypothesis that these dynamics would differ in mitochondria isolated from SkM of diabetic (DM) compared to control (C) mice. Mitochondria were isolated from hind limb SkM of mice treated with a high fat (HF) diet for 148 ± 1 days and low dose streptozotocin (STZ) to mimic type 2 diabetes. Mean DM plasma glucose was 408 ± 29 vs. 180 ± 7 mg/dl in C plasma (normal diet, no STZ). Respiration and ATP production on succinate alone by mitochondria of both the C and DM mice increased to a max at low ADP, following which respiration and ATP decreased with increasing [ADP]. As expected, respira-tion and ATP production continued to increase with increasing ADP if rot were present. On succinate alone, peak respiration and ATP production oc-

curred at higher [ADP] in the DM mitochondria; suggesting less RET and less inhibition by OA. The magnitude of ATP production was less in the DM mice whereas respiration and membrane potential (ΔΨ) were little affected; indi-cating that utilization of ΔΨ for ATP was impaired in the mitochondria of the DM mice. In summary, ATP production on succinate as substrate is impaired in mitochondria of the HF/STZ diabetic mice due in most part to decreased utilization of ΔΨ at any given ADP. Respiration occurs with a different dynamic relationship to ADP availability in SkM mitochondria of the DM mice.

Supported By: U.S. Department of Veterans Affairs

& 2020-PDownregulation of Heat Shock Protein Hsp90ab1 Isoform Is Asso-ciated with Metabolic Rewiring in Skeletal Muscle and Improves Glucose Homeostasis in DiabetesENXUAN JING, PRAGALATH SUNDARARAJAN, SUWAGMANI HAZARIKA, SA-MANTHA FOWLER, CHAHAN SHAH, STEPHANE GESTA, MICHAEL KIEBISH, VIVEK VISHNUDAS, RANGAPRASAD SARANGARAJAN, NIVEN NARAIN, Boston, MA

Hsp90ab1, a ATPase chaperone was identifi ed as a critical “master hub” infl uencing the diabetes phenotype by the use of a data driven Bayesian meta-bolic disease network generated by the interrogation of the biology under-lying multi-organ systems based diabetes model. Here we demonstrate that knock-down (KD) of Hsp90ab1 in primary human skeletal muscle myotubes is associated with signifi cant increase in glycolysis, beta-oxidation and mito-chondrial respiration. In addition, KD of HSP90ab1 is associated with a de-crease in PDH E1a phosphorylation. Furthermore, there was a trend of increase in HSP72 expression that is known to be down-regulated in skeletal muscle in type 2 diabetes. These results indicate a pivotal role of Hsp90ab1 in the regulation of skeletal muscle substrate metabolism. Hsp90ab1 is induced in skeletal muscle in response to high-fat diet, ASO mediated KD of Hsp90ab1 in high fat diet (HFD) fed C57B/6 mice signifi cantly improved glucose toler-ance and suppressed fed glucose levels, after 4 weeks treatment. This was accompanied by decreased muscle PDH-E1a phosphorylation, refl ecting an increased insulin sensitivity and carbohydrate substrate metabolism. Cardio-lipin species were signifi cantly increased while selective free fatty acids and acyl carnitines were decreased in muscle of Hsp90ab1 ASO HFD treated mice compared to control, suggesting an increase in mitochondria activity. Taken together, our data provides novel evidence that Hsp90ab1 isoform is a key regulator of skeletal muscle and systemic metabolism, and represents a viable target for potential treatment of diabetes. Benefi t of this presentation is the introduction to a novel data driven approach for identifi cation of targets and therapeutics using interrogative biology approach and the identifi cation of a specifi c isoform of HSP90 that is amenable for pharmacological manipulation as therapeutic strategy for treatment for diabetes.

& 2021-PTCA Cycle Intermediate Replenishment Did Not Improve Lipid Oxi-dation in Myotubes Established from T2D SubjectsMICHAEL GASTER, Odense, Denmark

Lipid oxidation and the TCA cycle fl ux are reduced while glucose oxida-tion is increased at basal in skeletal muscle of T2D subjects. The purpose of the present study was to evaluate whether increasing the TCA cycle fl ux would affect substrate oxidation, TCA metabolites and cellular ATP in human skeletal muscle cells.

Myotubes established from eight lean control subjects and eight obese type 2 diabetic subjects were treated with a) leucine/glutamine b) BCH a Glutamate dehydrogenase activator both replenish α-ketoglutarate (αKG) and c) BTA an inhibitor of the mitochondrial tricarboxylate carrier increasing citrate, for 16 h. Acetate-, palmitate (PA)- and glucose oxidation were stud-ied with labeled precursors while intracellular metabolites ATP, oxaloacetate (OAA), Citrate and αKG concentration were determined biochemically.

Diabetic myotubes at basal express a reduced TCA cycle fl ux (acetate oxi-dation) and complete PA oxidation while glucose and incomplete PA oxida-tion were increased compared to control myotubes. Intramyocellular OAA content was reduced while citrate was increased, and αKG and ATP content was unchanged compared to control myotubes. All treatment increased acetate oxidation (TCA fl ux), to the same extent in diabetic and control myotubes. Besides that BTA partly inhibited glucose oxidation was neither glucose nor PA oxidation signifi cantly affected by above treatments in the two groups. BTA treatment increased intracellular OAA and reduced citrate content but didn´t reached signifi cance. ATP was unaffected by treatment.

The present data indicates that the increased cataplerosis of citrate and the concomitant reduction of OAA may partly explain the reduced TCA cycle fl ux seen in T2D through loss of TCA intermediates. Although replenishing αKG and inhibition of citrate cataplerosis both enhanced the TCA cycle fl ux

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the lipid oxidation in diabetic myotubes was not concomitantly improved suggesting that they may partly be independently regulated.

2022-PChronic Infusion of Angiotensin-(1-7) Improves Insulin Sensitivity by Augmenting Peripheral Glucose DisposalIAN M. WILLIAMS, DEANNA P. BRACY, DAVID H. WASSERMAN, ITALO BIAG-GIONI, AMY C. ARNOLD, Nashville, TN

Overactivation of the renin-angiotensin system (RAS) is a well-known contributor to insulin resistance. Research has focused on the actions of an-giotensin (Ang) II, which reduces whole-body insulin sensitivity and impairs insulin-mediated suppression of hepatic glucose production. More recently, studies have shown that Ang-(1-7), a vasodilatory peptide that opposes Ang II actions, improves glucose tolerance and insulin sensitivity. The precise mechanisms and tissue-specifi c effects of Ang-(1-7) on insulin sensitivity in vivo, however, remain unclear. We tested the hypothesis that Ang-(1-7) improves insulin sensitivity by enhancing peripheral glucose disposal. Adult male C57BL/6 mice on standard chow diet received 3 weeks of Ang-(1-7) (400 ng/kg/min) or saline infusion via subcutaneous osmotic mini-pump. Hy-perinsulinemic(4 mU/kg/min)-euglycemic clamps were performed at the end of drug treatment in conscious, unrestrained mice. Ang-(1-7) treatment did not alter body weight, body composition, or fasting arterial glucose and in-sulin levels. Mice receiving Ang-(1-7) required a signifi cantly higher glucose infusion rate (GIR) to maintain euglycemia, indicating a marked improvement in insulin sensitivity [steady-state average GIR: 73±1 Ang-(1-7) vs. 40±4 mg/kg/min vehicle; p=0.003]. This improvement in insulin sensitivity was due solely to an increased rate of whole-body glucose disappearance [Rd: 64±12 Ang-(1-7) vs. 44±4 mg/kg/min vehicle; p=0.034], as insulin-mediated suppression of hepatic glucose output was unaffected. These data show that the insulin-sensitizing effects of Ang-(1-7) are due to enhanced insulin-stimulated peripheral glucose disposal. These fi ndings provide new insight into mechanisms by which Ang-(1-7) improves insulin action, reveal a novel pathway by which the RAS regulates metabolic homeostasis, and raise the possibility of developing Ang-(1-7) as a therapeutic target in the treatment of diabetes.

Supported By: 1K99HL122507-01A1, 5P01HL056693-18, 5U24DK059637-14

2023-PInsulin Resistance of Protein Anabolism Occurs in Lean, Glucose-tolerant Offspring of Persons with Type 2 Diabetes (T2D)SERGIO A. BURGOS, VIKRAM CHANDURKAR, MICHAEL A. TSOUKAS, STEPH-ANIE CHEVALIER, JOSE A. MORAIS, MARIE LAMARCHE, ERROL B. MARLISS, Montreal, QC, Canada, St. John’s, NL, Canada

Insulin resistance occurs in T2D offspring (T2D-O). We have shown con-current insulin resistance of both protein and glucose metabolism in T2D and obesity. We tested 7 (4W, 3M) T2D-O participants: lean (BMI 23±1 kg/m2), young (26±2 yr), nondiabetic (A1c 5.5±0.2%), and 2h post oral glucose (5.3±0.4 mmol/L). Control (C) participants (4W, 4M): BMI 21 ± 0.4, 25±1 yr, A1c 5.1±0.1, and 2h 5.6±0.4. All underwent 3h clamp protocols: hyperinsulinemic (40 mU/m2/min), euglycemic (5.5mmol/L) and isoaminoacidemic (total BCAA 320, µmol/L). Amino acids were clamped using 10% TrophAmine®. Kinetics were measured with 3[3H] glucose (in mg/kg LBM/min) and l-[13C] leucine (protein). Fasting glycemia and kinetic variables did not differ. Identical clamp decreases in endogenous Ra occurred (3.1±0.1 to 1.0±0.01, P<0.001, repeat-ed measures ANOVA). Glucose infusion rates were 9.8±0.8 in C vs. 6.6±0.6 in T2D-O (P<0.01). Rd showed a clamp increase to 10.7±0.8 in C vs. 7.6±0.6 in T2D-O (P<0.001), a group effect (P=0.013) and interaction (P=0.006). There were no group differences in fasting leucine kinetics (all µmol/kg LBM/min). In C vs. T2D-O, clamp leucine infusion rates were 1.14±0.08 vs. 0.91±0.10 (P=0.08). Similar increases occurred (P<0.001) in oxidation (to 0.92 vs. 1.05) and identical decreases in endogenous Ra (breakdown, to 2.20±0.07). How-ever, increases were less in T2D-O for both nonoxidative Rd (synthesis, to 2.42±0.06 vs. 2.06±0.08, P=0.016) and net balance (synthesis - breakdown, to +0.22±0.08 vs. -0.14±0.04, P<0.001, = 44% lower increment). Thus, we confi rmed insulin resistance of T2D-O, of glucose uptake, but not that of magnitude of Ra inhibition. We established that they have resistance of protein anabolism (synthesis and net balance), though not of breakdown. Hence, in these nonobese young adults, such resistance can be present, and may play a role in T2D susceptibility. Genetic, longitudinal, and mechanistic studies (including with prevention strategies) are required.

Supported By: Canadian Institutes of Health Research (MOP62889 to E.B.M.)

2024-PSkeletal Muscle Activation of the SRF/STARS Pathway Is Associ-ated with a Shift from Oxidative to Glycolytic MetabolismANA LUISA DE SOUSA COELHO, CHARLIE AOUN, ELIZABETH LI, MARY-ELIZA-BETH PATTI, Boston, MA

Striated muscle activator of Rho signaling (STARS) is a muscle-specifi c ac-tivator of SRF transcriptional activity. We reported that expression of STARS was increased by 2.5-fold in skeletal muscle of humans with T2D and cor-related with insulin resistance. Additionally, STARS expression was induced in muscle of HFD fed mice.

To determine if increased STARS expression contributes to dysregulated metabolism, we created transgenic mice overexpressing STARS (Tg-STARS) under the control of the muscle specifi c HSA promoter. Tg-STARS mice were born at Mendelian rates, but had ~14% reduction in body weight at weaning. Male Tg-STARS mice and WT littermates gained similar weight in response to a 60% HFD (begun at 8 weeks of age). Interestingly, Tg-STARS mice had normal fasting glucose and insulin levels, but improved glucose tolerance after 24 weeks of diet. Moreover, in vivo glucose uptake in TA muscle was increased in Tg-STARS mice.

To determine if the metabolic impact of STARS is cell autonomous, satel-lite cells were isolated and differentiated to myotubes. In agreement with the in vivo data, STARS overexpressing cells showed increased basal glu-cose uptake. Cellular bioenergetic analysis showed increased glycolytic rate and decreased mitochondrial respiration in Tg-STARS myotubes.

Together, these results suggest that overexpression of STARS, as in pa-tients with T2D, can augment glycolytic, relative to oxidative, metabolism. Such perturbations in tissue glucose metabolic fate may not only alter muscle metabolism but also alter whole-body glucose utilization. Thus, upregulation of STARS may represent a potentially adaptive response to tissue insulin resistance but may ultimately contribute to impaired oxidative metabolism.

Supported By: American Diabetes Association (7-12-BS-145 to M-E.P.); Sanofi U.S.; National Institutes of Health (R41DK096772-01A1)

2025-PGsa Signaling in Osteolineage Cells Regulate Body Adiposity by In-fl uencing Beige Adipogenesis via Irisin SecretionKEERTIK FULZELE, VAIBHAV SAINI, CHRISTOPHER DEDIC, FOREST LAI, YUHEI UDA, JORDAN SPATZ, MARC WEIN, PAOLA DIVIETI PAJEVIC, Boston, MA

Target ablation of osteolineage cells (osteoblasts or osteocytes) in mice leads to loss of body fat and metabolic abnormalities. However the intracel-lular signaling, endocrine factor(s), and target organ(s) of osteolineage cells are poorly understood. Specifi cally, the previously identifi ed bone-secreted insulin secretagogue undercarboxylated osteocalcin (ucOC) could not rescue the lean phenotypes of osteoblast defi cient mice. We found that mice lacking the stim-ulatory subunit of G-proteins (Gsa) in osteoblasts (OC-GsaKO) and/or osteo-cytes (DMP1-GsaKO), generated by Cre-loxP recombination, are lean similar to osteolineage cell defi cient mice. DMP1-GsaKO and OC-GsaKO mice had sig-nifi cantly decreased body weights beginning at 9-weeks of age. Gonadal and inguinal fat pads (GWAT and InWAT) were signifi cantly decreased in DMP1-GsaKO (77%) and OC-GsaKO (35%) mice whereas brown adipose tissue (BAT) and pancreas were not changed. Serum insulin levels were not altered and osteocalcin levels were reduced suggesting that the lean phenotypes of these mice were not dependent on ucOC. The DMP1-GsaKO mice were also resistant to high-fat diet induced obesity as assessed by DXA and fat pad weights. The lean phenotypes were associated with a dramatic increase in beige adipogen-esis in both GWAT and InWAT. Among the circulating factors that induce beige adipogenesis, we found that serum levels of irisin were signifi cantly increased in DMP1-GsaKO, and OC-GsaKO mice. To test direct cell-cell effects, we found that treatment with conditioned media (CM) from Gsα-defi cient osteocytic cell line (Ocy454-G2) signifi cantly increased irisin expression in myogenic C2C12 cells and UCP1 expression in primary adipocytes compared to the CM from control cells (Ocy454-D10). In summary, we show that Gsa-signaling is a criti-cal signaling pathway in osteolineage cells infl uencing body adiposity by tar-geting beige-adipogenesis, in part, via increased irisin expression.

Supported By: National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR060221)

2026-PSuccinate Supported Mitochondrial Respiration and ATP Produc-tion: Dependence on Respiratory StateFAN BAI, BRIAN D. FINK, LIPING YU, WILLIAM I. SIVITZ, Iowa City, IA

Mitochondrial respiration and ATP production on the complex II substrate, succinate, is generally believed to require inhibition of complex I; thereby, mitigating reverse electron transport (RET) and preventing accumulation

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of oxaloacetate (OA), a potent inhibitor of succinate dehydrogenase (SDH). However, studies are confounded since mitochondrial function during ATP synthesis is generally determined while ADP is consumed, resulting in a con-tinuously changing respiratory state. Here we utilized an ADP clamp along with novel NMR methodology for measuring ATP and metabolite accumula-tion to assess mouse hind limb skeletal muscle mitochondrial function on various substrates at fi xed respiratory states. Respiration and ATP produc-tion on succinate alone increased to a max at about 6-8 µM ADP, following which respiration and ATP production decreased with increasing [ADP]; a decrease likely associated with OA inhibition of SDH as suggested by me-tabolite detection by NMR. Concurrent measurements of reactive oxygen (ROS) and studies in the presence of a MnSOD mimetic or cyclosporin show that the deterioration in mitochondrial function on succinate was not related to ROS or to irreversible opening of the permeability transition pore. Calcium also had little effect. The decrease in succinate-supported respiration at higher ADP concentrations could be rescued upon late addition of rotenone, glutamate, TMPD plus ascorbate, or duroquinol, but not by addition of more succinate; showing that the capacity for electron transport through complex I and downstream complexes remained intact. In summary, we used novel ADP clamp methodology and NMR assessment to show that muscle mito-chondrial respiration and ATP production under conditions of RET is highly dependent on respiratory state (extent of ongoing oxidative phosphoryla-tion) and that respiration on succinate (often considered to require inhibition of complex I), may occur in robust fashion at low ADP concentrations.


2027-PAnti-infl ammatory Action of Interleukin-10 Protects Mice from Aging-mediated Decline in Energy Expenditure and Mitochondrial DysfunctionSEZIN DAGDEVIREN, DAE YOUNG JUNG, RANDALL H. FRIEDLINE, XIAODI HU, HYE-LIM NOH, TAEKYOON KIM, CAITLYN C. KEARNS, SHAYNA FISH, MARILIA M. LOUBATO, JASON K. KIM, Worcester, MA

Mitochondrial dysfunction is a major cause of aging, but the underlying mechanism is unclear. Our recent study found that infl ammation develops with aging and is causally associated with insulin resistance. Here, we performed a longitudinal analysis of energy balance using metabolic cages from 6 to 18 months (M) of age in mice with muscle-specifi c overexpression of IL-10 (MCK-IL10) and WT mice (n=6/group). Body weights were compa-rable between MCK-IL10 and WT mice throughout the study. At 6M, there was no difference in energy expenditure between groups. In WT mice, VO2 consumption rates gradually declined with aging and were signifi cantly re-duced by 18M (#; P<0.05 vs. 6M) (Fig. 1). In contrast, VO2 consumption rates in MCK-IL10 mice were not signifi cantly affected by aging and remained higher at 16M (*;P<0.05 vs. WT) and 18M. Muscle UCP3 mRNA levels were reduced by 62% in 18M WT mice, and this aging effect was blunted in MCK-IL10 mice (Fig. 2). Muscle glucose metabolism was signifi cantly increased in 18M MCK-IL10 mice, and these effects were associated with signifi cantly reduced IFN-γ and TNF-α levels in skeletal muscle of MCK-IL10 mice (Fig. 3 & 4). Overall, our fi ndings indicate that MCK-IL10 mice are protected from aging-mediated mitochondrial dysfunction and insulin resistance due to IL-10’s suppression of skeletal muscle infl ammation.


2028-PImpaired Capillary Tube Formation in Skeletal Muscle of Type 2 Diabetes Induced by Excessive Secretion of IL8 Involves Altered Signaling via the CXCR1/PI3K/MMP2 PathwayYIFAT AMIR LEVY, THEODORE P. CIARALDI, SUSAN A. PHILLIPS, ALEXANDER J. RYAN, SUNDER R. MUDALIAR, ROBERT R. HENRY, San Diego, CA, La Jolla, CA

Angiogenesis is a multistep process requiring endothelial cell (EC) acti-vation, migration, proliferation and tube formation. While IL8 can be pro-angiogenic, we found that elevated secretion of IL8 by myotubes (MT) from subjects with type 2 diabetes (T2D) reduced angiogenesis by human umbili-cal vein endothelial cells (HUVEC), resembling what is seen in T2D skeletal muscle (SkM). This lower vascularization was mediated through impaired ac-tivation of the PI3K-pathway. We sought to investigate additional elements that might mediate reduced angiogenesis in SkM of T2D patients.

HUVEC were exposed to levels of IL8 secreted by MT from non-diabetic (ND-IL8=2071 pg/ml) and T2D subjects (T2D-IL8=3280 pg/ml), and the in-volvement of components of the angiogenic response examined.

Cellular content of reactive oxygen species (ROS) and NO secretion by HU-VEC were similar after treatment with ND- and T2D-IL8, ruling out abnormal production of these as causes of impaired vascularization in T2D-SkM.

Since IL8 signaling could be mediated by the receptors CXCR1 and CXCR2, we added neutralizing antibodies against each receptor on HUVEC treated with T2D-IL8 and ND-IL8, and found that only CXCR1 mediated the response to IL8. Anti-CXCR1 inhibited tube formation with ND-IL8 to a greater extent than T2D-IL8 (p<0.005). A key modulator of angiogenesis is matrix metalo-proteinase-2 (MMP2). MMP2 secretion from HUVEC was higher after treat-ment with ND-IL8 compared to T2D-IL8 (P<0.05). Inhibition of MMP2 reduced tube formation to a greater extent with ND-IL8 vs. T2D-IL8 (p<0.005). IL8 regulation of MMP2 release is mediated via the PI3K-pathway, as LY294002 reduced IL8-induced MMP2 release to a greater extent with ND-IL8 vs.T2D-IL8 (p<0.05). These results suggest that high levels of IL8 secreted by T2D MT trigger reduced SkM capillarization via lower activation of a CXCR1-PI3K pathway, followed by impaired levels and activity of MMP2.


2029-PMice Lacking Cavin-1 (PTRF) Develop a Muscular Dystrophic Phe-notype Similar to That Caused by Mutations in Human Cavin-1SHI-YING DING, PAUL F. PILCH, Boston, MA

Caveolae are small invaginations of plasma membranes involved in many cellular processes and they are found in abundance in fat and muscle. Cav-in-1/PTRF (polymerase I transcription release factor) is a caveolar protein that has an essential role in the formation of caveolae structures. Human cavin-1 mutations cause loss of caveolae and lipo- and muscular dystrophy. Mice lacking cavin-1 are lipodystrophic, which results in systemic metabolic abnormalities including impaired glucose tolerance mediated in part by de-fects in adipocyte function. In the present work, we investigated the muscle phenotype caused by global deletion of cavin-1 in mice and its underlying molecular mechanism(s). Cavin-1 null mice had increased muscle mass, big-ger fi ber size and more centralized muscle cell nuclei. Elevated serum cre-atine kinase levels and increased muscle collagen content suggested the occurrence of muscular dystrophy and fi brosis. Treadmill running showed a decreased exercise capacity in the cavin-1 null mice. To assess the molecular mechanisms which contribute to the muscle phenotype, we examined the expression of key proteins involved in muscle fi ber integrity, stability and muscle repair function and found that lack of cavin-1 was linked to abnor-malities in the sarcolemmal dystrophin-glycoprotein complex, the T-tubule system and membrane repair complexes. We also observed impairments in the myostatin and insulin-like growth factor-1 (IGF-1) signaling pathways which are important players regulating key steps during muscle growth. Our data show that in addition to compromised adipocyte-related physiology in cavin-1 null mice, its absence also causes muscular dystrophy similar or iden-tical to that seen in humans with cavin-1 mutations. The data are consistent with a critical role for caveolae in maintaining muscle membrane integrity and normal function and in regulating muscle development, at least in part through myostatin/IGF-1 signaling pathways.

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2030-PImpaired Insulin-mediated ser1176 Phosphorylation of eNOS and Elevated NAD(P) Hoxidase Content in Skeletal Muscle Terminal Ar-terioles of Obese Zucker RatsMATTHEW COCKS, ANNIE E. STRIDE, RUTH MACDONALD, CHRISTOPHER S. SHAW, JANICE M. MARSHALL, SIMON M. POUCHER, ANTON J.M. WAGEN-MAKERS, Liverpool, United Kingdom, Birmingham, United Kingdom, Macclesfi eld, United Kingdom, Geelong, Australia

The ability of insulin to activate eNOS and dilate arterioles is known to control the delivery of glucose and insulin to muscle. While the production of superoxide by NAD(P)Hoxidase will scavenge nitric oxide and impair va-sodilation and nutrient delivery. The aim of this study was to identify the molecular mechanism that leads to a reduced muscle glucose uptake in obese Zucker rats (OZR). The m. tibialis anterior was dissected from 14 wk. old lean Zucker rats (LZR) and OZR in the fasted state (n=7) and 80min after the start of a hyperinsulinaemic-euglycemic clamp (HEC; n=7). Content and ser1176 phosphorylation of eNOS and NOX2 and P67Phox content (subunits of NAD(P)Hoxidase) were measured in the endothelial layer of skeletal muscle terminal arterioles and capillaries using immunofl uorescence microscopy. Total eNOS content and eNOS ser1176 phosphorylation in the fasted state were not signifi cantly different between LZR and OZR in terminal arterioles and capillaries. Insulin stimulation elevated terminal arteriole eNOS ser1176 phosphorylation in LZR (14%; P <0.05), while causing a signifi cant reduction in OZR (-28%; P <0.05). Insulin did not signifi cantly change capillary eNOS ser1176 phosphorylation in LZR (-7%), while causing a reduction in OZR (-31%, P <0.05). NOX2 and P67Phox content in terminal arterioles (NOX2 28%, P47Phox 21%; P <0.05) and capillaries (NOX2 31%, P47Phox 15%; P <0.05) were higher in OZR. Capillary density and number of capillaries per muscle fi bre were lower in OZR (P <0.05). The impairments in OZR coincided with a reduction in glucose infusion rate (LZR 95.8±14.1, OZR 39.7±17.9 mg/min/kg bw) during the fi rst 21min of the HEC and a 5-fold reduction in insulin sensi-tivity index (both P<0.05). The data suggest that a reduction in eNOS ser1176 phosphorylation during a HEC and an elevation in NAD(P)Hoxidase content in skeletal muscle terminal arterioles of OZR are associated with skeletal muscle insulin resistance.

Supported By: AstraZeneca; UK Biotechnology and Biological Sciences Re-search Council

2031-PThe Internal Radiation by Radioactive Cesium Exacerbates the High-Fat Diet-induced Insulin Resistance in Male Wister Rats, but Adiponectin Improves These AffectsSATORU YAMAZAKI, TSUYOSHI WATANABE, HIROAKI SATOH, Fukushima, Japan

Due to the Fukushima Daiichi nuclear disaster, chronic internal radiation exposure is a serious public concern in Fukushima. Because the physical half-life of radioactive cesium is around 30 years, and chronic internal radia-tion exposure is caused by long-term cumulative radiation exposure among residents in radiation-contaminated areas due to the sustained radiation contamination of locally grown produce.

In current study, we investigated the effect of internal radioactive cesium exposure on insulin sensitivity in male Wistar rats, whereby insulin sensitiv-ity was measured directly using the hyperinsulinemic-euglycemic glucose clamp studies (at 25 mU/kg/min insulin infusion rate) after a 8-h fast. Male Wistar rats were fed normal chow diet (NCD), or 60% high fat diet (HFD) containing with either radioactive cesium or not, for 5 weeks.

In the NCD fed rats, during the clamp studies, the glucose infusion rate (GIR), the clamp hepatic glucose output (cHGO) and insulin-stimulated glucose disposal rate (IS-GDR) were no signifi cant changes between two groups. On the other hand, in the HFD fed rats, the GIR and IS-GDR were signifi cantly decreased by 7.4% and 17.5%, respectively, in internal radio-active cesium exposure group compared to in the HFD control group. But cHGO was no signifi cant change between two groups. Consistent with the clamp data, the insulin-stimulated phosphorylation of Akt was signifi -cantly decreased in skeletal muscle. Furthermore, in the clamp studies, the adenovirus mediated adiponectin overexpression rats exhibited increased GIR (17%) and IS-GDR (23%) compared to the HFD fed internal radioactive cesium exposure rats. In fact, the GIR and IS-GDR values were similar to those of HFD fed control rats.

In conclusion, the internal radiation by radioactive cesium exacerbates the HFD induced insulin resistance in male Wister rats, but adiponectin im-proves these affects.

2032-P

2033-PThe Effect of Lipopolysaccharide and Flagellin on Energy Metabo-lism in Human Skeletal Muscle CellsRAGNA H. TINGSTAD, FRODE NORHEIM, FRED HAUGEN, COLIN CHARNOCK, VIGDIS AAS, Oslo, Norway, Trondheim, Norway

The microbiota and its implications on human health has gained much fo-cus over the past few years and research into how it affects us and how we can manipulate it is booming. Diabetes type 2 (T2D) and obesity are linked, >80% of patients with T2D being overweight. Obesity in its own right is established as a low grade, chronic infl ammatory condition, but the cause is still debated. It is thought that translocation of bacterial products from the gut into blood might be a possible source. It has been shown that Gram negative bacteria derived lipopolysaccharide (LPS) levels in blood are el-evated in obese patients, and that a single meal rich in fat elevates blood LPS in healthy and obese patients. LPS is a potent inducer of the innate immune system by binding Toll-like receptor (TLR) 4. The aim of the current project is to determine if such bacterial components have an effect on the energy metabolism in human skeletal muscle cells. Human myoblasts from M. obliquus internus abdominis were differentiated into multinucleated myotubes and pre-treated with LPS or fl agellin for 24 hours before trapping CO2 for 4 hours with either [14C-U] glucose (1 µCi/ml, 100µM) or [1-14C]oleic acid (1 µCi/ml, 100µM) and analyzing expression of selected cytokines. Basal glucose and oleic acid uptake and oxidation were not affected by LPS or fl agellin. Acute addition of oleic acid reduced glucose oxidation by 10% in control cells, whereas oleic acid was not able to suppress glucose oxidation in cells exposed to LPS or fl agellin. About 20% of oleic acid taken up by the cells was oxidized and acutely added glucose reduced this to about 15% in control cells. In cells exposed to fl agellin, glucose did not suppress oleic acid oxidation. Both LPS and fl agellin stimulated myotube secretion of IL-6 and IL-8, whereas fl agellin also enhanced TNF-α secretion. In conclusion, bacterial products as LPS and fl agellin reduce the ability to switch between energy substrates and increases the production of pro-infl ammatory cytokines in human myotubes.

2034-PExchange Protein Directly Activated By cAMP (Epac)1-defi cient Mice Have Reduced Exercise CapacityWAI-KIN SO, YINGXIAN CHEN, BILLY K.C. CHOW, SOOKJA K. CHUNG, Hong Kong, China

Exchange protein directly activated by cAMP (Epac) mediates multiple ac-tions of G-protein coupled receptor/cAMP signaling in cells and is implicated in various developmental, physiological and pathological processes. Our lab-

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oratory generated mice carrying homozygous deletion of Epac1 isoform and reported that Epac1 KO (knockout) mice displayed metabolic defects includ-ing higher respiratory exchange ratio, insulin resistance and insensitivity, higher plasma triglyceride, and lower glucose-stimulated insulin secretion. Defects in skeletal muscle metabolism may have contributed to metabolic syndrome in Epac1-defi cient mice. Therefore, we have investigated the con-tribution of Epac1-defi ciency on the exercise intolerance since it is a good in-dication of skeletal muscle dysfunction. In extensor digitorum longus and so-leus muscles, we found that Epac1, but not Epac2, can be easily detected by real-time PCR and Western blot analysis. To test if ablation of Epac1 impacts physical exercise performance, wild type (WT) and Epac1 KO mice were ex-posed to exercise stress by subjecting them to graded treadmill test. Epac1 KO mice exhibited signifi cantly reduced exercise capacity when compared with WT mice, in terms of lower work done and shorter running distance before exhaustion. Exercise-enhanced insulin sensitivity and glucose uptake were impaired as evidenced by the altered AMPK and AKT activation and GLUT4 expression in Epac1 KO muscle. We next examined the expression of PGC-1α, a master transcription co-activator of genes involved in oxida-tive metabolism. Basal PGC-1α expression in soleus of Epac1-defi cient mice without exercise was lower than the WT counterpart. Moreover, Epac1 KO blunted exercise-induced PGC-1α expression as the extent of PGC-1α induc-tion in KO soleus was signifi cantly reduced. In summary, our fi ndings suggest Epac1 in skeletal muscle regulates PGC-1α expression in response to exer-cise stress, and Epac1-defi ciency impairs exercise performance.

Supported By: Research Grant Council General Research Fund (to S.K.C); Re-search Grant Council Collaborative Research Fund to B.K.C.C)

2035-PMetabolic and Transcriptomic Changes in Cultured Myoblasts from Low Birth Weight IndividualsNINNA S. HANSEN, LINE HJORT, CHRISTA BROHOLM, BRYNJULF MORTENSEN, SINE W. JØRGENSEN, MARTIN FRIEDRICHSEN, JØRGEN F.P. WOJTASZEWSKI, ALLAN A. VAAG, Copenhagen, Denmark

Individuals born with low birth weight (LBW) have an increased risk of developing type 2 diabetes (T2D) later in life. Skeletal muscle is the pre-dominant site of insulin-mediated glucose uptake, and previous animal and human studies have shown a range of constitutional and metabolic changes in muscle tissue potentially involved in the development of T2D in LBW subjects. Immature stem cell functions including abnormal differentiation potential and metabolic function could represent a common denominator of multiple organ dysfunctions linking LBW with risk of developing T2D. We studied glucose uptake, insulin signaling, myokine secretion and key tran-scriptional markers of cell maturity as well as mitochondrial genes through-out differentiation in cultured muscle satellite cells from 23 LBW and 16 normal birth weight (NBW) adult male individuals.

In the cultured myotubes established from LBW individuals, we found signifi cantly reduced insulin stimulated glucose uptake, decreased glucose transporter-4 (GLUT4) gene expression and decreased pAS160/AS160 insulin signaling protein levels compared to control NBW cells. Additionally, inter-leukine 6 release was increased during myoblast differentiation and the expression level of a selected set of mitochondrial OXPHOS genes including the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), previously reported to be down regulated in muscle from T2D patients, as well as the myosin heavy chain 2 gene expression, were also found to be signifi cantly down regulated in the LBW myotubes. The documented mo-lecular changes involved in muscle glucose homeostasis, mitochondrial gene expression and myoblast maturation could be due to a preprogrammed underlying change of the epigenetic machinery in the immature myoblast, which we will further address.

2036-POxymatrine Reduces Lipid Accumulation of Skeletal Muscle and Relieves Insulin Resistance Induced by High-Fat Diet in RatsGUANGYAO SONG, CHAO WANG, HUIJUAN MA, WENJIE FEI, Shijiazhuang, China

Oxymatrine (OMT), an alkaloid monomer isolated from sophora fl avescens ait, showed anti-cancer, anti-diabetes and anti-infl ammatory functions. Here, we determined the role of OMT on lipid metabolism in skeletal muscle.

Rats were fed with high-fat diet for 8 weeks, and then were given OMT treatment (100 mg/kg) for 8 weeks. Normal control rats were fed with regu-lar low fat diet. Insulin sensitivity was evaluated by conscious hyperinsuline-mic-euglycemic clamp. Body weight (BW), fasting blood-glucose (FBG), serum insulin (INS) and triglyceride (TG), skeletal muscle lipids and morphological changes were assessed. Lipid metabolic factors such as peroxisome prolif-

eratoractivated receptor α (PPARα) and β (PPARB), peroxisome proliferator-activated receptor gamma coactivator gene (PGC1α), fattyacid translocase (FAT), fattyacid binding protein 3 (FABP3), carnitine palmitoyltransferase 1 β (CPT1β), medium-chain acyl-CoA dehydrogenase (MCAD), AMP-activated protein kinase α (AMPKα) in skeletal muscle were detected by quantitative RT-PCR and Western-blot.

The results showed that high-fat diet increased the level of BW, FBG, INS and TG, and lead to insulin resistance and skeletal muscle lipids accumula-tion. While OMT treatment suppressed the level of BW, FBG, INS and TG, and relieved insulin resistance as well as skeletal muscle lipids accumula-tion. High-fat diet inhibited the expression levels of PPARα, PPARB, PGC1α, CPT1β, MCAD and AMPKα in rats skeletal muscles, while increased the expression of FAT and FABP3. OMT treatment markedly increased the ex-pression of PPARα, PPARB, PGC1α, CPT1β, MCAD and AMPKα, and further increased FAT and FABP3 expression.

These results suggested that OMT could be used to relieve lipid accumula-tion in skeletal muscle and increase insulin sensitivity, as maybe due to its stimulation of fatty acid transport and oxidation.

2037-PBreaking Up Sedentary Time Modulates Both the Contraction- and Insulin-stimulated Glucose Uptake Pathways in Skeletal MuscleAUDREY BERGOUIGNAN, CELINE LATOUCHE, MEDINI REDDY-LUTHMOODOO, ALAINA NATOLI, NEVILLE OWEN, DAVID DUNSTAN, BRONWYN A. KINGWELL, Aurora, CO, Melbourne, Australia

Interrupting prolonged sitting has been associated with a 24% reduction in postprandial plasma glucose as compared to uninterrupted sitting1. We studied potential skeletal muscle mechanisms accounting for this effect. Vastus lateralis biopsies were obtained from overweight adults 5hr after a standardized test meal under conditions of uninterrupted sitting and sitting with 2min of light intensity (LI) or moderate intensity (MI) treadmill walking every 20min (n=8) or after a 3d intervention of either the uninterrupted sit-ting or LI break treatment (n=5). Contraction- and insulin-mediated glucose uptake pathways and net glucose transport regulation were assessed by the absolute and relative amounts of phosphorylated and total (p/t) acetyl car-boxylase Co-A (ACC), Akt and AS160 proteins, respectively (Western blot). p/tACC was elevated by both.5hr LI (p=0.04) and MI (p=0.03) breaks, due to an increase in pACC (LI: p=0.04; MI: p=0.03). This elevation was no longer present after 3d of LI breaks. Neither 5hr LI nor MI breaks modifi ed p/tAkt. Three consecutive days of LI breaks did not change pAkt but increased tAkt 23 fold (p=0.02) such that p/tAkt was signifi cantly reduced below the unin-terrupted sitting levels (p=0.01). This result was mirrored in the downstream GS3Kβ with a decrease in p/tGS3Kβ (p=0.02). tAS160 was increased by all interruption conditions (only tended for 5hr LI breaks), suggesting increased capacity for glucose transport. There was a corresponding increase in mi-tochondrial complex V (ATPase, p=0.04), consistent with an enhanced ca-pacity for ATP production. In conclusion, while acute interruptions to sitting stimulate the contraction-mediated glucose uptake pathway, longer term in-creases in interruptions induce a transition to the insulin signaling pathway. These changes provide a mechanistic basis to explain improved postprandial glucose metabolism with regular interruptions to sitting time.

1Dunstan DW et al. Diabetes Care 2012. Supported By: Australian Endeavour Postgraduate Research Fellowship Award;

Australia National Health and Medical Research Council

2038-PSupplemental Oxygen Improves Mitochondrial Impairment in Sed-entary Adults with Type 2 DiabetesMELANIE CREE-GREEN, BRAD R. NEWCOMER, AMY G. HUEBSCHMANN, IRENE E. SCHAUER, SHAWNA MCMILLAN, MARK BROWN, KRISTEN J. NADEAU, TIM A. BAUER, JANE E.B. REUSCH, JUDITH G. REGENSTEINER, Denver, CO, Birming-ham, AL, Aurora, CO, Boulder, CO

Exercise impairment and mitochondrial dysfunction are present even in uncomplicated type 2 diabetes (T2D). The presence of and interrelationship between these abnormalities is signifi cant given the strong relationship be-tween physical fi tness, functional status and mortality. We hypothesized that mitochondrial dysfunction in T2D is mediated by decreased tissue oxy-gen delivery and improves acutely with supplemental oxygen (O2). Mitochon-drial function was assessed by 31-P MR spectroscopy during sub-maximal isometric calf exercise performed at room air (RA) and with supplemental O2. Mitochondrial function was modeled during exercise recovery by: 1) phosphocreatine and ADP time constants (PCR TC and ADP TC) which are affected by muscle blood fl ow and pH, 2) oxidative phosphorylation (Oxphos) which refl ects substrate availability and 3) QMAX which models maximal

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mitochondrial function. Results from 8 adults with T2D (57±3.4 years, BMI 32±2, HbA1C 5.3%) and 6 obese non-diabetic controls (OC) (42±5.9 years, BMI 31±2, HbA1C 6.2%) are shown in Table 1. Mitochondrial function was impaired in T2D relative to OC, and O2 improved the PCR TC and OxPhos. We conclude that in-vivo post-exercise oxidative metabolism is impaired in T2D, and related to limitations in tissue O2 delivery. Further work is needed to determine if other therapeutic approaches to improve O2 delivery will improve mitochondrial function in T2D.

Table 1. Oxygen Saturation and Mitochondrial Measurements.Obese Obese Obese Type 2

DiabetesType 2

DiabetesType 2

DiabetesRoom Air Oxygen P-Value

(OC RA vs. O2)Room Air Oxygen P-Value

(T2D RA vs. O2)P-Value

(T2D RA vs. OC RA)Oxygen Saturation (%) 96±1 99±1 0.005 94±1 99±1 0.001 0.171PCr TC (seconds) 26±4 26±3 0.95 40(32,47) 32(16,37) 0.055 0.034ADP TC (seconds) 19±2 20±2 0.739 28±2 22±3 0.109 0.012OxPhos (mM/sec) 0.17±0.08 0.17±0.03 0.894 0.10±0.03 0.18±0.07 0.038 0.064Qmax (mM/sec) 0.66±0.12 0.62±0.10 0.811 0.34±0.04 0.59±0.12 0.124 0.022

Supported By: American Diabetes Association (1-12-CT-64 to J.G.R.); National Institutes of Health-National Center for Research Resources (K23RR020038-05, 1R56DK088971 to K.J.N.); T32DK063687, 2K12HD057022, M01-RR00051 (to M.C-G.); U.S. Department of Veterans Affairs (to J.E.B.R.); CVP (to J.E.B.R.); IES (to J.E.B.R.); Denver Research Institute (to J.E.B.R.)

2039-PNutrient Excess and AMPK Downregulation in Incubated Skeletal Muscle and Muscle of Glucose Infused RatsKIMBERLY A. COUGHLAN, EDWARD W. KRAEGEN, NEIL B. RUDERMAN, ASISH K. SAHA, Boston, MA, Sydney, Australia

It has been established that a persistent increase in the ambient glucose concentration causes insulin resistance in skeletal muscle by down regulating AMP-activated protein kinase (AMPK). The mechanism by which it does so is unknown. We have previously shown that incubation for 1h with excess glucose or leucine causes insulin resistance in extensor digitorum longus (EDL) muscle by inhibiting AMPK. To examine the events that precede and follow these changes, studies were performed in rat EDL incubated with elevated levels of glucose or leucine for 30min-2h. Incubation in high glucose (25mM) or leucine (100µM) signifi cantly diminished AMPK activity by 50% within 30min, with further decreases occurring by at 1 and 2h. The cause of the initial de-crease in activity at 30min was unclear; however, the decreases at 1h were accompanied by phosphorylation of αAMPK at Ser485/491, and at 2h by de-creased SIRT1 expression and increased PP2A activity, all of which have previ-ously been shown to diminish AMPK activity. Glucose infusion in vivo, which caused several fold increases in plasma glucose and insulin, produced similar changes but with different timing. Thus, the initial decrease in AMPK activity observed at 3h was associated with changes in Ser485/491 phosphorylation and SIRT1 expression and increased PP2A activity was a later event. Finally, we found that the decrease in AMPK activity is associated with a decrease in two mitochondrial genes UCP3 and PGC1 when EDL was incubated for 4h in high glucose. These fi ndings suggest that both ex vivo and in vivo, multiple fac-tors contribute to fuel induced decreases in AMPK activity in skeletal muscle and the insulin resistance that accompanies it.


2040-POxymatrine Promote Fatty Acid Uptake and Oxidation via SIRT2/PPARD in Skeletal Muscle CellGUANGYAO SONG, CHAO WANG, HUIJUAN MA, YAXIN WANG, Shijiazhuang, China

Oxymatrine (OMT), an alkaloid monomer isolated from sophora fl avescens ait, has been shown to improve the energy homeostasis. In this study, we determined the role of OMT on lipid metabolism in skeletal muscle cell.

L6 skeletal muscle myoblasts differentiated into myotubes then were treated with OMT (0, 1, 2, 4, 8 mg/ml) and 0.25 mmol/L palmitate for 24h, free fatty acid uptake and oxidation were determined. Sirtuin 2 (SIRT2), peroxisome proliferatoractivated receptor delta (PPARD), peroxisome prolif-erator-activated receptor gamma coactivator gene (PGC1α), fattyacid trans-locase (FAT), fattyacid binding protein 3 (FABP3), carnitine palmitoyltrans-ferase 1 β (CPT1β) and acyl-Coenzyme A dehydrogenase, long-chain (LCAD) in skeletal muscle were detected by quantitative RT-PCR and Western-blot. Knock-down of SIRT2 or PPARD by RNA interference were applied to further ellucidate the regulation mechanism.

The results showed that OMT treatment promoted fatty acid uptake and oxidation, and resulted in induction of SIRT2 and PPARD as well as several PPARD target genes PGC1α, FAT, FABP3, CPT1β and LCAD which involved in the fatty acid uptake and oxidation pathway. The up-regulation of these genes as well as promotion of fatty acid uptake and oxidation was partly abolished by PPARD or SIRT2 knock-down. Knock-down SIRT2 inhibited PPARD and its target gene expression, while knock-down PPARD also down-regulated SIRT2. Thus, SIRT2/PPARD might act as a positive feedback in-volved in lipid metabolism.

In summary, our study suggested that OMT is an accelerator for fatty acid metabolism in skeletal muscle cell, and this action is due to the induction of SIRT2/PPARD and their downstream gene expression. These data implied the potential value of OMT in the treatment of metabolic disease.


Guided Audio Tour: Other Hormones—Central Nervous System Regula-tion (Posters: 2041-P to 2047-P), see page 17.

& 2041-PLeptin Signaling in the Lateral Parabrachial Nucleus Is Both Nec-essary and Suffi cient to Suppress Hyperglucagonemia in Uncon-trolled DiabetesTHOMAS H. MEEK, MILES E. MATSEN, VINCENT DAMIAN, JONATHAN N. FLAK, MARTIN G. MYERS, JR., MICHAEL W. SCHWARTZ, GREGORY J. MORTON, Se-attle, WA, Ann Arbor, MI

Recent evidence implicates a population of leptin receptor (LepR) neurons in the lateral parabrachial nucleus (PBN) in the counter-regulatory response (CRR) to hypoglycemia. Leptin action on these neurons, which co-express cholecystokinin (CCK) and project to the ventromedial hypothalamic nucleus, appears to limit the CRR in part by blunting glucagon secretion in response to hypoglycemia. Since central leptin action also normalizes elevated plas-ma levels of glucose, glucagon and ketones in uncontrolled diabetes (uDM), we sought to determine whether the PBN is a target for these leptin effects. After induction of uDM with streptozotocin (STZ), rats received a continu-ous infusion of either leptin (0.1ug/d) or its vehicle directly into the PBN. We found that although PBN leptin infusion had no effect on either blood glucose or food intake, it reduced both hyperglucagonemia (160±21 vs. 114±7 pg/ml) and ketosis (1152±349 vs. 466±102 umol/l; p<0.05 for each). Thus, lep-tin action in the PBN is suffi cient to lower glucagon and ketone body levels in rats with uDM, and surprisingly, this effect is dissociated from leptin’s glucose-lowering action. To determine whether leptin signaling in the PBN is required for these anti-diabetic effects, we infused leptin icv (1µg/d for 10 d) to both WT mice with uDM and diabetic mice with selective deletion of leptin receptors from CCK neurons, the principle leptin-responsive neu-ronal subtype in the PBN. Consistent with our previous observations, blood glucose levels were normalized in diabetic WT mice receiving icv leptin. Whereas the anti-diabetic effect of icv leptin was not diminished in mice with reduced PBN leptin receptor signaling, reversal of both hypergluca-gonemia and ketosis was markedly attenuated in these mice. These fi ndings strongly implicate leptin-responsive CCK neurons in the PBN in the effect of leptin to ameliorate hyperglucagonemia and ketosis, but not hyperglycemia, in the setting of uDM.

Supported By: DK089056

& 2042-POne Minute of Circadian-timed Daily Dopamine (DA) Administration at the Biological Clock for 2 Weeks Ameliorates Metabolic Syn-drome in Spontaneously Hypertensive Rats (SHR) Held on a High-Fat Diet (HFD)SHUQIN LUO, MICHAEL EZROKHI, YELENA TRUBITSYNA, YANG LI, ANTHONY H. CINCOTTA, Tiverton, RI

Preclinical studies indicate that a reduction in the normal circadian peak of DA activity at the onset of daily locomotor activity (LA) at the area of the hypothalamic suprachiasmatic nucleus (SCN) is characteristic of metabolic syndrome examined among various animal models, including HFD feeding. Furthermore, an induced loss of this circadian peak by DA neurotoxin lesion at the SCN area in normal animals induces metabolic syndrome. However whether circadian-timed DA administration at the SCN area in metabolic syndrome animals to restore this circadian peak activity can actually reverse metabolic syndrome has never been investigated and was the subject of this study. Male SHRs (12 wks old; BW: 272±3 g) were fed a HFD (60% saturated

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fat) for 4 weeks at which time rats were divided into 3 groups (N=8/group) and subjected to a one minute infusion of either vehicle (0.2 µl artifi cial cere-brospinal fl uid [CSF]) or dopamine (either 2 or 4 nm in 0.2 µl artifi cial CSF) at the unilateral SCN daily at the onset of daily LA for 2 wks while held on the HFD. Glucose tolerance tests (GTT) were performed after 2 wks of treatment at 48 hrs prior to sacrifi ce for assessments of liver lipid, body fat store, and plasma norepinephrine (NE) levels. Relative to control, both DA treatments reduced GTT glucose AUC (30% and 25% respectively, p<0.005) and insulin AUC (46% and 41% respectively, p<0.05) and increased insulin sensitivity (2.2 and 1.8-fold; Matsuda Method; p<0.05). Relative to control, SCN DA treatments reduced liver fat (by 44% and 45%; P<0.05), body weight (by 14% and 13%, P<0.05) and total abdominal fat (by 41% and 28%, P<0.05) without altering food consumption. Such dopamine treatment also reduced plasma NE by 36% (P<0.02). These fi ndings indicate that restoration of the circadian peak in dopamine activity at the biological clock ameliorates metabolic syn-drome in these rats without altering food consumption.

& 2043-PHypothalamic POMC Defi ciency Improves Glucose Tolerance De-spite Obesity and Insulin Resistance by Elevating Glycosuria via Reduced Renal Sympathetic ToneKAVALJIT H. CHHABRA, JESSICA M. ADAMS, NATHAN QI, MARCELO RUBIN-STEIN, MALCOLM J. LOW, Ann Arbor, MI, Buenos Aires, Argentina

The role of brain proopiomelanocortin (POMC), whose defi ciency causes obesity, in glucose homeostasis is unclear. Oral glucose- and intra-perito-neal insulin- tolerance tests revealed that obese hypothalamus-specifi c (hs) POMC-defi cient mice (20-24 week old) exhibited improved glucose tolerance (AUC: 34,749±884 vs. 46,191±852 mg/dl*min.) despite reduced insulin sensi-tivity (AUC, insulin tolerance test: 14,965±1297 vs. 10,243±334 mg/dl*min.) compared to their WT littermate (P<0.05). A frequently-sampled intravenous glucose tolerance test indicated that the improved glucose disposal in hs-POMC-defi cient mice occurs independently of insulin action, because glucose effectiveness was elevated compared to WT mice (0.07±0.006 vs. 0.05±0.01 min-1, P<0.05). To precisely determine the insulin-independent mechanism, we hypothesized that hsPOMC-defi cient mice might exhibit reduced re-nal glucose reabsorption. Indeed, we found signifi cantly higher 24 h urine glucose levels (0.5±0.2 vs. -0.3±0.03 mg, logarithm of raw values) associ-ated with a 20% reduction in renal-cortical GLUT2 expression in the mutant compared to WT mice (P<0.05). Additionally, renal epinephrine (0.09±0.01 vs. 0.2±0.01 ng/mg tissue) and norepinephrine (0.2±0.02 vs. 0.3±0.03 ng/mg tissue) levels were signifi cantly lower in hsPOMC-defi cient mice versus controls (P<0.05). Interestingly, acute epinephrine (0.3 mg/Kg lean mass, i.p.) treatment abolished the genotype differences in glucose tolerance, glycosu-ria, and GLUT2 expression, suggesting the involvement of the sympathetic nervous system (SNS) in POMC-mediated effects on glucose homeostasis. These data indicate that hypothalamic POMC-defi ciency improves glucose tolerance by elevating glycosuria via low renal sympathetic tone. Thus, we have identifi ed a novel insulin-independent POMC/SNS/GLUT2 pathway, through which hypothalamic POMC regulates glycemia.

Supported By: National Institutes of Health (DK066604 to M.J.L), (DK068400 to M.J.L. and M.R.); Michigan Diabetes Research Center (DK020572); Michigan Nutrition and Obesity Research Center (DK089503)

& 2044-PCircadian Peak Dopaminergic Activity at the Biological Clock (Su-prachiasmatic Nucleus, SCN) that Potentiates Insulin Sensitivity Is Uniquely Attenuated by a High-Fat Diet (HFD)YANG LI, YAHONG ZHANG, SHUQIN LUO, ANTHONY CINCOTTA, Tiverton, RI

Seasonal loss or experimental removal of the daily peak in the circadian rhythm of dopamine (DA) level at the area of the SCN in rodents precipitates insulin resistance via its altered modulation of the neuroendocrine axis. To defi ne dopaminergic neurophysiological changes at the SCN area involved in the transition of insulin sensitive to resistant state, we investigated the impact of a HFD in rats on the circadian variation in a) local peri-SCN release of DA and b) electrophysiological responses to peri-SCN applied DA. Hypo-thalamic SCN DA D2 receptor sites in rats were mapped via specifi c binding of [125I]-iodosulpride and found to be located at the immediate lateral peri-SCN regions that overlapped with the medial preoptic area. Microdialysis studies of this area demonstrated a circadian peak in DA release at one hour after dark onset (night) (2-fold increase vs. one hour after light onset [day], P<0.0001) that was abolished by the HFD. In subsequent studies, electro-physiological responses of glutamate-activated SCN neurons to local DA ad-ministration at this peri-SCN site of the DA D2 receptors were performed at night or day. The SCN neuronal response sensitivity to DA inhibition at night

(EC50= 3.0± 0.5 mM, n=5) was 4 times greater than during the day (EC50= 14.5± 1.3 mM, n=5) (p<0.005), coincident with the peak in DA release. This effect was from DA D2 not D1 receptors. Importantly, HFD fed rats for 4 weeks had a much reduced DA activity response at night versus controls at night (EC50 = 6.1 ± 1.0 mM vs. 3.0 ± 0.5mM, P<0.01). These fi ndings highlight a unique neurophysiological shift at the SCN in response to a HFD typifi ed by attenuation of the circadian peak of both presynaptic DA release and postsynaptic DA response in peri-SCN neurons that is contributive to insulin resistance suggesting this shift represents a “new normal” response state rather than “pathology” to regulate neuroendocrine modulation of metabo-lism.

& 2045-PChronic eNAMPT Administration Induces a T2D-like Phenotype in MiceJULIUS KIESWICH, MUHAMMAD M. YAQOOB, PAUL W. CATON, London, United Kingdom

Serum levels of extra-cellular nicotinamide phosphoribosyltransferase (eNAMPT) are commonly elevated in type 2 diabetes (T2D), where raised eNAMPT levels strongly correlate with declining β-cell function. This implies a pathophysiological role of eNAMPT. Consistent with this, we have shown that eNAMPT immuno-neutralisation improves glucose homeostasis in high fat fed mice (HFD).

However, the precise role of eNAMPT in T2D pathophysiology is unclear. Several studies have examined the effects of eNAMPT on insulin secretion and sensitivity, but these have largely examined acute effects of eNAMPT, which do not accurately represent conditions likely to be found in T2D.

To examine the chronic effects of eNAMPT, C57Bl/6 mice were admin-istered recombinant eNAMPT (5 ng/ml), NMN (the putative product of the eNAMPT NAD-biosynthetic reaction; 500 mg/kg bw) or saline equivalent daily for 14 days (IP; n = 5/group).

eNAMPT administration increased serum eNAMPT levels from 1.6 ± 0.1 to 6.0 ± 1.2 ng/ml, similar to levels observed in HFD mice. eNAMPT administra-tion led to elevated blood glucose levels, impaired glucose tolerance (IPGTT), whole-body insulin resistance (HOMA-IR), and decreased insulin sensitivity (QUICKI). eNAMPT impaired β-cell function, demonstrated by a lack of com-pensatory insulin secretion in response to insulin resistance. The function of eNAMPT is unclear, having been attributed pro-infl ammatory, insulin-mi-metic and NAD-biosynthetic functions. Consistent with a pro-infl ammatory effect, eNAMPT administration led to increases in serum MCP1 levels. How-ever, the effects of eNAMPT were not mimicked by NMN administration, im-plying that eNAMPT effects are not mediated by NAD biosynthetic actions. Moreover, we did not observe any insulin-mimetic effects of eNAMPT.

We report that eNAMPT administration induces a T2D phenotype in mice. This implies that raised serum eNAMPT levels could play a key role in the pathophysiology of human T2D and that eNAMPT is a potential therapeutic target for treatment of T2D.

& 2046-PFox01 Differentially Regulates Both Normal and Diabetic Wound HealingCHENYING ZHANG, CHEN TIAN, SARAH ALSADUN, JASON LIM, GUANGYU DONG, DANA T. GRAVES, Beijing, China, Philadelphia, PA

Healing is delayed in diabetic wounds. Forkhead box O-1 (FOX01) is a transcription factor that is activated in diabetic wounds. We investigated the hypothesis that FOX01 played an important role in delayed diabetic wound healing. Small dermal wounds were created in experimental mice with keratinocyte-specifi c FOX01 deletion (K14.Cre+.FOX01L/L) and matched control littermates (K14.Cre-.FOX01L/L). Mice were rendered diabetic by streptozotocin injection. In normal mice FOX01 deletion reduced wound closure examined grossly or histologically (P<0.05) but in diabetic mice FOX01 deletion enhanced wound healing (P<0.05). In vitro FOX01 deletion signifi cantly reduced keratinocyte migration in standard media, which was rescued by addition of transforming growth factor-beta1 (TGFb1) (P<0.05). Chromatin immunoprecipitation (ChIP) assays showed that FOX01 interacted with the TGFb1 promoter and reporter assays demonstrated that FOX01 induced TGFb1 transcription (P<0.05) in normal media. In contrast FOX01 failed to interact with the TGFb1 promoter or induce TGFb1 transcription in high glucose media (P>0.05). Instead, high glucose media stimulated FOX01 binding to SERPINB2 and CCL2O promoters to increase their transcription (P<0.05). The negative impact of high glucose on keratinocyte migration was rescued by either silencing FOX01 or by inhibiting SERPINB2 or CCL20. Thus, in normal conditions FOX01 promotes expression of TGFb1 but fails to induce TGFb1 in high glucose. Moreover, high glucose signifi cantly enhances FOX01

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induced expression of SERPINB2 and CCL20, both of which interfere with keratinocyte migration. These results demonstrate that FOX01 expression in keratinocytes is a key determinant of dermal re-epithelialization. This occurs through FOX01 positively or negatively modulating keratinocyte migration by its regulation of downstream targets based upon glucose levels.


& 2047-PCombining Exendin-4 Treatment with Metformin Attenuates Pros-tate Cancer GrowthYOKO TSUTSUMI, TAKASHI NOMIYAMA, TAKAKO KAWANAMI, YURIKO HAMA-GUCHI, TOMOKO TANAKA, TOSHIHIKO YANASE, Fukuoka, Japan

Recently, pleiotropic benefi ts of incretin therapy beyond glycemic control have been reported. Although cancer is one of the main causes of death in patients with type 2 diabetes, few reports describe the anti-cancer effects of incretin. We previously reported that Exendin-4 (Ex-4), a GLP-1 receptor agonist, attenuates prostate cancer growth (Nomiyama T, Diabetes 2014). On the other hand, metformin (Met) is well-known as not only a glucose lowering agent but also anti-cancer agent. In the present study, we exam-ined anti-cancer effect of Ex-4 and Met using prostate cancer model. In in vitro experiments, Ex-4 and Met signifi cantly decreased growth curve of LNCaP cell, prostate cancer cell line, and further attenuation was observed in combined treatment of Ex-4 and Met. BrdU assay revealed that both Ex-4 and Met decreased LNCaP cell proliferation signifi cantly and additionally. On the other hand, Met induced apoptosis in LNCaP cell, but Ex-4 didn’t. Fur-ther, we transplanted LNCaP cell into non diabetic athymic mice and treated them with control, Ex-4, Met or Ex-4 and Met for 6 weeks. Ex-4 and Met signifi cantly decreased tumor size, and further reduction of tumor size was observed by combined treatment signifi cantly (p<0.05). If we performed im-munohistochemistry of P504S, a marker of prostate cancer, P504S positive cells were dramatically reduced by Ex-4 and Met treatment compared with control (p<0.01). Interestingly, GLP-1 receptor expression was signifi cantly increased in tumor of mice treated with Ex-4 and Met compared with con-trol (p<0.05). In these mice, body weight and blood glucose level were not changed during the experiment. Serum prostate specifi c antigen level was signifi cantly decreased in combined group compared with control (p<0.05).

These data suggest that Ex-4 attenuates prostate cancer growth through inhibiting proliferation, and Met does though induction of apoptosis. Com-bined treatment of Ex-4 and Met further attenuates prostate cancer growth more than mono treatment.

Guided Audio Tour: Other Hormones—A Grab Bag (Posters: 2048-P to 2055-P), see page 17.

& 2048-PFibroblast Growth Factor 21 Prevents Glycemic Deterioration in Streptozotocin-induced DiabetesBILAL OMAR, BIRGITTE ANDERSEN, KIRSTEN RAUN, BO AHRÉN, Lund, Sweden, Måløv, Denmark

In type 1 diabetes, glycemic control is rapidly lost at onset as endogenous insulin production declines. Fibroblast growth factor 21 (FGF21) has been shown to lower blood glucose levels in models of type 2 diabetes in an insu-lin independent manner. We aimed therefore to determine if the deteriora-tion of glycemic control that occurs early after the onset of insulin-defi cient diabetes can be blunted by treatment with recombinant FGF21. Normal C57BL/6J mice made diabetic by a single high dose injection of streptozo-tocin (STZ) were randomized to receive twice daily subcutaneous injection of vehicle or recombinant human FGF21 at doses of 0.3 and 1.0 mg/kg for 10 days. Body weight was recorded daily and fi ve hour fasted glucose, insulin, glucagon, free fatty acids and ketones were determined at 6 and 10 days post-randomization. FGF21 treatment resulted in slightly lower total body weight in the 1.0 mg/kg BID group which was signifi cant (p < 0.05), when cal-culated as change from baseline, from study day 3 until the end of the study. Fasting plasma glucose increased after STZ in untreated diabetic mice by approximately 50 percent, but this increase was prevented with FGF21 at 0.3 mg/kg BID (p=0.0033). In contrast, at 1.0 mg/kg BID, FGF21 did not prevent the increase in plasma glucose after STZ due to an apparent stimulatory effect of FGF21 1.0 mg/kg BID on plasma glucagon. Five hour fasting plasma glucagon was 4-fold higher in the diabetic group treated with FGF21 1.0 mg/kg BID than in untreated diabetic mice (p=0.007). This effect was not seen for the group treated with FGF21 0.3 mg/kg BID. There were signifi cant re-ductions in fi ve hour fasted plasma free fatty acids with FGF21 treatment in the 1.0 mg/kg BID group (p=0.0095). In conclusion, FGF21 prevents glycemic

decline in streptozotocin diabetic mice, at intermediate doses, and at higher dose an increase in basal plasma glucagon blunts the glycemic effect. FGF21 has potent lipid and weight lowering properties even in a mouse model of insulin defi cient diabetes.

Supported By: Novo Nordisk A/S

& 2049-PThe Role of Subclinical Infl ammation for the Early Time Course of Glycemic Control, Insulin Sensitivity, and Secretion in Newly Diag-nosed Type 1 and Type 2 DiabetesKATHARINA S. WEBER, BETTINA NOWOTNY, KLAUS STRASSBURGER, MARIE-CHRISTINE SIMON, GIOVANNI PACINI, JULIA SZENDROEDI, KARSTEN MUES-SIG, CHRISTIAN HERDER, MICHAEL RODEN, Düsseldorf, Germany, Padova, Italy

Infl ammatory processes are involved in the progression of insulin resis-tance and beta-cell dysfunction in pre-diabetic individuals and can therefore contribute to the development of diabetes. However, the role of biomarkers of subclinical infl ammation in the early time course of type 1 (T1D) and type 2 diabetes (T2D) is largely unknown. In a prospective study, new-onset T1D (n=42) and T2D (n=94) patients underwent detailed metabolic characteriza-tion within the fi rst year after diabetes diagnosis and 2 years thereafter. Associations between changes of markers of subclinical infl ammation with changes of glycemic control, insulin sensitivity and secretion as measured by intravenous glucose tolerance and glucagon stimulation testing were assessed using multivariable linear regression analysis. Associations were adjusted for diabetes type, age, sex, body mass index (BMI), smoking status, and (for indices of insulin secretion) also for glucose disappearance rate, as well as for 2-year changes of BMI, smoking status, and glucose-lowering medication. Patients exhibited good metabolic control both at baseline and after 2 years (mean HbA1c between 6.6±1.2 and 6.8±1.2%). Two-year increases in high-sensitive C-reactive protein, interleukin-18, soluble E-se-lectin, and soluble intercellular adhesion molecule-1 were associated with absolute increases of HbA1c by +0.4%, +0.5%, +0.8%, and +1.5% (all P<0.01; changes per doubling of cytokine levels between baseline and 2-year follow-up). A comparable 2-year increase in sICAM-1 associated with a relative decrease in fi rst-phase insulin secretion (-34.2%) and pre-hepatic beta-cell function (-48.0%, both P<0.05). These data indicate that - after the onset of diabetes - subclinical infl ammation relates to worsening of glycemia and that endothelial activation may contribute to decreasing beta-cell function.

Supported By: German Center for Diabetes Research

& 2050-PTumour Necrosis Factor a Impairs Glucagon-Like Peptide 1 Secre-tionJEFFREY GAGNON, MEGHAN SAUVÉ, WEN ZHAO, HOLLY M. STACEY, STEFFEN T.S. BOLZ, PATRICIA L. BRUBAKER, Toronto, ON, Canada

Increased circulating concentrations of pro-infl ammatory cytokines in-cluding tumour necrosis factor α (TNFα) are observed in obesity. This infl am-mation causes widespread dysregulation in glucose metabolism. In humans, obesity is also associated with reduced nutrient-stimulated secretion of the intestinal incretin hormone, glucagon-like peptide 1 (GLP-1). We hypothe-sised that TNFα plays a direct role in the impairment of GLP-1 secretion from the enteroendocrine L- cell, and that blocking TNFα can restore both GLP-1 secretion and glucose homeostasis. Firstly, L-cells were shown to express the TNFα receptor subytpe-1, in both the human NCI-H716 L-cell model and mouse ileal sections. Treating NCI-H716 cells with TNFα for 24 hours led to a reduction in proglucagon mRNA expression (p<0.05) and GLP-1 cellular content (p<0.05), but did not affect cell viability. Furthermore, NCI-H716 cells pre-treated with TNFα for 24 hours no longer responded to the GLP-1 secre-tagogue, glucose dependent insulinotropic polypeptide. This effect was reversed by co-incubation with the NFκB inhibitor, 5-aminosalicylic acid, known to lie downstream of the TNFα receptor. Mice given a high fat diet (HFD) for 8 weeks had impaired glucose tolerance as well as increased TNFα mRNA expression in both fat and ileal tissue. The impairment in glucose tol-erance was reversed in mice treated with the anti-TNFα biological, Etaner-cept, 6 times over 2 weeks. In primary intestinal cultures from these animals, HFD-control mice had impaired GLP-1 secretion and this was not observed in the HFD-Etanercept cultures (p<0.05). In conclusion, TNFα directly impairs GLP-1 secretion at the level of the intestinal L-cell, an effect that is reversed by anti-TNFα therapy in association with improved glucose tolerance.

Supported By: Canadian Institutes of Health Research; Canadian Diabetes Association

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& 2051-PBetter Antilipolytic Effect of Insulin and Reduced Adipose Tissue Glucose Uptake after Exenatide Injection during PET-OGTTPATRICIA IOZZO, GIUSEPPE DANIELE, MARJORIE L. MOLINA-WILKINS, DEM-ETRIO CIOCIARO, JACK LANCASTER, CURTIS TRIPLITT, EUGENIO CERSOSIMO, PETER FOX, NICOLAS MUSI, DEVJIT TRIPATHY, RALPH A. DEFRONZO, AMALIA GASTALDELLI, Pisa, Italy, San Antonio, TX

GLP-1 receptor agonists (GLP-1RA) act on multiple tissues beyond the pancreas, including liver and adipose tissue. Recent studies in mice dem-onstrated that Exenatide (EX) infl uences adipose tissue lipid metabolism by enhancing fatty acid oxidation and inhibiting lipolysis thereby reducing fatty acid fl ow to the liver.

The aim of this study was to evaluate the acute effects of EX injected subcutaneously before OGTT on adipose tissue lipolysis and glucose uptake, i.e., the fi rst step for TG synthesis in fat.

15 male subjects (12 with IGT and 3 with newly diagnosed diabetes, age=56±8 y, BMI=29±1 kg/m2, HbA1c=5.7±0.1%) underwent 2 oral glucose tests (OGTT 75 g), with double blind injection of EX (5 mcg) or placebo (PLC) 30 min before OGTT. Adipose tissue glucose uptake (AT-GU) was measured by PET following injection of 18FDG (5mCi) at T=0 and with image acquisition during hours 0-1 of the OGTT. Peripheral lipolysis (RaGLY) and glucose fl uxes were measured by U-2H-glycerol and 6,6-2H-glucose tracers. Adipose tissue insulin resistance index (AT-IR) was calculated as FFAxINS.

After EX mean plasma glucose0-60min (107±6 vs. 138±8 mg/dl) and Insulin-0-60min (17.5±3.2 vs. 24.7±3.8 mU/l, all p<0.02) concentrations were reduced in EX vs. PLC. Despite lower insulin concentrations, FFA suppression (11% vs. 7%) and RaGLY (3.7±0.3 vs. 3.9±0.4 µmol/min kg) were similar in EX and PLC (p=ns). AT-IR during OGTT was improved after EX (12±3 vs. 19±4 mmol/l mU/l, p=0.009) indicating enhanced antilipolytic effect of insulin. AT-GLU was reduced in EX vs. PLC (11±1 vs. 15±1 nmol/min ml, p=0.01), explained by the reduced RaGLU (11.9±1.4 vs. 19.9±1.5 µmol/min ml, p<0.001) secondary to the decrease in plasma insulin and glucose levels. Whole body insulin-stimu-lated glucose clearance during OGTT was similar in EX and PLC studies.

Conclusion: EX improved adipose tissue insulin sensitivity by enhancing insulin’s antilipolytic effect without affecting whole body insulin-mediated glucose disposal.

Supported By: Amylin Pharmaceuticals/Bristol-Myers Squibb Foundation/AstraZeneca

2052-P

& 2053-PThe Thioredoxin/Peroxiredoxin Family Member Adiporedoxin Reg-ulates Protein Secretion in Adipocytes In Vitro and In VivoMARK P. JEDRYCHOWSKI, LIBIN LIU, KALYPSO KARASTERGIOU, TOVA ME-SHULAM, SHIYING DING, YUANYUAN WU, COLLETTE LAFLAMME, MI-JEONG LEE, STEVEN P. GYGI, SUSAN K. FRIED, PAUL F. PILCH, Boston, MA

Adipocytes are robust secretors of physiologically important proteins, in particular adipokines such as adiponectin (Adpn) and resistin, and also col-lagens. We have identifi ed a novel peroxiredoxin family member, localized to the endoplasmic reticulum (ER), that we call adiporedoxin (Adrx) because it is expressed at much higher levels in fat cells than in any other cell type. Proteins with active site -CXXC- domains such as Adrx are involved in ER redox regulation and disulfi de bond formation, and the latter are required for the assembly and secretion of collagen and Adpn, and many other se-creted proteins. In vitro Western blot and proteomic analysis of Adrx over and under expressing murine cells showed increased and decreased secre-tion (increased cellular retention) of all disulfi de-linked proteins detected, including all 3 Adpn isoforms. In confi rmation of the in vitro knockdown ex-periments, mice null for Adrx expression have reduced circulating (secreted) Adpn, notably the high molecular weight, most biologically active form, and their adipose tissue was virtually free from fi brosis as assessed by trichrome staining. A proteomic comparison of wild type and Adrx null adipose tissue revealed greater cellular retention of several adipokines, including Adpn and resistin, as well as an average 35% reduction in the 16 collagen species detected. The knockout mice show modest hyperinsulinemia, increased expression of adipocyte ER stress proteins and infl ammation, the latter as indicated by increased adipocyte cytokine mRNA. In humans, adipose tissue Adrx and adiponectin mRNA levels are tightly correlated, and proteins levels are negatively correlated with the infl ammatory marker, p-JNK. Knockdown of Adrx in human adipocytes results in inhibition of Adpn secretion, just as in murine cells. Together these data support the notion that Adrx plays a criti-cal role in human and murine adipocyte biology and consequent regulation of organismal metabolism.

& 2054-PCharacterizing the Distribution of Enteroendocrine Cells in Patients with Type 2 Diabetes and Nondiabetic ControlsTINA JORSAL, NICOLAI A. RHEE, JENS PEDERSEN, SARA L. JEPSEN, CAMILLA D. WAHLGREN, BRYNJULF MORTENSEN, LOUISE S. DALBØGE, PETER VILMAN, HAZEM HASSAN, JAKOB W. HENDEL, STEEN S. POULSEN, JENS J. HOLST, TINA VILSBØLL, FILIP K. KNOP, Hellerup, Denmark, Copenhagen, Denmark, Hørsholm, Denmark

Hormones secreted by endocrine cells in the gastrointestinal (GI) tract play pivotal roles in regulation of appetite and glucose homeostasis. But knowledge of the distribution of enteroendocrine cells in the human GI tract is sparse, and it is unknown whether an alteration is seen in type 2 diabetes (T2D).

We investigated the distribution of endocrine cells along the entire GI tract in 12 patients with T2D and 12 age and BMI-matched non-diabetic con-trols. Upper and lower double-balloon enteroscopies with mucosal biopsy retrieval at 30 cm intervals were performed. Using immunohistochemical staining, we determined the distribution of cells positive for chromogranin A, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), somatostatin, prohormone convertase (PC) 1 and PC2.

In both groups, the density of GLP-1-positive cells increased signifi cantly along the small intestine and colon, whereas the density of GIP-positive cells dropped along the small intestine. Changes in the density of cells positive for somatostatin, chromogranin A, PC1 and PC2, respectively, were also found along the GI tract. We found signifi cantly higher density of chromogranin A-positive cells in the small intestine of controls compared to T2D patients, whereas the density of chromogranin A-positive cells in colon was greater in T2D patients. The controls showed higher density of GLP-1 and PC1-positive cells in the small intestine. GIP, PC2 and somatostatin densities did not differ signifi cantly between the two groups.

In a unique human biopsy material from the entire GI tract of patients with T2D and matched non-diabetic controls, we confi rmed the notions of GIP and GLP-1-positive cell distribution and also observed signifi cant differ-ences in the distribution of enteroendocrine cells between controls and T2D patients. The physiological and/or pathophysiological implications of these differences remain to be elucidated.

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& 2055-PBeta Cell-originating HDL-small RNA Intercellular Communication Is Altered in Type 2 DiabetesLESLIE A. ROTETA, CARINE BEYSEN, QUANHU SHENG, PRAVEEN SETHUPATHY, SCOTT TURNER, KASEY C. VICKERS, Nashville, TN, Emeryville, CA, Chapel Hill, NC

We have previously reported that high-density lipoproteins (HDL)-transport functional microRNAs (miRNA); however, high-throughput small RNA sequenc-ing (smRNAseq) has revealed that miRNAs represent only a small fraction (4%) of human small RNAs (smRNA) on HDL. Strikingly, a majority of HDL-smRNAs are non-human (58%) and most (95%) of the human smRNAs align to unan-notated loci. Nevertheless, we found 43 signifi cantly altered miRNAs on HDL isolated from type 2 diabetes (T2D) subjects compared to controls; however, we found >1000 signifi cant changes to non-miRNA smRNAs on HDL, suggest-ing that the robust extracellular RNA response to T2D is likely conferred by non-miRNA HDL-smRNAs. These include many smRNAs-derived from small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), tRNAs, rRNAs, and Y RNAs. Moreover, we found that pancreatic beta cells export distinct sets of novel smRNAs to HDL, which are then delivered to recipient endothelial cells and hepatoma cells and loaded onto cellular Argonaute (2/3)-RNA-induced si-lencing complexes, as determined by PAR-CLIP high-throughput sequencing. Based on T>C mutations representing transfer and in silico alignments, our re-sults suggest that many of HDL-smRNAs are regulating mRNA targets similar to miRNAs, the most active being smRNAs-derived from snRNAs. Moreover, results suggest that many beta cell-originating HDL-smRNAs are likely re-pressing genes critical to lipid and glucose metabolism in the liver. In addition to mRNA targets, we also found evidence that beta cell HDL-smRNAs are also targeting other long RNAs, including primary miRNAs, snoRNAs, scaRNAs, vault RNAs, and long no-coding RNAs in recipient endothelial cells and hepa-toma cells. Here, we demonstrate a HDL-smRNA intercellular communication network originating in pancreatic beta cells that likely targets endothelial cells and the liver controlling gene expression associated with energy metabolism that is altered in T2D.

2056-PPancreatic-derived Factor Impaired Glucagon-Like Peptide-1 Pro-duction from the Enteroendocrine L CellXIAOPEI CAO, FENGHUA LAI, HUIMEI LIN, YANBING LI, HAIPENG XIAO, Guang-zhou, China

Backgound: Pancreatic-derived factor (PANDER) was a pancreatic islet- specifi c cytokine that cosecretes with insulin. However, its biological function remains largely unknown to date. We recently showed that intestine might be a novel target tissue of PANDER. The aim of this study was to clarify if PANDER impacts the production of glucagon-like peptide-1 (GLP-1).

Methods: We employed adenoviral gene delivery to develop a murine model of PANDER overexpression after receiving control/high fat diet for 16-weeks, and treated the mouse intestine L cell line GLUTag cells with recombinant PANDER protein.

Results: Overexpression of PANDER in the C57/Bl6 mice reduced glu mRNA expression and impaired GLP-1 secretion. This effect was more high-lighted in high fat diet (HFD) fed mice which were overexpressed PANDER. In GLUTag cells line, PANDER exposure led to decreased glu mRNA expression and GLP-1 secretion in a dose- and time-dependent manner, while did not af-fect the viability of cells as well as activation of the apoptosis protease, cas-pase-3. Moreover, PANDER blocked insulin- induced and oleoylethanolamide (OEA)-induced GLP-1 secretion by inhibitting insulin signaling-Wnt pathway and cAMP-PKA pathway, respectively.

Conclusions: Our fi ndings fi rstly indicated that the intestinal L cell was responsive to PANDER and elevated PANDER level in vitro and in vivo result in impaired GLP-1 production indicating a crosstalk between pancreas and intestine.

Supported By: National Natural Science Foundation of China (81370907)

2057-PMetreleptin in Patients with Partial LipodystrophyNEVIN AJLUNI, MOAHAD DAR, JOHN XU, TAISIA ISUPOV, ADAM NEIDERT, ELIF A. ORAL, Ann Arbor, MI, Greenville, NC, Gaithersburg, MD, Fort Washington, PA

Lipodystrophy (LD) is a rare condition characterized by complete (general-ized) or incomplete (partial) adipose tissue loss, ectopic lipid deposition, and severe metabolic abnormalities (extreme insulin resistance, diabetes, and hypertriglyceridemia) that often respond poorly to conventional treatments. Metreleptin is the fi rst approved treatment for generalized LD, but effi cacy and safety in partial LD is not established. We report our fi ndings in patients with

partial LD (n=23) treated with metreleptin (mean dose 0.08 mg/kg/day) for 1 y from an ongoing sponsor-initiated open-label study with no entry criteria for baseline (BL) leptin. Most patients were female (96%), aged ≥18 y (100%) with a medical history of hepatic steatosis (82%), diabetes (77%), and hypertrig-lyceridemia (59%), and taking glucose- or lipid-lowering agents (73.9% each). Mean (SD) BL A1C, fasting plasma glucose (FPG), and triglycerides (TG) were 7.9% (1.5), 140.5 mg/dL (58.6), and 401.9 mg/dL (537.1). After 1 y of treatment (n=8), the mean (SE) changes from BL were −0.9% (0.6) for A1C, −42.0 mg/dL (22.4) for FPG, and −119.8 mg/dL (84.1) for TG. A1C and TG changes trended higher in patients with higher BL values (Figure). The most frequently reported adverse events were nausea (39.1%), hypoglycemia (26.1%), and urinary tract infection (26.1%). Improved A1C, FPG, and TG were observed in some but not all metreleptin-treated patients on background medications.

Supported By: AstraZeneca; National Institute of Diabetes and Digestive and Kidney Diseases (to N.A., E.A.O., A.N.)

2058-PIntestinal Lipid Sensing Does Not Play a Role in the Regulation of Glucose Metabolism in HumansROBYN A. TAMBOLI, FENG SHA, REEM M. SIDANI, VANCE L. ALBAUGH, EMILY A. ECKERT, NAJI N. ABUMRAD, Nashville, TN

The gastrointestinal (GI) tract plays a critical role in the metabolic re-sponse to a meal. Nutrient receptors in the intestine initiate hormonal and neuronal responses that regulate whole-body nutrient homeostasis. Rodent studies suggest that intestinal lipid sensing regulates glucose metabolism through a gut-brain-liver axis. We investigated whether intestinal lipid sens-ing regulates glucose metabolism in humans. A cohort of 8 lean subjects received a continuous intraduodenal (ID) infusion of olive oil (0.4 kcal/min) for 90 min followed by co-infusion of lipid and the topical anesthetic benzo-caine for 90 min. A second control cohort of 6 lean subjects did not receive ID lipid or lipid/benzocaine. Hepatic glucose production was measured by IV infusion of [6,6-2H2] glucose and glucose oxidation by ID infusion of [U-13C] glucose. Circulating lipid and glucose levels and pancreatic and GI hormone responses were assessed. ID infusion of lipid did not alter plasma free fatty acids, triglycerides, or insulin, which remained at levels similar to the control

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group, suggesting that the lipid was not absorbed and did not exert systemic effects on glucose metabolism. Lipid infusion had no effect on hepatic glu-cose production or glucose oxidation. Secretion of GIP, GLP-1, and PYY in-creased in response to lipid infusion but remained unchanged in the control group. CCK levels did not change in either group. Addition of benzocaine to the lipid infusion did not alter any of the parameters assessed, suggesting no involvement of afferent neuronal signaling. These results indicate that intestinal lipid sensing does not regulate hepatic glucose metabolism or glucose oxidation in lean subjects, but plays an important role in regulat-ing secretion of GI hormones. Additional studies are underway to further explore the role of intestinal neuronal signaling in the metabolic response to nutrient ingestion.

Supported By: P30DK 058404, DK100431

2059-PLeptin Enhances Metabolic Responses to Feeding EntrainmentLAURA SCOLARO, KYRA JEFFERSON-GEORGE, XIAOYAN YIN, CHARLES ADDO-YOBO, FRED ANOKYE-DANSO, REXFORD S. AHIMA, Philadelphia, PA

Disruption of sleep-wake cycles and timing of feeding alters energy me-tabolism and circadian rhythms, and predisposes toward obesity and dia-betes. We have developed a feeding entrainment model, in which mice are restricted to feeding during the dark and light periods. We hypothesized that leptin sensitivity modulates the behavioral and metabolic responses to the timing of feeding. To test our hypothesis we studied C57BL/6J wild type mice and l/l mice harboring a mutant exon 18b of LRb containing a substitution of Tyr985 with leucine that abolishes the phosphorylation of this site, blocks SHP/SOCS3 recruitment and enhances leptin signaling. We confi rmed that WT mice had no signifi cant response to a sub-threshold treat-ment of leptin (2mg/kg injected ip at 6 pm), whereas food intake and body weight were signifi cantly reduced in l/l mice. We compared the phenotypes of WT and l/l mice fed a high fat (45%) diet ad libitum (LD-fed), or restricted to feeding during the light (L-fed) or dark (D-fed) periods. The LD-fed and D-fed WT mice were more prone to diet- induced obesity than L-fed WT mice. In contrast, the l/l mice were resistant to obesity regardless of their feeding schedule. Food intake, energy expenditure and locomotor activity were signifi cantly lower in l/l mice. The respiratory exchange ratio (RER) was increased in l/l mice, indicating an increase in glucose oxidation. Hypotha-lamic proopiomelanocortin (POMC) expression was increased in the L-fed l/l mice, while expression levels of neuropeptide Y (NPY) and agouti-related protein (AGRP) were not altered signifi cantly. Ongoing studies will determine how the timing of feeding affects other central and peripheral leptin targets that mediate energy homeostasis and insulin sensitivity.

Supported By: American Diabetes Association (7-13-BS-004 to R.S.A); P01-DK049210, P30-DK19525

2060-PExploring a Potential Mechanism of FGF21 Resistance during ObesityKATHLEEN R. MARKAN, MEGHAN C. NABER, LILA PELTEKIAN, MATTHEW J. POTTHOFF, Iowa City, IA

Pharmacological administration of FGF21 ameliorates metabolic dysfunc-tion in multiple obese animal models. However, during obesity circulating FGF21 levels are signifi cantly elevated suggesting a state of FGF21 resis-tance. FGF21 signals in an endocrine manner via a receptor complex con-sisting of a classical FGF receptor and a co-receptor termed beta-klotho. Beta-klotho is expressed in a limited number of metabolic tissues, including adipose tissue, and is absolutely required for FGF21 signaling. To investigate potential mechanisms contributing to FGF21 resistance during obesity, we performed expression profi ling of beta-klotho in numerous tissues of C57/B6 mice on a high fat diet for varying amounts of time (0 to 16 weeks). We observed a marked decrease in white adipose tissue (WAT) beta-klotho pro-tein levels from mice fed high fat diet for 2 weeks. WAT beta-klotho protein was undetectable in mice fed a high fat diet for 12 weeks compared to lean controls. Plasma profi ling from the time-course revealed that circulating FGF21 levels were inversely correlated with WAT beta-klotho protein lev-els. Circulating FGF21 progressively and signifi cantly increased in parallel to plasma insulin levels over the 16 week high fat diet time-course. These data suggest a relationship between WAT beta-klotho expression, FGF21 sensitivity and insulin resistance during obesity. To determine whether maintenance of adipose beta-klotho expression would prevent FGF21 resis-tance during diet induced obesity, we generated a transgenic mouse model capable of expressing physiological levels of beta-klotho protein specifi cally in adipose tissue (Adipo-beta-klotho TG). The metabolic phenotyping of this

novel mouse model during diet induced obesity provides new insight into the mechanism(s) contributing to FGF21 resistance.

Supported By: Edward Mallinckrodt, Jr. Foundation

2061-PAn Optimized Novel GLP-1-GIP Receptor Dual Agonist with Potent Effects on Body Weight and Glucose Control in Mice Has the Poten-tial for Once-Weekly Administration in HumansCARSTEN B. KNUDSEN, JENS R. DAUGAARD, PERNILLE T. SHELTON, LISE GIEHM, MARIA A. DERYABINA, JACOB U. FOG, PIA NOERREGAARD, Glostrup, Denmark

Analogs of the incretin hormone GLP-1 are being used for the management of T2D and are often accompanied by modest weight loss. Approaches to enhance the anti-obesity effect are being actively pursued. One strategy to enhance the effi cacy of GLP-1 agonists includes its co-administration with other endogenous hormones e.g. glucagon. Recently, it has been demon-strated that the incretin hormone GIP enhances the weight loss induced by GLP-1.

Here we report the characterization of a novel balanced GLP-1-GIP recep-tor dual incretin (DI) agonist, ZP-DI-70, with selectivity >1000 fold over the glucagon receptor. ZP-DI-70 activated the human GLP-1 and GIP receptors in a cAMP assay (HEK293 cells) with EC50 values of 29 and 16 pM, respectively. Pharmacokinetic (PK) characterization was performed in mice and monkeys and the terminal half-life’s following IV administration was estimated to 17h and 68h, respectively. Simple two species allometric scaling of the PK indi-cates that the compound is suitable for once-weekly dosing in humans. The effects of ZP-DI-70 on body weight and glucose control were investigated in mice. ZP-DI-70, administered SC once every third day, dose-dependently reduced body weight in DIO mice. Dosing with 3 nmol/kg led to a relative weight loss of 13 % over 3 weeks of treatment. When given SC 22h prior to a glucose challenge, ZP-DI-70 (5 nmol/kg) signifi cantly reduced blood glucose levels 15-180 min after an IP bolus of glucose in diabetic db/db mice.

These results suggest ZP-DI-70 as a promising candidate for the treat-ment of T2D with superior body weight lowering effect compared to existing therapies. The in vivo profi le of the compound further suggests that ZP-DI-70 could be used as a convenient once-weekly treatment.

2062-PGlucagon-Like Peptide-1 Receptor: A Novel Receptor in PlateletsHASSAN KAHAL, AHMED ABURIMA, BENJAMIN SPURGEON, KATIE S. WRAITH, MARYAM GHAVIDELDARESTANI, FARAN RIZVI, ROGER G. STURMEY, ERIC S. KILPATRICK, STEPHEN L. ATKIN, KHALID M. NASEEM, Hull, United Kingdom, Doha, Qatar

Few studies have examined the effects of glucagon-like peptide-1 (GLP-1) analogues on atherothrombotic risk. Here we report that blood platelets express the GLP-1 receptor (GLP-1R) identifi ed through a combination of biochemical and functional techniques. GLP-1R mRNA expression in human platelets was confi rmed by reverse transcriptase polymerase chain reaction (RT-PCR). Immunoblotting demonstrated the presence of a 56kDa protein in platelets and megakaryocytes, which was indistinguishable in terms of size from GLP-1R in endothelial cells. Liraglutide, a GLP-1R agonist, inhibited collagen and thrombin induced platelet aggregation, which was associated with increased protein kinase A (PKA) activity. Importantly, the inhibition produced by liraglutide was reversed by GLP-1R specifi c antagonist exendin 9-39. The results suggest that platelets express the GLP-1R and that lira-glutide inhibits platelet activation at least partially through the activation of GLP-1R. Subsequently, treatment with GLP-1R agonist, liraglutide, may have a cardioprotective effect.

2063-PRegional Variation in Expression of Enzymes That Synthesise Incre-tin Hormones and Glucagon in Lean and Morbidly Obese HumansLAURA E. CLARIDGE, NEHA V. VALIYAPURAYIL, NAM Q. NGUYEN, CHRISTOPHER K. RAYNER, TONGZHI WU, JENNA E. BURGESS, NEKTARIA PEZOS, RICHARD L. YOUNG, Adelaide, Australia

We recently reported increased release of glucagon and glucose-de-pendent insulinotropic polypeptide (GIP) in response to enteral glucose in morbidly obese humans, while glucagon-like peptide-1 (GLP-1) release was decreased [1]. These changes may promote hyperinsulinemia and hypergly-cemia. Jejunal expression of prohormone convertase-1 (PC-1, PCSK1), the proteolytic enzyme for GIP and GLP-1 production, is decreased in morbidly obese patients with type 2 diabetes compared to those without diabetes [2] which may contribute to hyperglycemia. However, regional expression of PCSK1, and of PCSK2 (PC-2, the proteolytic enzyme for glucagon produc-tion), has not been evaluated in relation to obesity. Endoscopic biopsies

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were collected from the duodenum of lean (5M:1F, 46±9y, BMI: 25±2 kg/m2) and obese subjects (1M:5F, 46±5y, BMI: 46±6 kg/m2), while left colon biop-sies were collected separately from lean (4M:5F, 60±4y, BMI: 24±1 kg/m2) and obese subjects (3M:5F, 57±4y, BMI: 40±3 kg/m2), none of whom had diabetes. PCSK1 and PCSK2 transcript levels were assessed by qPCR. In lean subjects, PCSK1 transcripts were 5-fold higher (P<0.001) in duodenum than left colon, while PCSK2 transcripts - present at half the level of PCSK1 in the duodenum (P < 0.001) - were 8-fold higher in duodenum than left colon (P<0.001). There were no signifi cant differences in PCSK1 or PCSK2 transcript distribution or abundance between lean and morbidly obese subjects.

In conclusion: (i) duodenal L-cells may have greater capacity for incretin hormone biosynthesis and release, despite greater density of L-cells in the colon; (ii) the relatively high level of duodenal PCSK2 expression supports the concept of gut-derived glucagon production in humans; and (iii) transcrip-tional differences in PCSK1 or 2 do not underlie reduced GLP-1 production or augmented glucagon release in human obesity.

1. Nguyen QN et al. 2015 J Clin Endocrinol Metab (Epub).2. Rohdon F et al. 2014 Obes Surg 24:2075-81. Supported By: Royal Adelaide Hospital

2064-PAutocrine BDNF Signaling Promotes GLP-1 Secretion in Entero-endocrine L-CellsALEXANDER M. EFANOV, TAMER COSKUN, LIBBEY S. O’FARRELL, KRISTER BOKVIST, RUTH E. GIMENO, SAMREEN K. SYED, Indianapolis, IN

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is mostly found in the neurons, where it supports survival, growth and differentiation. BDNF also plays an important role in regulation of nutri-ent metabolism. Specifi cally, BDNF administration in rodents improves glu-cose and lipid metabolism, energy balance and feeding behavior. In addition to brain, some peripheral tissues also express substantial levels of BDNF, which include cells of the gastrointestinal tract. We studied the expression and function of BNDF and its high affi nity receptor TrkB in enteroendocrine GLUTag cells. GLUTag cells, an enteroendocrine L-cell model, express the preglucagon gene and secret glucagon like peptide 1 (GLP-1), a gut incre-tin hormone critical for regulation of nutrient homeostasis. RT-PCR analysis of GLUTag cells, mouse hypothalamus and mouse ileum demonstrated that GLUTag cells expressed BDNF mRNA at levels higher than those for hypo-thalamus or ileum. TrkB mRNA expression was also found in GLUTag cells and mouse ileum, albeit at levels lower than in hypothalamus. BDNF protein was detected in media from GLUTag cell cultures. BDNF was released by GLUTag cells through a regulated secretory pathway and was co-secreted with GLP-1. Exogenously added BDNF promoted increases in GLP-1 secre-tion in GLUTag cells at both basal and stimulated conditions. BDNF induced elevation in GLP-1 secretion was blocked with inhibitors of TrkB and PI3 ki-nase pathways. Knockdown of endogenous BDNF and TrkB gene expression with specifi c siRNAs resulted in inhibition of GLP-1 secretion in GLUTag cells. Based on these fi ndings, in enteroendocrine L-cells BDNF may serve as an autocrine factor that is co-secreted with GLP-1 and exerts a positive feed-back regulation of GLP-1 secretion.

2065-PDifferential Effect of Pioglitazone, Exenatide, and Combination of Pioglitazone and Exenatide on Plasma Metabolites in Type 2 DiabetesALBERTO O. CHAVEZ, RODOLFO GUARDADO-MENDOZA, STEPHEN VARVEL, RYAN ALONZO, KARYNE VINALES, ANDREA HANSIS-DIARTE, RALPH A. DE-FRONZO, DEVJIT TRIPATHY, San Antonio, TX, León, Mexico, Richmond, VA, Phoe-nix, AZ

Recent studies suggest that alpha-hydroxybutyrate (α-HB) is an early marker of insulin resistance. We examined the effect of Pioglitazone (PIO) and combined therapy with Exenatide and Pioglitazone (PIO-EX) on plas-ma metabolites in T2DM. We studied 30 T2DM subjects (age=55±3 yrs; BMI=34.9±5; FPG=167±10 mg/dl; HbA1c= 8.3±0.4%) who were randomized to receive: (i) (EXE 10ug bid, n=11), (ii) (PIO, 45 mg/d, n=10), or (iii) PIO+EXE (n=9) for 24 weeks. Before and after treatment subjects received OGTT and 2-step hyperglycemic (+125 and +400mg/dl) clamp followed by IV arginine (5g) bolus. We measured top-ranking insulin sensitivity metabolites, includ-ing plasma alpha-HB, linoleoyl-GPC (LGPC), oleic acid, before and after treat-ment with PIO, EXE and PIO+EXE. PIO + EXE caused a greater reduction in FPG, 2-h glucose and HbA1c compared to PIO or EXE alone (all p<0.05). PIO improved Matsuda index (MI) from 4.9 ± 0.8 to 7.9 ± 1.5 (p<0.05). PIO + EXE therapy increased MI of insulin sensitivity from 2.7 ± 0.5 to 5.9 ±0.6 (p<0.05). Pioglitazone decreased fasting FFA (0.643±0.06 to 0.376±0.07µM, p=0.001).

There was no signifi cant difference in plasma metabolite at baseline. At the end of study PIO treated individuals had a signifi cantly lower α-HB (6.31± 0.8 vs. 4.8± 0.7 µg/mL, p=0.04) and oleic acid (72 ±5.4 vs. 53.7±6.8µg/mL, p=0.03). Plasma LGPC levels were not affected by PIO. There was a trend to a reduced α-HB with PIO+EXE (7.18± 0.6 vs. 5.7± 0.8 µg/mL, p=0.056) though there was no signifi cant changes in plasma metabolites in patients on EXE group. In PIO treated subjects α-HB correlated with insulin secretion insulin sensitivity index (r=0.643, p=0.04).

Conclusion: PIO therapy modulates novel metabolites related to lipid me-tabolism and oxidative stress which could represent a fundamental mecha-nism by which PIO improves insulin sensitivity. EXE has no effect on plasma α-HB and likely improves glycemic control primarily by enhancing insulin secretion.

2066-PDifferent Effects of Meal and Incretin Hormones GIP and GLP-1 on Splanchnic Organs’ Blood Flow in Healthy and Morbidly Obese SubjectsJUKKA KOFFERT, HENRI HONKA, SAILA KAUHANEN, NOBUYUKI KUDOMI, AN-DREAS LINDQVIST, NILS WIERUP, LEIF GROOP, PIRJO NUUTILA, Turku, Finland, Kagawa, Japan, Lund, Sweden, Malmö, Sweden

Interplay between splanchnic blood fl ow, incretin secretion and glycemic homeostasis after meal ingestion is complex. This study’s objectives were to evaluate effects of meal and incretin hormones on blood fl ow in different splanchnic tissues and glycemic changes in healthy lean and obese individu-als. Therefore 11 healthy lean subjects participated in a mixed meal inges-tion (MMS), GIP-infusion (2 pmol/kg/min) studies, and GLP-1-infusion (0.75 pmol/kg/min) studies. Eight age-matched morbidly obese patients with type 2 diabetes participated in MMS and GIP-infusion studies prior to their bariat-ric surgery. Intestinal, pancreatic and hepatic blood fl ow and blood volumes were measured at 0, 20 and 50 min using PET/MRI with [15O]H2O and [15O]CO during each session. After MMS, duodenal blood fl ow remained unchanged, while jejunal blood fl ow increased by 40% in lean and 120% in obese sub-jects (from 0.4±0.2 to 0.7±0.4 ml ml-1min-1, P < 0.05). Plasma levels of GIP and GLP-1 increased similarly in obese and lean subjects together with increased insulin secretion. Pancreatic blood fl ow increased by 20 % in healthy (from 1.8±0.5 to 2.1±0.8 ml ml-1min-1, P < 0.05) but not in obese subjects. Simultane-ously, liver blood volume decreased by ~10% (from 29±7 to 25±76 ml 100g-1 of tissue, P < 0.05) in both groups. GIP-infusion increased jejunal blood fl ow by 140% (P < 0.01) in healthy and by 90% in obese (P < 0.06), whereas it decreased pancreatic blood fl ow by 24% (P < 0.01) in healthy and by 27% (P < 0.05) in obese subjects. Also, hepatic blood volume decreased by 9% (P < 0.01). Infusion of GLP-1 to healthy subjects decreased duodenal, jejunal and pancreatic blood fl ow (P range < 0.01 to < 0.05). To conclude, the splanchnic blood fl ow is redistributed after meal ingestion, preparing the intestine for nutrition absorption. Incretin hormones have partly counteracting effects on vascular changes in different splanchnic organs.

Supported By: Academy of Finland

2067-PBetatrophin: A New Potential Biomarker of Beta-Cell Function in Diabetes?ROBERTO LUPI, PAOLA URSOLEO, SILVIA DEL GUERRA, ERICA SELVAGGI, STE-FANO DEL PRATO, Pisa, Italy

Background: The potential effect of betatrophin as a stimulator of beta cell proliferation is a matter of debate and information of the hormone of the protein in human metabolic disease is scanty. We have measured serum betatrophin (BT) levels in diabetic and non-diabetic subjects.

Materials and Methods: We recruited 24 non-diabetic (ND; 52±15 yrs; 12M/12F; BMI 24.2±0.9 Kg/m2), 22 type 1 diabetic (T1DM; 41±4 yrs; 10M/12F; BMI 24.7±1.5 Kg/m2), 15 pancreas-transplanted T1DM (TX; 45±2 yrs; 6M/9F; BMI 22.7±3.2 Kg/m2) and 65 type 2 diabetic (T2DM 61±17 yrs; 27M/38F; BMI 29.5±6.7 Kg/m2) subject. BT levels were determined by ELISA and C-peptide by IRMA method. LPL expression was determined by Real-Time PCR and DOCK6 SNPs rs1541922 (C/T) and rs892066 (C/G) were genotyped using TaqMan Allelic Discrimination Assays on DNA from circulating blood cells.

Results: In the population as a whole, BT levels were inversely associated with age (r=0.380, p<0.0001) and HBA1c (r=0.260, p=0.039). A direct correla-tion was also apparent with HDL levels (r=0.382, p=0.0022). Excluding T1DM patients from the analysis, BT was directly correlated with C-peptide (r=0.396, p=0.0006) and blood glucose (r=0.319, p=0.0305) concentration. BT was higher in T1DM (798.3±19.43 pg/ml) and lower in T2DM (80.74±6.60 pg/ml) as com-pared to ND (384.27±25.14 pg/ml; all p<0.05 by Bonferroni’s test). In TX, BT concentration returned to levels similar to those of ND (477.81±19.65 pg/ml).

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On the contrary, LPL mRNA expression was lower in T1DM (0.56±0.10 2^-ΔCt) and higher in T2DM (2.59±0.31 2^-ΔCt) as compared to ND (1.65±0.16 2^-ΔCt; all p<0.05). In the whole population CC, CT and TT allele frequency was 0%, 53% and 47%, respectively while GG, GC and CC was 0%, 62.7% and 37.3%. There was no apparent difference in BT levels according to genotype.

Conclusions: The correlation between BT and C-peptide concentrations, together with normalization of BT in TX patients, suggests a potential role of the hormone as a biomarker of beta cell function (or mass).

2068-P

2069-PGlucolipotoxicity of GLP-1 SecretionHYE IN KIM, DAE-HEE KIM, YOU-BIN LEE, SEUNG-EUN LEE, YOUNG NAM KIM, SANG-MAN JIN, KYU YEON HUR, MOON-KYU LEE, Seoul, Republic of Korea

There have been many reports on the concept of glucolipotoxicity in pan-creatic beta cells and insulin target tissues which could decrease insulin se-cretion and/or insulin sensitivity. GLP-1 secreted from intestinal L-cells could stimulate glucose-induced insulin secretion from pancreatic beta cells and could decrease plasma glucose levels in patients with type 2 diabetes. It has not been shown whether glucolipoxicity would happen in GLP-1 secretion.

L cell lines (NCI-H716) were exposed to glucose (5.6 and 25 mM) or oleic acid (0.5 mM) for 72-96 hours and GLP-1 secretion in response to glucose was measured. Cell viability was measured by MTT assay and ATP level was also measured. In in vivo experiments, male Sprague-Dawley rats were divided into 4 groups and cannulations were done into jugular veins and ca-rotid arteries. After recovery, animals were infused with normal saline, glu-cose, intralipid, and glucose+intralipid for 3-6 hours, respectively, followed by mixed meal tolerance test (MMTT) for 2 hours.

Ninety-six hour exposure to high glucose (25 mM) signifi cantly decreased glucose-stimulated GLP-1 secretion from L-cells compared to normal glucose exposure (p<0.01). 96 hour exposure to oleic acid (with 5.6 mM glucose) also signifi cantly decreased glucose-stimulated GLP-1 secretion (p<0.05), but there was no additional change in GLP-1 secretion with oleic acid + 25 mM glucose exposure. There was no signifi cant change in cell viability, but there was a signifi cant decrease in ATP level when incubated with 25 mM glucose (p<0.05). In in vivo experiments, there were signifi cant decreases in GLP-1 level during MMTT in glucose, intralipid, and glucose+intralipid treated groups compared to the control group (p<0.01), and also signifi cant increases in AUCglucose in the same three groups compared to the control (p<0.01)

These results suggest that glucolipotoxicity could be demonstrated in GLP-1 secreting intestinal L-cells in vitro and glucose and/or intralipid-infused rats in vivo. Further studies are warranted as to the detailed mechanisms.

2070-PPostprandial Plasma Concentrations of Individual Bile Acids and FGF19 in Patients with Type 2 DiabetesDAVID P. SONNE, SAMUEL VAN NIEROP, FRÉDÉRIC M. VAZ, MAARTEN R. SO-ETERS, TINA VILSBØLL, FILIP K. KNOP, Hellerup, Denmark, Amsterdam, Nether-lands

Bile acids exert regulatory effects on lipid and carbohydrate metabolism by activating the nuclear farnesoid X receptor (FXR) in the gastrointestinal tract and liver. FXR activation in the ileum leads to secretion of fi broblast growth factor-19 (FGF-19), a gut hormone, which may be implicated in post-prandial glucose metabolism. We aimed to characterize postprandial plasma concentrations of individual bile acids and FGF-19 in patients with type 2 diabetes (T2D) and matched controls.

Twelve bile acids (conjugated and unconjugated) and FGF-19 concentra-tions were measured in plasma from 15 patients with T2D (mean duration of T2D: 7.5 years (range 6-20); age: 59.4±9.6 years (mean ± SD); BMI: 28.0 ± 2.2 kg/m2; HbA1c: 7.5±1.4%) and 15 healthy age, gender and BMI-matched con-trol subjects (age: 59.7±10.0 years; BMI: 27.9±2.0 kg/m2; HbA1c: 5.2±0.2%) undergoing 4 separate “meal” tests: a 75g-OGTT and 3 isocaloric (500 kcal) and isovolemic (350 ml) liquid meals with low, medium and high fat content, respectively.

Basal and postprandial concentrations of FGF-19 were comparable among the groups (P>0.05). Peaks occurred late (~3-4h after meal ingestion) and were more pronounced following the high fat meal. Total bile acid concen-trations increased with increasing meal fat content, peaked after 1-2h and were slightly higher in patients vs. controls (OGTT, low and medium fat meals (P<0.05); high fat meal (P=0.19)). These differences refl ected mainly glycine-conjugated forms of deoxycholic acid (DCA) and to a lesser extent cholic acid (CA) and ursodeoxycholic acid (UDCA), whereas chenodeoxycholic acid (CDCA) concentrations were comparable among the groups.

In conclusion, postprandial patterns of bile acids with FXR agonistic (CA, DCA and CDCA) and FXR antagonistic properties (UDCA) in T2D patients support the notion of a “T2D-bile acid” phenotype with possible pathophysi-ological implications. However, FGF-19 responses in T2D patients were not abnormal compared to controls.

2071-PFGF21 Is Not a Regulator of Bone Homeostasis in the Dio MouseMATTHIAS LOEHN, JOERG HAGER, MARTIN WALTER, DOMINIK HARTMANN, GERALD FISCHER, UWE SCHWAHN, THOMAS LANGER, OLIVER BOSCHEINEN, MARK SOMMERFELD, Frankfurt, Germany

Fibroblast growth factor 21 (FGF21) exerts diverse, benefi cial effects on energy balance and insulin sensitivity when administered systemically to rodents, monkeys or humans with diet-induced obesity (DIO) or diabetes. A recent report suggested that increased FGF21 levels by either gain-of-func-tion or pharmacological administration are associated with bone loss and FGF21 loss-of-function leads to a reciprocal increase of bone mass. The aim of the current study was to evaluate the effects of chronic FGF21 treatment on skeletal homeostasis.

Male C57BL/6J mice were fed a high-fat diet (HFD) or standard chow for 5 months. Before start of once daily i.p. treatment with 1 mg/kg human FGF21 or vehicle (PBS) groups were randomized on bodyweight. After 2 and 4 weeks of treatment samples of bone were collected, fi xed and preserved. Furthermore body weight, blood glucose, lipid profi le, and also concentration of bone biomarkers were analyzed during and at the end of the study. Quanti-fi cation of bone parameters was done by micro-computed tomography (µCT) imaging of femur.

4 weeks of treatment with FGF21 resulted in a 5% body weight reduction while vehicle treated obese control mice gained 17% weight. In addition FGF21 treatment normalized hyperinsulinemia, signifi cantly decreased tria-cylglycerides, total cholesterol and LDL-cholesterol, and resulted in normal-ization of leptin levels. Measurement of bone parameters by µCT revealed no change after 2 and 4 weeks FGF21 treatment in the diet-induced obese mice. Analysis of the bone biomarker PINP, DKK-1, SOST, OPG, and OCN by ELISA also did not result in any measurable changes versus the vehicle treated control.

Bone is a complex tissue and is constantly undergoing a complex process of renewal and remodeling involving many different factors and substances. However, the present study found no infl uence on bone mass or any bone biomarker after pharmacological treatment of DIO mice with FGF21.

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2072-PPlasma C1q/TNF-related Protein-9 and Adiponectin Levels Are Dif-ferentially Associated with Obesity and Metabolic Risk Factors in Patients with Type 2 DiabetesTAKESHI SAKURA, TOMOAKI MORIOKA, MARIKO ASADA, HIROKAZU MORI-GAMI, YUKO YAMAZAKI, KOKA MOTOYAMA, KATSUHITO MORI, SHINYA FUKU-MOTO, TETSUO SHOJI, MASANORI EMOTO, MASAAKI INABA, Osaka, Japan

C1q/TNF-related protein (CTRP)-9, a highly conserved paralog of adi-ponectin (ADN), is expressed predominantly in adipose tissue and has pro-tective effects on obesity, diabetes and atherosclerosis in rodents. How-ever, no report is available on the metabolic effect of CTRP-9 in human type 2 diabetes (T2D). This study was aimed to compare and examine the clinical associations of plasma CTRP-9 and ADN levels with metabolic parameters in patients with T2D. We included 457 patients with T2D in this study (males, 58%; median age, 65 years; BMI, 25.0 kg/m2). Fasting plasma CTRP-9 lev-els and total ADN levels were measured by ELISA. Plasma CTRP-9 levels (median, 16.8 µg/mL) were higher in females than in males, and were not correlated with plasma ADN levels (median, 6.1 µg/mL). Both plasma CTRP-9 and ADN levels were positively correlated with age, systolic blood pressure, serum creatinine, and urinary albumin. Whereas plasma ADN levels were negatively correlated with BMI, HOMA-R, triglycerides and uric acid, and positively with HDL-cholesterol, plasma CTRP-9 levels were positively corre-lated with BMI, triglycerides and uric acid, negatively with HDL-cholesterol, but not with HOMA-R. In multiple regression analyses, systolic blood pres-sure, serum creatinine, triglycerides, uric acid were positively, and male sex and HDL-cholesterol were negatively associated with plasma CTRP-9 levels. On the other hand, BMI, triglycerides were negatively, and serum creatinine and HDL-cholesterol were positively associated with plasma ADN levels. In summary, plasma CTRP-9 levels were affected by sex and renal function, and contrary to the case of plasma ADN levels, plasma CTRP-9 levels were unfavorably associated with obesity and metabolic risk factors in patients with T2D. These results suggest that CTRP-9 and ADN have different effects on the regulation of obesity and glucose/lipid metabolism in human T2D.

2073-PSGLT1/2 Inhibition Increases GLP-1 and Glucagon Release but Does Not Slow Gastric EmptyingSIMON A. HINKE, FUYONG DU, THOMAS KIRCHNER, NATHANIEL WALLACE, WENSHENG LANG, KEITH T. DEMAREST, JEAN WHALEY, YIN LIANG, Spring House, PA

Sodium-glucose co-transporters, SGLT1 and SGLT2, play dominant roles in intestinal and renal glucose absorption and re-uptake respectively. Slow-ing enteral glucose absorption and increasing urinary glucose excretion via a dual specifi city SGLT1/2 inhibitor improves glycemic control in diabetes. In vitro and in vivo pharmacology of dual SGLT1/2 inhibitor CmpdA (rSGLT1 IC50: 47nM; rSGLT2 IC50: 17nM) was reported at the 2014 ADA Meeting (1049-P). Our current data examines the acute effect of CmpdA on GLP-1, glucagon and gastric emptying during OGTT in Sprague-Dawley rats. Oral CmpdA (10mg/kg) signifi cantly reduced fasting (Veh: 98.0±3.5mg/dl; Cmp-dA: 73.3±1.5mg/dl) and peak post-challenge glycemia (Veh: 165.7±12.4mg/dl; CmpdA: 78.4±4.9mg/dl). Plasma tGLP-1 levels were signifi cantly elevated after glucose challenge (3.8±0.5-fold vs. control), warranting evaluation of CmpdA engagement of GLP-1’s therapeutically relevant effects: insulin stimulation, glucagon inhibition, and reduction in gastric emptying. Hence, we examined the effect of 1 and 10mg/kg CmpdA on glycemic excursion, 13C6-glucose absorption, gastric emptying, insulin, glucagon, GLP-1 and GIP, during an OGTT in SD rats (n=8/group). Detection of plasma 13C6-glucose showed inhibition of intestinal absorption by CmpdA during the 2h OGTT; dif-ferential effects on incretins were observed: plasma GIP AUC was reduced by 20.9% and 89.2% by 1 and 10mg/kg CmpdA, whereas tGLP-1 levels were augmented 5.5-6.1X in compound treated animals. Despite the signifi cant increase in circulating GLP-1, plasma acetaminophen appearance showed a compound-related increase in gastric emptying (~25-28%; P<0.05). Insuline-mia following the glucose challenge was signifi cantly blunted, while gluca-gon levels were enhanced at the highest dose of CmpdA, correlating well with glycemia. Whether plasma glucagon is impacted by a direct effect of SGLT inhibition on the alpha cell needs to be further evaluated.

2074-PGhrelin Is Not controlled by Efferent Vagal Activity Elicited by Mod-ifi ed Sham Feeding in HumansSIMON VEEDFALD, ASTRID PLAMBOECK, BOLETTE HARTMANN, CAROLYN DEACON, ANDRÉ WETTERGREN, LARS B. SVENDSEN, SØREN MEISNER, CLAUS HOVENDAL, TINA VILSBØLL, FILIP K. KNOP, JENS J. HOLST, Copenhagen, Den-mark, Hellerup, Denmark, Odense, Denmark

The orexigenic hormone ghrelin is secreted from endocrine cells in the gastric fundus, and plasma levels increase during fasting and decrease post-prandially. We evaluated the importance of efferent vagal activity for the regulation of ghrelin.

Plasma from truncally vagotomized individuals (TRUN, n=8), cardia-re-sected individuals (CARD, n=8) and matched controls (CONT, n=8) studied by modifi ed sham feeding (MSF) were analyzed. As part of the cardia resec-tions the esophageal plexus was severed rendering the CARD de facto vago-tomized. Both the TRUN and CARD had also received pyloroplasties. The stimulus used for the 15 min-MSF was an appetizing breakfast consisting of an egg omelet, bacon, bread, butter, cheese, marmalade, yoghurt, fresh fruit, pancakes, orange juice and tea or coffee). Participants were instructed to sample, chew and spit out all elements. The effi cacy of vagal activation was evaluated by measuring pancreatic polypeptide (PP) levels. Total ghrelin and PP plasma levels (mean±SEM) were measured by radioimmunoassay. At the time of peak cephalic activation (t = 15 min, i.e. end of MSF), PP levels in the CONT (34.8±8.1 pM) differed from values at t = -15 min (19.8±4.3 pM, P=0.0002) and t = 0 min (22.8±6.7 pM, P=0.003). At these time points there were no differences in the TRUN (27.8±11.7 pM) vs. (32.3±12 pM, P=0.40) and (30.5±10.8 pM, P=0.71), or in the CARD (18.0±2.5 pM) vs. (16.0±2.3 pM, P=0.88) and (19.0±4.9 pM, P=0.94).

Fasting (t = -15 min) ghrelin levels were 883±106, 471±77, and 655±51 pg/mL, while t = 15 min levels were 881±92, 486±95 and 663±60 pg/mL, for CONT, TRUN and CARD, respectively). Ghrelin levels remained unchanged at all time points in all groups (P>0.20). We conclude, that plasma levels of ghrelin are not acutely infl uenced by efferent vagal activity.

2075-PAdipose-Tissue Specifi c Deletion of Dipeptidyl Peptidase-4 (DPP-4) Enhances M2 Macrophage Markers and Results in Smaller Adipo-cytes Under HFDTANIA ROMACHO, IRA INDRAKUSUMA, DIANA RÖHRBORN, TAMARA R. CASTA-ÑEDA, TOMAS JELENIK, JÜRGEN WEISS, HADI AL-HASANI, MICHAEL RODEN, HENRIKE SELL, JÜRGEN ECKEL, Düsseldorf, Germany

DPP4 is an important therapeutic target for type 2 diabetes mellitus. We recently characterized this serine protease as a novel adipokine potentially linking obesity to the metabolic syndrome. By using a unique adipose-tissue (AT) specifi c DPP4 KO mouse model, we aimed to elucidate the role of adi-pose DPP4 in high fat diet-induced obesity.

Mice with deletion of the DPP4 gene in AT were generated using the cre/loxP system under control of the aP2 promoter on a C57BL/6J background. Body composition of male mice on chow diet and HFD for 24 weeks was performed using NMR. Serum DPP4 was measured by ELISA. Adipocyte size was quantifi ed by immunohistochemistry in subcutaneous (sWAT) and epididymal white adipose tissue (eWAT). Furthermore, gene expression was assessed in both depots by qRT-PCR.

KO mice gained signifi cantly more weight, fat and lean mass compared to WT mice on HFD. However, body fat percentage and body length remained similar. No differences in body composition were found on chow diet. Serum DPP4 was signifi cantly lower in KO animals on both diets. Within the HFD group, the KO mice showed a marked shift in the adipocyte size distribu-tion towards smaller adipocytes. In both fat depots, differentiation markers remained similar whereas the M2 macrophage markers mannose receptor 1 and interleukin 10 were signifi cantly upregulated in KO animals compared to WT under HFD. In eWAT, interleukin 6 and monocyte chemotactic protein 1 were signifi cantly increased in KO mice. Serum DPP4 correlated signifi cantly with adipocyte size in subcutaneous and epigonadal AT while negatively with serum adiponectin.

Although KO animals gained more weight and fat mass under HFD chal-lenge, the M2 macrophage markers were elevated. Taken into account that KO animals display smaller adipocytes under HFD, these fi ndings point towards a benefi cial role for DPP4 deletion in adipose tissue remodeling during HFD.

Supported By: European Foundation for the Study of Diabetes; European Com-mission (ADDIO-PIEF-2012-328793)

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2076-PA Novel Immune-based Approach for Measurement of the Anorec-tic Gut Hormone Oxyntomodulin: Changes after Gastric Bypass Sur-geryNICOLAI J. WEWER ALBRECHTSEN, SIGNE TORANG, DANIEL HORNBURG, REIDAR ALBRECHTSEN, CHARLOTTE JANUS, SARA LIND JEPSEN, SIMON VEED FALD, FELIX MEISSNER, MATTHIAS MANN, CAROLYN DEACON, JENS JUL HOLST, BO-LETTE HARTMANN, Copenhagen, Denmark, Munich, Germany

Oxyntomodulin (OX) arises from differential processing of proglucagon and may inhibit appetite and increase insulin secretion in humans. Reliable measurement of OX is challenging due cross-reactivity with glucagon and glicentin. Our aim was to quantify OX in plasma from gastric bypass pa-tients.

The sensitivity and specifi city of 4 commercial and 1 prototype OX kit(s) were evaluated. Four kits (cross)-reacted to an equal extent with OX, glicen-tin and glucagon, but 1 prototype kit showed low cross-reactivity (<20%) and was subjected to further analysis. To confi rm antibody-specifi city, a cell line (HEK293) was constructed to express OX or glicentin and stained with the N-terminal antibody used in the prototype kit and the N-terminal anti-body used in a commercial glicentin kit. Plasma from gastric bypass patients (n=18) were measured using the OX prototype- and the glicentin kit. Pooled plasma (n=10) from the same subjects and extracts of small-intestinal bi-opsies from healthy subjects (n=4) were further evaluated by fractionation (HPLC), immune-based detection and mass-spectrometry based proteomics.

The N-terminal antibody used in the OX prototype kit bound OX but not glicentin and the N-terminal antibody from the glicentin kit bound glicentin but not OX. HPLC of pooled plasma and extracts of small-intestinal biopsies followed by immune-based detection documented cross-reactivity to gli-centin (18±3%). Mass-spectrometry based proteomic of pooled plasma and extracts of small-intestinal biopsies identifi ed amino acid sequence(s) corre-sponding to OX and glicentin, respectively. Plasma levels of OX and glicentin increased signifi cantly (>5 fold) 1 and 6 month after RYGB. Glicentin levels were signifi cantly higher, before and after RYGB, compared OX.

Our study indicates that OX is fully processed in the human small intestine and that levels are 5 fold increased after gastric bypass. The prototype kit may be used for measuring OX in human plasma.

Supported By: Novo Nordisk Foundation Center for Basic Metabolic Research; University of Copenhagen; Danish Council for Independent Research (DFF-1333-00206A); Augustinus Foundation (14-0962); European Molecular Biology Organiza-tion; European Foundation for the Study of Diabetes

2077-PRenal Extraction and Acute Effects of Glucagon-Like Peptide-1 on Central and Renal Hemodynamics in Type 2 Diabetic MalesALI ASMAR, LENE SIMONSEN, MEENA ASMAR, STEN MADSBAD, JENS JUUL HOLST, ERIK FRANDSEN, CÉDRIC MORO, THOMAS JONASSEN, JENS BÜLOW, Copenhagen, Denmark, Toulouse, France

We previously demonstrated a signifi cant acute increase in cardiac output during an intravenous infusion of native glucagon-like peptide-1 (GLP-1) in healthy males due to a simultaneous increase in stroke volume and heart rate. Despite signifi cant renal extraction of GLP-1 by ~ 55%, renal hemody-namics and sodium excretion were unaffected during GLP-1.

In the present study, we elucidated similar effects of native GLP-1 in type 2 diabetics (n=8) under salt standardized conditions on two different occa-sions in random order.

During a 3-hour infusion of either GLP-1 (1.5 pmol kg-1 min-1) or saline, car-diac output was continuously estimated non-invasively, concomitantly with intra-arterial blood pressure and heart rate. Renal plasma fl ow, glomerular fi ltration rate, and uptake/release of hormones and ions were measured by Fick’s Principle after catheterization of a renal vein. Urine collection was conducted throughout the experiments at voluntary voiding, and subjects remained supine.

During GLP-1 infusion, systolic and diastolic blood pressure remained unchanged throughout the experiment. Heart rate increased signifi cantly whereas cardiac output remained unchanged. Renal plasma fl ow and glom-erular fi ltration rate as well as renal sodium and lithium excretion were not affected by GLP-1. Renal extraction of intact GLP-1 was 50%, while 30% of the primary metabolite GLP-1 9-36 amide was extracted during GLP-1 infu-sion.

In conclusion, like in healthy subjects, an acute administration of GLP-1 in type 2 diabetic subjects led to a positive chronotropic effect, but in contrast to healthy subjects, cardiac output did not increase in type 2 diabetics. Renal hemodynamics and sodium excretion were not affected in spite of signifi -

cant extraction of both the intact hormone and its primary metabolite during acute administration of GLP-1.

Supported By: Danish Heart Foundation

2078-PThe Role of Syntaxin-1a in Glucagon-Like Peptide-1 Secretion from Adult Mouse Intestinal L-CellsHOLLY M. STACEY, STEPHEN J. HALE, FIONA M. GRIBBLE, HERBERT Y. GAISANO, PATRICIA L. BRUBAKER, Toronto, ON, Canada, Cambridge, United Kingdom

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from dis-tal intestinal L-cells. Due to the glucose-lowering actions of GLP-1, GLP-1 receptor agonists and GLP-1 degradation inhibitors have been proven to be favorable type II diabetes treatments. Another potential therapeutic approach lies in enhancing endogenous GLP-1 release. Although signaling pathways promoting GLP-1 secretion have been extensively characterized, little is known about the precise downstream mechanism of GLP-1 exocyto-sis. We hypothesized that Syntaxin-1a, a core SNARE protein, is essential for exocytosis of GLP-1 from primary murine intestinal L-cells. Using immunocy-tochemistry, 1.1 ± 0.1% of adult mouse ileal cells (AMIC) in culture expressed GLP-1. AMIC cultures also expressed Syntaxin-1a, in both GLP-1-positive and -negative cells. GLP-1 secretion was enhanced by 10µM and 50µM forskolin/IBMX, by 2.1- to- 2.3-fold of control (p < 0.05, p < 0.001), respectively. GLP-1 secretion was also stimulated by 1µM glucose-dependent insulinotropic pep-tide (1.5-fold of control) and 15µM oleoylethanolamide (to 3.5-fold of control, p < 0.001). To specifi cally examine the role of Syntaxin-1a in GLP-1 secretion, AMIC cultures were generated from Syntaxin-1afl /fl mice and subsequently infected with adenovirus-cre-recombinase (AdV-iCre) or empty adenovirus (AdV-RFP) for 2 days, followed by analysis of Syntaxin-1a expression and GLP-1 release. Preliminary qPCR analysis demonstrated almost complete knockdown of Syntaxin-1a mRNA by AdV-iCre. Syntaxin-1a-cre L-cells ex-hibited a 1.3-fold increase in basal secretion, but the response to 15µM OEA was completely abrogated. Thus, Syntaxin-1a plays an essential role in GLP-1 secretion from the primary, adult mouse ileal L-cell.

Supported By: Natural Sciences and Engineering Research Council of Canada

2079-PDo Young Men with Low Birth Weight and Increased Risk of Type 2 Diabetes Exhibit Changes in Their Endocrine and Metabolic Circa-dian Rhythm(s)?CHARLOTTE BRØNS, PERNILLE NERLOE, YAN CHEN, MARTIN FRIEDRICHSEN, ALLAN VAAG, Copenhagen, Denmark, Lund, Sweden

A disrupted circadian rhythm and a low birth weight (LBW) are both as-sociated with increased risk of type 2 diabetes (T2D). We have shown that young men with LBW compared with normal birth weight (NBW) controls exhibit elevated fat oxidation and decreased glucose oxidation rates dur-ing nighttime. We therefore studied 24-hour profi les of plasma glucose, free fatty acids (FFA), triglycerides (TG), insulin, C-peptide, leptin, resistin, ghre-lin, incretin(s) (GLP-1, GIP) and infl ammatory markers (TNF-α, IL-6) in LBW and NBW men.

Twenty-four young (mean age 23.2 years), lean men born at term were in-cluded; 13 men were born with LBW (BW≤10th percentile) and 11 with NBW (BW 50-90th percentile). All subjects were standardized with regards to physical activity and provided with food for 3 days prior to and during the ex-amination. Twenty-fi ve blood samples were collected over a 24-hour period.

Despite lower birth weight, the LBW men did not differ from NBW controls with respect to adult body composition. Repeated measurements were used to analyze circadian variations and differences between groups. Plasma glucose and glucagon levels were both elevated in LBW subjects (P=0.03), only glucose varied over the 24-hour period (P<0.0001). Furthermore, plasma resistin levels were signifi cantly increased in the LBW subjects (P=0.003) with no signs of diurnal variations. Plasma FFA, TG, insulin, C-peptide, leptin, GLP-1, GIP, TNF-α and IL-6 levels displayed signifi cant diurnal variations with no differences between groups. Plasma ghrelin levels did not display circa-dian variations and did not differ between groups.

In conclusion, the increased nocturnal fat oxidation rate and plasma glu-cose levels in young LBW men is not associated with any major changes in the circadian rhythm of plasma FFA or TG levels, nor in key metabolic hor-mones. Increased plasma resistin levels may contribute to the development of insulin resistance and subsequently T2D in young men with LBW.

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2080-PTargeting of Endothelial FoxO1 Activity by Apelin Regulates Fatty Acid UptakeHYUNG CHUN, CHEOL HWANGBO, JINGXIA WU, BIKRAM SHARMA, IRINNA PAPANGELI, GWANG-WOONG GO, ARYA MANI, KRISTY RED-HORSE, Guilford, CT, New Haven, CT, Stanford, CA

The endothelium is emerging as a key regulator of tissue fatty acid uptake and transport. Such regulatory mechanisms have key potential roles in dis-ease conditions such as diabetes and insulin resistance. Recent studies have identifi ed the GPCR signaling mediated by the ligand apelin and the receptor APLNR to enhance glucose utilization in skeletal muscle and adipose tis-sue; however, the mechanism of such protective metabolic effects remains poorly defi ned. Here we demonstrate that APLNR is preferentially expressed in the microvascular endothelial cells of adult skeletal and cardiac muscles and adipose tissues. The endothelial cell based APLNR signaling was found to be critical for apelin function, as removal of endothelial cells from skeletal muscles led to abrogation of apelin induced Akt and AMPK phosphorylation. Moreover, we found that endothelial apelin stimulation leads to phosphory-lation and inactivation of the key metabolic transcription factor FoxO1. We identifi ed a critical mediator of fatty acid transport, FABP4, to be inversely regulated by apelin/APLNR and FoxO1, where disruption of apelin signaling leads to its increased expression, and disrupted FoxO1 leads to signifi cant decrease in its expression. Moreover, we found that tissue fatty acid uptake is signifi cantly elevated in the hearts, skeletal muscle, and adipose tissues of Apln KO mice, whereas Foxo1 ECKO mice have signifi cantly decreased fatty acid uptake in these tissues. Lastly, we found that the increased tissue fatty acid uptake in Apln KO mice can be reversed by either concurrent en-dothelial Foxo1 deletion or treatment with the FABP4 inhibitor BMS309403. Our fi ndings shed key mechanistic insights into the endothelial basis of ape-lin’s metabolic effects, a ligand mediated mechanism that determines en-dothelial regulation of fatty acid uptake and transport, and can lead to novel therapeutic strategies against diabetes and insulin resistance.

Supported By: American Diabetes Association (1-14-BS-035 to H.C.); National Heart, Lung, and Blood Institute

2081-PChronic Exposure of Islets to High Glucose Upregulates Nav1.3 Ex-pression and Glucagon Secretion In VitroMING YANG, RUTH CHU, YING GE, ARVINDER K. DHALLA, Fremont, CA

Voltage-gated Na+ channels in pancreatic α-cells play an important role in generating electrical activity for glucagon secretion. It has been shown that α-cells from STZ-diabetic mice have increased Na+ current density, ac-tion potential amplitude and fi ring frequency, suggesting that hyperglycemia may regulate Na+ channel expression and function. However, the effects of high glucose on Na+ channel expression and function in islets have not been studied. We determined the effects of chronic exposure to high glucose on Na+ channel expression and glucagon secretion in pancreatic islets.

Experiments were done in islets from SD rats or humans. Gene expression was determined by qPCR. Glucagon secretion was measured by an ELISA kit. Rat islets were exposed to 5.5, 11 or 33 mM glucose for 3 days. Nav1.3 gene expression signifi cantly increased by 71±16 and 83±22% in 11 and 33 mM vs. 5.5 mM glucose. Reverting glucose levels to 5.5 mM for 3 days led to reversal of up-regulated Nav1.3 expression. Acute treatment with 30 µM veratridine (1h), a Na+ channel activator, increased glucagon secretion in rat islets chronically exposed to 5.5 mM glucose by 54±23% compared to baseline. Veratridine-induced glucagon secretion was much greater (382±90 and 495±71% from baseline) in islets exposed to 11 and 33 mM glucose, respectively. In human islets, chronic exposure to high glucose also signifi -cantly increased Nav1.3 expression by 38±16 and 29±12% in 11 and 33 mM, respectively, vs. 5.5 mM glucose. Veratridine (30 µM) increased glucagon secretion by 211±86% from baseline in islets chronically exposed to 5.5mM glucose, whereas this increase was 401±142 and 408±102% from baseline in islets exposed to 11 and 33 mM glucose, respectively. No changes were observed in expression of other Na+ channel isoforms in both rat and human islets.

Data shows that islets chronically exposed to high glucose have increased Nav1.3 expression and function, suggesting that Nav1.3 may play an impor-tant role in progression of diabetes.

2082-PGPR40-mediated Decrease in Fasting Plasma Glucose Is Associ-ated with GLP-1 SecretionYANYUN CHEN, MIN SONG, JONATHAN RILEY, JAMES V. FICORILLI, KATH-LEEN R. JONES, JAYANA LINESWALA, DONALD JETT, RICHARD ZINK, STEVEN D. KAHL, ANJANA PATEL LEWIS, NICHOLE A. REYNOLDS, TRAVIS SHOCKLEY, CHAHRZAD MONTROSE-RAFIZADEH, HSIU-CHIUNG YANG, CHAFIQ HAM-DOUCHI, OVER CABRERA, ANNE REIFEL MILLER, Indianapolis, IN

It is known that synthetic agonists of GPR40 (FFAR1) lower post-prandial glucose (PPG) by stimulating GDIS during glucose and mixed meal challenges. Recent results from clinical and preclinical studies show that fasting plasma glucose (FPG) levels are also lowered following administration of a GPR40 agonist. However, the mechanism for this GPR40-mediated event remains unknown. Here, we provide evidence in rodent models of insulin resistance (IR) that the decrease in FPG is linked to GLP-1 secretion.

Two potent and effi cacious GPR40 agonists (A1 and A2) were adminis-tered orally to DIO mice 60 minutes prior to an oral glucose tolerance test (OGTT). Statistically signifi cant (SS) decreases in FPG were seen with both compounds at the 0 time point of the OGTT (just prior to the oral glucose bolus) along with decreases in PPG. When the more potent and effi cacious agonist (A2) was administrated orally to WT and GPR40 KO mice 60 minutes before an OGTT, decreases in FPG and PPG were seen in the WT mice but not in the GPR40 KO mice. No evidence of hypoglycemia (blood glucose ≤ 70 mg/dl) was detected at any time point during these studies. To explore the mechanism responsible for decreased FPG, A2 was administered orally to C57Bl/6 mice with blood samples collected at 15, 30, 90 and 180 minutes. GLP-1 levels were signifi cantly elevated at all time points. Additional stud-ies were performed in primary human islets perifused with 30 nM GLP-1 in 2.8 mM glucose showing that GLP-1 stimulates insulin secretion during normal glycemic conditions. An OGTT was then performed in DIO mice with a GLP-1 antagonist plus A2. Results from these studies showed that the GLP-1 antagonist completely reversed decreases in FPG and partially reversed decreases in PPG during the OGTT.

In conclusion, the GPR40 mediated decrease in FPG seen in rodent models of IR is associated with GLP-1 secretion suggesting that GPR40 agonists with signifi cant GLP-1 secretion may provide additional, safe glycemic con-trol by decreasing FPG without producing hypoglycemia.

2083-PChronic High Glucose Load Induces Dysregulation of Glucagon Se-cretion through Impaired Insulin SignalingDAN KAWAMORI, TAKASHI KATSURA, ERI AIDA, HIDEAKI KANETO, TAKAAKI MATSUOKA, IICHIRO SHIMOMURA, Suita, Japan

The pathophysiological signifi cance of glucagon is getting widely recog-nized, since its dysregulated secretion in diabetic state impacts on glycemic status. Nonetheless, underlying mechanism(s) are still obscure. Previously we identifi ed the insulin signaling in α-cells as one of the central regula-tors of glucagon secretion in vivo (Cell Metab. 2009). Given the clinical sig-nifi cance of insulin resistance, possible deterioration of insulin signaling in α-cell is anticipated to be important for dysregulated glucagon secretion. In this research, by utilizing an established glucagon secreting cell line in R1G, we found that chronic high glucose load to the cells induces dysregulation of glucagon secretion through impaired insulin signaling. Specifi cally, chronic pre-incubation under high glucose (25 mM, 12 h) induced reduced glucagon secretion by low glucose stimulation (2.5 mM, 30 min) and paradoxically el-evated secretion by high glucose (25 mM), which are similar to well known phenomenon in diabetic conditions. As underlying molecular feature, Akt phosphorylation was reduced in both basal and acute insulin-stimulated state. Further investigation revealed an involvement of upregulated JNK and reactive oxygen species in this impaired signaling. Indeed, inhibition of JNK signaling normalized the glucagon secretion suggesting a ROS-JNK-Akt axis in the induction of abnormal glucagon secretion pattern by high glucose. Interestingly, another insulin signaling component, p42/p44 ERK phosphorylation was inversely promoted suggesting a possible involvement in alteration of α-cell mass in diabetes. These data indicate that insulin sig-naling in α-cells can be impaired by chronic hyperglycemia and subsequent metabolic stresses, consequently, abnormal glucagon secretion and glyce-mic perturbations are generated. The research directs a clue to clarifying the mechanism as well as a development of new therapeutic approach to diabetes targeting glucagon.

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2084-PAbnormal Protein Phosphorylation in Plasma from Type 2 Diabetic PatientsDANJUN MA, YUE QI, ABDULLAH MALLISHO, MICHAEL ALEXANDER. CARU-SO, DIVYASRI DAMACHARLA, XIANGMIN ZHANG, REBECCA TAGETT, SORIN DRAGHICI, RODNEY O. BERRY, NISHIT SHAH, MAJED ABDULLAH. ALHARBI, BERHANE SEYOUM, ZHENGPING YI, Detroit, MI

Currently, there are 26 million type 2 diabetic patients (10% adults) and 86 million prediabetic people (37% adults) in the U.S. Therefore, there is a critical need to discover effective biomarkers to predict the risk of type 2 dia-betes (T2D) and/or to provide targets for mechanistic study of T2D. Plasma contains rich information on the overall pathophysiology of various diseases, including T2D, since the blood circulates through nearly all tissues in the hu-man body, picking up proteins (either in its native form or post-translationally modifi ed form, such as phosphorylated proteins) that are released from their origin. Protein phosphorylation plays a key role in biological processes in a tissue-specifi c or organ-specifi c manner and abnormal abundance of pro-tein phosphorylation sites have been observed in many tissues or organs in various diseases. These protein phosphorylation sites with abnormal abun-dance may be released into plasma and serve as biomarkers for a specifi c disease. However, no studies on global protein phosphorylation site profi ling in plasma from T2D patients have been reported. Here, we reported the 1st comprehensive quantitative phosphoproteomics analysis of plasma from the lean (N=9) and T2D participants (N=10). A total of 1100 unique phosphosites (in 477 proteins) were identifi ed, which is the largest catalog of phosphory-lation sites determined in human plasma so far. Pathway analysis revealed multiple signifi cantly enriched pathways for these plasm phosphoproteins, such as calcium signaling, FXR/RXR activation, FOXA transcription factor networks, etc. Out of these 1100 phosphor-sites, 117 sites (in 58 proteins) showed at least 1.5 fold changes between lean and T2D groups. Further-more, fi fty sites (in 27 proteins) out of these 117 sites showed signifi cant changes between lean and T2D groups (P<0.05). These signifi cantly different phosphorylation sites in T2D plasma may serve as potential biomarkers for T2D or may provide targets for mechanistic study of T2D pathogenesis.

Supported By: American Diabetes Association (7-13-TS-35 to Z.Y.)

2085-PEffect of GLP-1-Gastrin Dual Agonist ZP3022 on Pancreas Gene Ex-pression in ZDF RatsJOLANTA SKARBALIENE, NILS BILLESTRUP, KELD FOSGERAU, Glostrup, Denmark, Copenhagen, Denmark

GLP-1-gastrin dual agonist ZP3022 has been shown to increase β cell mass and improve glycemic control in db/db mice and Zucker Diabetic Fatty (ZDF) rats. Here we investigated alteration in pancreas gene expression fol-lowing treatment with ZP3022 in ZDF rats.

Gene expression profi ling in whole pancreas from ZDF rats was performed to characterize genes differently regulated by 3 weeks of treatment (s.c., bid) with either ZP3022 (40 nmol/kg), exendin-4 (30 nmol/kg), or a combination of exendin-4 and gastrin17 (30 + 80 nmol/kg).

Microarray analysis revealed that treatment with ZP3022 exerted spe-cifi c effects on gene expression not observed when treating with exendin-4 alone or in combination with gastrin17. The MAPK signaling pathway was observed among the highest affected pathways, while also pathways re-lated to insulin signaling and secretion were regulated by ZP3022 treatment. When searching for differences in gene expression between ZP3022 and vehicle treated rats, it was observed that rats treated with ZP3022 had a higher expression of genes encoding for the specifi c β cell/endocrine cell markers, such as islet amyloid polypeptide (IAPP) and protein convertase 1/3 and -2 (PC1/3 and PC2). Consistent with these fi ndings, an expression of a gene encoding for transmembrane protein 27 (TMEM27) which in pancreas is mainly restricted to the β cells was also elevated in ZP3022 treated rats compared to the vehicle treated rats. Taken together, our data point to either enhanced/preserved β cell mass or increased synthesis of β cell specifi c markers in rats treated with ZP3022.

We conclude that the GLP-1-gastrin dual agonist ZP3022 produces a dif-ferent gene expression response compared to exendin-4 given alone or in combination with gastrin17 and may have therapeutic potential in the pre-vention/delay of β cell dysfunction.

2086-PPharmacokinetics and Pharmacodynamics of GLP-1-GIP Receptor Dual Agonist Peptides: From Once-Daily to Once-WeeklyMARIA A. DERYABINA, JENS ROSENGREN DAUGAARD, CARSTEN BOYE KNUD-SEN, PERNILLE TOFTENG SHELTON, JACOB ULRIK FOG, LENE JESSEN, PIA NOER-REGAARD, Glostrup, Denmark

Analogs of the incretin hormone GLP-1 are being used for the management of type 2 diabetes and are often accompanied by modest weight loss. Evi-dence from animal studies suggests that anti-obesity effi cacy of GLP-1 can be enhanced by co-administration with the incretin hormone GIP. We have designed and characterized novel balanced GLP-1-GIP receptor dual incretin (DI) agonists. The compounds are full dual agonists with EC50 values (cAMP assay with HEK293 cells) of 4-35 pM and 9-31 pM, for the human GIP and GLP-1 receptors respectively. Using different fatty acid chains in conjugation of peptides it was possible to vary the terminal half-lives of the compounds in mice from 2 to 20 hours (SC administration).

The in vivo pharmacodynamic effects of DIs were investigated in mice. DIs with both short and long half-lives signifi cantly reduced body weights of DIO mice during 3 weeks of study. While a short-acting DI was dosed SC twice-daily (5 nmol/kg), signifi cant body weight lowering effects were demonstrated for long-acting DIs with SC administration only every third day (3 nmol/kg) of the study. DIs with an extended half-life were further characterized for acute effects on glucose control. When dosed SC 22 h prior to a glucose challenge, DIs (0.5 and 5 nmol/kg) signifi cantly reduced blood glucose levels after an IP bolus of glucose in lean mice.

In conclusion, these pharmacokinetic and pharmacodynamic results dem-onstrate the possibility to prolong the activity of GLP1-GIP DIs. The data show promise for the future development of a possible once-weekly GLP-1-GIP ago-nist drug candidate for the treatment of type 2 diabetes and obesity.

2087-PIncreased Capacity for Intestinal Serotonin Release in Obese and Diabetic HumansRICHARD L. YOUNG, CHRISTOPHER K. RAYNER, NAM Q. NGUYEN, TONGZHI WU, JENNA E. BURGESS, AMANDA L. LUMSDEN, RAVINARAYAN RAGHUPATHI, NEKTARIA PEZOS, DAMIEN J. KEATING, Adelaide, Australia

Peripheral serotonin (5-HT), derived largely from intestinal enterochroma-ffi n (EC) cells, is an important regulator of hepatic gluconeogenesis [1] and adipose tissue lipolysis and thermogenesis [2]. Increased peripheral 5-HT is linked to poorer glycemic control in patients with type 2 diabetes (T2D), while polymorphisms in tryptophan hydroxylase (Tph1, which synthesises 5-HT) link with human obesity [3,4]. We assessed glucose-stimulated 5-HT release from the colon and primary EC cells in mice, and duodenal Tph1 ex-pression in healthy lean, obese, bariatric (RYGB) and T2D subjects. Plasma 5 HT concentrations after enteral glucose were also assessed in healthy lean and T2D subjects at euglycemia and hyperglycemia. Glucose above 100 mM triggered 5-HT release from colonic tissue in mice, while amper-ometry showed augmented release of 5-HT from individual vesicles. Tph1 transcript was higher in obese (1.3-fold, P=0.11) and T2D subjects (1.5-fold, P=0.04) and identical in RYGB patients and lean subjects. Levels correlated with age in all subjects (P=0.009), and with BMI (P=0.04) in the lean and obese. Tph1 transcript levels were unaffected by glycemic status in lean and T2D subjects, but tended to increase after luminal glucose only in T2D (euglycemia 1.7-fold, P=0.09). Plasma 5 HT correlated positively with BMI in healthy lean subjects at baseline (P=0.02), and increased substantially more after intraduodenal glucose in T2D than health (AUC 3-fold higher, P=0.04). In conclusion (i) peripheral 5 HT release is triggered by enteral glucose, via increased release from single vesicles, (ii) obese and T2D subjects have in-creased capacity for gut-5 HT production and release, while (iii) gut-5-HT capacity may be lowered following RYGB. These fi ndings support a key role of 5-HT in metabolic dysregulation in obesity and T2D.

1. Sumara G et al Cell Metab 2012 16:588-600.2. Crane JD et al Nat Med 2014 Dec 8 (Epub).3. Takahashi T et al Diabetes Res Clin Pract 2002 58:123-129.4. Kwak SH et al Obesity 2012 20:233-238.

2088-PIleal Transposition Increases L-Cell Secretion and Decreases Plas-ma Lipopolysaccharide Levels in RatsTAE JUNG OH, SE HEE MIN, CHANG HO AHN, EUN KY KIM, JIE EUN LEE, KYONG SOO PARK, YOUNG MIN CHO, Seoul, Republic of Korea

Ileal transposition (IT) is an experimental surgery to examine the effect of expedited delivery of unabsorbed nutrients to the distal small intestine. We examined the effect of IT on plasma lipopolysaccharide (LPS) levels, which is

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known to contribute to the pathogenesis of metabolic abnormalities associ-ated with obesity. Sprague-Dawley rats were underwent either IT (n = 9) or sham operation (n = 10). After 4 weeks, oral glucose tolerance tests (OGTT) were performed, and fasting plasma LPS and gut histology were analyzed. Food intake and body weight decreased in the IT group, but daily glucose levels were not different between groups. Fasting insulin levels and HOMA-IR were signifi cantly lower in the IT group. During OGTT, incremental area under the curve (AUC) of insulin and total AUCs of glucagon, total GLP-1, and peptide YY (PYY) were signifi cantly higher in the IT group. Gut histology showed that villi length and muscle thickness increased in the transposed ileum (IT) compared with the ileum in situ (sham). In the immunofl uorescence assay, densities of GLP-1-positive or GIP-positive cells were not different between groups but the density of GLP-1- and GIP-co-expressing cells, which are known as K/L-cells, was signifi cantly higher in the IT group (P = 0.021). Fasting plasma LPS levels were 5.6 ± 0.2 EU/mL in the IT group and 6.8 ± 0.1 EU/mL in the sham group (P = 0.002), and they were signifi cantly corre-lated with HOMA-IR (r = 0.755, P < 0.001). Interestingly, there was negative correlation between fasting plasma LPS and total AUC of PYY (r = -0.710, P = 0.001). In conclusion, this study recapitulates that IT surgery improves insulin sensitivity and enhances the secretion of distal gut hormones such as GLP-1 and PYY. In addition, plasma LPS levels were reduced after IT surgery even in euglycemic rats which might be related with the improvement of insulin sensitivity and the increase of PYY secretion.

2089-PSodium-Glucose Transporter 1 and 2 Are Involved in the GLP-1 Re-lease from Pancreatic Alpha CellsVERONICA SANCHO BORNEZ, ROBERTO LUPI, SABRINA PAPARO, ANGELA DAR-DANO, GIUSEPPE PENNO, STEFANO DEL PRATO, Pisa, Italy

It has been recently reported that human alpha cells express the sodium-glucose co-transporters and that the diabetic condition is associated with reduced expression of SGLT2 and compensatory increased expression of SGLT1 and glucagon genes. Moreover, SGLT2 silencing or inhibition by the SGLT-2 inhibitor dapaglifl ozin was associated with increased expression of the glucagon gene. We have tested whether this system also is involved in the recently reported regulation of GLP-1 secretion by the alpha cell. More-over, since glucagon/GLP-1 secretion by the alpha cell has been claimed to be under the control of the TCF7L2 signaling pathway, we have also evalu-ated its interaction of the two systems in the alpha cell.

We have studied TC1/6 alpha cells from a mice pancreatic cell line in the presence of low (LG, 5.5 mM) or high (HG, 16.7 mM) glucose concentrations. Experiments were repeated with and without phloridzin (50 µM), a non-se-lective SGLT inhibitor. mRNA and protein expressions of SGLT1, SGLT2 and TF7L2 were determined by RT-PCR and Western blot. Total GLP-1 secretion in culture medium was measured by ELISA.

At LG both mRNA and protein expressions of SGLT1 and 2, and TCF7L2 were all detectable. HG incubation was associated with an increase of both mRNA and protein expression of TCF7L2 (protein: +46±10%, p<0.001; mRNA 2.8±0.5 folds, p<0.001). Concomitantly, SGLT1 mRNA expression increased (2.3±0.5 folds, p<0.001) while SGLT2 decreased (0.48±0.09, p<0.001). Chang-es in mRNA expressions were associated with -34±11% (p<0.01) reduction in SGLT1 protein expression with no signifi cant changes for the SGLT2 protein. GLP-1 concentration in the medium increased by +25±3% (p<0.001) as com-pared to LG. SGLT1/2 inhibition by phloridzin did not affect GLP-1 release at LG (-10±6%, p=NS), while it was associated with a -30±3% reduction (p<0.001) as compared to LG.

These data suggest that GLP-1 release from alpha cells in response to high glucose is mediated by SGLT expression independently of TCF7L2 activation.

2090-PChronic GLP-1 Exposure Further Decreases Leptin Levels in Vago-tomized RatsTIAGO MORAIS, SOFIA S. PEREIRA, SARA ANDRADE, ÂNGELA MOREIRA, DU-ARTE MONTEIRO, MADALENA COSTA, BÁRBARA PATRÍCIO, MARCOS C. CAR-REIRA, FILIPE CASANUEVA, MARIANA P. MONTEIRO, Porto, Portugal, Santiago de Compostela, Spain

Long acting GLP-1 analogues are widely used in clinical setting for type 2 diabetes treatment. Autonomic diabetic neuropathy can compromise the functional integrity of the vagus nerve and potentially impair the action of the gut/brain axis hormones.

The aim of this study was to evaluate the role of the vagal pathways in mediating GLP-1 chronic effects.

Male Wistar rats submitted to truncal sub-diaphragmatic vagotomy (VGX) or sham procedure (SHAM) plus pyloroplasty, were randomized to receive

GLP-1 (3.5 pM/min/Kg) or saline intraperitoneally trough osmotic mini-pumps for 28 days (n=5/ group), and an additional group of SHAM rats (n=6) was pair fed to the VGX-Saline (PF). Food intake and body weight were monitored daily, energy expenditure measured by indirect calorimetry, while epidy-dimal white adipose tissue (WAT) and interscapular brown adipose tissue (BAT) were weighed and fasting levels of glucose, insulin, GLP-1, leptin and ghrelin were measured by ELISA at the end of the experiment.

GLP-1 levels were signifi cantly higher in SHAM-GLP1 and VGX-GLP1 rats. VGX and PF rats had signifi cantly lower food intake, body weight gain, per-centage of WAT and BAT when compared to SHAM rats, but presented no difference in energy expenditure. Glucose levels were similar in all groups, but insulin and HOMA-IR were lower in VGX and PF, while PF rats also had a signifi cantly higher levels ghrelin compared to Sham and VGX. No changes were observed in any of these parameters in result of GLP-1 exposure. Leptin levels were also signifi cantly lower in VGX and PF when compared to SHAM, however GLP1 exposure was responsible for an additional decrease leptin levels compared to saline in VGX rats.

The vagus nerve seems to participate in the regulation of leptin levels in response to chronic GLP-1. Our data suggests that the implications of auto-nomic neuropathy both in the central and peripheral effects of GLP-1 based therapies with ought to be further characterized to access the role of these drugs in this particular subset of patients.

Supported By: PTDC/SAU-NMC/115700/2009; Foundation for Science and Tech-nology (Fcomp-01-0124-FEDER-015893)

2091-PThe Signifi cance of DNA Methylation in Leptin Gene ExpressionKASUMI NAKAGAWA, MASASHI KURODA, MARI KONDO, NAGAKATSU HARA-DA, HIROSHI SAKAUE, Tokushima, Japan

3T3-L1 adipocytes are the most commonly used cells in experiment, but leptin expression level of 3T3-L1 adipocytes is remarkably low as compared with that of mouse white adipose tissue (WAT). The purpose of this study is to investigate whether leptin expression in 3T3-L1 adipocytes is associated with the regulation of DNA methylation. With pyrosequencing, we found CpG islands in leptin promoter region were highly methylated in 3T3-L1 adi-pocytes compared to WAT. To examine the effect of DNA demethylation on leptin expression, we exposed 3T3-L1 cells to 5-azacytidine (5-aza-C), DNA methyltransferase inhibitor. Although 5-aza-C-treated 3T3-L1 preadipocytes (3T3-L1AZ preadipocytes) have low methylation level of leptin CpG islands, they did not change leptin expression. However, after adipocyte differentia-tion, 3T3-L1AZ adipocytes showed prominently increased leptin mRNA with lowered methylation in CpG islands of leptin. We next performed knock-down of DNA methyltransferase 1 (DNMT1) using siRNA transfection to 3T3-L1 preadipocytes. The knockdown of DNMT1 lowered methylation level of CpGs in leptin promoter region and induced its expression after adipocyte differentiation. Finally, we investigated the involvement of DNA methyla-tion status in the increase of leptin mRNA in hypertrophic adipocytes. In vitro, 3T3-L1 adipocytes hypertrophied by long-term culture increased leptin mRNA level even in 5-aza-C-untreated 3T3-L1 cells. However, this culture condition did not affect DNA methylation status of leptin CpG sites. In vivo, methylation analysis of adipocytes from high fat-fed obese mouse showed slightly higher DNA methylation in leptin promoter than that in normal chow-fed mouse. In conclusion, DNA demethylation in preadipocytes induces lep-tin mRNA expression after adipocyte differentiation, but the mechanism by which leptin mRNA is increased in hypertrophic adipocytes is still unknown. The other modifi cations of the leptin gene promoter with the development of obesity may need further investigation.

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2093-PExendin-4, a Glucagon-Like Peptide-1 Receptor Agonist, Inhibits Breast Cancer GrowthCHIKAYO OTA, TAKASHI NOMIYAMA, SHIHO KOMATSU, TAKAKO KAWANAMI, YURIKO HAMAGUCHI, TOMOKO TANAKA, TOSHIHIKO YANASE, Fukuoka, Japan

Incretin therapy has emerged as one of the most popular treatment for type 2 diabetes. Exendin-4 (Ex-4), has received much attention, because of its’ tissue protective effects beyond glycemic control. We have previously reported that Ex-4 attenuates atheroma formation in apoE−/− mice (Araka-wa M, Diabetes 2010) and also reduces intimal thickening after vascular injury (Goto H, BBRC 2011). On the other hand, the most important cause of death of type 2 diabetes in Japan is cancer. In our previous report, we have investigated that GLP-1R is abundantly expressed in human prostate cancer and Ex-4 attenuates prostate cancer growth both in vitro and in vivo ex-periments (Nomiyama T, Diabetes 2014). On the other hand, breast cancer is one of popular cancers in female patients with type 2 diabetes and obesity, especially in western countries. Then, we next examined whether Ex-4 could attenuate breast cancer in the present study.

First, we observed abundant GLP-1R expression in 3 kinds of human breast cancer cell lines, such as MCF-7 cell, MDA-MB-231 cell and KPL-1 cell in both mRNA level and protein level. 0.1~10nM Ex-4 signifi cantly decreased breast cell number of breast cancer cells, in dose-dependent manner (p<0.01). Although Ex-4 did not induce apoptosis in breast cancer cells, BrdU assay revealed that Ex-4 attenuates cell proliferation of breast cancer cells dose-dependently (p<0.01). Further, we examined anti-breast cancer effect of Ex-4 in vivo. If we transplanted MCF-7 cells into non-diabetic nude mice subcu-taneously and treated them with Ex-4 for 6 weeks, we observed decreased tumor size of MCF-7 and serum CA15-3 level, a marker of breast cancer, in Ex-4-treated mice, but not statistical signifi cant. However, if we performed immunohistochemistry with Ki67, a marker of cell proliferation, Ki67 positive cells were signifi cantly reduced in tumor extracted mice treated with Ex-4 (p<0.01).

These data suggest that Ex-4 could attenuate breast cancer growth via inhibition of breast cancer cell proliferation.

2094-PCircadian-Timed Daily Activation of Supramammillary Nucleus (SuMN) Ameliorates Obesity/Insulin Resistance in High-Fat Diet-induced Insulin Resistant RatsYAHONG ZHANG, SHUQIN LUO, MICHAEL EZROKHI, YANG LI, YELENA TRUBIT-SYNA, ANTHONY H. CINCOTTA, Tiverton, RI

The SuMN sends strong projections to several metabolic control centers in the brain including the lateral septum, ventral medial hypothalamus and the biological clock, suprachiasmatic nuclei (SCN) where circadian dop-amine (DA) input activity from the SuMN contributes to the maintenance of insulin sensitivity in rodents. High fat diet (HFD) feeding that induces insulin resistance (IR) abolishes the circadian peak in both SuMN and peri-SCN dopamine neuronal activity. However, it is not known whether activa-tion of SuMN neurons is capable of reversing the obese-insulin resistant state of animals while maintained on a HFD and that was thus the focus of this study. Neuronal activation of the SuMN of HFD sensitive rats fol-lowing 3 months on a HFD (60% saturated fat by % kcal) (BW: 336±4.3 g) was accomplished through infusion of NMDA (0.5 mM/0.5 ul) and its coago-nist d-serine (1 mM/0.5 ul) into the SuMN daily at the onset of locomotor activity (corresponding to the circadian peak of DA activity at the SuMN and SCN area in insulin sensitive animals) for 2 weeks while maintained on such HFD. Relative to vehicle infused controls, such SuMN NMDA treat-ment reduced IR (as assessed by glucose tolerance test, 78% increase in Belfi ore ISI, p=0.038), retroperitoneal fat (27%, p<0.01), total abdominal fat (27%, p=0.027), and liver triglyceride content (27%, P=0.04) without changing body weight or HFD food consumption. Intra-SuMN NMDA sig-nifi cantly increased plasma adiponectin (219%, p=0.002) and reduced plasma leptin (62%, p=0.002) levels. In summary, circadian-timed daily activation of the SuMN ameliorates obesity, fatty liver and insulin resis-tance in animals held on a HFD without altering food consumption (of a HFD). Available evidence suggests that such a response may be in part due to circadian activation of dopaminergic input to the master clock SCN that regulates the neuroendocrine axis modulating peripheral fuel metabolism.

2095-PGlycated Hemoglobin Is More Important in Predicting Bone Mineral Density than Bone Related Parameters in Healthy Premenopausal WomenKALLIOPI KOTSA, ANASTASIA SACHINIDOU, PANAGIOTIS TSAKLIS, IOANNIS CHRISOGONIDIS, ANNA GOTZAMANI-PSARRAKOU, KIRIAKOS KAZAKOS, Thes-saloniki, Greece

Premenopausal Bone Mineral Density (BMD) is determined by genetic and other hormonal or skeletal factors related to bone turnover. The aim of this study was to investigate the effect of integrated glycemia as determined by glycated heamoglobin (HbA1c) on BMD and combine this effect with estab-lished skeletal determinants in a multiple regression model aiming to predict BMD in healthy premenopausal women.

Patients and Methods: Forty-eight healthy premenopausal women (age 41 +/- 7 years) (BMI 24 +/- 4 kg/m2) were included in the study. Women with a family history of osteoporosis, menstrual disorders, diabetes or metabolic syndrome and glucocorticoid treatment for >1 month in the past were excluded from the study. Anthropometric measurements, diet and ex-ercise questionnaires and plasma/serum measurements for glucose, insulin, HbA1c, parathyroid hormone (PTH), vitamin D (25(OH)D), Calcium and osteo-calcin were performed. The BMD of the lumbar spine and femoral neck was measured by dual energy X-ray absorptiometry. Multiple linear regression was used to assess best determinants of BMD.

Results: There was a statistically signifi cant positive correlation of HbA1c with femoral neck BMD (r=0,343, p<0,05) while no correlation was found for 25(OH)D (r=-0,016, p=0,915), PTH (r=-0,057, p=0,708) and osteocalcin (r=-0,108, p=0,506). When lumbar spine BMD was examined no correlation was found for HbA1c, 25(OH)D, PTH or osteocalcin. Moreover no correlation was found with blood lipid parameters or uric acid for either site of DEXA measurement.

Conclusions: In a multiple regression model the use of 4 independent vari-ables (HbA1c, PTH, 25(OH)D and osteocalcin) cannot predict the variability in femoral neck BMD of healthy premenopausal women. The only statistically signifi cant correlation is with HbA1c and increasing HbA1c by 1% predicts an increase in femoral neck BMD by 0,185 g/cm2.

2096-PLongitudinal Changes in Multiple Adipokines Are Associated with Increasing Adiposity, Worsening Insulin Resistance, and Deterio-rating β-Cell FunctionMARY HELEN BLACK, JUN WU, CORINNA KOEBNICK, ENRIQUE TRIGO, RICHARD M. WATANABE, THOMAS A. BUCHANAN, ANNY H. XIANG, Pasadena, CA, Los Angeles, CA

Higher pro-infl ammatory and lower anti-infl ammatory adipokine levels have been shown to be associated with obesity, insulin resistance and type 2 diabetes (T2D). While cross-sectional relationships have been reported, longitudinal data on changes in adipokines in relation to changes in insulin sensitivity and β-cell function are scant. We examined these relationships in a subset of 324 BetaGene participants with baseline and follow-up measures (mean age: 34.6±8.2 yrs; 73% female; mean follow-up: 4.6±1.5 yrs). Body fat was assessed by DXA; insulin sensitivity (SI) and β-cell function (DI) were estimated from FSIGTs with Minimal Model analysis. Adiponectin, IL-1β, IL-6, IL-18, leptin, lipocalin, MCP-1, resistin and TNF-α were assayed using Mil-lipore multiplex kits with magnetic bead panels; CRP was measured with ELISA. Generalized estimating equations were used to model associations between rates of change in adipokines and rates of change in adiposity, SI and DI, adjusted for age, sex and baseline body fat. Longitudinal decreases in adiponectin and increases in CRP, IL-6, IL-18, leptin, MCP-1 and resistin were associated with increasing body fat over time (all p<0.05). Consistent with these observations, decreasing adiponectin and increasing CRP, IL-6 and leptin were each associated with declining SI (all p<0.01). Increasing CRP, IL-6, IL-18 and leptin were also associated with decreasing DI (all p<0.05). Further adjustment for rate of change in SI explained 72%, 75%, 29% and 100% of each of the respective associations; after adjustment, decreas-ing DI remained marginally associated with increasing IL-18 (p=0.09). Thus, obesity-induced changes in adipokine levels are associated with worsening insulin resistance and deteriorating β-cell function. Longitudinal effects of adipokine dysregulation on β-cell compensation for insulin resistance are mediated largely through changes in insulin sensitivity over time.

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2097-PGIP Jejunal Expression and Circulating Levels Do Not Explain the Blunted Incretin Effect in Individuals with Type 2 DiabetesBLANDINE B. LAFERRERE, ROXANNE DUTIA, STREAMSON CHUA, PETROS BE-NIAS, DANIEL BORON-BRENNER, SARAH STANO, UZONNA MKPARU, JAMES MCGINTY, New York, NY, Bronx, NY

Glucose-dependent insulinotropic polypeptide (GIP), secreted from intes-tinal K-cells in response to nutrient ingestion, potentiates glucose-induced insulin secretion. The incretin effect, mediated in part by GIP, is impaired in type 2 diabetes (T2D). The goal of this study was to investigate differ-ences in expression, levels and function of GIP between subjects with and without T2D. Expression of sodium-glucose linked transporter 1 (SGLT-1) was also studied, as glucose-induced GIP secretion depends primarily on SGLT-1. Seventy fi ve obese individuals, with and without T2D were studied. In study 1, jejunal biopsies were obtained at the time of gastric bypass surgery in 21 subjects (10 with T2D) for GIP and SGLT-1 expression. In study 2, GIP levels and incretin effect on C-peptide were measured in response to a glucose challenge in 54 subjects (37 with T2D). Total GIP was measured by ELISA, C-peptide by RIA, and the incretin effect by comparing the C-peptide response to an oral glucose challenge versus an isoglycemic IV glucose clamp. Total RNA was isolated, followed by RT- qPCR for targeted gene analysis. BMI (mean 44±6 kg/m2) was not different between study 1 and study 2 (p=0.109) or between subjects with (n=47) and without (n=28) T2D (p=0.385). T2D control was worse in study 1 (8.2±1.7% vs. 6.7±0.9%, p=0.02). T2D status had no effect on GIP (1.0±5.0 vs. 3.4±6.7 arbitrary units (AU), p= 0.374) or SGLT 1 (1.0±0.6 vs. 1.2±0.5 AU, p=0.417) expression. Although GIP plasma concentrations either fasted (39.2±21.1 vs. 41.4±28.3 pg/ml, p=0.759) or af-ter oral glucose (AUC 103.6±31.3 vs. 109.2±44.0 pg/ml/min, p=0.594; peak 187.3±61.7 vs. 170.5±64.3 pg/ml, p=0.363) were not different in subjects with and without T2D, the incretin effect was blunted in subjects with T2D (9.8±28.6 vs. 28.6±10.7% p=0.014). In summary, Levels of expression of GIP in the jejunum and circulating levels of GIP do not differ according to T2D status, and are unlikely to explain the blunted incretin effect in T2D.

Supported By: American Diabetes Association (1-09-CR-34 to B.B.L.); R01DK067561

2098-PAcute Free Fatty Acid-induced Insulin Resistance Alters Plasma Amino Acid LevelsLISA CHOW, TYLER BOSCH, ANNE BANTLE, XUAN-MAI PERSSON, DOUG MA-SHEK, K. SREEKUMARAN NAIR, Minneapolis, MN, Rochester, MN

Chronic insulin resistance is associated with altered plasma amino acid (AA) levels. We determined whether acute free fatty acid (FFA) induced in-sulin resistance from lipid infusion may alter plasma AAs in trained (T) and sedentary (S) subjects.

14 T (self-report>45 min running 5d/wk) and 14 S (self-report<30min exer-cise/wk) participants were matched for age [22.3 + 0.4y (SE)], sex and BMI [22+ 0.5 kg/m2 (SE)] In the setting of a hyperinsulinemic-euglycemic clamp, each subject received either a 6 hr lipid (20% Intralipid: 90ml/hr) or glycerol (2.25 g/100 ml at 90 ml/hr) infusion. Nephramine (5.4%) was co-administered (0.6 µmolar of leucine per kg of FFM/min) to blunt the expected AA decline with insulin infusion. LC-Tandem mass spectrometry was used to measure plasma AAs and their metabolites (38 measured/subject) at baseline (0 hr) and end-infusion (6 hr). For statistics, we used a mixed model analysis.

At baseline, higher VO2max (49 vs. 39 ml/kg/min:p=0.0004), insulin sensi-tivity (12.2 vs. 8.8 mg glucose infused/kg FFM/min: p=0.005), fat free mass (53 vs. 43 kg:p=0.02) and aspartic acid (15.7 vs. 11.3 µmol/L:p=0.05) levels were observed in T than S participants. Compared with glycerol infusion, lip-id infusion increased FFA (peak level 616-694 µmol/L) and insulin sensitivity declined [-48%(T) vs. -47%(S)] with no training effect. Compared with glyc-erol infusion, lipid infusion, independent of training, was associated with higher levels of isoleucine, 3-methylhistidine, alpha-amino-N-butyric acid, citrulline, ethanolamine, glutamic acid, hydroxylysine, leucine, ornithine, phenylalanine, serine and taurine and lower levels of asparagine, cystine, sarcosine, and tryptophan (all p<0.05). We conclude that acute modest FFA elevation from lipid infusion not only alters insulin sensitivity, but also alters AA metabolism and that these effects are independent of training status.

Supported By: U24DK100469, UL1TR000135

2099-PImpaired Glucagon Secretion Contributes to Hypoglycemia Pheno-type in Hyperinsulinism Mouse ModelsPAN CHEN, MING LI, DIVA D. DE LEON, CHARLES A. STANLEY, CHANGHONG LI, Philadelphia, PA, Beijing, China

Impaired glucagon secretion in children with hyperinsulinism (HI) due to inactivating mutations in KATP channels may contribute to the severity of hypoglycemia, but it is not known if impaired glucagon secretion also occurs in other types of HI. We measured glucagon secretion (HTRF assay) in vivo in 3 HI mouse models and wild type (WT) mice, including β-cell specifi c glu-tamate dehydrogenase gain of function mutation transgenic (H454Y-GDH), short-chain 3-hydroxyacyl-CoA dehydrogenase knockout (SCHAD-/-), and KATP channel knockout (SUR1-/-).

After 16 hours of fasting, plasma glucose was lower in H454Y-GDH mice compared to WT (37±2 mg/dL vs. 56±3 mg/dL, p<0.01), due to increased insu-lin/glucose ratio (14±2 vs. 8±1, p<0.05) and inappropriate glucagon response to hypoglycemia (135±14 vs. 227±36 pg/ml, p<0.05). After 3 hours re-feeding in WT mice, glucose and insulin levels increased, and glucagon/glucose ratio decreased by 60%. In contrast, H454Y-GDH mice showed persistently low glucose levels after re-feeding (58±9 mg/dL vs. 121±8 mg/dL, p<0.01) with 3-fold higher insulin/glucose ratio, and unchanged glucagon/glucose ratio. SCHAD-/- mice had a milder hypoglycemia phenotype compared to H454Y-GDH mice, partly due to higher glucagon secretion in response to fasting (glucagon/glucose ratio: 7±1 vs. 4±1, p<0.05). After re-feeding, glucose in SCHAD-/- mice increased to levels similar to WT controls with normal sup-pression of glucagon secretion. SUR1-/- mice showed impaired glucagon secretion in response to fasting but normal suppression of glucagon after re-feeding.

Conclusion: Abnormal glucagon secretion may contribute to the severity of hypoglycemia in multiple genetic forms of HI. Although GDH gain of function is responsible for the hyperinsulinism in both the SCHAD-/- and H454Y-GDH mice, the different glucagon responses in these two HI models may refl ect a gain of function of GDH in α-cells as well as β-cells in SCHAD-/- mice.


2100-PChanges in Carbohydrate Metabolism Before and After H. pylori EradicationISABEL Mª CORNEJO-PAREJA, M. MAR ROCA-RODRIGUEZ, LAURA COÍN-ARANGÜEZ, ANA M GÓMEZ-PÉREZ, ARACELI MUÑOZ-GARACH, MARÍA MOLI-NA-VEGA, JUAN ALCAIDE-TORRES, CARLOS CLU-FERNÁNDEZ, LAURA VIÑUE-LA-GONZÁLEZ, LAURA MORA-NAVAS, ISABEL MANCHA-DOBLAS, FRANCISCO TINAHONES-MADUEÑO, Málaga, Spain

Introduction: Controversial relationship between H. pylori infection and components of the metabolic syndrome, including type 2 diabetes and its complications, have been described.

Objective: To evaluate changes in carbohydrate metabolism induced by a 75 g oral glucose tolerance test (75g-OGTT) before and after eradication of H. pylori by standard triple therapy.

Methods: We conducted a prospective study of 30 nondiabetic subjects colonized by H. pylori. Clinical data and levels of ghrelin and GLP1 were analyzed at baseline and post-OGTT before and after antibiotic eradication treatment.

Results: 80% were women. Average age was 48.7 ± 11.7 years and 46.7% had personal history of gastrointestinal disease. After eradication therapy, no signifi cant lower fasting glucose, total cholesterol and LDL cholesterol levels, higher insulin levels and better pancreatic reserve were observed. HbA1c and 120 Post OGTT glucose signifi cantly decreased (p = 0.009 and p = 0.026, respectively). Ghrelin and GLP-1 were analyzed in 11 patients. No sig-nifi cant differences in levels of ghrelin and GLP-1 pre and post-treatment were found. Signifi cant correlations between ghrelin with glucose and insu-lin pretreatment (positive) and after treatment (negative) were observed. In addition, negative correlations between glucose and GLP1 before and after treatment were found. 30% of patients required treatment with ranitidine, 80% completed correctly the treatment and 86,7% achieved the eradication of H. pylori.

Conclusions: 1) Signifi cant improvement in carbohydrate metabolism was observed after H. pylori eradication. 2) Signifi cant correlations between plasma glucose and insulin with ghrelin and GLP-1 were found. 3) More than 95% of patients achieve H.pylori eradication.

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Guided Audio Tour: Endoplasmic Reticulum Stress, Infl ammation, Mi-tochondrial Function, and the Pathogenesis of Obesity (Posters: 2101-P to 2108-P), see page 17.

& 2101-PLipogenesis and ER Stress Are Mediated in Part by USF1 Effects on Transcriptional Regulation of ILDR2KAZUHISA WATANABE, ELIZABETH J. MILLINGS, STUART G. FISCHER, CHARLES A. LEDUC, RUDOLPH L. LEIBEL, Shimotsuke, Japan, New York, NY

Immunoglobulin-like domain containing receptor 2 (Ildr2), encodes an endoplasmic reticulum (ER) single pass, trans-membrane protein that medi-ates aspects of hepatic lipid homeostasis and ER stress. Livers of B6 mice in which Ildr2 expression is reduced by adenovirus-mediated Ildr2 shRNA, be-come steatotic, whereas overexpression of Ildr2 mitigates hepatic steatosis in ob/ob mice. We investigated transcriptional control mechanisms of Ildr2 in an effort to identify the molecular predicates for the role of ILDR2 in these processes. We identifi ed a conserved 22 bp element with ERSE (ER Stress Response Element)-like features in the Ildr2 promoter. Within each ERSE constituent 22-mer there is a 7 bp sub-sequence - TCACGTG - containing overlapping core binding sites for the transcription factor USF1 (which infl u-ences lipid homeostasis), and for the ER stress transducers: ATF6 and XBP1. In HepG2 cells, expression levels of USF1 and ILDR2 increased in response to ambient glucose, and were also coordinately increased in livers of ob/ob mice or B6 mice fed a high-fat diet. In vivo adenovirus-mediated knockdown of Usf1 in B6 mice, reduced Ildr2 expression and tunicamycin administration decreased hepatic Ildr2 expression by 45% and was associated with hepatic steatosis. Transcription from the ILDR2 promoter is stimulated in cells over-expressing USF1 and suppressed in cells overexpressing ATF6 or XBP1. We propose that ER stress increases nuclear ATF6 and XBP1 that out-compete USF1 for occupancy of the Ildr2 promoter, thereby reducing Ildr2 expression and increasing hepatic lipid levels. ILDR2 may be a molecular mediator for the effects of ER stress on hepatic lipogenesis and steatosis.

& 2102-PUnrefi ned Grain-derived Gamma-oryzanol Improves β-Cell Dys-function via Suppression of Dopamine Receptor Signaling and En-doplasmic Reticulum Stress in MiceCHISAYO KOZUKA, MORITAKE HIGA, HIDEAKI TANAKA, CHITOSHI TAKAYAMA, MASAYUKI MATSUSHITA, SEIICHI OYADOMARI, MICHIO SHIMABUKURO, HI-ROAKI MASUZAKI, Okinawa, Japan, Tokushima, Japan

Our clinical and experimental studies study showed that γ-oryzanol, a major bioactive component of brown rice, prevents obesity and type 2 dia-betes. However, the underlying mechanism still remains unclear. In the pres-ent study, we explored the metabolically-benefi cal impact of γ-oryzanol on islet dysfunction. In murine isolated islets, γ-oryzanol activated the cAMP/cAMP-dependent protein kinase (PKA) pathway, thereby enhancing glucose-stimulated insulin secretion (GSIS) (insulin secretion, 25 mM glucose; 2.2-fold increase). Furthermore, exaggerated secretion of glucagon was remark-ably suppressed. Of note, in α-cell line, α-TC, γ-oryzanol had no effect on glucagon secretion, suggesting that γ-oryzanol acts on β-cells. Literature demonstrates that local dopamine D2 receptor (D2R) signaling prevents in-sulin secretion in islets and D2R is localized to β-cells. We therefore tested the possible involvement of D2R signaling in effects of γ-oryzanol on β-cell function. In islets from HFD-fed obese mice, D2R signaling was exaggerated in islets from mice fed HFD (e.g. Drd2 mRNA level; 2.0-fold increase), which reciprocally decreased by γ-oryzanol. Furthermore, γ-oryzanol-induced en-hancement of GSIS was completely suppressed by quinpirole, a potent D2R agonist. On the other hand, endoplasmic reticulum (ER) stress in β-cells ag-gravates GSIS, leading to apoptosis. We found that γ-oryzanol reduced ER stress in islets from HFD-fed mice (e.g. Chop mRNA level; 37% reduction). Also in tunicamycin(ER stress inducer)-treated MIN6 cells, γ-oryzanol im-proved GSIS and rescued from apoptosis. Taken together, γ-oryzanol aug-ments GSIS via the attenuation of local D2R signaling and protects β-cells against ER stress-induced apoptosis. Our data provide rationale of γ-oryzanol as a potential anti-diabetic agent.

Supported By: Japan Ministry of Education; Japan Society for the Promotion of Science

& 2103-P11β-HSD1 Inhibition Prevents Hypothalamic ER Stress Induced Ap-petite via Upregulating Akt SignalingSEONG-SU MOON, MINCHUL SEO, YOUNG-SIL LEE, SANG SOO KIM, Gyeongju, Republic of Korea, Busan, Republic of Korea

Hypothalamus has been appreciated to be the headmaster to regulate energy balance by coordinating peripheral homeostatic activities including appetite control. Hypothalamic endoplasmic reticulum (ER) stress is known to be increased in obesity. Pharmacologic or genetic induction of ER stress in the hypothalamus caused central leptin and insulin resistance, resulting in food intake increase and obesity, whereas reduction of ER stress signifi -cantly alleviated these metabolic derangements. 11β-HSD1 inhibition has been suggested as an emerging target for the treatment of diabetes and obesity. In this study, we investigated the effect of 11β-HSD1 inhibition on hypothalamic ER stress induced appetite.

Tunicamycin and palmitate were used for ER stress inducer. Primary rat embronal hypothalamic neurons were cultured for in vitro study. Injection of Cabenoxolone (CBX) and a specifi c 11β-HSD1 inhibitor (67500) into third ventricle of mice was performed using the stereotaxic apparatus. Western blotting, immunocytochemistry, and immumohistochemistry were used for measurement of protein expression such as ATF6, CHOP, cleaved capase3, Akt, αMSH, and AgRP etc.

ER stress increased αMSH expression and decreased AgRP expression and Akt signaling in hypothalamic neuron. CBX and 67500 treatments were shown to decrease ER stress and prevented downregulation of phospho-rylated Akt by ER stress. Silence of Akt abolished the effect of 11β-HSD1 inhibition on αMSH amd AgRP expression in hypothalamic neuron. Injection of CBX and 67500 into the third ventricle in C57/BL6 mice before tunicamycin treatment alleviated ER stress induced increase of αMSH and decrease of AgRP expression in hypothalamus.

In conclusion, the result of this study suggests that 11β-HSD1 inhibition has a preventive effect against Hypothalamic ER stress induced appetite via alleviating downregulaiton of Akt signaling by ER stress.

& 2104-PIdentifying a New Pathway to Regulate AMPK Activity Under Meta-bolic StressEIJIRO YAMADA, SHUICHI OKADA, RYO SHIBUSAWA, YUKO TAGAYA, AYA OSA-KI, YOKO SHIMODA, TSUGUMICHI SAITO, CLAIRE C. BASTIE, JEFFREY E. PESSIN, MASANOBU YAMADA, Maebashi, Japan, Bronx, NY

Metabolic imbalance is associated with diabetes and insulin resistance and these patho-physiologies are linked with dysfunctions of nutrient-sensors such as AMP-dependent protein kinase (AMPK). Additionally pro-infl ammatory cytokines such as TNFα, secreted from adipose tissue contrib-utes to chronic low-grade infl ammation and whole body insulin resistance. Previously we reported that Fyn knockout mice display increased energy expenditure and fatty acid oxidation due to increased AMPK activity in pe-ripheral tissues. More recently, we demonstrated that Fyn regulates AMPK activity not only indirectly via its action on LKB1, but also by direct modula-tion of AMPK activity through Y426 phosphorylation of the α subunit. To investigate how Fyn regulates AMPK activity, we made AMPK α-Y426F mu-tant and examine functional interactions of the α subunit with the β and γ subunits. Although co-immunoprecipitation demonstrated no signifi cant dif-ference in β and γ subunit binding, the α-Y426F mutant displayed increased kinase activity compared to the wild type α subunit. These data suggested that Fyn-dependent tyrosine phosphorylation of AMPK α subunit on Y426 regulates its intra-molecular activity.

We then examined the signaling crosstalk between Fyn and pro-infl am-matory cytokines on AMPK regulation. Acute treatment with TNFα (10ng/ml for 12 h) enhanced AICAR (2mM, 10min) dependent phosphorylation of the AMPK α subunit on the activation T172 site. In contrast, prolonged in-cubation with TNFα (24-36 hr) suppressed AICAR stimulated T172 α subunit phosphorylation. In parallel, TNFα increased Fyn tyrosine kinase activity and siRNA knockdown of Fyn prevented the chronic TNFa inhibition of AICAR-stimulated AMPK T172 α subunit phosphorylation. Taken together, these data suggest that prolonged stimulation of TNFα blunts AICAR dependent AMPK activation through Fyn-dependent tyrosine phosphorylation of AMPK α subunit.

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& 2105-PRole of Host Genetics in Antibiotic Effects on the Gut Microbiome, Infl ammation, and Insulin Signaling in Diet-induced ObesitySHIHO FUJISAKA, LYNN BRY, CLARY B. CLISH, JONATHAN DREYFUSS, SU-ZANNE DEVKOTA, SIEGFRIED USSAR, MASAJI SAKAGUCHI, MASAHIRO KONI-SHI, C. RONALD KAHN, Boston, MA, Cambridge, MA

We have previously shown that different strains of mice and mice from different vendors exhibit different rates of obesity following high fat diet (HFD). C57BL/6J mice from Jax Labs (B6J) and 129 mice from Taconic (129T) are obesity prone, while 129 mice from Jax (129J) are obesity resistant. This difference between 129 mice is at least a part, due to differences in the gut microbiome from these two vendors. To further explore the role of the micro-biome in obesity, we have treated these 3 strains of mice with either vanco-mycin (to kill gram+ bacteria), or metronidazole (to kill anaerobic bacteria), and challenged them with HFD. 16S rRNA sequencing showed both antibiot-ics changed microbiome composition in all strains. In B6J, which tend to be-come insulin resistant, antibiotics improved glucose metabolism, decreased serum TNFα levels, decreased infl ammatory markers in liver, adipose tissue and improved insulin signaling. This improvement of glucose metabolism could be reproduced by transferring gut bacteria isolated from antibiotics-treated donors to HFD-fed B6J. On the other hand, in 129J, which tend to be lean and insulin sensitive, antibiotics increased infl ammatory markers and impaired insulin sensitivity. Metabolomic analysis showed HFD and antibiot-ics changed serum metabolites including prominent change in bile acids. The bile acid receptor, TGR5, which was also decreased in liver of HFD-fed mice, was restored by metronidazole treatment. Furthermore, treatment of B6J with a TGR5 agonist lowered infl ammatory gene expression of peritoneal macrophages in response to LPS. Thus, antibiotic treatment modifi es the gut microbiome, and impacts on various metabolites and infl ammation induced by HFD, leading to improved insulin signaling and glucose metabolism. These effects are strain dependent and partly dependent on bile acids, indicating an important interaction of the gut microbiome with host genetics, bile acid metabolism and metabolic state.

Supported By: Sunstar Foundation

& 2106-PErythropoietin and High-Fat Diet-induced Brain Infl ammationSOUMYADEEP DEY, JENNIFER L. ANHUT, CONSTANCE T. NOGUCHI, Bethesda, MD, Macon, GA

High fat diet (HFD)-induced hypothalamic infl ammation causes dysregu-lation of energy homeostasis. Microglial cells activated by saturated fatty acid exposure respond by producing infl ammatory cytokines. Erythropoietin (Epo), known for its hematopoietic role, inhibits obesity-induced white fat in-fl ammation in mice, is also produced in brain by astrocytes, and its receptor (EpoR) is expressed in microglial cells. We hypothesized that Epo signaling in brain could regulate HFD-induced microglial activation and thereby maintain metabolic homeostasis. We observed that mice lacking EpoR expression in the brain (nestin-Cre; EpoRloxP/loxP) were more glucose intolerant after 4 weeks of HFD, but did not show any difference in weight gain and food intake compared to littermate controls. In contrast, intracerebroventricular Epo (ICV-Epo) administration in HFD-fed wild-type mice for 4 weeks resulted in lower fasting glucose levels compared to saline control, but no difference in hematocrit, indicating no peripheral hematopoietic effect. Interestingly, ICV-Epo mice gained less weight compared to saline control after 1 week, but showed similar weight gain after 2-4 weeks on HFD, and the same food intake throughout. ICV-Epo administration signifi cantly reduced brain IL-1b, TNFα, and SOCS3 mRNA and increased IL-10 mRNA compared to saline con-trol. We examined primary mouse microglial cells to assess direct Epo anti-infl ammatory response using palmitic acid (PA) that activates infl ammatory response. PA signifi cantly increased TNF-α, IL-1b, and SOCS3 mRNA, while co-treatment with Epo signifi cantly reduced TNF-α and IL-1b mRNA expres-sion and showed an increased trend in IL-10 mRNA expression. In summary, these studies suggest an important extrahematopoietic and homeostatic function of Epo in brain infl ammation and metabolism.

Supported By: National Institute of Diabetes and Digestive and Kidney Diseases

& 2107-PColon Macrophage Regulates Insulin Sensitivity under High-Fat DietJUN NAKAE, YOSHINAGA KAWANO, NOBUYUKI WATANABE, TETSUHIRO KI-KUCHI, SANSHIRO TATEYA, YOSHIKAZU TAMORI, NORIKO KODANI, NOBUKO GOTO, MOTOKO MATSUZAKI, LISA OHIRA, AYAKO SHIGETA, MASASHI ONODE-RA, MASATO KASUGA, HIROSHI ITOH, Tokyo, Japan, Kobe, Japan, Osaka, Japan

Recently, it has been reported that the gut microbiome play important roles in the regulation of glucose and energy homeostasis. However, little is known about physiological role of intestinal macrophage on glucose me-tabolism under high fat diet (HFD).

We investigated effects of HFD on gut immunity in age-matched C57Bl6/J mice fed HFD. The length of colon from mice fed 4 weeks HFD was short-ened and the weight of Cecum was decreased compared with from mice fed normal chow diet (NCD). Moderate epithelial injury, decreased number of goblet cell and moderate infi ltration of mononuclear cell in colon were observed in mice after 4 weeks of HFD. The gene expression of Ccr2, F4/80, IL1β, and Nlrp3 were increased in colon. The concentration of IL1β in portal vein from HFD mice was signifi cantly increased compared with NCD mice. Analysis of macrophage cell population in colon from mice after 4weeks of HFD showed a higher population of F4/80+CD11b+CD11c-TNFα high cell than those of NCD mice. These data suggested that chronic infl ammation in colon occurred in early phase of HFD.

To investigate the pathological roles of colon macrophage in HFD, we gen-erated macrophage-specifi c Ccr2 knockout (M-Ccr2KO) mice. After 10 weeks of HFD, the body weight and food intake of M-Ccr2KO were similar to control mice. However, the glucose tolerance and the insulin sensitivity of M-Ccr2KO were signifi cantly improved compared with control mice. Insulin stimulated phosphorylation of Akt in M-Ccr2KO Liver was signifi cantly increased com-pared with control mice. The gene expression of F4/80, Ccr2, IL1β, and Nlrp3 in colon from M-Ccr2KO were signifi cantly decreased. The concentration of IL1β in portal vein of M-Ccr2KO was signifi cantly decreased. Moreover, in epididymal fat, liver, muscle, and small intestine, no signifi cant differences were observed in gene expression profi le of infl ammatory macrophage. These data suggested that inhibition of colon macrophage infi ltration could protect from diet-induced insulin resistance in early phase of HFD.

& 2108-PNovel Role of eNOS Co-factor Tetrahydrobiopterin for Mitochon-drial Regulation in Adiposity and Energy HomeostasisYASUO OGURI, YOSHIHITO FUJITA, ABULIZI ABDUKADIER, AKIO OBARA, AKIKO OHASHI, FUTOSHI FURUYA, TORU FUKUSHIMA, HIROYUKI HASEGAWA, MASA-YA HOSOKAWA, NOBUYA INAGAKI, Kyoto, Japan, Tokyo, Japan, Osaka, Japan

Endothelial nitric oxide synthase (eNOS) dysfunction is known to be in-volved in glucose intolerance, insulin resistance and progression of obesity. Tetrahydrobiopterin (BH4) is an essential co-factor of eNOS that regulates eNOS activity. We reported previously that BH4 has a glucose-lowering ef-fect as well as an insulin-sensitizing effect. However, whether BH4 itself regulates glucose, lipid, and energy metabolism is not known. In the pres-ent study, we investigated the role of BH4 in metabolism using hph-1 mice, in which GTP-cyclohydrolase I, a rate-limiting enzyme of BH4 syntheses is defi cient, and which exhibit a marked reduction in BH4 levels. Compared with control mice of the same background, hph-1 mice showed glucose intol-erance, insulin resistance, and increased lipid accumulation in liver tissues under normal diet. Gene expression related to glucose and lipid metabolism such as AMP kinase, CPT1, and p70-S6 kinase, a down-stream target of mTOR, were altered in liver tissues of hph-1 mice. Under high fat diet for 4 weeks, body weight and fat accumulation in liver and adipose tissue were increased in hph-1 mice. Rectal temperature as a marker of thermogenesis was decreased in hph-1 mice, we subsequently examined the function of brown adipose tissue (BAT). We found decreased mitochondrial mass, de-struction of cristae, and decreased expression of UCP1 and PGC-1α in BAT of hph-1 mice. The changes in mitochondrial function were also observed in liver. Thus, defi ciency of BH4 induces glucose intolerance, insulin resistance, and progression of obesity in these mice, strongly suggesting that BH4 medi-ates adiposity and energy homeostasis through mitochondrial function. BH4 is therefore a potential target for treatment and prevention of metabolic disorders.

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Guided Audio Tour: Central Pathways Regulating Metabolism (Posters: 2109-P to 2116-P), see page 13.

& 2109-PLesion of Dopaminergic Afferent Neurons Communicating with the Biological Clock Induces Metabolic Syndrome in RatsANTHONY H. CINCOTTA, MICHAEL EZROKHI, YELENA TRUBITSYNA, SHUQIN LUO, Tiverton, RI

The daily peak in dopaminergic neuronal activity at the area of the biologi-cal clock (hypothalamic suprachiasmatic nuclei [SCN]) is diminished in obese/insulin resistant vs. lean/insulin sensitive animals. Moreover, systemic dop-amine agonist therapy administered at the appropriate time of day to restore this daily peak in SCN dopaminergic activity ameliorates this insulin resis-tance. However, the impact of targeted lesioning of dopamine (DA) neurons specifi cally at the area of (and that communicate with) the SCN upon overall metabolism and blood pressure (BP) regulation in normal animals has not been investigated and was the focus of this study. Female Sprague-Dawley rats (10 wk old; BW: 220±3 g) were subjected to either DA neuron neurotoxin lesion by bilateral intra-cannular injection of 6-hydroxydopamine (2-4 µg/side, N=14) or vehicle treatment (N = 8) at the area surrounding the SCN at 20 minutes post protryptyline ip injection (20 mg/kg) (to spare noradrenergic and serotonergic neuron damage). At 16 wks post-lesion rats were subjected to a glucose tolerance test (GTT, 3 g glucose/kg BW) and blood pressure measurement prior to sacrifi ce 48 hrs later. Relative to vehicle treatment, SCN DA neuron lesioning increased weight gain (16%, P<0.005), uterine and retroperitoneal fat weight (40% and 85% respectively, P<0.05), fast-ing plasma insulin and leptin levels (117% and 71%, P<0.05), GTT AUC insu-lin and glucose (74% and 11% respectively, P<0.05), and insulin resistance (67% - Matsuda Index, p<0.02) without altering food consumption during the test period. SCN dopamine lesioned rats were also hypertensive relative to controls (systolic BP rise from 156±5 mmHg to 173±5 mmHg, P<0.02; heart rate increase from 367±11 to 400±12 BPM, P<0.05). These fi ndings suggest that reduced dopaminergic neuronal activity in neurons at the area of and communicating with the SCN contributes signifi cantly to the development of metabolic syndrome, independent of effects on feeding.

& 2110-PEffects of Kainic Acid Treatment on Nrf2/HO-1-Defense Pathway in the Hippocampus of Mice with Chronic High-Fat DietROK WON HEO, DONG HO KANG, HYUN JOO SHIN, JAE GEUN KIM, HWAJIN KIM, GU SEOB ROH, Jinju, Republic of Korea, Incheon, Republic of Korea

Obesity has deleterious effects on the brain, and metabolic dysfunction may exacerbate the outcomes of seizures and brain injuries. However, it is unclear whether obesity affects excitotoxicity-induced neuronal cell death. The purpose of this study was to investigate the effects of a high-fat diet (HFD) on neuroinfl ammation and endoplasmic reticulum (ER) stress in the hip-pocampus of kainic acid (KA)-treated mice. Mice were fed a HFD or normal diet for 8 weeks and then received a systemic injection of KA. HFD-fed mice showed hypercholesterolemia, insulin resistance, and hepatic steatosis. HFD-fed mice also showed greater susceptibility to KA-induced seizures, an increased number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, neuroinfl ammation, and ER stress. Further-more, we found that KA treatment increased HFD-induced Nrf2 and HO-1 expression in the hippocampus, suggesting a possible neuroprotective role of Nrf2/HO-1. Taken together, these fi ndings imply a causal relationship between metabolic dysfunction and excitotoxicity-induced neuronal cell death.

& 2111-PInsulin Activates ERK1/2 in the Striatum to Regulate Energy Balance and Hepatic Glucose ProductionGISELE CASTRO, LAÍS WEISSMANN, NATÁLIA MENDES, PAULA QUARESMA, MÁRIO JOSÉ ABDALLA. SAAD, PATRÍCIA OLIVEIRA. PRADA, Campinas, Brazil

Insulin activates PI3K/AKT in the hypothalamus to regulate food intake (FI) and hepatic glucose production. Extra-hypothalamic regions are also sensitive to insulin to regulate FI and glucose homeostasis. Striatum (STR) is one of the key region in the reward system, which express abundant insulin receptor (IR). However, whether insulin (INS) in this region contributes to energy homeostasis is unknown. Herein, we aimed to investigate whether (1) INS in the STR alters FI in control animals and which pathway is involved in this response; (2) whether obesity affects INS signaling in the STR. And whether (3) MAPK inhibition, by using pharmacological inhibitor (PD98059), affects energy metabolism and glucose production in control animals. INS injection in the STR reduced 8,12,24h-FI in rats on chow. This result was

associated with higher IR/ERK phosphorylation, but not PI3K/Akt activation, suggesting that insulin-induced reduction in FI is dependent of ERK1/2 path-way in the STR, unlike what occurs in the hypothalamus. There was a reduc-tion in ERK1/2 phosphorylation in response to INS in the STR of obese ani-mals, associated with a lack of anorexigenic insulin effect. Chronic inhibition of ERK1/2 in STR increased fat mass, FI, blocked the anorexigenic effect of INS and altered glucose tolerance, insulin sensitivity and oxygen consump-tion of animals on chow diet. Moreover, chronic treatment with PD98059 in the STR reduced fasting blood glucose and this result was associated with a decrease in PEPCK gene expression in liver, and lower blood glucose levels in response to pyruvate intraperitoneal injection, suggesting that the chronic inhibition of ERK1/2 in the STR may regulate hepatic glucose production. In summary, our results suggest that STR is an important site of INS action to control feeding and energy balance via IR/ERK1/2 pathway and this signal is impaired in obese animals. In addition, chronic ERK1/2 inhibition in the STR increases adiposity and may alter hepatic glucose production.

& 2112-PGLP-1 Analogs Treatment Improves Insulin and Leptin Action/Sig-naling in the Hypothalamus and Amygdala by Decreasing Adiposity and Food Intake in Dio MiceGIOVANNA T. RICARDO, LAÍS WEISSMANN, PAULA GABRIELE F. QUARESMA, THAYANA O. MICHELETTI, GISELE CASTRO, PATRICIA O. PRADA, Campinas, Bra-zil, Rio Claro, Brazil, Pirassununga, Brazil

Obesity and type 2 diabetes mellitus (T2DM) are major public health prob-lems. Gut-derived hormones, such as glucagon-like peptide 1 (GLP1), con-tribute to control food intake (FI). GLP1 and its analogs such as liraglutide are used to treat T2DM and are also critical regulators of energy balance. The GLP1 receptors are expressed in the hypothalamus and amygdala, how-ever it is not known whether liraglutide treatment may improve leptin and insulin signaling/action in the hypothalamus and amygdala. So, the aims of the present study are: 1) investigate whether liraglutide treatment alters adiposity and insulin sensitivity; 2) investigate whether liraglutide treat-ment may infl uence the action of insulin and leptin in the hypothalamus and insulin in the amygdala in vivo, by measuring FI after 4, 8, 12 and 24 hours after stimulation with these hormones; 3) investigate whether liraglutide treatment may infl uence protein expression and degree of phosphorylation of intracellular proteins involved in signal transmission of insulin (IR, AKT and FOXO-1) and leptin (OBR and STAT3) in hypothalamus after stimulation with these hormones. DIO mice were treated for 10 days with liraglutide intraperitoneal (200 µg/kg/day) or vehicle. Liraglutide treatment decreased adiposity, fasting glycemia, decreased oxygen consumption and UCP-1 pro-tein expression in brown adipose tissue and improved insulin sensitivity. Also, liraglutide treatment decreased FI and increased IR, AKT and FoxO1 phosphorylation were observed in response to intracerebroventricular (ICV) insulin in the hypothalamus and amygdala. And, liraglutide treatment de-creased FI, increased leptin receptor, STAT3 phosphorylation in response to ICV leptin in the hypothalamus. Our results suggest that GLP1 improves insulin and leptin action/signaling in the hypothalamus and amygdala of DIO mice, which may contribute to the reduction of FI, adiposity and the hedonic value of the food.

& 2113-PElevation of Norepinephrine (NE) Activity at the Ventromedial Hy-pothalamus (VMH) of Normal Rats Induces the Obese Hypertensive Insulin Resistant State without Altering FeedingSHUQIN LUO, MICHAEL EZROKHI, YELENA TRUBITSYNA, YANG LI, ANTHONY CINCOTTA, Tiverton, RI

We have previously identifi ed increased NE activity at the VMH, a fuel sensing center that regulates sympathetic function, as a common occur-rence among a wide variety of animal models of insulin resistance. To es-tablish a cause-effect relationship between elevated VMH NE activity and metabolic syndrome this study investigated the impact of chronic exogenous administration of NE site-specifi cally to the VMH on peripheral metabolism and blood pressure in young healthy Sprague-Dawley (SD) rats. Female SD rats (10 wks old; BW: 238±1 g) were equipped with osmotic pumps to contin-uously deliver vehicle (N=8) or NE (N=8) at a rate of 25 nmol/hr to the VMH to raise endogenous VMH NE levels to those observed in metabolic syndrome rats. Following 3-wks of such treatment, blood pressure and glucose toler-ance test (GTT) measurements were taken prior to sacrifi ce and the analy-ses of body fat levels, liver lipids and plasma metabolites and hormones. Relative to controls, VMH NE-treated rats exhibited an increase in basal plasma glucose (23%, P<0.001) and insulin (2.6-fold, P<0.05), GTT glucose and insulin area under the curves (46% and 67% respectively, P<0.05), and

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insulin resistance (62%, Matsuda Index, P<0.005). VMH NE treatment also increased BW by 9%, abdominal fat weight by 65%, and liver lipid content by 48% relative to control without altering food consumption (P<0.05). VMH NE infusion also increased both plasma glucagon and NE levels by 2.7-fold (P<0.05). Furthermore, VMH NE infusion induced hypertension relative to controls (systolic blood pressure rise from 142±4 mmHg to 176±6 mmHg, P<0.01; diastolic blood pressure rise from 102±4 mmHg to 125±6 mmHg, P<0.01). These fi ndings indicate that increased VMH NE levels associated with metabolic syndrome can actually be causative in the genesis of obesity, insulin resistance, fatty liver and hypertension, independent of any effect on feeding.

& 2114-PHypothalamic Bone Morphogenetic Protein Receptor 1A (BMPR1A) Regulates Energy BalanceKRISTY L. TOWNSEND, CHRISTOPHER MADDEN, LINDSAY MCDOUGALL, MAGDALENA BLASZKIEWICZ, DOMENICO TUPONE, MATTHEW D. LYNES, YUJI MISHINA, PAUL YU, SHAUN MORRISON, YU-HUA TSENG, Orono, ME, Portland, OR, Boston, MA, Ann Arbor, MI

Energy balance and body weight are regulated by complex processes in the brain, and our understanding of the relevant signaling pathways is still evolving. The main anorectic neurons of the hypothalamus contain pro-opi-omelanocortin (POMC). We have previously shown that bone morphogenetic protein 7 (BMP7) acts centrally to reduce appetite. Now we demonstrate that a type 1 BMP receptor, BMPR1A, is co-localized with POMC neurons and ablation of BMPR1A in those neurons results in hyperphagia with a lack of obesity, even on a high-fat diet. This was due to a compensatory increase of BMPR1A expression in non-POMC neurons of the hypothalamus of the knock-out mice, accompanied by an increase in energy expenditure and improved cold-tolerance. Brown adipose tissue (BAT) of knock-out animals showed greater thermogenic capacity and increased sympathetic innerva-tion, and removal of the sympathetic drive to BAT by surgical denervation resulted in hyperphagic weight gain in the knock-out mice. Wild-type mice injected intracerebroventricularly (i.c.v.) with BMP7 displayed an acute in-crease in energy expenditure and rats injected i.c.v. with BMP7 displayed a specifi c increase in sympathetic nerve activity (SNA) in BAT, along with increased BAT temperature and energy expenditure. In these animals, block-ade of type 1 BMP receptor signaling by pre-treatment with a pharmaco-logical inhibitor blunted the ability of BMP7 to increase energy expenditure or BAT SNA. Together, these data demonstrate an important novel role for hypothalamic BMP signaling in the regulation of appetite and activation of brown fat-mediated energy expenditure.

Supported By: American Diabetes Association (1-14-JF-55 to K.L.T.)

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& 2116-PHypothalamic Deletion of Apolipoprotein J (ApoJ) Causes Severe Obesity in MiceJI A. SEO, MIN-CHEOL KANG, MICHELLE CHUNG, JEONG HO KIM, SOO HYUN HONG, LEANDRO MAURA, INES S. LIMA, YOUNG-BUM KIM, Seoul, Republic of Korea, Boston, MA

Our work demonstrates that intracerebroventricular injection of recombi-nant ApoJ protein decreases food intake and body-weight homeostasis in mice. Studies with liver-specifi c ApoJ-defi cient mice demonstrate that the liver is a major sourcing organ of circulating ApoJ, evidenced by the fi ndings that no ApoJ in sera is found, whereas the ApoJ level in CSF and the hypo-thalamus is normal. These data suggest that hypothalamic ApoJ involved in anorectic action may originate from the brain but not from the periphery. Sup-porting this, previous fi ndings reveal that circulating ApoJ cannot cross the blood-brain barrier because of the existence of a saturated system. To deter-mine the physiological role of hypothalamic ApoJ in energy metabolism, we in-jected adeno-associated virus encoding Cre recombinase (AAV-Cre) bilaterally into the hypothalamic arcuate of loxP-fl anked ApoJ mice (ApoJfl ox/fl ox mice). Control mice were injected with AAV encoding GFP (AAV-GFP). Immunoblot-ting analysis indicated that ApoJ expression levels in the hypothalamus were reduced by ~80% in AAV-Cre injected ApoJfl ox/fl ox mice. After 14weeks of AAV injection, body weight (30.7 ± 2.5, AAV-GFP vs. 43.03 ± 2.2, AAV-Cre, P < 0.01) signifi cantly increased in AAV-Cre injected ApoJfl ox/fl ox mice compared to control mice. These effects are most likely due to increased food intake. In parallel, total fat mass measured by an MRI also increased in AAC-Cre injected ApoJfl ox/fl ox mice. Concurrently, blood glucose and serum insulin levels were greatly elevated in AAV-Cre injected ApoJfl ox/fl ox mice. These effects are thought to be secondary, due to increased adiposity. Our data suggest that ApoJ action in the hypothalamus is critical for normal body-weight homeo-stasis and energy metabolism, and may offer a novel target for the treatment of obesity-related metabolic diseases. Currently, studies with hypothalamic neurons-specifi c ApoJ-defi cient mice are being investigated.

Supported By: American Diabetes Association (7-12-BS-094 to Y-B.K.)

2117-PFatty Acid Binding Protein 5 (FABP5) Regulates Diet-induced Obesi-ty (DIO) via GIP Secretion from Enteroendocrine K-cells in Response to Fat IngestionKIMITAKA SHIBUE, SHUNSUKE YAMANE, NORIO HARADA, AKIHIRO HAMA-SAKI, KAZUYO SUZUKI, ERINA JOO, KANAKO IWASAKI, DANIELA NASTESKA, TAKANARI HARADA, YOSHITAKA HAYASHI, YASUHIRO ADACHI, YUJI OWADA, RYOICHI TAKAYANAGI, NOBUYA INAGAKI, Kyoto, Japan, Nagoya, Japan, Kitaky-ushu, Japan, Yamaguchi, Japan, Fukuoka, Japan

Gastric inhibitory polypeptide (GIP) is an incretin released from enteroen-docrine K-cells in response to nutrient intake, especially fat. GIP is one of the contributing factors inducing induce fat accumulation that results in obesity. A recent study shows that fatty acid-binding protein 5 (FABP5) is expressed in murine K-cells and is involved in fat-induced GIP secretion. We investigated the mechanism of fat-induced GIP secretion and the impact of FABP5-related GIP response on diet-induced obesity (DIO). Single oral ad-ministration of glucose and fat resulted in 40% reduction of GIP response to fat but not to glucose in whole body FABP5 knockout (FABP5-/-) mice, with no change in K-cell count or GIP content in K-cells. In an ex vivo experiment us-ing isolated upper small intestine, oleic acid induced only a slight increase in GIP release, which was markedly enhanced by coadministration of bile and oleic acid, together with attenuated GIP response in the FABP5-/- sample. FABP5-/- mice exhibited 24% reduction in body weight gain and body fat mass under high-fat diet compared to wild-type (FABP5+/+) mice; the dif-ference was not observed between GIP-GFP homozygous knock-in (GIPgfp/gfp)-FABP5+/+ mice and GIPgfp/gfp-FABP5-/- mice, in which GIP is geneti-cally deleted. These results demonstrate that bile effi ciently amplifi es fat-induced GIP secretion and that FABP5 contributes to the development of DIO in a GIP-dependent manner.

2118-PCdc2-like Kinase 2 (CLK2) in the Hypothalamus Is Crucial for Energy and Glucose MetabolismPAULA GABRIELE F. QUARESMA, LAÍS WEISSMANN, ANDRESSA SANTOS, AL-EXANDRE MATOS, ISCIA LOPES-CENDES, MÁRIO SAAD, FERNANDO SIMABU-CO, PATRICIA O. PRADA, Campinas, Brazil, Limeira, Brazil

Cdc2-like kinase 2 (CLK2) was recently shown to have profound effects on liver metabolism. It is regulated by insulin in a PI3K/AKT manner to control gluconeogenesis and hepatic glucose production. Insulin and leptin act in

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the hypothalamus to control energy homeostasis and glucose metabolism. However, whether CLK2 is regulated by insulin and leptin in the hypothala-mus and participates in the control of energy homeostasis and glucose me-tabolism has not been established yet. Thus, our aims were to investigate: 1) the regulation of hypothalamic CLK2 in response to insulin and leptin in control and obese mice; 2) whether chronic CLK2 inhibition in the hypothala-mus affects energy homeostasis; 3) whether chronic CLK2 overexpression in the hypothalamus affects energy homeostasis and 4) whether hypothalamic CLK2 regulates hepatic gluconeogenesis. CLK2 phosphorylation occurred in response to refeeding, insulin and leptin in the hypothalamus and was depended of the PI3K/Akt (insulin) or JAK2 (leptin). Hypothalamic CLK2 phos-phorylation in response to insulin was suppressed in diet-induced obesity and db/db mice. Chronic inhibition of CLK2 by siRNA or by pharmacologic inhibitor TG003 in the hypothalamus increased adiposity, food intake, as well as NPY and AgRP gene expression, and decreased POMC and CRH gene expression, energy expenditure and UCP-1 levels in brown adipose tissue. In addition, a lack of hypothalamic CLK2 increased fasting blood glucose, hepatic glucose production measured by pyruvate test and PEPCK gene ex-pression in the liver. Furthermore, overexpression of CLK2 in the ventrome-dial hypothalamus (VMH) by adenovirus in obese animals increased energy expenditure, decreased food intake and adiposity. Overexpression of CLK2 in VMH of obese mice also improved peripheral insulin sensitivity and fasting blood glucose levels. In conclusion, we provided evidences that hypotha-lamic CLK2 is crucial to maintain energy and glucose homeostasis.

Supported By: FAPESP; CNPq; INCT

2119-PRobust Antiobesity and Metabolic Effects of a GLP-1/Glucagon Co-agonist Peptide in Rodents and Nonhuman PrimatesANISH KONKAR, BALAJI AGORAM, MARIA BEDNAREK, MARIA FRITSCH-FRE-DIN, JOSEPH GRIMSBY, SIMON HENDERSON, DAVID HORNIGOLD, RON JACK-SON, LUTZ JERMUTUS, CRISTINA RONDINONE, Gaithersburg, MD, Cambridge, United Kingdom, Mölndal, Sweden

Oxyntomodulin (OXM), the endogenous GLP-1/Glucagon co-agonist pep-tide, is secreted from intestinal L-cells in response to meals. OXM reduces body weight in obese subjects by decreasing food intake and increasing energy expenditure. In addition, it produces direct acute glucoregulatory ef-fects in type 2 diabetic patients. Clinical utility of this hormone, however, is limited due to its short lifetime in circulation, as it is readily cleaved by the enzyme DPP-IV. We have synthesized stable analogs of oxyntomodu-lin with varying ratios of GLP-1: glucagon activity which are palmitoylated and suitable for once-daily administration. MEDI0382 is a highly potent and selective agonist of the human GLP-1 and glucagon receptors and is highly protein bound. It also displays similar potency at the rodent and cynomolgus monkey receptors. Twice daily dosing with MEDI0382 for 1 month in male diet-induced obese (DIO) mice dose-dependently reduced body weight gain by 15.3%, 23.7% or 24.1%, at 14.8, 25.9 or 40.7 µg/kg (s.c.), respectively, as compared with vehicle-treated DIO mice. A signifi cant improvement in glucose tolerance was observed in these mice in an oral glucose tolerance test. In diabetic db/db mice, a single dose of MEDI0382 reduced glucose excursion following an intraperitoneal glucose challenge. Repeated daily dosing of MEDI0382 for 2 months in cynomolgus monkeys resulted in dose-dependent reductions in body weight of 5% to 12% compared to starting bodyweights at the end of the treatment period. Bodyweights returned to baseline within 2 weeks of cessation of treatment but thereafter stabilized at a lower level than concurrent vehicle-treated monkeys. In summary, our data indicates that a GLP-1/Glucagon co-agonist shows signifi cant promise as a weight loss drug in overweight and obese subjects. In addition, the co-agonist has the potential to produce glucose-lowering in type 2 diabetics.

2120-PPyruvate Dehydrogenase Kinase Is Required for the AdipogenesisSUNG-WOO KIM, DONGWOOK KIM, HYEON-JI KANG, BYUNG-GYU KIM, KEUN-GYU PARK, IN-KYU LEE, Daegu, Republic of Korea

Obesity has known to cause metabolic disease by disrupting mitochondrial function. Accumulating data suggested that energy imbalance by the mito-chondrial dysfunction was closely linked to the onset of metabolic disease. Mitochondria are the major organ balancing energy sources between fat and glucose, which is important step to maintain cells healthy. However, the de-tailed mechanism on how mitochondrial dysfunction cause metabolic disease still remains to be elucidated. Here, we identifi ed Pyruvate dehydrogenase kinase (PDK) in mitochondrial matrix is essential for mitochondrial function by regulating adipogenesis. PDKs have four different isoforms and their expres-sion patterns are dependent on tissue types and environmental stimulation.

Interestingly, we found that expression level of PDK1, 2 and 4 was signifi cantly elevated in matured adipose tissue fraction as compared with stromal vas-cular fraction (SVF) obtained from subcutaneous and epididymal fat tissues and those expression patterns were confi rmed in primary adipocyte culture. To explore molecular mechanism, we used 3T3-L1 cells. Unexpectedly, we found that only PDK1 and 2 was strongly elevated during adipogenesis, but not PDK4. As expected, overexpression of either of PDK1 or PDK2 strongly increased adipogenesis, accompanied with increased adipogenesis markers such as SREBF1, FASN, c/EBPα and PPARγ. In contrast, knocking down of both PDK1 and 2 using siRNA completely inhibited adipogenesis, along with decreased expression of adipogenesis markers. Furthermore, maximal oxygen consump-tion ratio was increased by silencing of both endogenous PDK1 and 2, leading to increased spare respiratory capacity (SRC). In addition, lactate concentra-tion in media was dramatically decreased by silencing both PDK1 and 2 during adipogenesis. Taken together, these results suggest that PDK could regulate adipogenesis by regulating mitochondrial function and be therapeutic targets for the patients with metabolic disease.

2121-PA Synthetic PAI-1 Inhibitor, TM5441, Protects High-Fat Diet-induced Adipocyte Injury in MiceLINGJUAN PIAO, INJI JUNG, TOSHIO MIYATA, HUNJOO HA, Seoul, Republic of Korea, Sendai, Japan

Obesity is one of the most prevalent chronic diseases worldwide, and dys-regulated adipocyte function plays an important role in obesity-associated metabolic disorder. Plasminogen activator inhibitor-1 (PAI-1), a 50kDa glyco-protein, is a major physiological inhibitor of tissue-type and urokinase-type plasminogen activator. Plasma PAI-1 levels is increased in obese subjects and PAI-1 null mice show improved insulin sensitivity under high-fat and high-sucrose diet-induced metabolic stress. The present study examined the pre-ventive effect of TM5441, a novel orally active PAI-1 inhibitor that does not cause bleeding episodes) on high fat diet (HFD)-induced adipocyte dysfunction. 10-week-old C57BL/6J mice were fed a normal diet (ND; 18% of total calories from fat) or HFD (60% of total calories from fat) for 10 weeks, and TM5441 (20 mg/kg oral gavage) was daily administered with the initiation of HFD. TM5441 prevented HFD-induced body weight gain and insulin resistance in epididymal white adipose tissue (WAT). TM5441 upregulated the expression of uncoupling protein-1 and mitochondrial biogenesis factors in WAT, suggesting increased energy expenditure. TM5441 also attenuated macrophage infi ltration along with decreased expression of HFD-induced proinfl ammatory cytokines, such as monocyte chemotactic protein-1 and inducible nitric oxide synthase. In 3T3-L1 adipocytes, TM5441 pretreatment inhibited TNFα-induced dysregulation of adipokine expression and insulin resistance. Microarray analysis revealed that exogenously administered PAI-1 affected cellular processes, including lipid metabolism, hypoxia, cell cycle, and the mitogen-activated protein kinase, and p53 signaling pathways, indicating direct role of PAI-1 on adipocyte function. Taken together, our data suggest that TM5441 may become a novel therapeu-tic agent for obesity and obesity-related metabolic disorders.

2122-PEffects of a Long-Acting Formulation of Exenatide on Glucose and Weight Control in Type 2 Diabetic Rhesus MonkeysZUNYUAN YANG, LI GONG, WEN ZENG, ZUNWEI YAO, MICHAEL XU, Chengdu, China, Suzhou, China

Site-specific pegylation of exenatide (PB-119) has been reported to main-tain in vitro activity and prolonged plasma duration. This study aimed to evaluate the effect of PB-119 on glucose and weight in type 2 diabetic rhe-sus monkeys. A placebo-control, 4-weeks effi cacy study was performed in 20 type 2 diabetic monkeys, which were divided into 4 groups and received subcutaneous injection of PB-119 and vehicle once a week. Fasting plasma glucose (FPG), 2h postprandial glucose (2hPPG) and body weight was mea-sured weekly. Compared with baseline values, after monkeys were treated by 20µg/kg, 10µg/kg, and 5µg/kg PB-119 for 4 weeks, FPG decreased by an average of 22.8%, 15.4% and 14.1% respectively, 2h-PPG decreased by an average of 30.4%, 22.5% and 18.0% respectively, and HbA1c decreased by an average of 5.9%, 5.6%, 5.0% respectively. The daily food consump-tion decreased by an average of 22.1%, 9.2% and 14.3% respectively, body weight decreased by an average of 7.7% (p<0.05 vs. control group), 2.73% and 3.54%, respectively. PB-119 also reduced the abdominal subcutaneous fat volume by an average of 42.1%, 42.2% and 39.0% respectively (p<0.05 vs. control group), and reduced the intra-abdominal fat volume by an average of 20.8%, 20.8% and 16.9% (p<0.05 vs. control group).

In conclusion, this study indicated that PB-119 can control glucose and body weight and may provide a therapy choice for obese diabetic patients.

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2123-PSmall Molecule Inhibitors of Infl ammation Delay the Progression of High-Fat Diet-induced Nonalcoholic Fatty Liver Disease in Male C57BL6/J MiceASHLEY PATTON, DOUG GOETZ, STEPHEN BERGMEIER, RAMIRO MALGOR, JEAN THUMA, KELLY D. MCCALL, FRANK L. SCHWARTZ, Athens, OH

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of both metabolic and infl ammatory diseases, and it has become pervasive worldwide. Infl ammation, including infl ammation resulting from free fatty acid (FFA) activation of toll-like receptor (TLR) signaling, has been suggested to be an essential component of the pathophysiology of NAFLD. High fat diets (HFDs) promote an increased uptake and storage of FFAs and triglycer-ides (TGs) in hepatocytes, which initiates steatosis and induces lipotoxicity and infl ammation. We evaluated the effi cacy of novel small molecule inhibi-tors of infl ammation developed in our laboratory, including a TLR inhibitor [phenylmethimazole (C10)], to delay and/or prevent HFD-induced steatosis in a C57BL/6J mouse model of high fat diet (HFD)-induced NAFLD. The mice were fed a HFD (60% fat, 20% protein, 70% carbohydrate) and divided into 6 groups (N=5 for each group): sham injection (stress control), DMSO (vehicle control), phenylmethimazole (C10), COB-187, COB-204, and COB-214. Each compound was administered once daily at a dosage of 1mg/kg for 6 weeks. Histological examination of the liver of the mice in this study using hemo-toxylin and eosin (H&E) staining as well as hepatic triglyceride quantifi cation revealed that mice treated with C10, COB-187, COB-204, and COB-214 had less steatosis than mice in the DMSO and sham control groups, however the degree of inhibition of steatosis varied. COB-214 had the greatest inhibitory activity. Thus, our data indicate that these novel small molecule inhibitors of infl ammation delay and/or prevent the progression of HFD-induced steatosis in mice and may thus be useful for the treatment of NAFLD.

2124-PJNK2-Defi cient Heart Develops Severe Insulin Resistance follow-ing Chronic High-Fat Feeding in MiceJONG HUN KIM, DAE YOUNG JUNG, HYEKYUNG HA, RANDALL H. FRIED-LINE, SEZIN DAGDEVIREN, XIAODI HU, KEVIN S. HSU, KUNIKAZU INASHIMA, KAVITHA MURALIDHARAN, ADAM R. WENDE, E. DALE ABEL, KI WON LEE, ROGER J. DAVIS, JASON K. KIM, Worcester, MA, Daejeon, Republic of Korea, Iowa City, IA, Seoul, Republic of Korea

Insulin resistance plays an important role in diabetic heart failure, a lead-ing cause of mortality in type 2 diabetes. The c-Jun NH2-terminal kinase (JNK) signaling pathway regulates glucose metabolism in a cell-autonomous manner, but the role of JNK in cardiac insulin resistance is unknown. Mice with heart-selective deletion of JNK2 (MHC-JNK2-/-) were born without anomaly and showed normal insulin sensitivity on chow diet. Male MHC-JNK2-/- (KO) and MHC-Cre (WT) mice (n=9~10/group) were fed a high-fat diet (HFD) for 8 wks, and hyperinsulinemic-euglycemic clamps were conducted to measure glucose metabolism in awake mice. Following HFD, both groups of mice became obese and developed insulin resistance (Fig. 1). Despite similar obesity, glucose infusion rates were reduced by ~40% in HFD-fed KO mice as compared to HFD-fed WT mice (Fig. 2). Whole body glucose turnover was also further reduced in KO mice, and this was partly due to a 43% decrease in insulin-stimulated glucose uptake in the heart (Fig. 3 and 4). Additionally, glucose uptake in brown adipose tissue was signifi cantly reduced in HFD-fed KO mice. Overall, these results indicate that JNK2-defi cient heart develops more severe insulin resistance after HFD and suggest an important role of JNK2 in the regulation of myocardial glucose metabolism.


2125-PThe Interferon Activated Gene Ifi 202b Promotes Obesity by Improv-ing Adipocyte Differentiation and Triglyceride StorageMANDY STADION, HEIKE VOGEL, HANS-GEORG JOOST, ANNETTE SCHÜR-MANN, Nuthetal, Germany

Obesity is a polygenic metabolic disease and the major risk factor for type 2 diabetes (T2D). In an intercross population of the New Zealand Obese (NZO) mouse, a model for polygenic obesity and T2D, with the lean and di-abetes-resistant C57BL/6J (B6) strain, Ifi 202b was identifi ed as an obesity gene by positional cloning. Due to a loss of function mutation in B6 mice the interferon activated transcriptional modulator Ifi 202b is not expressed in tissues of B6 mice but present in white adipose tissue, liver and skeletal muscle of NZO mice. We hypothesize that Ifi 202b participates in obesity and insulin resistance of NZO mice. Male mice of a recombinant congenic line ex-pressing Ifi 202b (NZO allele) or lacking Ifi 202b (B6 allele) where fed a high-fat diet (45 kcal% from fat) and body composition and glucose metabolism were determined. Moreover, the role of Ifi 202b in adipocyte differentiation and triglyceride storage was investigated by adenoviral-mediated overexpres-sion of the gene in 3T3-L1 fi broblasts. Ifi 202b expressing NZO-allele carriers developed a signifi cantly higher fat mass than corresponding B6-allele car-riers which was accompanied by a marked adipocyte hypertrophy. Besides Ifi 202b expressing mice exhibited elevated plasma insulin levels as well as a mild insulin resistance during an oral glucose tolerance test in week 22. Adenovirus-mediated overexpression of Ifi 202b in 3T3-L1 fi broblasts result-ed in an improved differentiation into adipocytes indicated by an increased expression of adipogenesis markers (e.g. Pparγ, Fabp4, Plin1). Furthermore, we detected a signifi cantly higher amount of triglycerides in Ifi 202b overex-pressing preadipocytes. Accordingly, Ifi 202b transgenic B6 mice exhibited elevated fat mass and increased fasting insulin concentrations. We con-clude that Ifi 202b promotes fat accumulation by an induction of adipogenic genes, thereby driving obesity and insulin resistance.

Supported By: German Center for Diabetes Research

2126-PGalectin-9 Defi ciency Ameliorates Adiposity and Glucose Toler-ance in Mice with Diet-induced ObesityTOMOKAZU NUNOUE, SANAE TESHIGAWARA, SATOSHI YAMAGUCHI, YUSUKE SHIBATA, MASAFUMI TENTA, AKIHIRO KATAYAMA, KAZUTOSHI MURAKAMI, ATSUKO NAKATSUKA, JUN EGUCHI, JUN WADA, Okayama, Japan

Galectins belong to a unique family of mammalian lectins which have specifi c binding activity to β-galactoside-containing carbohydrates. Galec-tin-9 (Gal-9), a well-known ligand for T-cell immunoglobulin mucin-3 (Tim-3), induces apoptosis of CD4+Tim-3+ T helper 1 (Th1) cells and thus negatively regulates Th1 immune responses. The polarization of Th1 cytokines and M1 macrophages in visceral white adipose tissue (WAT) closely links to insulin resistance in metabolic syndrome. The line of evidence prompted us to in-vestigate whether Gal-9 participates in chronic infl ammation under obese state and analyze Gal-9 defi cient (Gal-9 KO) and wild type C57BL/6J mice (WT). On a standard chow (STD), there were no signifi cant differences in body weight and adiposity between Gal-9 KO and WT. In contrast, body weight was signifi cantly suppressed, and WAT mass as well as adipocyte size were signifi cantly reduced in Gal-9 KO on high fat and high sucrose diet (HFD). The glucose tolerance was signifi cantly improved in Gal-9 KO; how-ever, the adipose tissue infl ammation such as crown-like structure and M1/M2 polarity of macrophages revealed by fl ow cytometry were not altered. Since Gal-9 is abundantly expressed in both immune cells and adipocytes, we performed bone marrow transplantation (BMT) between Gal-9 KO and WT in order to confi rm whether the defi ciency of Gal-9 in immune cells or so-matic cells are responsible for the phenotype. By analyzing the body weight and adiposity of BMT mice, we found out that the suppression of obesity

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in Gal-9 KO depends on the defi ciency of Gal-9 in somatic cells, but not in immune cells. These results suggest that Gal-9 has a novel function in adio-pocyte biology and it may open a new avenue to understand pathogenesis of obesity and type 2 diabetes.

2127-PAblation of Sweet Taste Receptor (STR) Signaling Protects from the Metabolic Derangements of High-Fat Diet (HFD) FeedingGEORGE A. KYRIAZIS, KATHLEEN SMITH, RICHARD E. PRATLEY, Orlando, FL

STRs on the tongue mediate gustatory sweet sensing, but emerging physi-ological roles in the gut, pancreas and adipose tissue suggest their regulato-ry contribution in whole-body nutrient sensing that operates beyond nutrient uptake and metabolism. We showed that STRs on beta-cells are downregu-lated during HFD or in response to hyperglycemia, leading to basal insulin hyper-secretion. These fi ndings suggest a functional involvement of STRs in the development of diabetes. To shed light on the direct role of STR signal-ing in diet-induced obesity, WT and T1R2 knockout (KO; lacking STR signal-ing) male mice (n=10-14/group) were placed on a 12-week HFD (58% fat) or control diet (11% fat). KO mice on HFD have reduced fat mass (WT: 14.8±0.8 vs. KO: 10.7±0.9g; p<0.001) and preserve their lean mass despite demon-strating increased energy intake (WT: 10.8±0.2 vs. KO: 17.3±1.1kcal/day; p<0.0001). These effects are not attributed to intestinal fat malabsorp-tion, or to increased energy expenditure. Ablation of STRs also protected against HFD-induced hyperglycemia (WT: 204.0±5.0 vs. KO: 181.3±5.7mg/dl; p<0.05), partially due to enhanced insulin secretion and/or peripheral glu-cose metabolism. Moreover, KO mice have suppressed rates of fat oxida-tion (WT: 1045±27 vs. KO: 859±37g/kg/h; p<0.001), reduced muscle TGs, and are protected from liver lipid accumulation (cholesterol WT: 1.8±0.4 vs. KO: 1.1±0.1microg/mg; p<0.05 and triglyceride WT: 34.1±3.5 vs. KO: 17.1±1.9 mi-crog/mg; p<0.0001) likely altering energy distribution. Accordingly, STRs are upregulated in the adipose tissue of WT mice in response to HFD and their expression positivity correlates with fat mass (r2=0.30; p<0.01) and glucose intolerance (r2=0.21; p<0.05). Collectively, these data highlight a critical un-recognized role of STR signaling in the regulation of glucose homeostasis and exemplify the signifi cance of glucose sensing during the development of metabolic disease independent of its metabolism.

2128-PIRF4, a New Partner of PGC-1α, Plays a Role in ThermogenesisXING XING KONG, EVAN D. ROSEN, Boston, MA

Brown fat can reduce obesity through the dissipation of calories as heat. Control of thermogenic gene expression occurs via the induction of various co-activators, most notably PGC-1α. In contrast, the transcription factor partner(s) of these co-factors are poorly described. We recently have iden-tifi ed interferon regulatory factor 4 (IRF4) serves as a key molecular node in virtually all of the metabolic actions of adipose tissue. Specifi cally, we have demonstrated that IRF4 regulates lipolysis and lipogenesis in white and brown adipocytes, thermogenesis in brown adipocytes, and M2 polarization in adipose-resident macrophages. Here we demonstrate that IRF4 can coor-dinate PGC-1α to manipulate thermogenesis in BAT. First, we found that IRF4 can interact with PGC-1α, by employing Co-IP and GST-pull down assays. Moreover, IRF4 activated the mouse UCP1 promoter, which was mediated by a putative interferon-stimulated response element (ISRE). This activity can be strongly stimulated by co-transfected with PGC-1α. Chromatin im-munoprecipitation assay confi rmed that IRF4 bound to the identifi ed ISRE. Finally, cold, β-agonists, or forced expression of PGC-1α are unable to cause thermogenic gene expression in the absence of IRF4. Our results indicate that IRF4 functions as a partner of PGC-1α and mediates the PGC-1α effects on thermogenesis.

Supported By: American Heart Association

2129-PThe Cannabinoid Receptor-1 Is an Imaging Biomarker of Brown Adi-pose TissueOLOF ERIKSSON, KIRSI MIKKOLA, DANIEL ESPES, KIRSI A. VIRTANEN, SARITA FORSBÄCK, LAURI TUOMINEN, MERJA HAAPARANTA-SOLIN, JARMO HIETALA, OLOF SOLIN, PIRJO NUUTILA, Turku, Finland, Uppsala, Sweden

The existence of deposits of brown adipose tissue (BAT) in human adults was recently confi rmed. Its role in the human metabolism in unknown but could be substantial. Inhibition of the Cannabinoid Receptor 1 (CB1) has been associated with activation of BAT thermogenesis and weight loss. Clearly, the role of peripheral and central CB1 in the activation of BAT merits further investigation. Here we developed a technique for quantifying CB1 in BAT by positron emission tomography (PET).

Sections of human and rat BAT and subcutaneous white adipose tissue (WAT) were immunofl uorescently (IF) stained for CB1 and UCP-1. We found that CB1 was co-localized with UCP-1 in BAT (Fig. A-C), but neither protein was found in WAT.

Binding of the radiolabeled and clinically available CB1 antagonist [18F]FMPEP-d2 to BAT in vivo and in vitro was assessed in human and rat by PET. Binding of the radiotracer to BAT sections (but not WAT) in vitro was high and displaceable by pretreatment with rimonabant. Deposits of BAT in human and rat had signifi cant binding of [18F]FMPEP-d2 in vivo, indicating high CB1 receptor density (Fig. D-E). WAT deposits were negative, consistent with the IF staining and in vitro results. We conclude that [18F]FMPEP-d2 PET can quantify CB1 density non-invasively in vivo. CB1 is thus a promising imaging biomarker for assessing the presence of BAT deposits, as well as for eluci-dating the mechanism of CB1 antagonist mediated weight loss in humans.

2130-PGLP-1 Receptor Agonist Protects against Obesity by Stimulating Li-polysis and Fatty Acid Oxidation in White Adipose Tissue through Activation SIRT1FEN XU, BEISI LIN, KEJING ZENG, ZONGLAN CHEN, ZHUO LI, HUA LIANG, HAIXIA XU, JIANPING WENG, Guangzhou, China

Accumulating evidences have been pointed to the signifi cant role of GLP-1 and its mimetics in contributing to suppression of weight gain and obesity, but the precise molecular mechanisms responsible for these interactions remains largely unknown. We herein try to dissect the underlying mecha-nisms of GLP-1 receptor agonist exenatide’s effects on lipid metabolism in white adipose tissue. In HFD challenged C57BL/6 mice, exenatide could sig-nifi cantly reduce adiposity and improve lipid metabolism especially lipolysis. However, in exenatide-treated SIRT1+/- mice, adiposity was still obvious and adipocytes were more hypertrophy compared to wild-type mice under HFD challenging. It inspired us to examine the SIRT1-dependent lipolytic effect of exenatide in adipocytes. We found that lipid droplets were reduced and lipolysis was increased in adipocytes by exenatide/exendin-4 treatment both in vivo and in vitro, accompanied with increased SIRT1 level or activ-ity. Whereas the above response diminished in adipocytes when absence of SIRT1. As a result, SIRT1 targets of fatty acid oxidation gene profi le contain-ing UCP1, PPARα, and PGC-1α were all increased by exenatide/exendin-4 but were abolished when lack of SIRT1. Collectively, our data highlight that GLP-1 receptor agonist, as a promising novel regulator of adipocyte energy homeostasis by enhancing lipolysis and fatty acid oxidation through activa-tion SIRT1, provides a potential strategy in the treatment of obesity and metabolic disorders.

Supported By: National Natural Science Foundation of China (81300705)

2131-PDeletion of Epsins in Adipocytes Attenuates Obesity in High-Fat Diet-induced Animal ModelYUNZHOU DONG, SCOTT HAHN, LILI YU, TIMOTHY GRIFFIN, HONG CHEN, Okla-homa City, OK

Epsins are adaptor proteins implicated in endocytosis which modulate angiogenesis and embryogenesis. The aim of current study is to investigate the role of epsins in a high fat diet-induced obesity animal model. We have generated inducible double knockout (iDKO) mice for epsin genes globally by driving the expression of cre recombinase. Our data show that iDKO mice fed on HFD were drastically resistant to body weight (BW) gain. The expres-sion of epsins is upregulated in ob/ob mice or HFD-induced obese models. Glucose tolerance test and insulin tolerance test indicate that insulin sen-sitivity is greatly better in iDKO mice. Lipid profi le of serum suggests that

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triglyceride (TG) and LDL/VLDL are not signifi cantly changed in iDKO mice. In liver, TG content was dramatically reduced in iDKO mice. Furthermore, mitochondrial respiratory exchange ratio, oxygen consumption and energy expenditure are similar in iDKO mice compared to controls on HFD. Relative food intake (normalized to BW) for iDKO mice is the same as controls. Leptin and insulin levels in circulation are signifi cantly reduced in iDKO mice on HFD. Mechanistically, deletion of epsins in adipocyte using aP2-cre gener-ated the similar phenotype as iDKO mice and the expression of adipogetic genes PPAR-g, CEBPα/β, aP2 and Fox2 are all downregulated revealed by qPCR. The development of epididymal white fat in epsin-/-/aP2-cre mice is drastically attenuated. In vitro adipocyte differentiation using 3T3-L1, MEF and SVF cells in epsin-defi cient genetic background is signifi cantly attenu-ated in Oil Red O staining analysis, accompanying with the reduced adipoge-netic gene expression. Furthermore, we have identifi ed that epsins directly bind to IRS1 to stabilize the IRS/IR/IGF-R1 complex. We conclude that epsins are critical regulator of obesity, and loss of epsins attenuates HFD-induced obesity through a novel mechanism by the disruption of the insulin signaling dependent adipogenesis.

Supported By: Oklahoma Center for the Advancement of Science and Technol-ogy (AH14-056 to Y.D.); American Heart Association (12SDG8760002 to Y.D.); R01HL-093242, R01HL118676, P20RR018758 (to H.C.)

2132-PQuantitative Comparative Proteomic Analysis of Serum Proteins Associated with the Progression of Diabetes in db/db MouseMAYU KYOHARA, JUN SHIRAKAWA, AYUKO KIMURA, HISASHI HIRANO, YA-SUO TERAUCHI, Yokohama, Japan

The db/db mice, an obese diabetes model, show transient beta cell pro-liferation, insulin resistance, and diabetic complications with age. Here we sequentially analyzed serum proteins in db/db mice and db/+ mice at 4, 8, 12, and 24 weeks of age (n = 4) by comparative proteomic analysis using a label-free mass spectrometry. Among 212 identifi ed proteins, 26 proteins were increased and 48 proteins were decreased signifi cantly (p<0.05) in db/db mice compared to db/+ mice at 4 weeks of age. Serum adiponectin levels were markedly decreased in db/db mice after 24 weeks of age (0.10-folds, p<0.001). Apo C-III levels were gradually elevated in db/db mice with age. CRP and RBP-4, proteins involved in infl ammation and insulin resistance, were increased in db/db mice through all the experiments from 4 weeks to 24 weeks of age. Hence the results of serum proteomic analysis in this study clearly refl ected dyslipidemia, chronic infl ammation, and insulin resistance of db/db mice. To identify causal factors for insulin resistance, beta cell fail-ure, or diabetic complications, we focused on the proteins whose expression levels were signifi cantly changed only before development of severe diabe-tes, namely at 4-8 weeks of age. There were 5 up-regulated proteins such as Pigment epithelium-derived factor (PEDF) and Pon1, and 11 down-regulated proteins such as CD5L and IGFBP2 in db/db mice at 4 weeks-old, but not at older ages. Since PEDF is reportedly related to HOMA-IR of obese or dia-betes patients, increased serum PEDF level may indicate the presence of insulin resistance in the pre-diabetic phase in db/db mice. We also identifi ed 7 up-regulated proteins (e.g., SerpinA6, PLTP) and 4 down-regulated proteins (e.g., Apo B-100, MCG116526) only in both 4 and 8 weeks-old db/db mice sera. The functions of other identifi ed proteins have been mostly unknown in diabetes, suggesting that some of those might be novel regulators of dia-betes, obesity, beta cell proliferation, and diabetic complications.

2133-PA Homodimer Scan Identifi es PEGylated PYY3-36 Conjugates with Greatly Improved Potency and SelectivityJAMES LEONARD, DEREK STEINER, VERONICA MORENO, WILMELENNE CLAP-PER, KATHERINE FIGUEROA, TERRANCE BARRETT, CELIA JENKINSON, REBECCA PICK, ARTHUR SUCKOW, RONALD SWANSON, Spring House, PA, San Diego, CA, Carlsbad, CA, Gaithersburg, MD

PEGylation is a commonly used strategy to extend the half-life of biologi-cally active peptides, but often results in markedly reduced potency. More-over, peptides such as PYY3-36 are not suitable for recombinant fusion ap-proaches due to required post-translational modifi cations. We employed a cysteine scan of PYY3-36 in combination with conjugation to monofunctional and bifunctional linear 30 kDa PEG molecules to generate a series of PYY-PEG monomers and homodimers. A small number of cysteine-substituted PEGylated PYY3-36 homodimers were equipotent to native PYY3-36, and in all cases were several-fold more potent than the corresponding PEGylated monomer. G9C substituted PYY3-36 PEG homodimer (“PYY-HD”) was selected for further characterization.

In addition to its robust potency, PYY-HD was signifi cantly more Y2R-selective than native PYY3-36, with reductions in potency observed at Y1R and Y5R. In high-fat diet-induced (DIO) mice, s.c. administration of PYY-HD reduced food intake acutely. When compared to its corresponding mono-meric PEG analog, PYY-HD was more potent at reducing food intake in DIO mice. Sustained reductions in body weight were observed with daily s.c. administration of PYY-HD for 2 weeks in DIO mice.

The effi cacy of PYY-HD was also assessed in combination with the GLP1 receptor agonist liraglutide. Acutely, PYY-HD alone had no effect on glucose tolerance, but surprisingly amplifi ed the effi cacy of liraglutide. The improved effi cacy was observed in association with enhanced plasma levels of in-sulin. Two-week co-administration of PYY-HD and liraglutide in db/db mice mediated very robust improvements in body weight and fasting plasma glu-cose levels, relative to either agent alone, and could not be replicated with pair-feeding to the combination treatment group. Collectively, these data show that PEG homodimerization via site-specifi c conjugation of PYY3-36 is an effective strategy to produce long-acting analogs with greatly improved potency.

2134-PEndogenous Catalase Protects Adipocyte Injury in High Fat-fed MiceLINGJUAN PIAO, HYUNJI KIM, HUNJOO HA, Seoul, Republic of Korea

The incidence of obesity and obesity-associated diseases has become a worldwide epidemic. Increased oxidative stress in adipocyte is an important mechanism of obesity-associated metabolic disorder. Catalase, a major per-oxisomal antioxidant, is highly expressed in the white adipose tissue (WAT) as well as livers. Adipose catalase expression was signifi cantly suppressed in obese mice, indicating its association with obesity. We, therefore, hypoth-esized that endogenous catalase would play an important role in adipocyte fi tness. Catalase knockout (KO) and age-matched wild type (WT) C57BL/6J male mice were fed a normal diet (18% of total calories from fat) or a high fat (HF, 60% of total calories from fat) diet. At 12 weeks after HF feeding, body weight was signifi cantly higher in HF-fed catalase KO mice than in HF-fed WT mice. Absolute subcutaneous and epididymal WAT from HF-fed catalase KO mice weighed more compared to those from HF-fed WT mice. This change was consistent when corrected by body weight. Consistently, phosphorylation of hormone-sensitive lipase which catalyzes triglyceride to diacylglycerol and free fatty acids, was downregulated in HF-fed catalase KO mice than HF-fed WT mice. Macrophage infi ltration estimated by F4/80 immunostaining and mRNA expression was the highest in HF-fed catalase KO mice. Plasminogen activator inhibitor-1 (PAI-1) in WAT as well as plasma of HF-fed catalase KO mice was higher than those in HF-fed WT mice. To dis-sect the role of endogenous catalase in adipocyte, we treated differentiated 3T3-L1 adipocyte with 3-aminotriazole (3-AT), an inhibitor of catalase activ-ity. 3-AT dose-dependently downregulated mRNA expression of adiocyte triglyceride lipase and adiponectin but upregulated PAI-1 and TNFa mRNA expression. Collectively, these data suggest that endogenous catalase may play an important role in maintaining adipocyte fi tness under HF-induced metabolic stress.

2135-PDietary Soy Isofl avones Improve Insulin Sensitivity and Reduce Adi-posity in Female Low-Fit Rats, Independent of Ovarian StatusTERESE M. ZIDON, BRIDGET E. UPTON, REBECCA J. WELLY, YOUNG-MIN PARK, KATHERINE S. WAINRIGHT, REBECCA J. SCROGGINS, STEVEN L. BRITTON, LAU-REN G. KOCH, JAUME PADILLA, VICTORIA J. VIEIRA-POTTER, Columbia, MO, Ann Arbor, MI

Ovarian hormone loss reduces energy expenditure (EE) and is associ-ated with obesity and insulin resistance (IR), increasing risk of type II dia-betes. Phytoestrogens (e.g., soy isofl avones, SI) improve postmenopausal symptoms including metabolic and cardiovascular parameters. The purpose of this study was to determine whether increased dietary SI attenuates ovariectomy (OVX)-associated metabolic dysfunction in rats bred for low intrinsic aerobic fi tness (LCR), susceptible to develop IR after OVX. Female LCR rats (n=40; 20 wks old) received OVX or sham (SHM) surgery and were fed either a high SI (600ppm) or control (<15ppm, C) diet creating: SHM/C, SHM/SI, OVX/C, and OVX/SI (n=10/grp). SI did not reduce energy intake, yet attenuated weight gain by ~14% among SHM and ~5% among OVX (no ovar-ian status x line interaction), with OVX gaining more weight than respective SHM controls (main effects of OVX vs. SHM and C vs. SI, p<0.05). These differences were corroborated by body composition via EchoMRI and fat pad weights at the end of the study (C vs. SI and OVX vs. SHM, p<0.01 for all). 18 wks post-surgery, rats were assessed for spontaneous physical activity

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(SPA) and EE via metabolic chambers. Although SI did not increase SPA in either group, total and resting EE relative to body mass increased with SI (C vs. SI, p<0.05). Glucose tolerance tests with insulin response performed 20 wks post-OVX revealed IR via Matsuda index was completely prevented by SI and also improved among SHM groups (C vs. SI and OVX vs. SHM, both p<0.01). In conclusion, increased SI reduces adiposity, likely via increased EE, and improves insulin sensitivity in both OVX and SHM LCR female rats. Analyses are underway to identify tissue-specifi c molecular mechanisms by which SI offer metabolic protection.

Supported By: Missouri University Center for Botanical Interaction Studies (to V.J.V-P.)

2136-PInhibiting ERRa Blocks Liver Steatosis Induced by PTEN LossBANGYAN L. STILES, YANG LI, CHIEN-YU CHEN, LINA HE, NI ZENG, Los Angeles, CA

The prevalence of obesity has markedly increased over the past two de-cades. Obesity is recognized as a key factor in the development of diabetes and cardiovascular diseases, and more recently cancer. In this project, we intended to understand the role of Estrogen-related receptor α (ERRα), a master regulator that orchestrate mitochondria response to modulate me-tabolism. Mitochondrial dysfunction has been attributed to be a major cause for fatty liver development. However, loss of ERRα, a master transcriptional factor for maintenance of mitochondrial function, led to resistance to high fat induced obesity rather than increased lipid deposition. Using a liver-con-ditional Pten deletion model where the activation of its downstream PI3K/AKT signaling led to fatty liver, we investigated whether and how ERRα reg-ulates the lipid accumulation in the liver. We found that PTEN loss resulted in a dramatic induction of ERRα leading to increased reactive oxygen species (ROS) production, which may propagate lipid oxidation, mitochondrial dam-age and lipid accumulation. In addition, when ERRα expression is attenu-ated with siRNA or inhibited by a small molecule polyamide that interrupts ERRα binding to DNA, we observed signifi cant downregulation of lipogenic regulatory genes. Furthermore, delivery of the small molecule polyamide to inhibit ERRα action in vivo remarkably rescued the fatty liver phenotype and reduced the hepatic triglyceride level. Thus, our study highlights ERRα’s crucial role in regulating mitochondrial bioenergetics and underscores its therapeutic potential in lipid disorders.


2137-PMyeloid Cell-specifi c HIF-1 alpha Deletion Protected against Insu-lin Resistance with Increased Angiogenesis in High Fat-fed MiceAKIKO TAKIKAWA, ARSHAD MAHMOOD, ALLAH NAWAZ, TOMONOBU KADO, SATOKO SENDA, KEISUKE OKABE, ISAO USUI, KAZUYUKI TOBE, Toyama, Japan

Adipose tissue becomes hypoxic as obesity progresses. We investigated the role of hypoxia-inducible factor (HIF)-1α, a key factor to hypoxic condi-tions, in myeloid cells using myeloid cell-specifi c Hif-1α knockout mice (Mye-HIF-1α KO). Mye-HIF-1α KO mice and control mice were analyzed after 18 week HFD treatment. Mye-HIF-1α KO mice showed improved glucose toler-ance with lower serum insulin concentrations and improved insulin sensitiv-ity. Mye-HIF-1α KO mice have fewer crown-like-structures in adipose tissue along with the reduced expression of CD11c, an M1 macrophage marker, hypoxia-related genes and infl ammatory genes. In the liver, expression of F4/80 and CD11c but not an M2 macrophage marker CD206, was markedly decreased as well as gluconeogenic genes in KO mice. We also investigated hypoxic condition in adipose tissue of the mice. Pimonidazole, a hypoxic probe, was less accumulated and tissue perfusion was increased in adipose tissue from Mye-HIF-1α KO mice than those from control mice. The gene expression of angiogenic factors except VEGF, such as Ang1, FGF1 and FGF2 was elevated in the adipose tissue from Mye-HIF-1α KO mice. To clarify the mechanism of the increase in angiogenesis in Mye-HIF-1α KO mice, we sep-arated macrophage, endothelial cells and PDGFRα-possitive adipocyte pro-genitors from epididymal adipose tissue and performed quantitive PCR. The gene expression of angiogenic factors including Ang1, FGF1, FGF2 and FGF10 was signifi cantly increased in the adipocyte progenitor fraction but not mac-rophages nor endothelial cells. Adipocyte progenitors are a possible sources of angiogenic factors in adipose tissue of obese mice. In conclusion, HIF-1α in myeloid cells contributes to the development of pathological expansion of adipose tissue and insulin resistance with inducing infl ammatory response and suppressing angiogenesis mediated by adipose tissue hypoxia.

2138-PA Role for Wnt Signaling in the Reversal of Metabolic Disturbances after Gastric Bypass in Obese MiceROMAN VANGOITSENHOVEN, BART VAN DER SCHUEREN, JOAO PAULO MON-TEIRO CARVALHO MORI CUNHA, MATTHIAS LANNOO, CHANTAL MATHIEU, LUT OVERBERGH, MICHAEL MARIS, Leuven, Belgium

Gastric bypass surgery has benefi cial effects on obesity-induced meta-bolic disturbances, but the underlying mechanisms remain unclear. As Wnt genes are both positively and negatively correlated with obesity-induced adipose tissue infl ammation and insulin resistance, our main aim is to reveal a potential role for Wnt signaling in the reversal of these metabolic conse-quences after gastric bypass surgery in mice.

8-weeks old male C57Bl/6 mice were fed a high fat diet (60 kcal% fat) for 14 weeks, followed by either Roux-en-Y gastric bypass (RYGB; n=13) or Sham surgery (Sham; n=10). After surgery, mice were maintained on a high fat diet for another 8 weeks (Sham group was pair fed with the RYGB group). Using qRT-PCR, the mRNA expression profi le of Wnt ligands and its antagonists (i.e. secreted Frizzled-related proteins (sFRP)) was determined in epididymal white adipose tissue of RYGB and Sham mice.

8 weeks post-surgery, Sham mice returned to their pre-surgery weight (43.4 g ± 6.9), whereas RYGB mice showed a 43% reduction in body weight, accompanied by improved metabolic parameters, as compared to Sham mice (P < 0.01). Epididymal white adipose tissue of RYGB mice showed a 75% and 94% decrease in sFRP4 and sFRP5 mRNA expression levels respectively as compared to Sham mice (P <0.01). Additionally, a trend towards decreased mRNA expression of Wnt5a and Wnt5b could be detected in RYGB mice as compared to Sham mice (P = 0.1).

In conclusion, RYGB surgery strongly reduced the expression level of the secreted Wnt antagonists sFRP4 and sFRP5 in white adipocyte tissue. As secreted proteins are interesting drug targets, a potential role for these se-creted proteins in the reversal of metabolic disturbances after RYGB surgery will be studied more in depth.

Supported By: FWOG.0857.13

2139-PHuman Kallistatin Predisposes to Adiposity but Preserves Insulin Sensitivity in MiceJULIA REINKE, DIANA WILLMES, JOSPEH TIO, JEFFREY D. MCBRIDE, JIAN-XING MA, ANDREAS L. BIRKENFELD, Berlin, Germany, Dresden, Germany, Oklahoma City, OK

The circulating serine proteinase inhibitor Kallistatin (KST), also known as SERPIN A4, has antiinfl ammatory and antioxidative properties. Recent studies in humans observed a negative association between plasma KST levels and body fat and markers of glucose homeostasis. Whether or not KST has direct effects on human metabolic control remained unclear. To elucidate possible metabolic actions, transgenic mice overexpressing hu-man KST systemically (hKST-TG) and littermate control wildtype mice where studied on a regular chow. Human plasma KST levels were 6.8±3.1 µg/ml in hKST-TG and not detectable in littermate-control mice. Body weights (bw) were similar between hKST-TG and littermate-control mice up to an age of 24 weeks. However, body composition analysis, using nuclear magnetic res-onance (NMR)-spectroscopy, revealed an increased fat mass and reduced lean mass in hKST-TG mice (16.83±3.29% body fat (bf), hKST-TG, 11.89 ± 3.71% bf, littermate-control mice, P<0.01). Moreover, we observed increased epigonadal (1.1±0.2% of bw, hKST-TG, 0.6±0.3% of bw, littermate-control, P=0.002), subcutaneous (0.5±0.2% bw, hKST-TG, 0.3±0.1% bw, littermate-control, P=0.001), and perirenal white adipose tissue mass (0.2±0.1% bw, hKST-TG, 0.1±0.1% bw, littermate-control, P<0.01), in hKST-TG mice in line with the NMR results. The respiratory exchange ratio (RER) (0.88±0.06, hKST-TG, 0.94±0.06, littermate-control, P<0.01), food intake (2.5±1.1 g, hKST-TG, .4±1.1 g, littermate-control, P<0.01) and punctual physical activity where reduced in hKST-Tg mice. Interestingly, IPGTTs yielded similar glucose and insulin excursion curves between groups. In summary, overexpression of hu-man KST predisposed mice fed a regular chow to adiposity and had marked effects on energy homeostasis, while whole body glucose tolerance was preserved. These data suggest that KST may play a role in the regulation of human energy homeostasis. Further studies are needed to determine under-lying molecular mechanisms.

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2140-PC2 Domain-containing Protein CDP138 Is Involved in Body Weight RegulationQIONG L. ZHOU, YE SONG, JUN-YUAN HUANG, ZHENWEI GONG, ANDRIA G. SHARMA, DANIELLE CONNEELY, KAVIN ZHU, ZHEN Y. JIANG, Boston, MA, Or-lando, FL

CDP138 is a C2 Domain containing Phosphoprotein that has been indicated as an Akt downstream target and is involved in insulin-regulated GLUT4 trans-location and glucose transport in cultured 3T3 L1 adipocytes. Here we report impaired lipid metabolisms in mice with whole-body deletion of CDP138. In this study, CDP138-/- mice and age- and sex-matched WT mice wild type (WT) mice were fed with normal chow diet (NCD) or high-fat diet (HFD, 60% of fat calo-ries). At 22 weeks old with NCD feeding, CDP138-/- mice average body weight was 29,38 gram with 9.82 gram of fat, while average body weight of WT mice was around 22.37 with 3.48 gram of fat. After feeding with a HFD for 8 weeks, CDP138-/- mice average body weight reached 42.54 gram, but WT mice aver-age body weight only reached to 31.48 gram. The signifi cant difference in body weight between CDP138-/- mice and WT mice is mainly due to more fat ac-cumulation in CDP138-/- mice. Metabolic cage analysis revealed that compared to WT mice, CDP138-/- mice had signifi cant lower energy expenditure and oxy-gen consumption during both light and dark periods on NCD. Biochemically, we analyzed the lipolysis pathway in both brown and white adipose tissues from CDP138-/- and WT mice and found that hormone sensitive lipase (HSL) phospho-rylation was signifi cantly decreased in CDP138-/- mice compared to WT mice. In addition, intracellular cAMP levels in BAT from CDP138-/- mice were signifi cant lower compared to WT mice under both NCD and HFD conditions.

Taken together, these data suggest that CDP138 plays an important role in mouse lipid metabolism.

Supported By: American Diabetes Association (7-11-BS-72 to Z.Y.J.); National Institute of Diabetes and Digestive and Kidney Diseases

2141-PKlk7 Plays a Role in Body Fat Regulation and Insulin SensitivityANNE KUNATH, MATTHIAS KERN, MICHAEL STUMVOLL, MATTHIAS BLÜHER, NORA KLÖTING, Leipzig, Germany

Previous studies showed that benefi cial effects on glucose metabolism of vaspin are at least in part mediated through inhibition of the protease Kallikrein 7 (Klk7). Based on these fi ndings, we investigated the physiologi-cal relevance of the Klk7-vaspin system in mice with a targeted whole-body disruption of Klk7 in vivo.

We generated a whole-body Klk7 knockout mouse (Klk7-/-) and systematically characterized the consequences of Klk7 defi ciency on body weight, fat mass, serum concentrations of leptin, basal metabolism, food intake and parameters of glucose and lipid metabolism under chow and high fat diet conditions (HFD).

Klk7 defi ciency causes signifi cant changes in body fat content, basal metabolism, spontaneous activity, food intake as well as leptin serum con-centrations. At an age of 30 weeks, Klk7-/- mice have signifi cantly lower body fat (7.2±2.9%) than the controls (9.9±2.7%) (p<0.05). Moreover, Klk7-/- mice have signifi cantly lower serum leptin concentration (25.9±6.2ng/ml) in relation to the controls (42.3±2.5ng/ml) (p<0.05). In female Klk7-/- mice, food intake (4.3±0.5g/day) was signifi cantly lower compared to the controls (4.7±0.3g/day, p<0.05). Although glucose tolerance was indistinguishable between Klk7-/- and control mice of both genders, male Klk7-/- mice display a signifi cant improved insulin sensitivity compared with littermate controls at an age of 24 weeks (p<0.05). Both genders of Klk7-/- mice showed a dif-ferent basal energy expenditure at day and night phase compared with con-trols. Klk7 knockouts are not protected from diet induced obesity but male knockouts remained still insulin sensitive after HFD.

Our data indicate that Klk7 plays a previously unrecognized role in body weight regulation, food intake, insulin sensitivity as well as basal- and glu-cose metabolism. The mechanisms, how Klk7 disruption affects these traits need to be explored in further studies.

Supported By: German Federal Ministry of Education and Research (to M.B., N.K.)

2142-PEffects of Teneligliptin on Glucose and Energy Metabolism in a Mice Model of Postmenopausal ObesityAZUSA SAMESHIMA, TSUTOMU WADA, TETSUO ITO, AYAKA KASHIMURA, RIKA YONEZAWA, HIROSHI TSUNEKI, TOSHIYASU SASAOKA, Toyama, Japan

Decrease of serum estrogen in menopause is closely associated with the development of visceral obesity and onset of type 2 diabetes in women. Recently, incretin based therapies can be used as a novel approach for the treatment of type 2 diabetes. However, the effects of DPP-4 inhibitor in postmenopausal women are currently not well studied. Therefore, we

investigated the impact of the novel DPP-4 inhibitor, teneligliptin, on body weight, glucose metabolism, fat accumulation, and energy expenditure in a mice model of postmenopausal obesity.

Eight-week-old female C57Bl/6J mice were ovariectomized (OVX) or sham operated. After 1 week of recovery, sham mice were fed standard diet (Sham), and OVX mice were fed high fat diet for 12 weeks (OVX-HF). For the treatment group, teneligriptin at 60 mg/kg/day was administrated with drinking water in ovariectomized and high fat diet-fed mice.

OVX-HF mice showed increased body weight and accumulation of both visceral and subcutaneous fat. OVX-HF mice demonstrated marked eleva-tions of blood glucose during OGTT. In contrast, administration of teneliglip-tin markedly attenuated body weight gain, fat accumulation, and improved glucose intolerance without changing food consumption. In fl ow cytom-etry analysis, marked accumulation of CD11c-positive M1 macrophage in perigonadal fat was signifi cantly attenuated by the treatment. Importantly, decreased energy consumption in both dark and light phase, reduced loco-motor activity in dark phase, and lowered core body temperature in OVX-HF mice were ameliorated by teneligliptin.

These results indicate that teneligliptin effectively improved glucose me-tabolism in postmenopausal obese mice by amelioration of lowered energy metabolism and body fat gain. Since reduced energy metabolism is common physiology of menopause, teneligliptin appears to be a benefi cial anti-diabetic drug for the treatment of type 2 diabetes with postmenopausal obesity.

2143-PAblation of Pi3-kinase γ Increases Energy Expenditure through Brown Fat ActivationLUIZ O S. LEIRIA, ANDREY DOS SANTOS, MARCOS L. BRIOSCHI, ALEXANDRE GABARRA. OLIVEIRA, GUILHERME Z. ROCHA, RODRIGO M. MARIN, MAURO M. TEIXEIRA, YU-HUA TSENG, MARIO J. SAAD, Campinas, Brazil, São Paulo, Brazil, Belo Horizonte, Brazil, Boston, MA

Phosphatidilinotol-3 kinases (PI3Ks) constitute a family of lipid kinases that regulates a wide range of essential cellular processes. The Class1B PI3K consists of only one member, the PI3Kγ, which operates downstream of G protein-coupled receptors (GPCRs) and is activated by trimeric G protein Gβ,γ subunits. Deletion of PI3Kγ results in prevention against diet-induced obesity. In the present study, we aimed to investigate whether PI3Kγ regu-lates brown adipose tissue (BAT) function and energy expenditure. We used PI3K p110γ knockout mice (PI3Kγ-/-) aging 12 to 14 weeks and their respective aged matched C57BL/6 wild type mice as a control group. PI3Kγ-/- mice dis-played reduced weight gain and improved glucose homeostasis. Darkening of BAT was visible to the eye in the PI3Kγ-/-, that was confi rmed by histologi-cal analysis through either haematoxylin & eosin staining or increased UCP1 staining in slices of BAT from PI3Kγ-/- mice. mRNA expression of the genes involved in the thermogenic program, such as Ucp1, Prdm16, and Cidea, was increased in the BAT of PI3Kγ-/- mice (P<0.05). PI3Kγ-/- animals also exhib-ited increased VO2 and CO2 consumption measured by indirect calorimetry (CLAMS), while no changes in food intake was observed. Moreover, thermo-graphic measurements showed that BAT temperatures were signifi cantly el-evated in PI3Kγ-/- mice in both room temperature (22.5ºC) and cold exposed (4ºC, 3h) conditions (P<0.05). Rectal temperature of PI3Kγ-/- remained higher under cold exposure in comparison with the WT group (P<0.05). PI3Kγ-/- mice were protected against obesity and insulin resistance induced by high-fat-diet. Under high-fat feeding, PI3Kγ-/- mice exhibited darkening of BAT, and increased mRNA expression of the genes involved in thermogenic program in the BAT. In conclusion, this study unravels a role for PI3Kγ-/- as a negative regulator of the BAT function, and its inhibition/deletion represents a poten-tial way to induce BAT activation to improve energy expenditure.

Supported By: Fundação de Amparo à Pesquisa do Estado de São Paulo

2144-PIdentifi cation of Novel Adipocyte Progenitor Cells Expressing Stem Cell Markers and Adipocyte Specifi c Proteins in Stromal FractionKOICHIRO TAGUCHI, KAZUO KAJITA, YOSHIHIRO KITADA, ICHIRO MORI, TAKA-HIDE IKEDA, MASAHIRO YAMAUCHI, TATSUO ISHIZUKA, HIROYUKI MORITA, Gifu, Japan

We have reported that small adipocytes expressing proliferative activ-ity contribute to growth of adipose tissue. Moreover, small round cells ex-pressing adipocyte specifi c proteins such as adiponectin, leptin, aquaporin 7 and Glut4 (small adipocytes: SA) were detected in the stromal fraction. In this study, we studied the feature of SA. We also found SA in 3T3-L1 pre-adipocytes. SA were confi rmed by uptake of EdU and survived in soft agar medium or fetal bovine serum free medium. SA formed a colony when they were cultured with fi broblast-like cells. SA was distinguished as a popula-

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tion whose mean diameter was under 10 µm from fi broblast-like cells whose diameter was over 15 µm by fl ow cytometric analysis. In addition, SA ex-pressed preadipocyte factor-1 (Pref-1), CD 105, CD24 and stem cell antigen-1 more markedly than fi broblast-like cells. The expression levels of adipocyte specifi c genes such as PPARγ2, FABP-4, adiponectin and leptin in SA were approximately the same as those in the intermediate of the mature adipo-cytes and stromal vascular cells. The expression levels of Pref-1 and CD105 in SA were equivalent to those in stromal vascular cells. Adiponectin and leptin were detected in the medium in which 3T3-L1 pre-adipocytes were cultured. SA accumulated lipid when stimulated by pioglitazone only. In con-clusion, we have identifi ed novel adipocyte progenitor cells, named as SA, which exhibit the property of both stem cells and adipocytes, and easily differentiate into adipocytes. SA might secret adipokines as endocrine cells and play a role as an adipocyte precursor in adipose tissue.

2145-PAdipose-specifi c Dipeptidyl Peptidase-4 (DPP-4) Knockout Mice Display Improved Fasting Insulin and Cholesterol Levels Despite Increased Weight Gain on HFDHENRIKE SELL, DIANA RÖHRBORN, IRA INDRAKUSUMA, TOMAS JELENIK, TA-MARA R. CASTAÑEDA, HADI AL-HASANI, TANIA ROMACHO, MICHAEL RODEN, JÜRGEN ECKEL, Düsseldorf, Germany

DPP-4 is a protease cleaving incretins, and therefore a target for treating type 2 diabetes. In addition, DPP-4 is an adipokine that is increased in the obese state and correlates with parameters of the metabolic syndrome. The aim of this study is the role of adipose tissue (AT)-derived DPP-4 in the develop-ment of obesity-associated metabolic diseases with a unique animal model.

We generated AT-specifi c DPP-4 knockout mice using an aP2-Cre trans-gene with the Cre-lox system. Metabolic phenotyping of mice on chow diet and HFD (60% kcal fat) over 24 weeks was performed using metabolic cages, NMR, glucose and insulin tolerance tests (ipGTT, ipITT), and eugly-cemic hyperinsulinemic clamps. Serum DPP-4, DPP-4 expression in mature adipocytes and stroma-vascular fraction (SVF) as well as plasma levels of glucose, insulin, triglycerides and cholesterol were measured.

DPP-4 expression in mature adipocytes from KO mice was reduced up to 65% (p<0.01) with unchanged expression in the SVF. Serum DPP-4 was sig-nifi cantly lower in KO animals on both diets. KO mice gained signifi cantly more weight, fat and lean mass on HFD. However, body fat percentage and length remained similar. No infl uence of the genotype on total energy expenditure, respiratory quotient and spontaneous physical activity was observed. ipGTT, ipITT and clamps were affected by HFD but not by genotype. On the other hand, fasting insulin and HOMA-IR were signifi cantly lower in KO mice on HFD despite unchanged insulin sensitivity. Cholesterol was reduced in KO mice on chow and on HFD compared to WT but triglyceride levels were similar.

Our model demonstrates that AT is a major source of DPP-4 in mice. Although KO mice under HFD gain more weight and fat mass, this is not followed by decreased glucose tolerance. However, HFD-induced hyperin-sulinemia is reduced in KO mice indicating that AT DPP-4 could be a player in HFD-induced metabolic disturbances.

Supported By: European Foundation for the Study of Diabetes; European Com-mission (ADDIO-PIEF-2012-328793)

2146-PA Novel Dual Amylin and Calcitonin Receptor Agonist (KBP-089) for the Treatment of ObesitySOFIE GYDESEN, SARA T. HJULER, KIM VIETZ. ANDREASSEN, MORTEN A. KARSDAL, KIM HENRIKSEN, Herlev, Denmark

There is an urgent medical need for treatments which substantially re-duce body weight, thereby affecting the metabolic syndrome. Amylin and/or calcitonin receptor agonists such as pramlintide and davalintide have shown promise on weight reduction in preclinical models and clinical settings, me-diated through reduced food intake and increased energy expenditure, albeit with limited effi cacy on glucose homeostasis.

KBP-089 is a dual amylin- and calcitonin receptor agonist, based on struc-ture activity analysis of the peptide backbone of amylin and calcitonin, se-lected on acute food intake, body weight and glucose control. We evaluated KBP-089 as a treatment for obesity in high fat diet (HFD) rats, as well as fasting plasma glucose and HbA1c in ZDF rats.

1., HFD rats were treated with KBP-089 s.c., at 0.625, 1.25, 2.5 µg/kg and vehicle. 2., we compared two doses of KBP-089 (1.67 and 5.0 µg/kg) to similar davalintide doses in HFD rats. 3., we tested KBP-089 (5 µg/kg for 4 weeks, 20 µg/kg for additional 4 weeks) in ZDF rats.

8 weeks KBP-089 treatment resulted in a dose dependent and sustained 17.4% vehicle corrected weight loss in the 2.5 µg/kg group (p<0.001), while

pair-feeding resulted in a 5.3% body weight reduction. KBP-089 signifi cantly reduced weight of visceral fat depots. Treatment with KBP-089 and Daval-intide (5µg/kg) resulted in a 15.6% and 5.0% vehicle corrected weight loss respectively, and a corresponding reduction in epididymal adipose tissue for KBP-089, but not davalintide. KBP-089 lowered fasting plasma glucose and by 5.3 mM (p<0.05) and HbA1c by ~2.0% (p<0.05) compared to vehicle.

In conclusion, KBP-089 induced and sustained a signifi cant weight loss and reduced adiposity in HFD rats. The mode of action was a combination of decreased food intake and increased energy expenditure. In addition, KBP-089 improved glucose homeostasis, in contrast to davalintide, underscoring the potential of KBP-089 as an anti-obesity agents with additional benefi ts on glucose control.

Supported By: Danish National Research Foundation

2147-PPharmacological Activation of the TRs Elicits a Functional Conver-sion of White to Brown FatKEVIN PHILLIPS, Houston, TX

The functional conversion of white adipose tissue (WAT) into a tissue with brown adipose tissue (BAT)-like activity represents an intriguing strategy to combat obesity and metabolic disease. We demonstrate that thyroid hor-mone receptor (TR) activation by a synthetic agonist markedly induces a pro-gram of adaptive thermogenesis and uncoupled respiration in subcutaneous WAT of obese mice with a correspondent amelioration of obesity and insulin resistance. Surgical denervation of BAT has no effect on systemic thermo-genesis indicating that classical BAT makes no measurable thermogenic contribution, yet, all metabolic increase is lost in Ucp1-KO mice, suggesting that the induction of thermogenesis is mediated exclusively by WAT. TR ago-nist treatment was also found to strongly increase uncoupled respiration in white adipocyte cell culture, indicating that TR mediated browning of WAT is cell autonomous. These data demonstrate that TR activation can elicit facul-tative thermogenesis in WAT, implicate the TRs in the browning of WAT, and establish the profound pharmacological potential of this action.

2148-PRoscovitine Induces Browning of White Adipose TissueHONG WANG, STEPHEN R. FARMER, Boston, MA

Brown adipose tissue dissipates energy through heat and functions as a defense against cold and obesity. PPARγ (peroxisome proliferator-activated receptor gamma), is a functioning receptor for white-to-brown fat conver-sion, however, the underlying mechanisms remain unclear. Previous stud-ies demonstrated that deacetylation of lysines 268 and 293 in PPARγ by Sirt1 induced browning of white adipose tissues in mice. Our data show that preventing the phosphorylation of S273 through modifi cation to alanine or treatment of adipocytes with a Cdk5 inhibitor, roscovitine, induces brown gene expression in white adipocytes in vitro. Both roscovitine and S273A modifi cation enhances the binding of PPARγ to regulatory regions of brown genes and promotes its interaction with Sirt1 and prdm16 while dislodg-ing NCoR. Treatment of mice with roscovitine protects against diet-induced obesity and promotes the formation of beige adipocytes in subcutaneous WAT. Identifying compounds that regulate the post-translational modifi ca-tion of PPARγ may represent a plausible therapeutic approach for the treat-ment of obesity and diabetes by browning white adipose tissues.

2149-PSustained Glucagon-Like Peptide (GLP)-1 Receptor Activation Pre-vents High-Fat Diet (HFD)-induced Endothelial Dysfunction, Vascu-lar Insulin Resistance, and Muscle Capillary RarefactionWEIDONG CHAI, FEI YAN, KEVIN W. AYLOR, EUGENE J. BARRETT, ZHENQI LIU, Charlottesville, VA

Diabetes and obesity are associated with endothelial dysfunction, vas-cular insulin resistance, and capillary rarefaction which contribute to the pathogenesis of cardiovascular complications. GLP-1 and its analogs are able to acutely cause vasodilation. To examine whether sustained GLP-1 re-ceptor activation improves endothelial function, vascular insulin responses and angiogenesis in the insulin resistant state, adult male SD rats were fed a HFD (60% calories from saturated fat) ± liraglutide (200 µg/kg twice daily, SQ) for 4 wks. Additional rats were fed a chow diet for 4 wks as controls. Distal saphenous artery was dissected and its vasodilatory responses to acetylcholine (Ach, endothelium dependent), sodium nitroprusside (endothe-lium independent) and insulin were determined. Muscle capillary density (capillaries/fi ber) was quantifi ed using immunofl uorescent staining. Muscle AMPK phosphorylation and VEGF mRNA expression were measured. Com-pared with controls, HFD feeding decreased both Ach (10-6 M) and insulin

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(10-8 - 10-6 M)-induced vasodilation by 20% and up to 35% respectively

(p<0.05 for all) without affecting the vasodilation response to sodium nitro-prusside. These were associated with a signifi cantly reduced capillary den-sity (~25%, p<0.01) and VEGF mRNA expression (~50%, p<0.05) in muscle. Liraglutide treatment induced a marked increase in AMPK phosphorylation and VEGF mRNA expression (p<0.05 for all) in muscle and fully restored Ach- and insulin-induced vasodilation and muscle capillary density. We conclude that sustained GLP-1 receptor activation with liraglutide improves endothe-lial function, restores vascular insulin responses, and muscle capillarization in the insulin resistant state. This may help improve muscle insulin sensitiv-ity and reduce cardiovascular complications in patients with type 2 diabetes receiving incretin-based therapy.

Supported By: American Diabetes Association-Novo Nordisk (7-09-Novo-11 to Z.L.); National Institutes of Health

2150-PObesity Impairs Muscle Function and Skeletal Muscle Extracellu-lar MatrixCHARMAINE TAM, JOSEPH POWER, TANIA MARKOVIC, CHRISTINE YEE, MARCO MORSCH, SUE MCLENNAN, STEPHEN TWIGG, Sydney, Australia

Skeletal muscle extracellular matrix (ECM) remodeling has been proposed as another feature of the pathogenic milieu associated with obesity and metabolic dysfunction. Whether muscle ECM is associated with impaired physical function in obese conditions is unknown. We developed a preclini-cal model to assess for muscle function-structure presence and relation-ships.

C57BL/6 mice were fed high-fat diet (HFD) or regular chow for 5, 10 and 25 weeks. Non-invasive physiological tests (hang wire, hang mesh and grip strength) to assess neuromuscular function and motor coordination were performed. To account for the effects of body weight, hang wire and hang mesh test results were multiplied by weight. Genes related to ECM struc-ture [collagens (COL)1, COL3, COL6A2, secreted protein and rich in cysteine (SPARC)], growth factors [transforming growth factor (TGF)B1, TGFB2, con-nective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and muscle function [dystrophin (DMD (Dp147), calpain 3 (CPN3), β-dystroglycan (DAG1)] were measured in gastrocnemius muscle using real-time PCR. Two-way analysis of variance was performed to examine changes in physical function and gene expression with group and time effects.

Compared with chow, HFD mice had impaired muscle strength and lower COL1, COL3, COL6, VEGF and CPN3 gene expression (all P<0.05). At 5 weeks, greater muscle COL3 (r2=0.48, P=0.02) and COL6 gene expression (r2=0.41, P=0.03) were each associated with physical impairment (hang wire) in high-fat fed animals; these associations were not seen at the later time points of 10 or 25 weeks.

In conclusion, muscle function and motor co-ordination tests revealed that high-fat fed mice had impaired performance, which after only 5 weeks was associated with greater muscle ECM accumulation in large muscle. Our fi nd-ings contribute to the growing body of evidence underlining the signifi cance of skeletal muscle ECM in metabolic and whole-body dysfunction.

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2152-PPaternal Obesity and Epigenetic Modifi cation of Metabolic and Be-havioral Profi le in OffspringFELICIA V. NOWAK, YIZHU ZHANG, ALEXIS ZONTINI, GHADIR SINDI, YURIY SLY-VKA, Athens, OH

Offspring of obese parents have increased risk of obesity; this risk dou-bles if both parents are obese. Clearly, there is a paternal contribution to this phenomenon. We hypothesize this contribution is due not only to learned behavior, but also has inherited epigenetic components. Male C57BL/6 mice were fed a low fat diet (LFD, 10% fat) or high fat diet (HFD, 45% fat) for 12 weeks then mated to females fed LFD. Pups were fed regular chow and tested at 20 days, 6 weeks, and 6 and 12 months of age. Body composition was determined by NMR. Body weight/length, swimming and running tests, food preference (LFD vs. HFD) and serum leptin ELISA were done. Offspring of LFD (Gr 1) and HFD-fed fathers (Gr 2) were compared. Gr 2 male offspring were heavier than Gr 1 males at 6 weeks, 6 and 12 months. Gr 2 female offspring weighed less with shorter body length than Gr 1 females at 12 months. Gr 2 male offspring had a higher % body fat at 6 months then male offspring from Gr 1. Male offspring in Gr 2 had higher HFD consumption at 6 weeks than male offspring in Gr 1. This group also ate more HFD than their female littermates. Gr 2 female offspring had higher levels of serum leptin at 6 months than Gr 1 females. At 6 weeks of age, Gr 2 males voluntarily ran longer than Gr 1 males. Females ran more than males in Gr 1 at 6 weeks and in Gr 2 at 6 and 12 months. When subjected to involuntary swim at 6 weeks, males in Gr 2 spent less time in the chamber and less time fl oating than Gr 1 males. At 6 weeks Gr 1 males also spent more time fl oating than Gr 1 females. Thus at a young age, Gr 2 males are heavier, with higher % fat and exhibit several modifi able behaviors different from offspring in Gr 1, includ-ing shorter passive fl oat times, higher HFD consumption, when available, and increased voluntary running. Voluntary running, fl oating time, serum leptin and HFD consumption are regulated in part by CNS neurotransmit-ters, neuromodulators and their receptors. Effects of paternal dietary fat on several components of this reward circuity in offspring are currently being investigated.

2153-P14-3-3ζ Controls Adipogenesis through Inhibitory Actions on Hedgehog SignalingGARETH E. LIM, TOBIAS ALBRECHT, KARNJIT SARAI, JASON LEE, SUNITA SIN-HA, COREY NISLOW, JAMES D. JOHNSON, Vancouver, BC, Canada

Adipocyte differentiation is mediated by complex transcriptional net-works, requiring precise spatial and temporal control of key factors. We pre-viously identifi ed the molecular scaffold, 14-3-3ζ, as a critical regulator of adipogenesis in vitro and in vivo, but its exact function was not fully known. 14-3-3ζ can control the localization of transcription factors, suggesting a regulatory role on the adipogenic transcriptome. Thus, we used RNA-Seq, followed by GSEA, to compare the transcriptome of differentiating control and 14-3-3ζ-depleted 3T3-L1 pre-adipocytes and discovered negatively enriched gene sets (FDR adjusted q=0.001), such as cell cycle regulation and fat cell differentiation. Knockdown of 14-3-3ζ prevented 3T3-L1 cells from entering S-phase (25% vs. 8%, p<0.01). Levels of Cdkn1b/p27Kip1, a regulator of G1-S transition, decreases upon differentiation, but 14-3-3ζ knockdown increased p27Kip1 (4-fold, p<0.05) in undifferentiated cells and prevented differentiation. Knockdown of p27Kip1 in 14-3-3ζ-depleted cells rescued the defect in differentiation. Binding motifs for Gli effector proteins, which mediate the inhibitory actions of hedgehog signaling on adipogen-esis, were detected in the Cdkn1b promoter (+950-450kb), and luciferase promoter assays showed enhanced transcriptional activity of this region fol-lowing knockdown of 14-3-3ζ. Pull-down of 14-3-3ζ revealed interactions with the Gli3 isoform during differentiation, and a 66% decrease (p<0.05) in Gli3 abundance on the Cdkn1b promoter was detected by ChIP-qPCR. SiRNA against 14-3-3ζ increased Gli3 levels (1.5-fold, p<0.05), and 14-3-3ζ knock-down enhanced hedgehog signaling transcriptional activity. Knockdown of Gli3 in 14-3-3ζ-depleted cells attenuated p27Kip1 expression and complete-ly rescued adipocyte differentiation. Collectively, these data reveal novel regulatory roles of 14-3-3ζ on hedgehog signaling and adipogenesis and suggest its potential as a therapeutic target for the treatment of obesity.

Supported By: JDRF; Canadian Diabetes Association

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2154-PEosinophil-derived Cytokine Associates with Preserved Insulin Sensitivity in Adipose TissueELENA A. DE FILIPPIS, ELISABETH JACOBSEN, TING LI, JAMES J. LEE, LAW-RENCE J. MANDARINO, Scottsdale, AZ, Tempe, AZ

Crosstalk between the innate immune system and metabolism is increas-ingly appreciated. Eosinophils (EOS) accumulation in adipose tissue (AT) is required for the maintenance of systemic glucose homeostasis and insulin sensitivity. 12 week old C57Bl6J (WT), ΔdblGATA EOS-less (ΔGATA) and hy-pereosinophilic IL-5 overexpressing transgenic mice (NJ.1638), were placed on standard (STD) and 60% high fat (HFD) diets for 6 weeks to investigate the metabolic interaction between EOS and AT. After diets, mice had eug-lycemic clamps to measure glucose turnover. NJ.1638 on HFD gained less fat mass than WT or ΔGATA (1.8±0.2 vs. 3.8±0.6 or 3.7±0.6 g, respectively, P<0.05). On STD ΔGATA required lower rates of glucose infusion during hy-perinsulinemia to maintain euglycemia than WT or NJ.1638 (39±6 vs. 58±4 or 57±4 ug/(g.min), P<0.05 for effect of genotype). On HFD, NJ.1638 mice were protected against HFD-induced insulin resistance for Rd. Analysis of perigonadal AT revealed a signifi cant increase in Glut-4 protein in NJ.1638 compared to WT (2.47 fold increase vs. WT HFD, P<0.05). Eosinophils may preserve insulin sensitivity in part through the release of cytokines. Animals with excess of EOS also had trend to increase mRNA level for IL-4, IL-13 and IL-6 compared to other genotypes and a statistical decrease in mRNA for Resistin (83% decrease in NJ.1638 vs. WT, P<0.001). We treated 3T3-L1 mature adipocytes with two doses (10 or 100 ng/ml) of IL-6 for 24hrs in vitro to address the role of EOS-derived cytokines on AT metabolism. As shown in the animal studies, IL-6 treatment led to a decrease in mRNA level for Resistin (about 65% vs. untreated cells, P<0.05). Collectively, these data showed that under HF diet hypereosinophilia results in lower fat gain and that adipose tissue eosinophils contribute to the preservation of whole body glucose homeostasis. In particular, the data suggest that eosinophil-derived cytokines such as IL-6 may have a direct role in maintenance of healthy, insulin sensitive adipose tissue.

2155-PIncreased 11β-HSD1 and H6PDH Expression in Adipose Tissue May Contribute to Glucocorticoid-induced Adipogenesis and Abdominal Obesity in MiceCHAOYING YAN, XIWEN LIU, GUADALUPE NAVARRO, ADAKU UME, CARL SIMS, KABIR LUTFY, THEODORE FRIEDMAN, YANJUN LIU, Changchun, China, Los An-geles, CA

Long-term glucocorticoid exposure increases the risk of developing obe-sity and diabetes. Prereceptor activation of intracellular glucocorticoid availability in target tissue by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) coupled with hexose-6-phosphate dehydrogenase (H6PDH) is an important mechanism mediating the development of obesity and metabolic syndrome. We explored whether tissue-specifi c modulation of 11β-HSD1 and H6PDH expression in adipose tissue mediates glucocorticoid-induced abdominal fat accumulation and assessed the effect of a glucocorticoid re-ceptor antagonist, RU486, on the key adipogenesis genes and glucocorticoid metabolism in adipose tissues in mice exposed to corticosterone (CORT). Our results showed that RU486 treatment of CORT-exposed mice reduced the increased adipose 11β-HSD1 level with concomitant reduction of fat H6PDH expression. Decreased expression of 11β-HSD1 was correlated with the suppression of C/EBP and PPAR gamma mRNA levels, which are the key transcriptional regulators for adipogenesis that were elevated in adipose tissues of CORT-treated mice. RU486 treatment also reduced abdominal fat pad weight gain and also improved body weight and hyperglycemia in CORT-exposed mice. These fi ndings suggest that induction of adipose 11β-HSD1 and H6PDH expression may contribute to glucocorticoid-induced adipogen-esis and obesity.

Supported By: National Institute of Diabetes and Digestive and Kidney Diseases (SC1-DK087655, SC1-DK104821 to Y.L.)

2156-PGastric Bypass Surgery in Mice Does Not Change Islet Size nor CompositionROMAN VANGOITSENHOVEN, MICHAEL MARIS, JOAO PAULO MONTEIRO CAR-VALHO MORI CUNHA, MATTHIAS LANNOO, LUT OVERBERGH, CHANTAL MA-THIEU, BART VAN DER SCHUEREN, Leuven, Belgium

Gastric bypass surgery improves type 2 diabetes, but the weight loss in-dependent mechanisms remain elusive. We set out to unravel a potential benefi cial role of gastric bypass surgery on islets of Langerhans.

Eight week old male C57Bl/6 mice were fed a high fat diet (60% kcal fat) for 14 weeks and subsequently subjected to Roux-en-Y gastric bypass (RYGB; n=16). Weight matched (WMS; n=13) and pair fed sham operated mice (PFS; n=14) were used as controls. 8 weeks post-surgery, mice were phenotyped by intraperitoneal glucose tolerance test (IPGTT) and indirect calorimetry. Pancreatic tissue was analyzed by immunohistochemistry.

Eight weeks after surgery, RYGB and WMS mice showed abundant weight loss, decreased fasting glucose and insulin levels, as well as improved glu-cose tolerance as compared to obese PFS mice (Table 1). Additionally, energy expenditure was higher in RYGB (2022 ± 145 kJ/day) as compared to WMS (1756 ± 284 kJ/day, P < 0.05) and PFS (1863 ± 195 kJ/day, P 0.11). Pancreatic islet size, as well as insulin, glucagon and somatostatin positive area per islet were similar between all groups.

In summary, RYGB surgery improves glucose homeostasis and increases energy expenditure, but has no effect on size or cell composition of islets of Langerhans. As improved beta cell function has been reported after RYGB, RNA-Seq analysis of isolated islets is currently ongoing in order to reveal the underlying molecular mechanisms.

Table 1. Metabolic Parameters and Islet Characteristics (Mean ± SD, * P < 0.05 vs. RYGB).

RYGB WMS PFSBody weight (g) 27.6 ± 2.5 28.6 ± 1.5 43.4 ± 6.9 *Fasting glycemia (mmol/l) 10.5 ± 0.9 9.8 ± 0.8 13.4 ± 1.3 *Fasting insulin (pmol/l) 187.2 ± 30.1 309.1 ± 96.7 574.9 ± 115.4 *IPGTT (AUC) 32804 ± 1344 31814 ± 2290 59723 ± 1354 *Islet size (µm²) 12626 ± 12614 11878 ± 13358 14577 ± 13192Insulin pos. area (% of islet area) 69.5 ± 5.0 69.3 ± 7.4 68.7 ± 5.5Glucagon pos. area (% of islet area) 11.9 ± 5.7 10.0 ± 3.9 12.8 ± 5.7

Supported By: FWOG.0857.13

2157-PThe Extracellular Matrix Regulates Adipose Function and Expan-sionMARCELLA K. VAICIK, MALLORY MORSE, ALEN BLAGAJCEVIC, ERIC BREY, RON-ALD COHEN, Chicago, IL

Obesity is a global epidemic that contributes to the increasing medical bur-dens related to type 2 diabetes, cardiovascular disease and cancer. The extra-cellular matrix (ECM) has been shown to regulate the development and func-tion of numerous tissues and organs. An understanding of the role the ECM plays in adipose tissue function and expansion could lead to new therapeutics that eliminate or reduce obesity-associated morbidity and mortality. Laminin 4 is upregulated during adipogenesis and is present in the ECM surrounding fully differentiated adipocytes. However, there is little understanding of its function in adipose tissue. We have found that mice with a null mutation of the laminin α4 gene (Lama4−/−) exhibit reduced weight gain and fat mass accumulation in response to both aging and high-fat diet when compared to wild-type (Lama4+/+) animals. However, the underlying mechanisms by which Lama4 regulates fat mass have not yet been defi ned. We have now found that physical activity and food intake does not differ between Lama4+/+ and Lama4−/− mice. However, Lama4−/− mice have a signifi cantly increased meta-bolic rate at 25C (room temperature) and 16C (cold) compared to Lama4+/+ mice. Interestingly, Lama4−/− mice exhibit signifi cantly increased UCP-1 expression in subcutaneous adipose tissue [18.79±4.97% UCP-1 positive compared to 2.62±1.63% (n=5, p≤0.01)]. In contrast, in thermoneutral conditions at 30C both Lama4+/+ and Lama4−/− mice exhibit equivalent metabolic rates. These results suggest that beiging of subcutaneous adipose tissue in Lama4−/− mice may lead to decreased adipose tissue accumulation and potentially improved metabolic function. Thus, alterations in laminin composition suggest that the ECM plays a role in modulating cellular behavior in adipose tissue expansion in a temperature- and depot-specifi c manner.

Supported By: University of Chicago Diabetes Research and Training Center (P30DK020595)

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2158-PA Novel Monoacylglycerol Acyltransferase 2 (MGAT2) Inhibitor Re-duces Body Weight and Fat Mass in Mice with Dietary ObesityIPPEI KAWANISHI, Kawasaki, Japan

Monoacylglycerol acyltransferase 2 (MGAT2) is one of 3 MGATs sharing sequence homology, which is exclusively expressed in the intestine. MGAT2 catalyzes the fi rst step of triacylglycerol resynthesis by which monoacylg-lycerol and fatty acyl-CoA are converted to diacylglycerol in small intestinal epithelial cells. Therefore, MGAT2 activity is assumed to be crucial for di-etary fat absorption. Since MGAT2 knockout mice on a high-fat diet show weight loss, increased energy expenditure compared to wild type mice, and no phenotypic abnormalities, MGAT2 seems an attractive target for treat-ment of obesity.

We have identifi ed novel low-molecular weight synthetic chemical com-pounds that inhibit human MGAT2 with IC50 values of 10 nM or less. Oral administration of IMG0240, one of these inhibitors, reduced the elevation of plasma triglyceride levels after fat loading in normal mice. Once-daily oral administration of IMG0240 (30 mg/kg) for 3 weeks resulted in signifi cant weight loss of 7.3% and reduction of fat mass without a change of lean mass in mice with dietary obesity. IMG0240 administration reduced hepatic triglyceride levels, but fecal triglycerides were unchanged after treatment. The deep body temperature of IMG0240-treated mice (determined by an em-bedded intraperitoneal thermometer) was increased during the light period. These data suggest that increased energy expenditure, especially during the light period, contributes to weight loss with IMG0240 treatment.

Therefore, pharmacological blockade of MGAT2 alleviates fat accumula-tion by decreasing fat absorption and increasing energy expenditure, similar to the fi ndings in knockout mice. This novel MGAT2 inhibitor (IMG0240) could be a potential anti-obesity drug.

2159-PNaturally Increased AMPK and pAMPK in the Wild Type MRL Mouse Strain Causes High-Fat Diet ResistanceNATHAN W. ROBERTS, AARON J. MULL, MAGDALIS GONZALEZ-VEGA, AHLKE HEYDEMANN, Chicago, IL

We have been studying the MRL mouse strain because of its superhealing and muscular dystrophy-mediated fi brosis resistance. During our investiga-tions into the fi brosis resistance we identifi ed that the MRL skeletal muscle tissues have greatly increased AMPK and pAMPK compared to all other mouse lines assessed. Because increasing pAMPK (by exercise or Met-formin treatment) is benefi cial for Type 2 Diabetes Mellitus (T2DM) we hy-pothesized the wild type MRL mice would be resistant to high fat diet (HFD) mediated T2DM. We have recently published the data describing systemic T2DM resistance to 12 weeks of HFD of the MRL mice. We are now present-ing data which aims to uncover the mechanisms behind this resistance.

In addition to the increased skeletal muscle pAMPK we have now identi-fi ed that liver and adipose tissues also have naturally increased AMPK and pAMPK, which increase even further in response to HFD. The liver increase in pAMPK is despite an increase in the pAKT inhibitor, describing an overrid-ing pAMPK activating mechanism.

We now also describe cardiac resistance to HFD in the MRL mice. By echocardiography the MRL cardiac function does not deteriorate, while the control C57Bl/6J mice present with diastolic dysfunction and cardiomyocyte hypertrophy. Surprisingly the western blot of heart tissue demonstrates a decrease in AMPK and pAMPK levels compared to control mouse strains.

MRL mice contain 2 non-synonymous heteroplasmies (a mitochondria and cell containing more than one mitochondrial genome, Sachadyn, 2008). We also have exciting innovative data which demonstrates a correlation between the percentage of the MRL mitochondrial genome and improved resistance to HFD.

As the MRL mice are resistant to all effects of HFD, including cardiomyo-pathy, we are very interested in continuing the search upstream to identify the proximal cause for the unique, resistant metabolism.

Supported By: National Institutes of Health (R01HL102322)

2160-PThe Protracted In Vivo and In Vitro Effects by the Novel Dual Amylin and Calcitonin Receptor Agonist, KBP-089, Do Not Correlate with the Observed PK Profi le In Vivo KIM VIETZ. ANDREASSEN, SOFIE GYDESEN, SARA T. HJULER, MORTEN A. KARSDAL, KIM HENRIKSEN, Herlev, Denmark

Novel dual amylin and calcitonin receptor agonists (DACRAs) are currently in development as novel potential treatment possibilities for obesity. Under-standing the relationship between the observed effects in vitro and in vivo

and the PK profi le in vivo would assist in understanding the mode of action of DACRAs for treatment of obesity. Hence, the aim of the study was to investigate the relation between the PK profi le and the effi cacy of the novel DACRA KBP-089 both in vitro and in vivo.β-arrestin recruitment in vitro: human U2OS cell line heterologously ex-

pressing the human CTRa (DiscoverX) and β-arrestin signaling was investi-gated up to 72 hours of stimulation. Food intake in High fat Diet (HFD) rats: Sprague Dawley rats were on high fat diet for ten weeks prior to testing. Food intake was assessed for 4-72 hours after s.c. administration of 5 µg/kg DACRAs. PK profi ling: Plasma samples were collected at baseline and 10, 20, 30, 40, 80 and 120 min after injection and PK profi le was determined with an sandwich ELISA targeting both ends of KBP-089.

KBP-089 demonstrated a protracted β-arrestin response over 72 hours of stimulation. Human calcitonin (hCT), rat amylin (rAMY) and davalintide did not demonstrate a protracted response. Compared to vehicle, KBP-089 attenuated food intake in HFD rats by >90% (p<0.001) after 24h, >70% (p<0.001) after 48h, and returned to vehicle levels after 72h. Neither hCT, rAMY nor davalintide attenuated food intake past 4h.

In contrast, the concentration of injected KBP-089 peaked after 10-20 min after injection, and was completely cleared from plasma after 2 hours.

A protracted DACRA response exists beyond what is measured in plasma, as KBP-089 was completely cleared from circulation after 2h. Hence, this study provides insights into differences in in vitro and in vivo PK and PD responses between novel DACRAs that could further assist in the develop-ment of new improved agonists for the treatment of obesity.

Supported By: Danish National Research Foundation

2161-PLeontopodium Improved the Cognitive Function of Diabetic RatsMEI-HUA QU, LU-JUAN WANG, FENG-BIN WANG, Weifang, China

Study the effect of Leontopodium in improving the cognitive of diabetic rats and the changing of the ultrastructure of neurons in the hippocampus CA1of the diabetic rats. Experimental diabetic rats were established by high-glucose-high-fat diet and low dose streptozotocin injection to Wistar rats. 20 diabetic rats were randomly divided into diabetic control group and Le-ontopodium treatment group. 10 Wistar rats were used as wild type control group. The fasting blood glucose was measured every 2 days. Morris water maze was used to check the change of cognitive function. Electron micro-scope was used to observe the ultrastructure of neurons in hippocampus CA1 of the 3 groups. Results showed that Leontopodium decreased fast-ing blood glucose signifi cantly comparing the diabetic rats with(15.36±1.08)mmol/L vs. (19.23±3.12) mmol/L (p< 0. 01) when treated with leontodium orally at the dose of 1.95g/kg weight/day for 6 weeks. When examed by Morris water maze, the escape latency of the Leontopodium treatment group decreased signifi cantly from 42.58±9.50 S to 30.83±11.15S (P < 0. 05) and the times striding over the platform increased signifi cantly from 1.60±1.07 to 2.60±0.69 (P < 0. 05 ) comparing to the diabetic control animals. That means the leontodium treatment improved the cognitive function of the diabetic rats. The ultrastructure neurons of hippocampus showed that neurons of the diabetic rats showed irregular shape, nuclear membrane to thin and de-pressed, chromatin condensation and margination, and most mitochondria exhibited swelling and vacuole degeneration. Neurons of the Leontopodium treatment animals’ showed regenerated, nuclear membrane becoming regu-lar, chromatin condensation and swelling mitochondria was signifi cantly reduced. All the results showed that Leontopodium decreased fasting blood glucose of the diabetic rats, improved the learning and memory abilities, and reduced phathologic ultrastructure changes and protected neurons in diabetic rats.

Supported By: Natural Science Foundation of Shandong Province, China (ZR2009DM024, ZR2011HM043); Higher Educational Science and Technology Program of Shandong Province, China (J11LF33)

2162-PMicro-RNA Regulation Responses to Cardiac Ischemia/Reperfu-sion Injury Differ between Lean and Obese SwineDANIEL J. SASSOON, JOHNATHAN D. TUNE, ADAM G. GOODWILL, JILLIAN N. NOBLET, B.P. HERRING, JEANETTE N. MCCLINTICK, KIEREN J. MATHER, India-napolis, IN

Obesity is associated with worse functional and survival outcomes af-ter cardiac ischemia. We examined micro RNA (miR) expression changes induced by ischemia-reperfusion injury (IRI), comparing diet induced obe-sity/metabolic syndrome (MetS) Ossabaw swine versus normal chow-fed controls. Reversible ligation of the left circumfl ex artery allowed a 30 min occlusion, followed by 2 hours of reperfusion. The heart was then arrested

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and myocardial biopsies were collected from normally perfused and isch-emic myocardium and fl ash frozen for miR analysis. MiR microarrays showed signifi cantly different miR expression in both the normally perfused and ischemic regions of MetS hearts when compared with lean non-ischemic and ischemic regions. Interestingly, miR expression was not signifi cantly different in lean non-ischemic vs. ischemic tissue, whereas 11 miRs were differentially regulated in MetS non-ischemic vs. ischemic territories. The differentially expressed miRs have predicted effects on transcription of genes relating to cell survival, apoptosis, autophagy, fi brosis, infl ammation, and angiogenesis (Target Scan v6.2). These observations suggest effects of obesity to modulate ischemia-induced changes in specifi c regulatory path-ways relating to cardiovascular function and cell survival. This pathway to discovery may uncover novel therapeutic targets to combat obesity-associ-ated heart disease.

Supported By: National Institutes of Health (HL117620), (TL1TR000162 to A.S.)

2163-PGlucagon-Like Peptide-1 Receptor Agonist Activates Brown Adi-pose Tissue Function and Browning in Mice via NOS and STAT3 PathwaysSHANMEI SHEN, LING-JUN SUN, DA-LONG ZHU, HUAN SHI, MING-YUE HU, YAN BI, RAN MENG, YA-LI JING, LU JIN, TIAN TIAN, Nanjing, China, Shanghai, China

Although factor that impacts central regulation of BAT thermogenesis is GLP-1, its direct action on BAT and signal pathways resposible for these in-teractions remain largely unknown. Here we examined in vitro the molecular basis of GLP-1R agonist exenatide on BAT function and browning as well as possible roles of NOS and STAT3. Primary brown adipocytes and white adi-pocytes in mice were cultivated in vitro, then the cells were divided as fol-lows: (1) the cells were treated with different concentrations of exenatide: 10-9 mol/L and 10-8 mol/L; (2 ) the cells were treated with 2 types of inhibtors (NOS inhibtor, 10-3 mol/L L-NAME; STAT3 inhibtor, 10-6 mol/L S3I-201) and GLP-1R antagonist (10-7 mol/L exendin 9-39), which were all added into the high level of exenatide for 5 days; (3) untreated cells were served as a con-trol. Exenatide administraion led to an increase in the expression of specifi c genes (UCP1, CIDEA, PGC1-α), differentiation genes (PRDM16, PPAR-γ) in brown adipocytes. Notably, upregulation of UCP-1, PGC1-α, PRDM16, and PPAR-γ were also observed in white adipocytes. Furthermore, exenatide stimulated BAT function partly through NOS pathway, as evidenced by in-creased eNOS and iNOS expression levels together with NOS protein level, and the effect on BAT specifi c genes was suppressed by the supplement with NOS antagonist L-NAME. In contrast, STAT3 pathway was mainly in-volved in the regulation of WAT. In summary, our results provide insight into the molecular basis of GLP-1R agonist effect on BAT function and browning in WAT, and suggest a benefi cial role for NOS and STAT3 pathways. Thus, exenatide may constitute a potential therapeutic tool for the treatment of obesity.

2164-PDipeptidyl Peptidase-4 (DPP-4) Inhibitor Linagliptin Prevents Aortic Stiffening and Vascular and Cardiac Diastolic Dysfunction Caused by Western Diet Feeding in Female MiceVINCENT G. DEMARCO, ANNAYYA R. AROOR, GUANGHONG JIA, JAVAD HABIBI, MONA GARRO, ZHE SUN, GERALD A. MEININGER, ADAM WHALEY-CONNELL, JAMES R. SOWERS, Columbia, MO

Aortic stiffness, endothelial dysfunction and diastolic dysfunction (DD) are cardiovascular (CV) abnormalities seen in obesity associated with con-sumption of high fat/fructose western diet (WD). Moreover, CV dysfunction is increasingly prevalent in obese women. Herein, we examined whether the DPP-4 inhibitor, linagliptin (LINA), improves these outcomes in WD fed female C57BL/6 mice. Four week old mice were fed control diet (CD) or WD with or without LINA for 16 weeks, after which pulse wave velocity (aortic stiffness) (PWV), echocardiography (diastolic function), atomic force micros-copy (endothelial stiffness) and wire myography (aortic vascular reactivity) were performed. Compared to CD mice, WD mice exhibited 21% and 353% higher PWV and endothelial stiffness, respectively. WD induced DD, indicat-ed by impaired septal wall motion (E’/A’ ratio), fl ow propagation velocity (Vp), left atrial fi lling pressure (E/Vp ratio), prolonged isovolumic relaxation time (IVRT) and impaired myocardial performance index (MPI). These vascular and cardiac abnormalities were prevented by LINA. LINA also prevented WD-induced impairments in acetylcholine-, sodium nitroprusside-, and insulin-mediated aortic vascular relaxation. These results show that LINA exerts CV protection in a translational model of obesity.

Table 1. WD Induces Aortic Stiffness and Diastolic Dysfunction that is Pre-vented by LINA. *P<0.05 vs. CD, † vs. WD-LINA. Control Diet (CD), N=6-8; Western Diet (WD), N=6-9; WD plus LINA, N=4-10.

Stiffness Measures Cardiac Function ParametersGroups PWV

m/s)Endothelial

kPaE’/A’ Vp

cm/sE/Vp IVRT

msMPI

CD 3.39±0.09 2.83±0.41 2.05±0.05 62±2 14.4±0.7 15±1 0.40±0.02WD 4.11±0.09* 9.99±0.77* 1.44 ±0.19* 42±4* 22.3±1.7* 22±1* 0.66±0.03*WD-LINA 3.44±0.11† 3.46±1.04† 1.97±0.08† 55±2† 15.5±1.2† 17±1† 0.45±0.03†

Supported By: Boehringer Ingelheim Pharmaceuticals, Inc.

2165-PSodium Glucose Transporter Type 2 (SGLT2) Inhibitor, Empaglifl ozin, Improves Diastolic Function in Female Diabetic db/db MiceVINCENT G. DEMARCO, ANNAYYA R. AROOR, RAVI NISTALA, MONA GARRO, ERIC W. MAYOUX, ADAM WHALEY-CONNELL, JAMES R. SOWERS, Columbia, MO, Biberach, Germany

Dysglycemia, proteinuria, vascular stiffness, and diastolic dysfunction (DD) are abnormalities seen more frequently in the obese/diabetic population. Lean premenopausal women are protected from CV disease compared to men, but protection is compromised in obese/diabetic and premenopausal women. SGLT-2 inhibitors (SGLT-2i), which increase urinary glucose/sodium excretion to lower HbA1c and blood pressure, are emerging as unique diabetes thera-pies. We examined whether the SGLT-2i, empaglifl ozin (EMPA), improves dys-glycemia, proteinuria, aortic stiffness and DD in obese/diabetic female db/db mice. Eleven week old mice were fed a diet with or without EMPA (10mg/kg/day) for 5 weeks. In vivo diastolic function (echo), aortic stiffness (pulse wave velocity, PWV), proteinuria and HbA1c were evaluated. HbA1c and proteinuria were elevated (P<0.001) in control (Db-C) mice vs. treated mice (Db-EMPA) (HbA1c: 10.3±0.2 vs. 7.8±0.4%; µAlbumin/Creatine: 169±28 vs. 61±4). Db-C exhibited DD that was improved in Db-EMPA as indicated by improved septal wall motion (E’/A’), fl ow propagation velocity (>Vp), LV fi lling pressure (<E/Vp), isovolumic relaxation time (<IVRT) and myocardial performance (<MPI). PWV was elevated in Db-C but was unaffected by EMPA. These results show that EMPA exhibits CV/renal protective effects in obese diabetic female mice.

Table. Db/db Mice Exhibit Diastolic Dysfunction that is Improved with Empaglifl ozin. *P<0.05 vs. Db-C.

E’/A’ Vp (cm/s) E/Vp IVRT (ms) MPI PWV (m/s)Db-C 1.13 ± 0.08 43 ± 4 22.3 ± 2.5 21.0 ± 1.5 0.54 ± 0.03 4.6 ± 0.3Db-EMPA 2.06 ± 0.06* 57 ± 3* 15.5 ± 1.2* 15.1 ± 0.5* 0.40± 0.01* 4.9 ± 0.4

Supported By: Boehringer Ingelheim Pharmaceuticals, Inc.

2166-PLYPLAL1, an Obesity-associated Gene Is Not Required for Adipo-cyte DifferentiationWEIQIN CHEN, XINNUO LEI, HONGYI ZHOU, Augusta, GA, Martinez, GA

Obesity and its associated morbidities represent one of the major and most rapidly expanding health epidemics in the world. Recent genome-wide association studies (GWAS) have identifi ed several variants in LYPLAL1 gene that are signifi cantly associated with central obesity preferentially in females. However, the exact function of this gene in adipose tissue develop-ment and obesity remains completely uncharacterized. We found murine Ly-plal1 gene demonstrated a depot and sex-specifi c expression profi le in white adipose tissues (WAT), and was signifi cantly reduced in the epididymal and retroperitoneal fats in a murine model of high fat diet induced obesity (DIO). Lyplal1 mRNA was mildly up-regulated during adipogenesis and enriched in mature adipocytes. However, overexpression and knockdown of Lyplal1 did not signifi cantly perturb adipocyte differentiation and triacylglycerol accu-mulation. These data highlight a depot-specifi c marked reduction of Lyplal1 transcripts in diet induced obesity but a dispensable role of Lyplal1 in adi-pose tissue development.

Supported By: Georgia Regents University

2167-PObesity-induced Insulin Resistance and Altered Gut Microbiota in Mice Lacking Interleukin-27HIROTSUGU SUWANAI, TOMONOBU SAWADA, HIROKI YOSHIDA, TAKASHI KA-DOWAKI, KOHJIRO UEKI, Tokyo, Japan, Saga, Japan

The onset of type 2 diabetes is induced by chronic infl ammation and in-sulin resistance. In Recent years, gut microbiota have been shown to be the important culprit of the low-grade infl ammation, a key component of the

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pathogenesis of diabetes. The previous studies have reported that Inter-leukin 27 (IL-27), which has important immunoregulatory functions, is a key cytokine to maintain the mucosal barrier in gut. In the present study, we in-vestigated as to whether IL-27 is involved in insulin resistance. Diet-induced obese mice lacking IL-27 receptor (il27raKO) exhibited marked hyperinsuline-mia and abnormal glucose tolerance. without alteration of body weight com-pare to the control mice. The infl ammatory cytokines including IL-1b, IL-6 and MCP1, were induced in expression analysis of liver associated with changes in gut microbiota; suppression of Bacetroides and marked increase in En-terobacteriaceae. Administration of broad-spectrum antibiotics to il27raKO mice resulted in improving insulin resistance to much larger extent than the control mice. Taken together the fi ndings suggest that IL-27 is a key cytokine to integrate the homeostasis of gut microbiota and suppress chronic infl am-mation leading to metabolic syndrome.

2168-PHigh Vitamin D Intake Decreases Adiposity and Prevents Diabetes in Mice Fed a High-Fat DietIGOR N. SERGEEV, Brookings, SD

Vitamin D-dependent cellular Ca2+ signaling can mediate the link between obesity and type 2 diabetes (T2D); moreover, low vitamin D status is consid-ered a risk factor for these diseases. We have recently shown that hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) induces Ca2+ signals associated with activation of Ca2+-dependent apoptotic proteases in mature adipocytes and that increased intakes of vitamin D and calcium decrease body weight gain in high fat diet-induced obese (DIO) mice via induction of Ca2+-mediated apoptosis of adipose tissue. We have also demonstrated that 1,25(OH)2D3 induces synchronous Ca2+ oscillations in pancreatic β-cells, which tempo-rally pattern oscillatory insulin release from these cells. In this study, we ex-amined effects of increased vitamin D intake in a high fat DIO mouse model of pre-T2D, characterized by obese phenotype, elevated blood glucose and impaired glucose tolerance, on development of adiposity and diabetes. Mice fed high fat diet with high vitamin D3 content demonstrated a decreased weight of white adipose tissue depots and improved blood markers related to adiposity, T2D, and vitamin D status. Specifi cally, fasting plasma glucose and insulin concentrations in DIO mice were signifi cantly decreased, ap-proaching levels found in normal-weight, non-obese control, whereas adi-ponectin concentration in these mice demonstrated an increasing trend. DIO was accompanied by low vitamin D status (decreased plasma 25(OH)D) and a decreased concentration of 1,25(OH)2D3. High vitamin D3 intake induced a signifi cant increase in plasma concentrations of 25(OH)D and 1,25(OH)2D3, indicating high vitamin D nutritional status and normal vitamin D hormonal status. The results obtained demonstrate that high vitamin D intake is ef-fective in decreasing blood glucose and insulin in DIO and that the hormonal mechanism of this effect can involve 1,25(OH)2D3. These fi ndings also imply that increased vitamin D intake may contribute to the prevention of obesity and T2D.

Supported By: U.S. Department of Agriculture (2009-35200-05008, SD00H533)

2169-PInsulin Action Contributes to C-Fos Induction by NPY in the Para-ventricular Hypothalamic NucleusSHENGJIE FAN, JANANI DASHINAMOOTHY, EUN RAN KIM, QINGCHUN TONG, Houston, TX

It is well established that NPY increases food intake. However, the spe-cifi c brain regions that mediate NPY effects on feeding are not yet clear. The paraventricular hypothalamic nucleus (PVH) is thought to be one of major sites that mediate the NPY action since NPY induces abundant C-Fos expres-sion in the PVH. However, activation of PVH neurons, as indicated by C-Fos, is thought to inhibit rather than stimulate feeding. We therefore examined whether NPY directly induces C-Fos in the PVH or indirectly through other mediators. Our studies showed that C-Fos induction by NPY was dramati-cally reduced in mice with type 1 diabetes. Associate with this, NPY effects on feeding were also blunted in type 1 diabetes. In addition, icv insulin also produced signifi cant C-Fos expression in the PVH. Thus, insulin action con-tributes signifi cantly to C-Fos induction by NPY.


2170-PChronic Inhibition of the Insulin Receptor in the Amygdala Leads to Hyperfagia, Decreased Energy Expenditure, and Obesity in Control AnimalsFERNANDA M. CHAVES, PAULA GABRIELE F. QUARESMA, TAMIRES ZANOTTO, GISELE CASTRO, LAÍS WEISSMANN, ANDREY DOS SANTOS, ALEXANDRE HI-LÁRIO BERENGUER. DE MATOS, MÁRIO JOSÉ ABDALLA. SAAD, PATRICIA O. PRADA, Campinas, Brazil, Pirassununga, Brazil, Rio Claro, Brazil

The hypothalamus contributes to maintain body weight through a balance between food intake and energy expenditure. Insulin signaling and action induce anorexia and they are well described in the hypothalamus. Although most studies have focused on the hypothalamus, evidence has shown that the dopamine reward system, including amygdala also modulates energy metabolism. Several studies have shown that the central nucleus of the amygdala (CeA) has a potential role participating in the control of food in-take in response to acute insulin injection. However, it is not known whether chronic absence of insulin signaling in the amygdala will alter energy metab-olism. Thus, the aim of the present study is to investigate whether chronic inhibition of the insulin receptor (IR) by siRNA in the amygdala affects en-ergy metabolism. Our results showed that siRNA reduced 90% the expres-sion of IR in vitro and in vivo (into amygdala of control mice). Control mice were implanted with unilateral cannula attached to minipump aimed to CeA. The Chronic inhibition (14 days) of IR by siRNA signifi cantly increased body weight and fat mass of control mice. The increased adiposity was a result of hyperfagia and lower oxygen (O2) consumption measured by Oxymax/CLAMS. Hyperfagia was associated with a reduction on Akt phosphoryla-tion and an increase in NPY (neuropeptide Y) and a decrease in POMC (proo-piomelanocortin) and CRH (corticotrophin releasing hormone) gene expres-sion in the amygdala. The reduction on O2 consumption was associated with lower UCP-1 (Uncoupling protein 1) gene expression in BAT (brown adipose tissue), suggesting lower thermogenesis. Thus, the results suggest that in-sulin signaling in the CeA plays a key role in the control of energy metabolism and its inhibition leads to hyperfagia, lower energy expenditure and obesity in control animals.

OBESITY—HUMAN

Guided Audio Tour: Factors Contributing to the Development of Obesity and Impact of Intervention Studies (Posters: 2171-P to 2178-P), see page 17.

& 2171-PObesity Risk in the Preschool Years among U.S. ChildrenSHIVANI A. PATEL, SOLVEIG A. CUNNINGHAM, MICHAEL R. KRAMER, K.M. VEN-KAT NARAYAN, Atlanta, GA

Little is known about incident obesity, a potential risk factor for adult dia-betes, in the preschool years. We report obesity risk and its predisposing factors among preschool age children in the U.S. using data from a nationally representative cohort, the Early Childhood Longitudinal Study-Birth Cohort of 2001.

We defi ned normal weight (inclusive of underweight), overweight, and obesity using age-appropriate CDC body mass index cut-points for n=3,400 children at ages 2 y and 5 y. Three-year cumulative incidence (i.e., risk) of obesity was estimated among children who were not obese at age 2 y. Odds ratios (OR) compared incident obesity by child and family factors.

At age 2 y, 15.9% of U.S. children were obese. The risk of obesity in the preschool years, from age 2 y to 5 y, was 12.1%. Children who were over-weight compared to normal weight at 2 y were more often obese at 5 y (OR 2.7, 95% CI 1.6-4.4). Incident obesity at 5 y was higher for children who were Hispanic (2.3, 1.2-4.6) or Black (2.1, 1.1-3.6) vs. White; from low vs. high income families (2.1, 1.2-4.0); had birth weight > 2500 g vs. low birth weight (6.6, 2.1-21.3); and whose mothers were obese vs. non-obese pre-pregnancy (2.0, 1.1-3.8). Of overweight 2 year-olds, more than 1 in 4 who were Hispanic, non-Hispanic Black, low-income, or were born to obese mothers became obese by age 5.

Efforts to reduce obesity might focus on overweight children, especially from race/ethnic minorities and low income families, as early as age 2 y.

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& 2172-PBody Mass Index and Long-Term Cardiovascular Mortality among 2.3 Million AdolescentsGILAD TWIG, HAGAI LEVINE, GAL YANIV, ADI LEIBA, NEHAMA GOLDBERGER, ESTELA DERAZNE, DORIT TZUR, ARNON AFEK, ARI SHAMISS, ZIONA HAKLAI, JEREMY KARK, Ramat Gan, Israel, Jerusalem, Israel

Our objective was to assess the association of body mass index (BMI) in 16-19 year-old Israeli adolescents with subsequent cardiovascular mortality. BMI of 2,298,130 adolescents (60% males, age 17.4±0.3 years), measured prior to military service between 1967 and 2010, was grouped according to the U.S. CDC percentiles and linked with national mortality records. The primary outcomes of the study were mortality due to coronary heart disease (CHD), sudden death (SD), and cerebrovascular events (CVA). Cox propor-tional hazards models were applied.

During 45,729,521 person-years of follow-up (mean 19.9±12.0 y, maximum 44 y) there were 1497, 528 and 893 deaths from CHD, CVA and SD, respec-tively. There was a graded increase in CHD mortality from the 5th-25th BMI percentiles onwards. Obesity (>95th percentile) was associated with hazard ratios of 5.1 (95% CI=4.0-6.4, p<0.001), 2.5 (95% CI=1.6-3.9, p<0.001) and 2.1 (95% CI=1.5-3.0, p<0.001) for CHD, CVA and sudden deaths, respectively, after adjustment for sex, age, birth year, education, socioeconomic status, country of origin and height. For the latter model, overweight (85th-95th BMI percentiles) was associated with HR of 3.1 (95% CI=2.5-3.7, p<0.001), 1.7 (95% CI=1.2-2.4, p=0.002) and 1.4 (95% CI=1.1-1.8, p=0.02) for CHD, CVA and sudden deaths, respectively. Findings persisted when the analysis was restricted to those with unimpaired health status (n=1,669,687, including 50,002 obese) and when the follow-up ceased at 45 years of age. When BMI of 17.5-19.9 kg/m2 was used as a reference, BMI of 20-22.4 kg/m2 was already associated with increased adjusted CHD mortality (HR 1.2, 95% CI= 1.1-1.4, p=0.006), and BMI of 22.5-24.9 kg/m2 with higher CVA and SD mor-tality (HR=1.3, 95% CI=1.1-1.7, p=0.02 and HR=1.4, 95% CI=1.1-1.6, p=0.002, respectively). Therefore, BMI in adolescence, well-within the currently ac-cepted normal range, predicts cardiovascular mortality. These fi ndings have important implications given the ongoing epidemic of childhood obesity.

& 2173-PHepatic Lipid Accumulation Is Mainly Determined by Environmental Factors: A Classic Twin StudyADAM L. JERMENDY, ZSOFIA D. DROBNI, TAMAS HORVATH, SZILARD VOROS, ADAM D. TARNOKI, DAVID L. TARNOKI, ANDREA BARTYKOWSZKI, VIKTOR VOROS, BELA MERKELY, PAL MAUROVICH-HORVAT, GYÖRGY JERMENDY, Buda-pest, Hungary, Richmond, VA

Abdominal adipose tissue and non-alcoholic fatty liver disease have been linked to increased cardiovascular morbidity and mortality. Nevertheless, the role of genetic and environmental factors in the hepatic lipid accumulation is unclear. Our goal was to evaluate the genetic and environmental impact on the hepatic lipid accumulation within a cohort of healthy twin pairs.

We have investigated 210 twin subjects with 256-slice CT-scanner (Bril-liance iCT, Philips Healthcare, Best, The Netherlands), of whom 63 were mo-nozygotic (MZ) pairs (age: 55.7±9.7 years) and 42 were dizygotic (DZ) pairs (age: 58.1±8.7 years). We have assessed the CT-based waist circumference, subcutaneous (SAT) and visceral abdominal adipose tissue (VAT) areas at the level of L3/L4. Liver attenuation (LA) was determined by calculating the aver-age of three regions of interest (ROI) with an area of 300 mm2. The presence of hepatic lipid accumulation was defi ned as LA≤40 Hounsfi eld units (HU). To quantify phenotypic similarity, intra-pair correlations were calculated.

These correlations were broken down to additive genetic (A), common (C) and unique (E) environmental correlation components using structural equa-tion models.

Median SAT was 203.9 [IQR: 148.3-279.0] cm2, while median VAT was 147.1 [IQR: 89.1-212.8] cm2. The mean LA was 58.8±11.2 HU; hepatic lipid ac-cumulation was detected in 14.3% of the twins (30/210 cases). Correlations between SAT, VAT and LA values were as follows: rMZSAT=0.74, rDZSAT=0.29; rMZVAT=0.67, rDZVAT=0.22; rMZLA=0.34, rDZLA=0.16; all p<0.05. Strong herita-bility was found regarding SAT (A=74.0%) and VAT (A=66.8%), whereas ge-netic determination was weak regarding the average LA values (A=34.2%).

In contrast to abdominal fat compartment areas, a weak genetic depen-dence of hepatic lipid accumulation was found in our twin cohort, indicating that environmental factors and lifestyle characteristics are predominantly involved in the development of lipid accumulation in the liver.

Supported By: European Foundation for the Study of Diabetes

& 2174-PIn Adult Twins, Visceral Fat Accumulation Is More Dependent on Adiposity Thresholds than GeneticsTYLER A. BOSCH, LISA CHOW, SUSAN J. MELHORN, MARY WEBB, DANIELLE YANCEY, HOLLY CALLAHAN, MARY ROSALYNN B. DE LEON, VIDHI TYAGI, ELLEN SCHUR, Minneapolis, MN, Seattle, WA

In adults, visceral fat (VAT) accumulates after body fat (BF) exceeds sex-specifi c thresholds (%BF: M-23.4%, F- 38.3%). Using monozygotic (MZ) and dizygotic (DZ) twins, we examined the infl uence of genetics on regional fat distribution measured by dual-energy x-ray absorptiometry, above and be-low these thresholds.

Fifty-eight twin pairs (44MZ, 14 DZ) were recruited from the University of Washington Twin Registry. Segmented linear regression assessed a thresh-old between VAT mass and %BF. To assess the effect of genetics on VAT accumulation, Dunnett’s T3 compared MZ and DZ pairs split into two groups, whether the twin pairs were above the adiposity threshold or not.

Consistent with earlier studies, sex-specifi c %BF thresholds for VAT ac-cumulation were identifi ed (%BF:M- 22.1%, F- 37.6%). Table 1 presents the results for within pair differences of VAT accumulation depending on the adiposity threshold. DZ twins have larger differences when both twins are above threshold. If both twins are not above threshold, there are no sig-nifi cant differences in VAT accumulation between MZ and DZ twins, even though DZ twins have signifi cantly higher differences for %BF.

The results support sex-specifi c adiposity thresholds for visceral fat ac-cumulates. Using a twin study approach, we now shows that, while total BF is infl uenced by genetics, VAT accumulation depends on %BF above his or her sex-specifi c adiposity threshold.

Supported By: National Institutes of Health (R01DK089036, UL1TR000423, KL2TR000421, TL1TR000422); National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases (P30DK035816)

& 2175-PNot Performing an OGTT Results in Signifi cant Underdiagnosis of (Pre) Diabetes in an Overweight/Obese PopulationABRAHAM MEIJNIKMAN, AN VERRIJKEN, ILSE MERTENS, CHRISTOPHE DE BLOCK, LUC VAN GAAL, Antwerp, Belgium

Diabetes (DM) is underdiagnosed. Tests for diagnosis include fasting plasma glucose (FPG), a 2h-OGTT and HbA1c. HbA1c can be tested in non-fasting conditions. Therefore, General Practitioners almost no longer ex-

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ecute OGTT’s. We evaluated the performance of OGTT versus HbA1c in an overweight/obese population. 1241 overweight and obese subjects without a history of diabetes (M/F: 375/866, age 44±13 y, BMI 38.0±6.1 kg/m²) were tested for glucose tolerance status using FPG, OGTT and HbA1c. Anthropom-etry (CT visceral fat, VAT), HOMA-S and HOMA-B were determined. Exactly 46.8% were found to have pre-diabetes and 11.9% were newly diagnosed with diabetes (M/F=18.9/8.9%) using ADA criteria. In total, 10.1% subjects tested positive for diabetes using OGTT, whereas 1.9% subjects had an HbA1C ≥6.5% but did not fulfi ll OGTT criteria. Testing only HbA1c would have resulted in 78 (6.8%) subjects being diagnosed with diabetes, but in 70/148 subjects (47.3%) diabetes would have been missed if OGTT would not have been done. Of the 581 subjects with pre-diabetes 1.4% subjects had IFG, 30.5% had impaired glucose tolerance (IGT), 5.1% subjects had a combined IFG+IGT, and 9.8% had an isolated elevated HbA1c (5.7-6.4%). Of the 581 pre-diabetic subjects, 257 had an HbA1c <5.7%. Therefore, 44.2% subjects would have been wrongfully reassured as having a normal glucose status, when OGTT would not have been done. Logistic regression analysis with pre-diabetes as dependent variable identifi ed age (B=0.44, p<0.0001), BMI (B=0.086, p=0.002), VAT (B=0.003, p=0.046), HOMA-S (B=0.226, p<0.0001) and HOMA-B (B=-0.001, p=0.024) as independent risk factors. In an exclu-sively overweight/obese population 46.8% were diagnosed with pre-diabe-tes and 11.9% were newly diagnosed with diabetes. When not using OGTT, diabetes would have been missed in 5.5% of the total population and 19.3% would wrongfully be reassured as having a normal glucose tolerance. There-fore, we advocate to keep on performing an OGTT.

& 2176-PComparative Effectiveness of Diabetes Resolution in the Super Obese following Bariatric SurgeryJOHN MORTON, TARA MOKHRATI, HOMERO RIVAS, DAN AZAGURY, Stanford, CA

Diabetes is a common condition in the obese and bariatric surgery leads to DM remission in many patients. Among weight loss procedures, laparo-scopic Roux-en-Y gastric bypass (LRYGB) offers the highest rates of DM resolution. However, the impact of LRYGB on DM in the super obese (BMI ≥50 kg/m2) remains undetermined. This study aims to investigate diabetes remission following LRYGB in the super obese.

At a single academic institution, 1412 bariatric patients undergoing LRYGB participated in this prospective study. Of these, 415 (29.4%) were super-obese at pre-op and 997 (70.6%) were obese (BMI <50 kg/m2). Pre-op demographic, anthropometric, and standard lab data were collected. Review of participants’ medical record was undertaken to assess for pre-op DM. Rates of DM resolution at 12 mos post-op were assessed by hemoglobin A1C. Student t-test and Fisher’s exact analysis were used to compare con-tinuous and dichotomous variables, respectively. Analysis performed using GraphPad Prism 6.

As expected, super-obese participants had signifi cantly higher pre-op weight (344.4 vs. 263.6 lb) and BMI (56.4 vs. 43.0 kg/m2) than obese (all p’s <0.0001). At 12 mos post-LRYGB super-obese lost less weight and had higher BMI than obese counterparts (all p’s <0.0001). Prior to surgery, super-obese vs. obese populations had no difference in rate of DM (39.3 vs. 37.1%, p=0.47). 12 mos follow-up data were collected for 251 super-obese and 574 obese participants. Among the super-obese, 105 participants were noted to have DM either improve (22) or resolve (83), 12 reported DM had not resolved and 12 were unable to report. There was no difference in DM resolution/im-provement at 12 mos based on pre-op obesity classifi cation (p=0.32).

Diabetes commonly affects bariatric patients and is often improved fol-lowing LRYGB. This study found that pre-op BMI ≥50 kg/m2 was not found to impact DM resolution, super-obese patients can expect diabetes resolution comparable to the general bariatric population.

& 2177-PMaintenance of Blood Glucose Control and Stable Weight over 6 Months after a Very Low Calorie Diet in Short- and Long-Duration Type 2 DiabetesSARAH STEVEN, KIEREN G. HOLLINGSWORTH, AHMAD AL-MRABEH, MURIEL J. CASLAKE, ROY TAYLOR, Newcastle upon Tyne, United Kingdom, Glasgow, United Kingdom

We have previously demonstrated normalisation of blood glucose control in type 2 diabetes following a very low calorie diet (VLCD; 600-800 kcal/day). This study aimed to defi ne durability of blood glucose control over 6 months following acute weight loss in type 2 diabetes of short (<4yr; n=15) and long (>8yr; n=14) duration. The underlying pathophysiological mechanisms were assessed. Weight remained stable after an 8 week VLCD for 6 months in both groups (short: 99.0±3.7kg at baseline; 84.7±3.5 after VLCD; 84.7±3.7 at

6 months (p=1.00); and long: 96.9±3.8kg, 82.8±3.3, and 84.6±3.5 (p=0.12) re-spectively). At baseline, HbA1c was 7.2±0.2% and 8.6±0.4% in short and long groups respectively. In short group after VLCD, 12/15 individuals achieved HbA1c <6.5% and 11/12 (73% of the group) maintained this at 6 months. In long duration group after VLCD, 2/14 achieved HbA1c <6.5% and 3/14 (21% of the group) at 6 months.The initial fall in hepatic triacylglycerol (measured by the 3-point Dixon magnetic resonance method) was maintained:11.9±2.2, 2.4±0.2, 2.3±0.3% and 8.2±1.4, 2.0±0.1 to 2.2±0.3% at baseline, after VLCD and at 6 months in short and long groups respectively. Correspondingly, the improvement in hepatic insulin sensitivity was durable (hepatic IR in-dex short: 2063.4 (552.6-6601.9), 939.1 (304.7-2200.9), 754.5 (292.4-3723.2) µmol/min-1/kgffm

-1/pmol/l; long: 1219.2 (424.9-2743.9), 718.1 (156.4-1388.4), 767.5 (166.4-2397.2) µmol/min-1/kgffm

-1/pmol/l). Fasting insulin was 17.4 (3.9-48.9), 7.9 (2.4-22.9), 7.6 (2.2-31.6) mU/l (short) and 7.0 (4.1-31.9), 5.4 (1.4-14.8) to 5.9 (1.2-19.0) mU/l (long). Following a VLCD in both long and short duration type 2 diabetes the improvements in glucose control, hepatic triacylglycerol and hepatic insulin sensitivity are maintained. This study confi rms the dura-bility of the pathophysiologic changes which permit return of normal blood glucose control in people with previous type 2 diabetes.

Supported By: Newcastle National Institute for Health Research; Novo Nordisk UK Research Foundation

& 2178-PRelationships between Insulin Resistance, Brain Activity, and Pro-spective Weight Changes in a Community Sample of AdultsANIA M. JASTREBOFF, MARC N. POTENZA, CHERYL LACADIE, DONGJU SEO, KWANGIK A. HONG, RAJITA SINHA, New Haven, CT

Obesity is associated with metabolic perturbations and changes in brain reward pathways. Whether these peripheral and central adaptations are re-lated to future weight gain is unknown. We hypothesized that fasting insulin and insulin resistance would be predictive of future weight gain and that neural responses to food cues would be predictive of weight changes ir-respective of BMI. We prospectively followed 58 individuals of varying BMI (18.7-39.5kg/m2) and age (18-48y), assessing baseline BMI, fasting plasma insulin (FPI), insulin resistance (IR) (by homeostatic model assessment, HO-MA-IR), and brain responses (by functional MRI) during personalized food cue and neutral situations using a validated, guided-imagery paradigm. Weight was remeasured after an average period of 8 months. On follow-up 48% of participants lost or maintained weight (WL) (mean (x̄) BMI change BMI -0.86kg/m2) while 52% gained weight (WG) (x̄ BMI change 1.39kg/m2). There was no signifi cant difference between WL vs. WG in baseline BMI (WL x̄ BMI 26.3kg/m2, range 19.7-39.5; WG x̄ BMI 28.4kg/m2, range 18.7-37.8; p=0.1), age (WL 27.7y; WG 27.8y; p=0.9), FPI (WL 12.3µU/ml, WG 14.4µU/ml; p=0.3), or HOMA-IR (WL 2.85, WG 3.38; p=0.3). Interestingly, FPI and HOMA-IR correlated positively with future increases in BMI in WG (FPI: r=0.38, p<0.05; HOMA-IR: r=0.41, p<0.03) but not in WL (FPI: r=0.33, p=0.8; HOMA-IR: r=-0.05, p=0.7). In contrast brain maps of food cue vs. neutral con-ditions, HOMA-IR and FPI were negatively correlated with bilateral activa-tion in the ventral striatum in WL, whereas no such correlation was observed in the WG. Elevated fasting insulin and insulin resistance may be associated with future weight gain irrespective of baseline BMI. Lower fasting insulin levels and insulin sensitivity may be associated with activation in reward brain regions such as the ventral striatum, potentially relating to attenuated consumption of food, contributing to weight loss or maintenance.

Supported By: UL1DE019586, PL1DA024859, 1K23DK101694, RL1AA017539

2179-PFunctional and Clonal Analyses of Human Brown and White Fat Progenitors Identify Markers that Predict Thermogenic Capacity of Mature AdipocytesRUIDAN XUE, MATTHEW LYNES, JONATHAN DREYFUSS, TIM SCHULZ, HONG-BIN ZHANG, TIANLIAN HUANG, KRISTY L. TOWNSEND, YIMING LI, HIROKAZU TAKAHASHI, LAUREN WEINER, ANDREW WHITE, MAUREEN LYNES, LEE RUBIN, LAURIE J. GOODYEAR, AARON M. CYPESS, YU-HUA TSENG, Boston, MA, Wo-burn, MA, Shanghai, China, Cambridge, MA

Obesity is a pandemic and major contributor to diabetes and other meta-bolic disorders. Brown adipose tissue (BAT) holds therapeutic potential for obesity and metabolic syndrome via increasing energy expenditure. Both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human popula-tions. In order to investigate the heterogeneous nature of the progenitor cell population in human BAT and WAT, we have generated clonal cell lines from human neck fat and characterized their adipogenic differentiation and metabolic function. Indeed, inter-subject differences not only exist in whole

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adipose tissue, but also exist in adipose progenitors and their derivative adipocytes. Importantly, despite the inter-subject variations, human brown adipocytes clearly possess great metabolic capacity. Combining a UCP1 reporter system and gene expression profi ling, we defi ned novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of mature adipocytes. Knocking out the positive UCP1 regulators in brown preadipocytes by CRISPRs markedly abolished the high level of UCP1 in mature brown adipocytes. Finally, we showed the ability to prospec-tively isolate adipose progenitors with great thermogenic potential. Taken together, these data provide new insights into the cellular heterogeneity in human fat and offer clinically relevant gene targets that mark thermogeni-cally competent preadipocytes.

Supported By: American Diabetes Association (7-12-BS-191 to Y-H.T.); National Institutes of Health (R01DK077097); National Institute of Diabetes and Digestive and Kidney Diseases (P30DK036836); Chugai Pharmaceutical Co., Ltd.

2180-PConsistent Improvement of Metabolic Parameters in Obese Patients with Type 2 Diabetes by the Endoscopically Delivered Duodenal-Jejunal Bypass Liner (DJBL)KATHARINA LAUBNER, NIKOLAOS PERAKAKIS, LAURA POTASSO, HENNING SCHWACHA, JOCHEN SEUFERT, Freiburg, Germany

The duodenal-jejunal bypass liner (DJBL) is an endoscopically delivered and removable device creating a duodeno-jejunal bypass thereby mimick-ing the effect of surcigal bariatric Roux-en-Y and Duodenal-Jejunal Bypass procedures. Therefore the DJBL provides important properties that may be exploited for the improvement of metabolism in obese T2Dm patients. We tested this hypothesis prospectively in 30 obese T2Dm patients.

30 subjects with T2Dm (80% f., mean age: 46,3 + 9,8y, mean diabetes duration 7,3 + 5,6y) on various medications (66,6% on insulin therapy) and obesity (mean BMI: 45,5 + 8,2kg/m²) were implanted with a DJBL under gen-eral anaesthesia. Anthropometric, metabolic and cardiovascular risk param-eters were monitored together with adverse events every 3 months. After 12 months the DJBL was removed as per license.

Average HbA1c reductions were 1,0% for the overall patient popula-tion (p=0,004), and 1,2% for patients with baseline HbA1c values of >7,0% (p<0,0001), despite simultaneous reduction of antidiabetic medication. Mean BMI was reduced from 45,5 kg/m² to 38,1 kg/m² (p=0,05), corresponding to an excess weight loss of 30,8%. LDL and parameters of NAFLD decreased as well. The most common AEs were abdominal pain and nausea especially in the fi rst two weeks after implantation. We observed one duodenal perfora-tion as a serious complication. The DJBL was removed early in 7 subjects (abdominal pain, dysfunction, migration, pancreatitis, need of anticoagula-tion). As of now, follow up data from few patients after explantation are available, but HbA1c and BMI (seems to be preserved after 3 month (6,1% e.g. 39,1 kg/m²).

The DJBL is a safe and effective endoscopic device that improves gly-cemic control, body weight and related metabolic and cardiovascular risk-parameters in the majority of obese T2Dm patients. The DJBL may represent an alternative or adjunct to bariatric surgery to treat type 2 diabetes mellitus and obesity.

2181-PAdipose Nicotinamide N-methyltransferase Expression Is In-creased in Omental Adipose Tissue of Morbidly Obese Women and Correlates with Infl ammatory Markers and Insulin ResistanceMURAT KILICARSLAN, BARBARA B. KAHN, BARBARA A. DE WEIJER, JOHANNES A. ROMIJN, UNGA A. UNMEHOPA, MARIETTE T. ACKERMANS, IGNACE A. JANSSEN, FRITS BERENDS, ARNOLD VAN DE LAAR, LEX HOUDIJK, SUSANNE E. LA FLEUR, MIREILLE J. SERLIE, Amsterdam, Netherlands, Boston, MA, Arnhem, Netherlands, Alkmaar, Netherlands

Recent studies have shown that Nicotinamide N-methyltransferase (NNMT) is increased in white adipose tissue in obesity and type 2 diabetes mellitus (T2DM). The exact mechanism by which adipose NNMT is increased is unknown. Previous studies have shown that factors secreted by mac-rophages increase NNMT expression and activity in, among others, glioma cells and endometrial stromal cells. As obesity is characterized by increased macrophage accumulation with infl ammation in adipose tissue, we hypoth-esized that the increased NNMT expression in obesity is associated with the presence of adipose-tissue macrophages and infl ammatory markers. To test this hypothesis, we studied expression of NNMT and infl ammatory markers in subcutaneous adipose tissue (SAT) and omental adipose tissue (OAT)) in obese women undergoing bariatric surgery (mean BMI 45 [39-51] kg/m2) and 6 lean controls undergoing elective cholecystectomy (mean BMI 22 [20-24]

kg/m2). In the obese subjects, we performed a hyperinsulinemic-euglycemic clamp to assess whole-body glucose uptake (rate of disappearance [Rd]). Rd inversely correlated with NNMT expression in OAT (rs = -0.534, P = 0.027) but not with NNMT expression in SAT. NNMT expression was higher in OAT (P = 0.019) but not in SAT in the obese subjects versus lean controls. Furthermore, NNMT expression in OAT correlated positively with expression of CD68 (P = 0.06), MIP1b (P = 0.05), IL10 (P = 0.004) and TLR4 (P = 0.007) while SAT NNMT expression correlated positively with MIP1b (P = 0.07), IL10 (P = 0.05) and TLR4 (P = 0.005). To conclude, these results show that adipose NNMT expression is increased in obesity and suggest that the increase may be associated with adipose-tissue infl ammation and insulin resistance that occur with the development of obesity.

2182-PRelationship of Adiposity and Insulin Sensitivity to Endothelial Function, Vascular Structure, and Arterial Stiffness in Pubertal and Postpubertal Children and AdolescentsJUSTIN R. RYDER, DONALD R. DENGEL, DAVID JACOBS, ALAN SINAIKO, AARON S. KELLY, JULIA STEINBERGER, Minneapolis, MN

Adiposity and insulin resistance in adults are associated with vascular dysfunction, but these relations are not well defi ned in children. We exam-ined the relations of adiposity and insulin sensitivity/resistance to endothe-lial function, vascular structure, and arterial stiffness in 252 children (mean age 15.1 ± 2.4 yrs; mean BMI-percentile 68.2 ± 26.5; Tanner stage 2-5). Body fat percentage (BF%) was measured using dual-energy X-ray absorptiometry (DXA) and visceral fat (VAT) was estimated with computed tomography (CT) and DXA. Insulin sensitivity/resistance was measured using euglycemic-hyperinsulinemic clamps. Vascular measurements were obtained for en-dothelial function - brachial artery fl ow-mediated dilation (FMD), vascular structure - carotid intima-media thickness (cIMT), arterial stiffness - carotid incremental elastic modulus (cIEM) and pulse wave velocity (PWV). Data were analyzed using ANCOVA and linear regression, adjusting for Tanner, sex, race, family relationship, and baseline artery diameter (for FMD analy-ses only). Regardless of measurement method (BMI, BF%, VAT), the highest adiposity group (split by tertiles) exhibited the highest FMD compared with lower adiposity groups (p<0.01 for all). Higher fasting insulin was associated with greater FMD (p=0.019, R2=0.222) independent of BF%; however, no as-sociation was found between insulin sensitivity and FMD. Greater cIMT was observed in the most insulin resistant tertile group (p=0.05), but no associa-tion was seen for cIEM and PWV with adiposity or insulin sensitivity. These fi ndings demonstrate a paradoxical relationship of FMD with adiposity and fasting insulin levels, but not insulin sensitivity in pubertal and post-pubertal youth. Research is needed to identify the mechanisms responsible for the vascular protection in pediatric obesity and the trajectory of transition to-wards vascular disease seen adulthood.

Supported By: National Institutes of Health (1UL1-RR033183, R01DK072124-01A3, R01CA113930-01A1, M01RR00400, UL1TR000114, T32-DK083250)

2183-PInsulin Sensitivity Indices Derived from Oral Glucose Tolerance Tests: Are They Valid after Roux-en-Y Gastric Bypass?KIRSTINE N. BOJSEN-MØLLER, CARSTEN DIRKSEN, NILS B. JØRGENSEN, VIGGO B. KRISTIANSEN, JENS-ERIK B. JENSEN, JENS J. HOLST, ERIK A. RICHTER, STEN MADSBAD, Hvidovre, Denmark, Copenhagen, Denmark

Insulin sensitivity indices obtained from oral glucose tolerance tests (OGTTs) are frequently used in diabetes research and provide an easily ob-tained measure of insulin sensitivity. Roux-en-Y gastric bypass (RYGB) in-duces extensive weight loss and improvements in insulin sensitivity when evaluated by the hyperinsulinemic clamp. Insulin sensitivity indices obtained from OGTTs have not been validated after RYGB and may be inaccurate due to surgical alterations of the gut with accelerated glucose absorption and increased insulin secretion.

We investigated insulin sensitivity in 20 obese subjects (10 with and 10 without T2D) before, 3 months (mo) and 1 year (y) after RYGB using a 4 h hy-perinsulinemic (40 mU/m2/min) euglycemic (5.5 mmol/L) clamp and a 2 h 75 g OGTT. Fat free mass (ffm) was assessed by DEXA. Matsuda and Oral Glu-cose Insulin Sensitivity (OGIS) indices were calculated from OGTTs and were compared to the clamp-derived Rate of disappearance (Rd) of [6,6-2H2]-glucose (in mg/min/kg ffm).

Before RYGB, both Matsuda and OGIS correlated signifi cantly with Rd (Pearson r=0.63, p<0.001 and r=0.62, p=0.005). After RYGB, the relative im-provements in Matsuda were substantially larger than improvements in Rd at 3 months (Matsuda:+86±17%, Rd:+36±13%, p<0.05) and 1 year (Matsu-da:+164±28%, Rd:+57±14%, p<0.01). Furthermore, relative changes in Mat-

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suda did not correlate signifi cantly to relative changes in Rd (3 mo: r=0.23, p=0.35, 1y: r=0.46, p=0.07). In contrast, relative changes in OGIS were com-parable to the relative changes in Rd (OGIS, 3 mo:+31±5.9%, 1 y:+46±5.9%, p=ns) and correlations between the relative changes were signifi cant (3 mo: r=0.61, p=0.007, 1 y: r=0.50, p=0.04).

In conclusion, insulin sensitivity indices obtained from oral tests may be inaccurate after RYGB. Compared to the gold standard hyperinsulinemic euglycemic clamp, OGIS seems to perform better than Matsuda index in as-sessing changes in insulin sensitivity within the fi rst year after RYGB.

Supported By: UNIK

2184-PThe ApoB/PCSK9 Ratio: A New Index for Metabolic Risk in HumansHANNY WASSEF, SIMON BISSONNETTE, NATHALIE SAINT-PIERRE, VALERIE LA-MANTIA, YANNICK CYR, MICHEL CHRÉTIEN, MAY FARAJ, Montreal, QC, Canada

PCSK9 loss-of-function (LOF) variants have shown cardiovascular protec-tion because they lower plasma LDL-C concentrations, yet the metabolic im-pact of PCSK9 concentration on glucose and fat metabolism remains unclear. We hypothesized that increased apoB-lipoprotein uptake in subjects with elevated plasma apoB but reduced PCSK9 might injure white adipose tissue (WAT) and provoke metabolic dysfunction.

Fourteen normoglycemic postmenopausal obese women underwent in-depth assessment of glucose and fat metabolism using 13C-triolein-labeled-high-fat meals and Botnia clamps. PCSK9 gene sequencing revealed that 8 women carried LOF variants including a potent variant Q152H, and uncov-ered a new variant (L14ins). Irrespective of the PCSK9 variant, the plasma apoB/PCSK9 ratio correlated signifi cantly with fasting TG (R=0.67), fasting and postprandial TG-content of TG-rich-lipoprotein (R=0.71 and R=0.70) and postprandial total TG (R=0.72) and 13C-TG (R=0.60). Plasma apoB/PCSK9 ratio also correlated negatively with gynoid fat function ex vivo (R=-0.63) and with insulin sensitivity (Rclamp: GIR=-0.56, M/I=-0.65). Concomitant evaluation of apoB with PCSK9, improved the association of apoB with WAT dysfunction and insulin resistance. On the other hand, plasma PCSK9, LDL-C or LDL-C/PCSK9 did not correlate signifi cantly with any index of metabolic risk.

In summary, a higher plasma apoB/PCSK9 ratio correlates with metabolic risks in normoglycemic obese women. These fi ndings are consistent with in-creased apoB-lipoproteins uptake into non-hepatic peripheral tissues induc-ing metabolic dysfunction. With the advent of chronic anti-PCSK9 therapy to reduce LDL-C, we hypothesise that the plasma apoB/PCSK9 ratio, not PCSK9 alone, provides a clinical index for early metabolic disturbances that may occur before normal fasting glucose is affected.

Supported By: Canadian Institutes of Health Research (MOP123409)

2185-PNo Effect of Long-Term High-Dose Resveratrol on VLDL-TG Kinetics, Insulin Sensitivity, and Liver Fat Content in Obese Men with Nonal-coholic Fatty Liver DiseaseMARIANNE KJAER POULSEN, BIRGITTE NELLEMANN SØRENSEN, HANS STØDKILDE-JØRGENSEN, STEEN BØNLØKKE PEDERSEN, HENNING GRØNBÆK, SØREN NIELSEN, Aarhus, Denmark

Non-alcoholic fatty liver disease (NAFLD) is an obesity-associated condi-tion caused by an imbalance between hepatic lipid input and output. Res-veratrol (RSV) reverses the features of insulin resistance and reduces liver fat content in animals with NAFLD. However, the effects of RSV are still controversial in humans. The objectives were to assess the long-term ef-fects of RSV on VLDL-TG kinetics, insulin sensitivity and liver fat content in upper-body obese men with NAFLD (NAFLD+).

Sixteen non-diabetic, obese (waist-to-hip ratio >0.9, BMI >28 kg/m2) NA-FLD+ men with similar BMI (31.1±1.7 vs. 34.1±4.4 kg/m2, P=.14) were ran-domized in a double-blinded, placebo-controlled clinical trial and treated with either high-dose RSV or placebo (PL) for 6 months. MR spectroscopy assessed liver fat content. 14C-labeled VLDL-TG and 3H-labeled palmitate and glucose tracers were applied in combination with indirect calorimetry and breath samples to assess turnover rates and substrate oxidation rates postabsorptively and during a hyperinsulinemic-euglycaemic clamp. DXA and MRI assessed body composition.

Liver fat content was similar in the two groups. However, the PL group had greater visceral and subcutaneous adipose tissue volumes compared with the RSV group (VAT: 7139±1554 vs. 5260±1234 cm3, P = .03; SAT: 10248±2836 vs. 7518±1645 cm3, P = .04). RSV did not improve the insulin-resistant changes in VLDL-TG kinetics associated with NAFLD (secretion: basal before (RSV vs. PL): 67.8±47.5 vs. 104.4±49.7, clamp before: 47.4±39.8 vs. 72.9±39.5, basal after: 61.6±41.5 vs. 110.6±54.7, clamp after: 42.7±30.0 vs. 64.3±34.1 µmol/min, P=.61). Likewise, no improvement in FFA and glucose

kinetics, body composition or liver fat occurred in the RSV compared with the PL group.

In conclusions, long-term supplementation of high-dose Resveratrol did not change the VLDL-TG kinetics, insulin sensitivity or liver fat content in a favorable manner.

2186-P

2187-PBetatrophin/ANGPTL8 Is a Novel ER Stress-inducible Hepatokine Increased in Nonalcoholic Fatty Liver DiseaseYONG-HO LEE, SO RA KIM, EUN YOUNG LEE, HYE JIN YOON, SOO HYUN KIM, GYURI KIM, EUGENE HAN, BYUNG-WAN LEE, EUN SEOK KANG, HYUN CHUL LEE, BONG SOO CHA, Seoul, Republic of Korea

Betatrophin/ANGPTL8 is a liver-secreted protein, recently identifi ed as a potent stimulator of beta cell proliferation in mice. However, regulation of betatrophin expression in human with non-alcoholic fatty liver disease (NAFLD) has not been determined. The aim was to investigate whether beta-trophin is a hepatokine that may be highly produced in subjects with NAFLD as well as in hepatocytes in vitro and mice in vivo. A total of 98 age-matched subjects with control, IFG and T2D were recruited. Serum betatrophin lev-els were examined by ELISA. NAFLD was diagnosed by either abdominal ultrasound or computed tomography. To further elucidate the regulation of betatrophin, we used HepG2 cells and various mice models with fatty liver. Serum betatrophin levels were signifi cantly elevated in subjects with NA-FLD compared with controls (0.97 ± 0.45 vs.0.60 ± 0.22 µg/L, P<0.001), even after stratifi cation for their diabetic status. Serum betatrophin positively correlated with BMI grouped into tertiles (r=0.22), fasting glucose (r=0.22), HbA1c (r=0.23), AST (r=0.23), ALT (r=0.21) and triglycerides levels (r=0.23), while was inversely related to age (r=−0.27). Treatment with palmitate or endoplasmic reticulum (ER) stress activator, tunicamycin, increased the ex-pression of betatrophin mRNA and protein in HepG2 cells in a time- and dose-dependent manner, whereas 4-phenylbutyrate, a chemical chaperone, blocked the induction of betatrophin. Moreover, betatrophin was highly increased in the liver of dbdb mice and mice with high-fat or methionine-choline defi cient diet. In conclusion, regardless of diabetic status, subjects with NAFLD had signifi cantly elevated circulating levels of betatrophin of which expression is highly induced in hepatocytes by palmitate or chemical mediated ER stress as well as in fatty liver from various mice models. This indicates that betatrophin may be used as a novel biomarker for detecting NAFLD (ClinicalTrials.gov No. NCT02285218).

Supported By: National Research Foundation of Korea (HI14C2476)

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2188-PmiR-148a Is Associated with Obesity and Modulates Adipocyte Dif-ferentiation of Mesenchymal Stem Cells through Wnt SignalingCHUNMEI SHI, CHENBO JI, XIRONG GUO, Nanjing, China

Obesity is the result of numerous, interacting genetic, behavioral, and physiological factors. Adipogenesis is regulated in part by several adipo-cyte-selective microRNAs (miRNAs) and transcription factors that regulate the proliferation and differentiation of human adipose tissue-derived mes-enchymal stem cells (hMSC-Ad). In the present study, we examined the role of adipocyte-selective miRNA in the differentiation of HMSC-Ad to adipo-cytes. We found that miR-148a, miR-26b, miR-30 and miR-199a levels were increased in differentiating hMSC-Ad. Of these miRNAs, only miR-148a had more effects on increasing PPRE luciferase activity than others. We further found miR-148a expression level increased in adipose tissue from obese people and in mice fed a high fat diet. miR-148a acted through suppression of its target gene, Wnt1, an endogenous inhibitor of adipogenesis, and ecto-pic expression of miR-148a accelerated differentiation and partially rescued Wnt1-mediated inhibition of adipogenesis. Analysis of the upstream region of the miR-148a locus identifi ed a 3 kb region that contained a functional CREB response element that was required for miR-148a expression in hMSC-Ad. These results suggest that miR-148a as a biomarker of obesity in human subjects and in a mouse model, which represents a CREB-modulated miRNA that acts to repress Wnt1, and hence promote adipocyte differentiation.

Supported By: National Key Basic Research Program of China (2013CB530604)

2189-PAdipocyte Iron Regulates Leptin and Food IntakeYAN GAO, ZHONGGANG LI, JUDITH SIMCOX, SOH-HYUN LEE, DEBORAH JONES, ROBERT C. COOKSEY, J. SCOTT GABRIELSEN, DONALD A. MCCLAIN, Salt Lake City, UT, Boston, MA

Previous reports have investigated role of iron in the regulation of appe-tite. Dietary iron supplementation is associated with growth and increased appetite in children. We therefore investigated the effect of iron on lep-tin, a hormone that is secreted primarily by adipose tissue and responsible for regulating food intake and energy homeostasis. The concentration of serum ferritin is negatively associated with serum leptin in patients with type 2 diabetes and in obese subjects with metabolic syndrome (r=-0.452, p=0.0002). Mice fed a high iron diet exhibited decreased leptin mRNA and protein. Consistent with the changes in leptin, dietary iron content was also directly related to food intake, independently of weight. Loss of the adipocyte iron export channel, ferroportin, in mice resulted in adipocyte iron loading and decreased leptin. Conversely, decreased levels of hepcidin in hereditary hemochromatosis result in increased adipocyte ferroportin ex-pression, decreased adipocyte iron, and increased leptin. Treatment of 3T3-L1 adipocytes with iron decreased leptin mRNA levels in a dose-dependent manner. We found iron negatively regulated leptin transcription via CREB activation. Two potential CREB-binding sites were identifi ed in the mouse leptin promoter region. Mutation of both sites completely blocked the effect of iron on promoter activity. We also found enrichment of phospho-CREB binding to those two sites by ChIP in 3T3-L1 adipocytes treated with iron. These fi ndings indicate that levels of dietary iron play an important role in regulation of appetite and fat metabolism through CREB-dependent modula-tion of leptin.

Supported By: U.S. Department of Veterans Affairs (2I01BX001140-05); National Institutes of Health (1R01DK081842)

2190-PAssociation of Plasma ApoB with Hyperinsulinemia and Insulin Re-sistance: Role of Dietary Fat Clearance and Adipose Tissue Func-tion in HumansVALÉRIE LAMANTIA, SIMON BISSONNETTE, NATHALIE SAINT-PIERRE, YANNICK CYR, ROBERT DUFOUR, HANNY WASSEF, MAY FARAJ, Montreal, QC, Canada

Plasma apoB predicts insulin resistance (IR) and T2D in humans. Delayed triglyceride (TG) clearance is known to promote lipotoxicity and IR. We recently reported that LDL, the major form of apoB-lipoproteins, directly impairs fat storage in human white adipose tissue (WAT) ex vivo. We hy-pothesized that the link between plasma apoB, IR and hyperinsulinemia is mediated through delayed dietary fat clearance in vivo.

We examined insulin secretion and sensitivity during a 1-hour intrave-nous-glucose tolerance test followed by a 3-hours hyperinsulinemic clamp in normoglycemic overweight/obese men and postmenopausal women (N=29, 45% men, BMI ≥ 27 kg/m2, 45-74 yrs). Postprandial dietary TG clear-ance was measured over 6 hours after the ingestion of a 13C-triolein-labeled high-fat meal. WAT function was measured as the ability of fasting hip WAT

samples, obtained by needle biopsy, to hydrolyze and store a synthetic 3H-TG substrate. Plasma apoB averaged 1.03 ± 0.28 g/L, and was signifi cantly as-sociated with higher IR (r=0.41) and 2nd phase insulin secretion (r=0.40) after corrected for fat mass (IR: r=0.55; 2nd phase insulin secretion: r=0.52). There was also an association trend of plasma apoB with 2nd phase C-peptide se-cretion (r=0.32, p=0.090). Plasma apoB correlated with delayed dietary TG clearance (r=0.53) and reduced WAT function (r=-0.48). Moreover, delayed dietary TG clearance was associated with higher IR (r=0.36), 2nd phase insu-lin secretion (r=0.42) and 2nd phase C-peptide secretion (r=0.45). As hypoth-esized, correcting for dietary TG clearance or WAT function eliminated the association of plasma apoB with IR and 2nd phase insulin secretion. There were no sex-differences in the direction of associations of plasma apoB with the metabolic outcomes.

In conclusion, the association of plasma apoB with IR and hyperinsuline-mia in overweight and obese subjects is mediated, at least in part, by de-layed dietary fat clearance and WAT dysfunction.


2191-PLocus Refi ning of the FTO Gene at Density of 1000 Genome Project and Haplotyping Allow a Better Understanding of the Gene Asso-ciation in Complex DisordersREDHA ATTAOUA, MIHAIL COCULESCU, CHRISTOPHE NORMAND, SARA HAY-DAR, DOAA FAKIH, MADALINA VINTILA, SINZIANA MURARU, NICOLETA BAC-ULESCU, DELPHINE CAZALEDES, PATRICK POUCHERET, FLORIN GRIGORESCU, MEDIGENE CONSORTIUM, Montpellier, France, Bucharest, Romania

FTO (fat mass and obesity associated) gene was associated with obesity and many metabolic abnormalities in populations of different ethnic origins, notably through a panel of SNPs located in the linkage disequilibrium (LD) block of intron 1. The effect of the FTO has been suggested to act through neighbouring genes such as IRX-3, highlighting potential gene-gene interac-tion at the FTO locus. We previously reported the association of FTO through the SNP rs1421085 (T/C) with metabolic syndrome (MetS) and the impaired glucose tolerance (IGT) in women from Central Europe with polycystic ovary syndrome (PCOS). The same pattern of association was observed through haplotyping strategy using 5 SNPs in intron1. As part of the European proj-ect MEDIGENE, we genotyped with customized gene-chip the FTO gene in a well characterized subpopulation of our samples (55 PCOS and 48 controls) at density of 1000 Genome Project (about 2800 SNPs). The sliding-window strategy (windows of 10 kb) applied on the entire gene (GoldenHelix soft-ware) allowed the reconstruction of 31662 haplotypes. Comparison of their prevalence in women with PCOS and controls showed that more than 100 haplotypes were 4 times more frequent in PCOS. They were widely distribut-ed through the gene, including regions far for intron 1. Two haplotypes, close to the 3’-UTR and containing SNPs rs2540769, rs75298050, rs2665275 were found at 28% in PCOS and 5% in controls. Our fi nding highlights the powerful role of locus refi ning and haplotyping strategy for a better characterization of FTO gene association and is a step ahead towards the comprehension of genetic association with complex disorders.

Supported By: European Commission (FP7-279171-1)

2192-PThe Impact of a Telephonic Wellness Coaching Program on Weight Loss: A Natural Experiments for Translation in Diabetes (NEXT-D) StudyJULIE A. SCHMITTDIEL, SARA R. ADAMS, NANCY GOLER, MINDY BOCCIO, RASHEL S. SANNA, DAVID J. BELLAMY, SUSAN D. BROWN, ROMAIN S. NEUGE-BAUER, HONG XIAO, ASSIAMIRA FERRARA, Oakland, CA

More than two-thirds of adult Americans are overweight or obese; this excess weight increases the risk of developing diabetes, and puts diabetes patients at higher risk for complications. We evaluated the impact of a tel-ephonic Wellness Coaching program offered by Kaiser Permanente North-ern California (KPNC) on the 12-month trajectory of patient body mass index (BMI) in 938 KPNC members who called the Wellness Coaching center to receive counseling on weight loss, healthy eating, or exercise from January 1-August 23, 2011. Patients were included in the study if they had at least one BMI in the electronic medical record in the 12 months leading up to, and in the 12 months after, the fi rst coaching session. The comparison group was a 20:1 propensity-score matched cohort; controls were matched with Well-ness Coaching participants based on baseline age, sex, race, BMI, smoking status, number of primary care visits, home KPNC facility, and diagnostic cost group (DxCG) score. We used interrupted time series analyses to ex-amine the impact of coaching on the trajectories of BMI in the 12 months before vs. the 12 months after initiating coaching in the intervention group,

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and for the same time period in their controls. Wellness Coaching partici-pants had a signifi cant upward trend in BMI in the 12 months before their fi rst Wellness Coaching session, and a signifi cant downward trend in BMI in the 12 months after their fi rst session (p<.01 for both). Based on this trend analysis, the average weight decrease after initiating Wellness Coaching for participants was 9.8 pounds. Matched controls did not have statistically signifi cant decreases in weight during the post-period. Wellness Coaching has a positive impact on BMI reduction that is both statistically and clinically signifi cant. Future research and quality improvement efforts should focus on disseminating the use of Wellness Coaching for weight loss in both diabetes patients and those at risk for developing the disease.

Supported By: Centers for Disease Control and Prevention; National Institute of Diabetes and Digestive and Kidney Diseases (U58 DP002721)

2193-PEffects of Weight Loss on Heme Ooxygenase-1 Tissue ExpressionsCLAUDIA RESS, ALEXANDER M. MOSCHEN, HELMUT WEISS, CLEMENS MOL-NAR, GUENTER WEISS, HERBERT TILG, SUSANNE KASER, Innsbruck, Austria, Salzburg, Austria

Heme oxygenase-1 (HO-1) expression has previously been shown to be signifi cantly higher in livers and visceral adipose tissues of insulin-resistant obese subjects than in lean or obese, insulin-sensitive subjects. Additionally, mice defi cient for liver or macrophage HO-1 are resistant to diet-induced in-sulin resistance and infl ammation. Mechanistically, HO-1 infl uences infl am-matory skewing and NFkB amplifi cation in macrophages and insulin signal-ing in hepatocytes.

Aim of this work was to elucidate the effects of weight loss following bariatric surgery on HO-1 expression in livers and subcutaneous adipose tis-sue extracts.

Twenty non-diabetic obese subjects were investigated before and 6 months after bariatric surgery. HO-1 mRNA expression levels were mea-sured in livers and subcutaneous adipose tissue extracts by fl uorescence-based real time PCR.

Six months after bariatric surgery mean weight reduction of 26 kg (BMI 42.51+/-3.95 kg/m2 vs. 34.96+/-4.66 kg/m2, p<0.01) was accompanied by a signifi cant increase of insulin sensitivity as estimated by the HOMA index (5.35+/-4.02 vs. 2.74+/-2.08, p<0.01). In parallel, hepatic HO-1 expression signifi cantly decreased by 39.5% (30.46+/-28.00 vs. 18.42+/-13.0, p=0.025). HO-1 mRNA expression levels in subcutaneous adipose tissue extracts, which were measured in a subset of 10 patients, signifi cantly decreased by 67.5% (27.20+/-25.42 vs. 8.83+/-6.78, p=0.01).

Increased HO-1 tissue expression in obesity is at least partially revers-ible by pronounced weight loss following bariatric surgery. Reductions of HO-1 expression in livers and subcutaneous adipose tissues might explain improvements in subclinical infl ammation and insulin resistance in weight-reduced patients.

Supported By: Austrian Federal Ministry of Science, Research and Economy; Austrian National Foundation for Research, Technology and Development

2194-PGastric Bypass Surgery in Severely Obese Women with Type 1 Dia-betes: Cardiometabolic Effects at 1 and 5 Years Post-surgeryROELAND J.W. MIDDELBEEK, TAMARRA JAMES-TODD, JERRY D. CAVALLERA-NO, DEBORAH SCHLOSSMAN, MARY-ELIZABETH PATTI, FLORENCE M. BROWN, Boston, MA

While the benefi ts of gastric bypass (GB) surgery in type 2 diabetes are well-recognized, the long-term effects of GB on body weight, glycemic con-trol, and cardiometabolic risk in severely obese women with type 1 diabetes have not been well studied. Severely obese women with confi rmed type 1 diabetes (n=10) were studied prior to GB and at 1 and 5 years post-surgery. Data on body mass index (BMI), hemoglobin A1c (HbA1c), blood pressure, cho-lesterol, triglycerides, and insulin requirements were collected from medical records, and paired t-tests were used to determine whether mean cardio-metabolic risk factors at 1 and 5 years post-surgery differed vs. baseline and over time. GB reduced BMI and insulin requirements at 1 and 5 years, but did not improve HbA1c at these time points. Interestingly, GB led to improve-ments in cardiometabolic factors, including systolic blood pressure (p=0.03) and triglycerides (p=0.004) at 1 year, with improved HDL at 5 years (p=0.04). No worsening of microalbuminuria or retinopathy was seen. We conclude that GB does not improve longitudinal glycemic control in severely obese women with type 1 diabetes. However, GB reduced body weight and im-proved cardiometabolic risk factors for up to 5 years post-surgery.

Table 1. BMI, HbA1c, and Cardiometabolic Risk Factors at Baseline, 1 and 5 Years after Surgery.

Baseline 1 Year p† 5 Year p‡ p§BMI (kg/m2) 43.5±7.5 29.3±5.4 <0.0001** 33.8±7.5 <0.0001** <0.01*HbA1c (%) 8.1±1.3 8.3± 1.4 0.47 9.8±1.9 0.15 0.26Systolic BP (mmHg) 123.6 112.6 0.003* 118.7 0.45 0.19HDL (g/dL) 61.5 63.0 0.8 80.5 0.04* 0.004*LDL (g/dL) 102.3 92.8 0.41 91.5 0.44 0.95Triglycerides (g/dL) 112.8 80.6 0.004* 111.2 0.12 0.29Microalbumin (mg/L) 62.2 21.5 0.34 14.2 0.29 0.07† 1 year compared to baseline, ‡ 5 years compared to baseline, § 5 years compared to 1 year, *signifi cant at p<0.05, ** signifi cant at p<0.001.

2195-PReduced Hepatic Insulin Sensitivity Is a Distinct Feature of Obese Humans with Impaired Fasting GlucoseKASPER W. TER HORST, PIM W. GILIJAMSE, BARBARA A. DE WEIJER, MARI-ETTE T. ACKERMANS, MAX NIEUWDORP, MAARTEN R. SOETERS, JOHANNES A. ROMIJN, MIREILLE J. SERLIE, Amsterdam, Netherlands

Elevated basal endogenous glucose production (EGP), impaired suppres-sion of EGP by insulin and reduced insulin-stimulated glucose disposal are cornerstones in the pathogenesis of hyperglycemia in patients with type 2 diabetes. We aimed to determine the contribution of these processes to impaired fasting glucose in obese non-diabetic adults.

We analyzed data from obese adults with normal fasting glucose (fasting glucose <5.6 mmol/l; 62 men, 25 women; age 49 ± 11 y; BMI 38 ± 6 kg/m2) or impaired fasting glucose (fasting glucose 5.6 - 7.0 mmol/l; 35 men, 9 women; age 53 ± 8 y; BMI 38 ± 6 kg/m2) who underwent a two-step euglycemic hyperinsulinemic clamp with [6,6-2H2]glucose infusion to assess hepatic and peripheral insulin sensitivity.

Fasting insulin (99 ± 46 vs. 114 ± 52 pmol/l, p = 0.115), glucagon (77 ± 24 vs. 70 ± 20 ng/l, p = 0.126), triglycerides (1.5 ± 0.9 vs. 1.4 ± 0.7 mmol/l, p = 0.525), C-reactive protein (8.1 ± 7.2 vs. 4.0±3.6 mg/l, p = 0.143) and EGP (14.6 ± 2.5 vs. 15.1 ± 3.4 µmol·kgFFM-1·min-1, p = 0.327) did not differ between subjects with normal or impaired fasting glucose. During the fi rst step of insulin infu-sion, insulin-mediated suppression of EGP was markedly reduced in subjects with impaired fasting glucose (68.1 ± 13.8% vs. 54.6 ± 16.6%, p < 0.001), indicating reduced hepatic insulin sensitivity. Peripheral insulin sensitivity, assessed as the rate of glucose disappearance during the second step of insulin infusion, was low in all obese subjects, but did not differ between both groups (26.6 ± 9.1 vs. 25.2 ± 9.3 µmol·kg-1·min-1, p = 0.320).

In conclusion, impaired insulin-mediated suppression of EGP, i.e. hepatic insulin resistance, is a distinct characteristic of obese non-diabetic adults with impaired fasting glucose. This indicates that the progression from compensated insulin resistance to prediabetes is associated with reduced hepatic insulin sensitivity, but no further reduction in peripheral insulin sen-sitivity.

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2197-PCold Exposure of Human Subcutaneous (SC) White Adipose Tissue (WAT) Results in Increased Meteorin-like and Il4 along with In-creased Adipose BeigingPHILIP A. KERN, BRIAN S. FINLIN, BEIBEI ZHU, ESTHER E. DUPONT-VERSTEEGDEN, Lexington, KY

Mammals adapt to the cold with an increase in thermogenesis, although the role of WAT beiging (versus BAT activation) has not been well studied. With cold exposure and seasonal temperature changes, human SC WAT increases the expression of numerous genes, including UCP1, PGC1α, and other markers consistent with the conversion of white to beige adipocytes (JCEM 99:2772, 2014). Studies in rodents have suggested a role for mete-orin-like (Metrnl), which is expressed by adipocytes and exercising skeletal muscle. In mice, Metrl stimulates adipose resident eosinophils (EOS) to express IL4, which causes alternative activation of macrophages, causing catecholamine-induced conversion of white to beige adipocytes. To test these concepts in humans, we examined the expression in WAT in fat biop-sies from the summer and winter. In the winter, UCP1 protein and RNA and PGC1α RNA were signifi cantly elevated, as were Metrl and IL4. Other adi-pose genes increased in winter included DIO2 and IRF4, a known transcrip-tional effector of thermogenesis. In addition, fat biopsies were performed before/after a 30 min cold exposure, and the increase in beiging genes was accompanied by increases in Metrl and IL4.

To dissect the cell type(s) responsible for IL-4 and Metrl, human stem cell adipocytes were exposed to 16C in culture for 30 min, followed by 4 hr at 37C, and this resulted in a 2-3 fold increase in PGC1α, UCP1 RNA and Metrl. We measured markers for macrophages, eosinophils, and mast cells, and mast cell carboxypeptidase was robust and signifi cantly increased in response to a cold stimulus. These data suggest that Metrl and IL4 help generate the beige signal in human adipose tissue, and that mast cells may be involved in the pathway. SC WAT represents an enormous depot in hu-mans: the upregulation of thermogenic capacity may be important in energy regulation, and could be a target for the treatment of obesity.

2198-PEffect of Body Mass Index (BMI) on Outcomes in T2DM Patients Ini-tiating Insulin Glargine by Concomitant Oral TherapyFRANCESCA PORCELLATI, LOUISE TRAYLOR, WOLFGANG LANDGRAF, GEREMIA B. BOLLI, Perugia, Italy, Bridgewater, NJ, Frankfurt, Germany

Previous studies suggest that BMI may modulate treatment outcomes in T2DM. This patient-level meta-analysis standardized and pooled data from 15 RCTs (fasting plasma glucose [FPG] target < 100 mg/dL) of ≥ 24 weeks. Patients (pts) were adults with T2DM, adding insulin glargine to existing oral antidiabetes drugs (OADs). Data were examined by BMI (< 25, ≥ 25 to < 30, and ≥ 30 kg/m2) and concomitant OAD (metformin [MET], sulfonylurea [SU], or MET + SU). Outcomes included A1C, FPG, insulin dose, and hypoglycemia.

Overall, 3,187 pts were included (BMI [kg/m2]: < 25, n=368; ≥ 25 to < 30, n=1,214; and ≥ 30, n=1,605); 52.7% male, mean age 57.7 years. Pts with lower BMI had longer T2DM duration (10.7 vs. 9.6 vs. 8.2 years for BMI < 25, ≥ 25 to < 30, and ≥ 30 kg/m2, respectively); baseline A1C values were similar (8.8% vs. 8.7% vs. 8.7%). A1C changes from baseline were similar overall (−1.4% vs. −1.5% vs. −1.5%) and by OAD (Table). FPG change (−81.6 vs. −74.1 vs. −71.4 mg/dL) and insulin dose (0.36 vs. 0.41 vs. 0.47 U/kg) differed by BMI. More pts with high BMI achieved A1C < 7.0% (36% vs. 45% vs. 48%). Hypoglycemia rates were highest in low BMI pts. Hypoglycemia rates in pts with BMI ≥ 25 kg/m2 were highest with MET + SU (Table).

Pts with high BMI were more likely to reach A1C target with less hypo-glycemia, than low BMI pts. Low BMI is a risk factor for hypoglycemia. SU increases hypoglycemia rates in pts with BMI ≥ 25 kg/m2 when in combina-tion with MET.

Table. A1C and Hypoglycemia Event Rate in Pts Initiating Insulin Glargine Stratifi ed by Concomitant OADs and BMI.

MET (n = 644) SU (n = 920) MET + SU (n = 1,623)< 25

kg/m2≥ 25 to

< 30 kg/m2 ≥ 30

kg/m2< 25

kg/m2≥ 25 to

< 30 kg/m2 ≥ 30

kg/m2< 25

kg/m2≥ 25 to

< 30 kg/m2 ≥ 30

kg/m2

A1C at baseline, mean, %A1C at Week 24, mean, %

8.67.1

8.77.0

8.87.0

9.17.7

8.97.5

9.07.6

8.57.2

8.67.1

8.67.1

A1C change from baseline to Week 24, mean, %

−1.5 −1.7 −1.7 −1.3 −1.4 −1.4 −1.4 −1.5 −1.5

Overall hypoglycemia with PG < 70 mg/dL, events/pt yeara

6.16 3.55 1.77 9.67 4.85 3.55 10.03 7.90 6.56

Nocturnal hypoglycemia with PG < 70 mg/dL, events/pt yearb

2.55 1.00 0.34 1.38 0.64 0.43 1.74 1.82 1.33

Severe hypoglycemia (third party assistance required), events/pt year

0.26 0.09 0.03 0.07 0.15 0.04 0.07 0.07 0.10

aIncluding severe events; bOccurring between 0:01 AM and 5:59 AM. PG, plasma glucose.

Supported By: Sanofi

2199-PDo Sucralose and Saccharin Promote Fat Accumulation in Cultured Human Adipose Derived Mesenchymal Stem CellsSABYASACHI SEN, CAROL ROUPHAEL, SARA HOUSTON, ALLISON C. SYLVETSKY MENI, KRISTINA ROTHER, Washington, DC

Artifi cial sweeteners are extensively used as alternatives for caloric sugars, particularly among individuals with obesity and diabetes. Recent studies suggest that artifi cial sweeteners may increase fat formation. Thus we investigated whether varying concentrations of sucralose and saccharin promote adipogenesis in human mesenchymal stem cells (MSCs). MSCs are multipotent cells, which differentiate into adipocytes, myoblasts, osteo-blasts or chondroblasts, depending on the cellular environment. We cultured MSCs in Adipogenic Media (Lonza, 5.5mM) with both sucralose and sac-charin separately (conc: 0, 0.45 or 4.5mM) for a total of 6 and 12 days. Next, the cells were stained with Oil Red O stain for lipolysis using a plate reader (520nm). A portion of non-stained cells was collected for RT-PCR.

Before lipolysis, microscopy was performed. With increasing sweetener concentration, more intracellular fat droplets were noted. Plate reader assay showed 1.8 fold more fat accumulation with sucralose (0 to 0.45mM) and by 1.2 fold with saccharin (0 to 0.45mM).

Our 12 day experiment, showed increased intracellular fat droplet accu-mulation with both sucralose and saccharin at 4.5mM concentration. Com-paring MSCs exposure to 0 and 0.45mM of sucralose and saccharin respec-tively, using RT-PCR, showed up-regulated PPAR-g (2.28 and 1.25 fold), C/EBPb (1.14 and 1.86 fold), C/EBPa (1.42 and 1.11 fold), adiponectin (2.16 and 3.17 fold), leptin (1.55 and 2.68 fold), SOD1 (1.59 and 2.22 fold), SOD2 (1.39 and 1.58 fold) and TNF alpha (1.36 and 1.2 fold), respectively.

We conclude that both sucralose and saccharin appear to promote fat accumulation noted by Oil Red O stain quantifi cation and upregulates ex-pression of genes involved in adipogenesis, infl ammation (TNFα) and anti-oxidant pathways (SODs). The increase noted in SOD is possibly an adaptive response to [O] presence. These fi ndings may have important public health implications and warrant further cellular, animal and human studies.

2200-PSuccessful Weight Loss Maintenance Includes Long-Lasting In-creases in the Appetite Inhibiting Hormones GLP-1 and PYY3-36EVA WINNING IEPSEN, JULIE REHNE LUNDGREN, OLUF PEDERSEN, TORBEN HANSEN, STEN MADSBAD, JENS JUUL HOLST, SIGNE SØRENSEN TOREKOV, Copenhagen, Denmark, Hvidovre, Denmark

Glucagon-like peptide 1 (GLP-1) and peptide YY (PYY3-36) are important mediators in post-meal satiety, but the effect of long-term maintenance of weight reduction in obesity on meal-induced GLP-1 and PYY3-36 secretion is sparsely investigated.

20 healthy obese individuals obtained a 12% weight loss by following an 8 week long low-calorie diet. After weight loss the participants were fol-lowed for 1 year in order to maintain weight loss. In case of weight gain, a low-calorie diet-product was allowed to replace up to two meals per day to sustain weight loss. Plasma GLP-1, PYY3-36 and ghrelin were measured prior to and up to 180 min. after ingestion of a 600 cal. liquid meal before, and after 8 and 52 weeks follow-up.

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The initial 12% body weight loss was maintained for 52 weeks. Total GLP-1AUC increased by 20% from before weight loss to after weight loss (p=0.007) and remained elevated by 23% at week 52 (p<0.0001) (Figure 1). Total PYY3-36AUC increased by 37% (p=0.001) and remained elevated at week 52 (p=0.004). Total ghrelin AUC increased by 18% after weight loss (p<0.0001), but by week 52 the increase was reduced to 14% (p=0.01).

GLP-1 and PYY3-36 levels rose after weight loss, consistent with reports of decreased levels in obesity, and remained elevated after 1 year of weight loss maintenance. Thus, successful weight loss maintenance includes long-lasting increases in GLP-1, PYY3-36 and ghrelin.

2201-PLiver Adiposity Is Associated with Hyperinsulinemia and Insulin Resistance Independent of Total, Central, and Muscle Adiposity in African Ancestry MenIVA MILJKOVIC, ALLISON KUIPERS, J. JEFFREY CARR, JAMES TERRY, ALAN PAT-RICK, YAORONG GE, SANGEETA NAIR, CLAREANN BUNKER, JOSEPH ZMUDA, Pittsburgh, PA, Nashville, TN, Scarborough, Trinidad and Tobago, Charlotte, NC

Emerging evidence indicates that fat accumulation in non-adipose tissues (ectopic adiposity) is associated with an increased risk of type 2 diabetes (T2D) independent of general obesity. Compared to white men, both obese and lean black men have less visceral and liver adiposity, while in contrast, they paradoxically have more skeletal muscle adiposity. It has been pro-posed that liver adiposity may not be as important a risk factor for T2D in black compared to white individuals, although comprehensive data on liver adiposity and T2D in African ancestry men is sparse. To test if liver adipos-ity is independently associated with glucose and insulin homeostasis, we simultaneously measured total body and trunk fat (kg) by DXA, and liver at-tenuation (HU, lower values indicate more fatty liver) and calf intermuscular adiposity (IMAT, cm3) by CT in 320 non-diabetic Afro-Caribbean men, aged 40-90 years. Insulin resistance was assessed by the homeostasis model as-sessment of insulin resistance (HOMA-IR) index. Mean (SD) liver attenuation was 59.5 HU (6.7), and only 1.6% of men had fatty liver defi ned as mean liver attenuation <40 HU. Liver attenuation was signifi cantly and inversely associ-ated with fasting serum insulin (r=-0.34) and HOMA-IR (r=-0.30) independent of age (P<0.0001). This association remained signifi cant (both P<0.009), after additional adjustment for total and trunk adiposity, IMAT, lifestyle risk fac-tors (current smoking, current alcohol intake, and physical activity), and self-reported health status. Liver attenuation was not associated with serum glucose in any model. Our fi ndings suggest that in African ancestry men liver adiposity may indeed be an important and independent fat depot contrib-uting to hyperinsulinemia and insulin resistance. Larger population-based studies are needed to quantify the individual and collective contributions of each ectopic fat depot to T2D risk among African ancestry individuals.


2202-PPancreatic Steatosis Is a Stronger Indicator of Central Adiposity Status than Impaired Glucose ToleranceVISHESH KHANNA, JAMES BENA, MARWAN HAMATY, ERICK REMER, SAN-GEETA R. KASHYAP, Cleveland, OH

Although emerging data suggests that fat accumulation in the pancreas leads to defects in insulin secretion and type 2 diabetes mellitus (T2DM), lim-ited clinical data on the relationship between intra-pancreatic fat (IPF) levels and glucose tolerance in humans exist. To test whether IPF is a marker of adiposity vs. T2DM development, we determined the relationship between IPF levels and glucose tolerance in subjects with normal glucose tolerance (NGT), pre-diabetes (PDM), and T2DM in a observational, cross-sectional analysis. 72 adults (39F/33M, age: 50.0y [22.0, 81.0], BMI: 28.3kg/m2 [16.0, 54.7], HbA1c: 5.7% [4.6, 9.2]) with NGT (n=34), PDM (n=24), and T2DM (n=11) who underwent a standard 75-g 2 hr OGTT and had a non-contrast abdomi-nal CT scan within 6 m between 2001 and 2010 were included. Associations of IPF (mean pancreatic attenuation/mean splenic attenuation) with BMI, age, gender, visceral fat area, subcutaneous fat area, FPG, 2-hour glucose, iAUC glucose (0-120min), HDL, and triglycerides were examined. Subjects were excluded if chart review revealed pancreatitis, pancreatic neoplasms, or if they previously had a splenectomy. NGT were younger, had lower BMI and visceral and subcut fat levels than PDM and T2DM (P<0.05).Median IPF attenuation levels were slightly lower (i.e. higher fat content) in PDM with both IGT+IFG ({0.61 [0.44, 0.75]} vs. NGT {0.89 [0.79, 0.97] and T2DM {0.80 [0.76, 0.94]}, p=0.08). IPF levels correlated strongly with age (ρ=0.48, p<0.001), BMI (ρ=0.30, p=0.01), visceral fat area (ρ=0.58, p<0.001), and sub-cutaneous fat (ρ=0.28, p=0.01), but did not differ with respect to gender or smoking status. IPF tended to relate to fasting (ρ=0.23, p=0.05) and 2-hour glucose levels (ρ=0.20, p=0.09), but not to HDL (ρ=-0.07, p=0.59) or triglyc-erides (ρ=0.08, p=0.53). In conclusion, IPF is a strong indicator of adiposity status, particularly the visceral fat depot, and may contribute to the develop-ment of impairments in glucose tolerance and pre-diabetes.

Supported By: The Endocrine Society

2203-PSubclinical Atherosclerosis in Normal Weight Obesity (NWO) Individuals Assessed by Pulse Wave Velocity and Coronary Ct AngiographyMIN-YOUNG LEE, SOHEE KIM, CHANHEE KYUNG, JONG SUK PARK, SEUNG-PYO LEE, HYE KYUNG KIM, CHUL WOO AHN, KYUNG RAE KIM, SHINAE KANG, Seoul, Republic of Korea

Subjects within normal BMI but with elevated body fat amount (normal weight obesity; NWO) show cardiometabolic dysregulation compared to the subjects with normal BMI and body fat amount (normal weight lean; NWL). In this study, we aimed to evaluate whether NWO individuals have higher rate of subclinical atherosclerosis compared to NWL using pulse wave ve-locity (PWV) and coronary computed tomography angiography (CCTA).

From a health check-up system, we identifi ed 2,078 normal weight (18.5 ≤ BMI < 25 kg/m2) subjects without any history of coronary artery disease. All patients underwent multi-frequency bioimpedance analyzer, CCTA and PWV. NWO was defi ned as normal BMI and highest tertile of body fat percentage by gender (men >25.3% and women >31.3%). All CCTA was taken using a 64-detector row CT. Plaque was defi ned as structure >1 mm2 within and/or adjacent to the vessel lumen and the plaque was classifi ed according to the presence/proportion of intraplaque calcifi cation.

A total of 283 subjects were classifi ed as NWO. NWO subjects demon-strated metabolic dysregulation compared to NWL individuals (fasting blood glucose, 99.1±15.9 vs. 95.7±17.5, p-value=0.001; LDL-cholesterol, 123.9±32.2 vs. 117.1±30.6, p-value=0.001). The NWO individuals showed higher PWV value than the NWL individuals (1474.0±275.4 vs. 1380.7±234.3, p-val-ue<0.001). Compared with the NWL subjects, the NWO subjects had higher prevalence of any coronary plaque (45.2% vs. 35.6%, p-value=0.002) which was largely driven by the difference in the prevalence of soft plaque (20.8% vs. 14.2%, p-value=0.003). When adjusted for multiple factors associated with atherosclerosis (age, sex, smoking status, blood glucose, blood pres-sure, lipid level), NWO itself was an independent predictor of the presence of soft plaque (odds ratio 1.498, 95% confi dence interval 1.072~2.094, p-value=0.018).

NWO individuals have a higher risk of subclinical atherosclerosis com-pared to NWL individuals.

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2204-PExpression of Lipolysis-related Genes in Subcutaneous Adipose Tissue of Obese Subjects with Type 2 Diabetes Mellitus: The Infl u-ence of Gastric Plication and Duodenal-Jejunal Bypass Liner ImplantationJANA KLOUCKOVA, MILOS MRAZ, ZDENKA LACINOVA, PETRA KAVALKOVA, ANNA CINKAJZLOVA, PAVEL TRACHTA, MARTIN MATOULEK, STEPAN SVACINA, MARTIN HALUZIK, Prague, Czech Republic

Lipolysis is one of the key metabolic processes determining the size of adi-pose tissue. Its proper stimulation is indispensable for weight reduction. In the present study we aimed at identifying the main lipolytic factors involved in metabolic chnges induced by bariatric surgery and duodenal-jejunal by-pass liner (DJBL) implantation.

Sixteen obese patients with type 2 diabetes mellitus (T2DM) undergoing gastric plication (GP) or implantation of DJBL and 14 healthy lean control (C) subjects were included into the study. Anthropometric, biochemical and hor-monal parameters were measured and subcutaneous adipose tissue (SCAT) from abdominal region was obtained at baseline and 6 or 10 months after the procedure. mRNA expression of 43 lipolysis-related genes was assessed in SCAT using quantitative Real-Time PCR.

Both procedures markedly decreased body weight (BMI 42.6±2.4 vs. 36.1±2.1, p<0.001 for GP and 43.5±1.1 vs. 40.5±1.0, p<0.001 for DJBL), im-proved glucose control (HbA1C 55.1±4.0 vs. 44.5±3.1 mmol/mol, p=0.037 for GP and 72.5±7.0 vs. 58.6±3.2, p=0.018 for DJBL) and in case of GP also partially ameliorated lipid profi le (HDL 1.1±0.1 vs. 1.3±0.1 mmol/l, p=0.033). At baseline, a number of lipolysis-related genes were down-regulated in SCAT of T2DM compared to C subjects including hormone-sensitive lipase and PPARγ (peroxisome proliferator-activated receptor γ). While decreasing the expression of the antilipolytic PIK3 (phosphoinositide-3-kinase) GP up-regulated the expression of several lipolytic factors (lipin-1, AZGP1, SREBP1), whereas DJBL only increased mRNA expression of lipin-1 and chemerin.

We conclude that GP and DJBL stimulate lipolysis in a slightly different way with GP being a more potent lipolysis activator which is in line with higher weight reduction associated with GP compared do DJBL.

Supported By: RVOVFN64165, IGANT14083-3, SVV260019/2014

2205-PLipocalin 2 Is Induced by Glucocorticoids and Produces Insulin Re-sistance in Human Adipose TissuePRASAD G. KAMBLE, MARIA J. PEREIRA, CHERNO O. SIDIBEH, SAM AMINI, MAGNUS SUNDBOM, JAN W. ERIKSSON, Uppsala, Sweden

A recently identifi ed adipokine, lipocalin 2 (Lcn2) was shown to be linked with obesity related metabolic disorders. Agents promoting insulin resistance (IR) like glucocorticoids (GC) induce Lcn2 expression which can be reversed by thiazolidinediones. However, the role of Lcn2 in glucose and lipid metabo-lism is not fully explored in humans. We aimed to study the role of Lcn2 in the regulation of glucose and lipid metabolism in human adipose tissue and the underlying mechanisms. Subcutaneous (SC) and omental (OM) adipose tissue obtained from non-diabetic subjects (5M/22F; age: 25-68 years old; BMI: 20.7-39.83 kg/m²) was incubated with or without dexamethasone (Dex) (0.03-3µM) or recombinant human (RH) Lcn2 (100 ng/ml). Gene expression was analysed by qPCR (n=7) and protein expression by Western blot (n=4). Isolated adipocytes were used for glucose uptake (GU) (n=8) and lipolysis (n=11) assays. Dex increases Lcn2 expression in a dose-dependent man-ner in both SC and OM depots (by ~150% and ~ 40%, respectively, p<0.05). RH-Lcn2 reduced the basal and insulin stimulated (1000 µU/ml) GU in SC adipocytes by 18 and 25% respectively (p<0.01). In addition, Lcn2 reduced the main glucose transporter GLUT4 protein content by ~35% in SC adipose tissue (p<0.05). Lcn2 did not affect basal or isoproterenol-stimulated lipoly-sis in adipocytes. Interestingly, PPARG gene expression decreased by ~45% (p<0.05), but the mRNA level of ADIPOQ was increased by ~45% (p<0.05). Lcn2 also increased the expression of GC downstream target gene CNR1 by ~35% (p<0.05). Our results suggest that Dex increases Lcn2 expression in human adipose tissue, a factor that inhibits GU and reduces expression of the GLUT4 in human adipocytes. In conclusion, the recently described adi-pokine Lcn2 can contribute to development of IR in human adipose tissue, and may be a mediator of metabolic adverse effects of GC excess. Further studies should explore the role of Lcn2 in obesity and T2D including effects in other tissues such as liver and muscle.

Supported By: SDA; AstraZeneca

2206-PThe Findrisc Score, Visceral Adiposity, Insulin Sensitivity, and Se-cretion Estimates Are Independent Predictors of Type 2 Diabetes in Obese SubjectsABRAHAM MEIJNIKMAN, AN VERRIJKEN, ILSE MERTENS, CHRISTOPHE DE BLOCK, LUC VAN GAAL, Antwerp, Belgium

The Findrisc score is a valuable tool to screen for patients at risk of develop-ing diabetes (DM), but it was never used in an exclusively obese population.

651 obese subjects (M/F: 193/458, BMI 38.2±6.1 kg/m², age 43±13 y) were tested for glucose status using OGTT and HbA1c. CT visceral fat, Findrisc score, HOMA-S and HOMA-B were determined.

Exactly 50.4% were found to have pre-DM and 11.1% were newly diagnosed with DM2, (M/F= 22.2/8.8%). Subjects without DM had a Findrisc score of 11±3, those with pre-DM 13±4 and subjects with de novo DM 15±5.

The sensitivity of the Findrisc when using 12 as cutoff value was 80.6%, the specifi city was 47.8%. Subjects with a Findrisc score ≥12 had more vis-ceral fat [203 (146-272) vs. 158.5 (105-210) cm², p<0.001], higher HbA1c [5.6 (5.4-5.9) vs. 5.4 (5.2-5.6)%, p<0.001], higher triglycerides [150 (107-200) vs. 117(86-168) mg/dl, p<0.001] and higher HOMA-S [3.8 (2.4-5.9) vs. 2.8 (1.8-4.5), p<0.001] compared to those with a Findrisc score <12. Fasting [89 (82-98) vs. 83 (78-90) mg/dl, p<0.001] and 2h post-OGTT glycaemia [152 (126-181) vs. 130 (111-148) mg/dl, p<0.001] also differed. Exactly 81% of the subjects with DM had a risk score ≥12. Logistic regression analysis with DM sta-tus as dependent variable identifi ed the Findrisc score (B=0.094, p<0.045), HOMA-S (B=0.27, p<0.001), HOMA-B (B=-0.005, p<0.004) and visceral fat (B=0.04, p<0.026) as independent risk factors. From the Findrisc model, age (B=-1.3, p<0.021), history of hyperglycemia (B=-0.863, p<0.006), and increased blood pressure (B=1.378, p<0.002) were identifi ed as independent risk factors, but not BMI, waist, physical activity, eating habits nor familial DM in this population.

In an obese population, 50.4% of the subjects were found to have pre-DM and 11.1% were newly diagnosed with DM2. The Findrisc score increased with worsening of glucose tolerance status and proved to be an independent predictor of DM status, as did HOMA-B, HOMA-S and visceral fat.

2207-PIn Overweight/Obese Men, Weight Loss Improves Both Muscle Capillary Recruitment and Whole Body Glucose Uptake in Response to InsulinYVO H. KUSTERS, CASPER G. SCHALKWIJK, ALFONS J. HOUBEN, JOS OP ‘T ROODT, PETER J. JORIS, RONALD P. MENSINK, EUGENE J. BARRETT, COEN D. STEHOUWER, Maastricht, Netherlands, Charlottesville, VA

Overweight and obesity are associated with impaired whole body glucose uptake (WBGU), and an increased risk of type 2 diabetes (T2DM) and cardio-vascular diseases (CVD). In skeletal muscle insulin can increase the number of perfused capillaries and enhance its own delivery and that of glucose to myocytes. This insulin-induced increase in perfused capillaries may be a determinant of WBGU. It is, however, unknown whether changes in WBGU following weight loss are related to changes in capillary recruitment. There-fore, we studied the effect of weight loss on WBGU and whether changes in capillary recruitment associate with changes in WBGU.

In a controlled trial with blinded analyses we randomized 53 non-smoking, overweight/obese men (waist circumference 102-110 cm; aged 18-65; no CVD or T2DM) to either an 8-week program (6 weeks low calorie diet (LCD), 2 weeks weight stable) or usual diet. During a 1 mU/kg/min euglycemic insulin clamp we estimated WBGU from the glucose infusion rate (GIR) and forearm muscle capillary recruitment as the change in microvascular blood volume (MBV) using contrast-enhanced ultrasound.

Fifty men completed the study, 3 dropped out. Baseline BMI was 29.9 ± 2.5 kg/m2 in the control group and 30.0 ± 1.7 kg/m2 in the LCD group. LCD reduced BMI by 3.0 ± 0.8 kg/m2, and BMI in controls did not change (+0.1 ± 0.4 kg/m2). The GIR increased by 1.3 ± 1.2 mg/kg/min with LCD and was unaltered (-0.1 ± 0.9 mg/kg/min) in controls. Also, MBV increased by 34.2 ± 50.2 % (p < 0.05) in the LCD group, but was unchanged (3.3 ± 32.6 %, p = 0.61) in controls, and changes in MBV were related to changes in GIR (r = 0.31, p < 0.05).

We conclude that, in overweight/obese men, weight loss improves both insulin-induced muscle capillary recruitment and WBGU. These changes are related to one another. These fi ndings support the hypotheses that impaired vascular actions of insulin contribute to metabolic insulin resistance in over-weight/obese individuals and that weight loss improves both.

Supported By: Top Institute Food and Nutrition

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2208-PVisceral Fat Area as a New Predictor of Short-Term Diabetes Re-mission after Roux-en-Y Gastric Bypass Surgery in Chinese Sub-jects with a Body Mass Index Less than 35 kg/m2HAOYONG YU, YUQIAN BAO, LEI ZHANG, YINFANG TU, WEIPING JIA, Shanghai, China

Background: Metabolic surgery has been proposed for inadequately con-trolled type 2 diabetes mellitus (T2DM) in association with obesity. How-ever, prediction of successful T2DM remission after surgery has not been clearly studied in Chinese patients.

Objective: Predicting the outcome in those with T2DM after metabolic surgery may help in patient selection.

Methods: A retrospective review of prospectively collected data of 68 ethnic Chinese with mean BMI 31.5 and T2DM were examined for the meta-bolic outcomes at 1 year after Roux-en-Y gastric bypass (RYGB). Visceral and abdominal subcutaneous fat areas were assessed using magnetic reso-nance imaging before and 1 year after RYGB. Remission was defi ned as a glycated hemoglobin (HbA1c) <6.5% and no medications at one year. Binary logistic regression analysis was used to identify predictors.

Results: At 1 year after surgery, the BMI in the study group decreased from 31.5±3.6 to 24.5±2.5kg/m2. Remission was achieved in 50 subjects (73.5%) at 1 year. Compared with patients in the non-remission group, patients in the remission group had a shorter duration of diabetes, lower preoperative HbA1C level, higher fasting C-peptide level and more visceral fat area (VFA). Preoperative body mass index (BMI) and waist circumference did not differ between the two groups.

Conclusion: The metabolic improvement in T2DM after RYGB in the mildly obese is greater with a shorter duration of diabetes, higher fasting C-pep-tide. Those who have more visceral adiposity may obtain greater benefi t from RYGB.

Supported By: Key Program of the Shanghai Municipality for Basic Research (11JC1409600)

2209-PPlasma Dihydroceramides Predict Future Type 2 Diabetes in Obese Persons from Mexican-American FamiliesHEMANT KULKARNI, MANJU MAMTANI, GERARD WONG, JACQUELYN M. WEIR, CHRISTOPHER K. BARLOW, THOMAS D. DYER, MICHAEL C. MAHANEY, ANTHONY COMUZZIE, LAURA ALMASY, PETER J. MEIKLE, JOHN BLANGERO, JOANNE E. CURRAN, Harlingen, TX, Melbourne, Australia, San Antonio, TX

Obesity and type 2 diabetes (T2D) are intimately interconnected. We studied whether the plasma lipidome can predict incident T2D in initially T2D-free obese persons of Mexican American ethnicity - a high-risk, minor-ity U.S. population.

We used data from obese (body mass index >= 30 kg/m2), T2D-free per-sons from the San Antonio Family Heart Study. Plasma lipidomic data (319 lipid species, 23 lipid classes) was based on a combination of high perfor-mance liquid chromatography and mass spectrometry. Using mixed effects Cox proportional hazards modeling in R (coxme) we tested the multivariable association of the 23 lipid classes (as sum of concentrations of all compo-nent lipid species within a class) with future T2D while accounting for the kinship structures. We inverse-normalized the lipid class sums and adjusted for age, age2, sex, waist circumference, systolic and diastolic blood pres-sure, total serum cholesterol, serum triglycerides, high-density lipoprotein cholesterol, and use of anti-hypertensive and lipid-lowering drugs.

Of the 280 obese persons included in this study, 75 (26.8%) developed T2D over 3,128.13 person-years of follow-up. Five lipid classes were signifi -cantly associated with future T2D: dihydroceramides (p 0.0002), ceramides (p 0.0081), alkenylphosphatidylethanolamines (p 0.0140), phosphatidylglyc-erols (p 0.0247) and phosphatidylserines (p 0.0340). In a model including these fi ve classes with the abovementioned covariates, only dihydrocer-amides (p=0.0069) were retained as signifi cant. This class continued to be signifi cantly associated with future T2D even after adjusting for prediabetes (based on the results of 2-hour oral glucose tolerance test; relative hazards for 1 SD change 1.52, 95% confi dence interval 1.08-2.13, p 0.0164).

We conclude that plasma dihydroceramides additively and independently predict future T2D in obese individuals over and beyond the routinely used clinical measures including oral glucose tolerance test.

Supported By: National Institutes of Health (R01DK082610, R01DK079169, R01HL045522, R37MH059490, 1R01DK088972-01)

2210-P

2211-PCould the Interleukin 1-beta System Mediate the Relation of ApoB-lipoproteins to Insulin Resistance and Hyperinsulinemia in Obese Subjects?SIMON BISSONNETTE, NATHALIE SAINT-PIERRE, YANNICK CYR, VALERIE LA-MANTIA, HANNY WASSEF, MAY FARAJ, Montreal, QC, Canada

Insulin resistance and compensatory hyperinsulinemia are known to increase type 2 diabetes (T2D) risk. To understand the etiology of these factors, we further investigated the significance of elevated apoB-lipoproteins which have been associated with increased risk of T2D and insulin resistance. Activation of the pro-inflammatory IL-1β system is another predictor of T2D risk. We assessed whether IL-1β mediates the relation of apoB-lipoproteins to insulin resistance and hyperinsulinemia in humans.

Eighty one non-diabetics (48 women and 33 men) aged 45 to 74 years underwent a 1-hour intravenous glucose tolerance test during which insu-lin secretion was measured, then followed by a 3-hours hyperinsulinemic-euglycemic clamp. Insulin resistance was expressed as the glucose infusion rate divided by the corresponding insulinemia over the last 30 minutes of the clamp.

Plasma apoB correlated positively with total (r=0.22), second phase insu-lin secretion (r=0.24) and insulin resistance (r=0.29), independent of adipos-ity. Fasting plasma IL-1Ra, an indicator of IL-1β activity, was also positively correlated with total (r=0.39) and second-phase insulin sensitivity (r=0.44),

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insulin resistance (r=0.46) and apoB (r=0.26). There were no gender differ-ence in these relations. After correction for IL-1Ra levels, all associations between apoB and insulin indexes were lost.

ApoB is associated to insulin secretion and insulin resistance in non-dia-betic overweight subjects. Furthermore, IL-1β system activation is a poten-tial mediator in this relation. Blocking this pathway could be a novel therapy for reducing T2D risk in this population.


2212-PMaintaining a Relatively Low Rate of Fatty Acid Mobilization from Adipose Tissue Helps Protect Obese Adults from Becoming Insulin ResistantDOUGLAS W. VAN PELT, LISA M. GUTH, ALEXANDER HINKO, JEFFREY F. HOROW-ITZ, Ann Arbor, MI

Excessive fatty acid (FA) mobilization from adipose tissue is often found in obesity, and is central to the development of insulin resistance. However, FA mobilization can vary considerably among seemingly homogenous obese adults. The primary aim of this study was to test the hypothesis that obese adults with relatively low FA mobilization rates are “protected” against developing insulin resistance. We measured fatty acid rate of appearance in plasma (FA Ra) via 13C-palmitate isotope dilution and insulin resistance using a hyperinsulinemic-euglycemic clamp after an overnight fast in 21 obese adults (body mass index [BMI]: 37±1 kg/m2). Across the entire cohort, we found FA Ra to be negatively correlated with insulin-mediated glucose disposal (R2=-0.33; p<0.01). We also divided our cohort into tertiles based on their FA Ra to compare subjects who had the lowest FA Ra (LOW-FA: ≤ 8.3 µmol/min/kg FM; n=7) to those with the highest FA Ra (HIGH-FA: ≥ 10.6 µmol/min/kg FM; n=7) - subjects exhibiting “intermediate FA Ra” were not included in this analysis. Subjects in LOW-FA and HIGH-FA groups were well-matched for BMI (38±2 vs. 38±2 kg/m2), fat mass (50±5 vs. 53±3 kg) and waist circumference (111±5 vs. 110±4 cm). Insulin-mediated glucose disposal was 45% higher in LOW-FA compared with HIGH-FA (5.8±0.5 vs. 4.0±0.6 (mg/min/FFM)/[µIU/ml] x 10-2; p=0.04). Our fi ndings suggest that maintaining a relatively low basal endogenous FA Ra may protect some obese adults from developing insulin resistance.

Supported By: National Institutes of Health (R01DK077966, P30DK089503, IL1RR02RR024986)

2213-PEarly Response to Liraglutide 3.0 mg in Adults with Overweight or Obesity and Type 2 Diabetes: Subanalysis of the SCALE Diabetes TrialMELANIE DAVIES, OFRI MOSENZON, RICHARD M. BERGENSTAL, ROBERT F. KUSHNER, TRINE V. SKJØTH, BIRGITTE CLAUDIUS, RALPH A. DEFRONZO, Le-icester, United Kingdom, Jerusalem, Israel, Minneapolis, MN, Chicago, IL, Søborg, Denmark, San Antonio, TX

Individuals with early weight loss (WL) are more likely to achieve and sus-tain WL after 1 year.

This post-hoc analysis of subjects on liraglutide 3.0 mg completing 56 wks of treatment (n=324) compared effi cacy and safety in early responders (ER; ≥4% WL at 16 wks) vs. early non responders (ENR; <4% WL at 16 wks).

846 subjects (age 54.9 yr, M 50%, BMI 37.1 kg/m2, A1C 7.9%, T2D duration 7.3 yr) were randomized (2:1:1) to liraglutide 3.0 mg, 1.8 mg, or placebo plus diet (500 kcal/day defi cit) and exercise for 56 wks (NCT01272232).

At Wk 56, ER (n=214) had greater WL than ENR (n=110), (-8.5% vs. -3.1%). Consistent with this, larger improvements in A1C, FPG, CV risk markers and QoL were seen in ER vs. ENR. Due to liraglutide’s direct effect on glycemia, A1C and FPG improved both in ER and ENR (Table).

Driven by higher frequency of nausea (36.0 vs. 22.7%) gastrointestinal AEs were more common in ER vs. ENR. Observed mean change in pulse was similar (1.7 vs. 3.1 bpm), as were rates of ADA documented symptomatic hypoglycemia (PG ≤3.9 mmol/L) for ER and ENR respectively (0.83 vs. 1.03 events/patient years of exposure). Severe hypoglycemia was rare and simi-lar between groups.

In conclusion, subjects who were ER to liraglutide 3.0 mg and completed 56 wks of treatment achieved greater weight loss and improvement in other clinical parameters, compared with ENR, with a largely similar safety pro-fi le.

Supported By: Novo Nordisk

2214-PTreatment Satisfaction with Different Weight Loss Methods among Patients with Type 2 Diabetes MellitusSHALOO GUPTA, ZHIXIAO WANG, Princeton, NJ, Woodcliff Lake, NJ

Treatment satisfaction with different weight loss (WL) methods can be a measure of perceived effectiveness and a driver for long-term commitment, which is essential in weight management. This study assessed treatment satisfaction associated with different WL methods among patients with type 2 diabetes mellitus (T2DM). Data were obtained from the 2012 National Health and Wellness Survey, an annual Internet-based survey demographi-cally representative of the U.S. adult population (N=71, 157). Overweight and obese patients (BMI ≥27) with T2DM were categorized as having a WL procedure (e.g., gastric bypass, gastric banding) or using a prescription medi-cation for WL (Sur/Rx), vs. using self-modifi cation WL techniques only (e.g., diet, exercise, over-the-counter medications, weight management programs, and WL supplements). Sur/Rx patients were matched to self-modifi cation patients on demographics, comorbidities, lifestyle behaviors, obesity class, etc., via propensity scores matching (1:2). Treatment satisfaction with cur-rent WL methods (1 [extremely dissatisfi ed] to 7 [extremely satisfi ed]) was assessed. Chi-square tests and ANOVAs were used to determine signifi cant differences after matching. Of the 5,726 patients (average age 59.5 years (SD=11.8), 40.1% female) included in the analysis, 49.6% took no current ac-tion to lose weight, 2.9% were in the Sur/Rx group and 47.5% were in the self-modifi cation group. The Sur/Rx group (vs. self-modifi cation) reported being extremely/very satisfi ed more frequently (46.6% vs. 22.7%, p<0.001). There was no difference in treatment satisfaction between those using Rx and those using Sur (p>0.05). This study indicates the need for more educa-tion on obesity awareness and management. The fi nding that treatment sat-isfaction was greater in patients with Sur/Rx than those with self-modifi ca-tion methods suggests the important role of Sur/Rx in weight management in addition to lifestyle modifi cation in patients with T2DM.

2215-PThe Effect of Why WAIT Method of Weight Management vs. Laparo-scopic Adjustable Gastric Banding on Cardiometabolic and Quality of Life Outcomes in Obese Patients with Type 2 Diabetes: A 1-Year Randomized Clinical TrialOSAMA HAMDY, ANN GOEBEL-FABBRI, DONALD C. SIMONSON, SU ANN DING, FLORENCIA HALPERIN, MARLENE WEWALKA, KATHLEEN FOSTER, KATHERINE KELLY, JENNIFER PANOSIAN, KERRI CLANCY, DAVID LAUTZ, ASHLEY VERNON, ALLISON B. GOLDFINE, Boston, MA, Concord, MA

Weight Achievement and Intensive Treatment (Why WAIT) is a 12-week multidisciplinary intensive lifestyle intervention program for weight man-agement and diabetes control designed for application in clinical practice. To compare the effectiveness of this program vs. bariatric surgery, 40 obese patients with type 2 diabetes (22 M/18 F; weight 109±15 kg; BMI 36.5±3.7 kg/m²; age 51±10 years; A1C 8.2±1.2%) were randomized to Why WAIT (WW; n=22) or laparoscopic adjustable gastric band (LAGB; n=18). At 12 months, weight loss after LAGB was higher than after WW (-13.5±1.7 vs. -8.5±1.6 kg respectively, p< 0.05) however, A1C reduction was similar (-1.2±0.3 vs. -1.0±0.3%). The improvement in blood pressure, triglycerides, LDL were not different between the 2 groups. Five-year risk of a coronary heart disease event, as determined by the UKPDS risk engine scores, decreased from 9.7±1.7% to 9.0±1.8% after WW and from 10.5±1.8% to 9.2±2.1% after LAGB, with no signifi cant difference between groups. Risk of fatal CHD events also decreased from 5.8±1.1% to 5.5±1.3% after WW and from 6.5±1.3% to 5.9±1.5% after LAGB, but these changes were not statistically signifi cant. Quality of life (QOL) scores (SF-36 physical health, mental health) changed

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minimally from baseline with no difference between groups. The Impact of Weight on QOL and Problem Areas in Diabetes improved signifi cantly from baseline in both WW and LAGB (p<0.01 for both), but the effects were simi-lar between groups. These fi ndings suggest that Why WAIT program and LAGB have similar benefi ts on diabetes control, cardiometabolic risk and QOL parameters. These results may be useful in guiding obese patients with type 2 diabetes when they explore their weight management options.

Supported By: National Institutes of Health (DK086918, DK095451, DK036836); Covidien; LifeScan, Inc.; Nestlé; Novo Nordisk

2216-PDifferential Effect of Weight Loss on Altered Metabolite Levels and Response to a Glucose Load in Prediabetic Obese IndividualsNINA GEIDENSTAM, ANDERS P.H. DANIELSSON, PETER SPÉGEL, MARTIN RID-DERSTRÅLE, Malmö, Sweden, Gentofte, Denmark

Weight loss is an effective means of improving insulin sensitivity and glu-cose tolerance, reducing the risk of type 2 diabetes in obese subjects with impaired glucose tolerance (IGT). The metabolite changes refl ecting these im-provements are not known in detail. We investigated the infl uence of weight loss followed by weight stability on metabolite levels and response during an oral glucose tolerance test (OGTT). Fourteen obese individuals with IGT (BMI 44±6 kg/m2; [mean±SD]) lost weight with low-calorie diet (800-1,200 kcal/day; 102±26 days; BMI 36±6 kg/m2), followed by a weight maintenance phase (158±35 days; BMI 35±7 kg/m2). Serum samples were collected at three time points (0, 30 and 120 min) during OGTT at baseline, after weight loss when improvement of hepatic insulin sensitivity was noted, and after weight maintenance when improvement of glucose tolerance was noted. The identity of 58 serum metabolites was determined using gas chroma-tography/time-of-fl ight mass spectrometry. Decreased fasting levels of phe-nylalanine after weight loss were correlated with decreased BMI (r= 0.68, p=0.007). Enhanced glucose-elicited suppression of the aromatic amino ac-ids, observed after weight loss, was associated with improved insulinogenic index (tyrosine: r=0.72, p=0.01, phenylalanine: r=0.63, p=0.04). Fasting levels of palmitate and laurate decreased during weight loss and remained de-creased after weight loss maintenance. By contrast, fasting levels of oleate were not decreased until after weight maintenance. Several amino acids showed perturbed glucose-elicited responses that improved during weight maintenance. We conclude that weight loss and weight maintenance lead to differential improvements in glucose homeostasis of obese IGT subjects and this is mirrored by specifi c changes in the metabolome.

2217-PMetabolic Improvements Observed in Subjects Receiving Endobar-rier: A Pooled Analysis of Clinical TrialsJULIAN TEARE, CAREL W. LE ROUX, ELAINE CHIQUETTE, DAVID MAGGS, IG-NACE JANSSEN, London, United Kingdom, Dublin, Ireland, Lexington, MA, Arnhem, Netherlands

EndoBarrier therapy (EBT) involves the reversible endoscopic placement of an impermeable liner that anchors in the duodenum and bypasses the fi rst two feet of the upper bowel, thus replicating features of bariatric surgery. EBT in obese subjects with ≥ 1 comorbidity (diabetes, hypertension, hyper-lipidemia) or morbidly obese with or w/o comorbidities from 6 clinical trials (n=211, age 46 yrs, BMI 38.9 kg/m2) was evaluated.

Signifi cant metabolic improvements were observed with EBT (Table). At 12 months, 57% of subjects achieved ≥10% weight loss while 55% of those with T2DM reached an A1C of ≤7%. The improvement in glycemic control occurred despite a shift to decreased concomitant anti-diabetic medication use. Moreover, 37% of T2DM subjects achieved both a ≥0.5% A1C reduction and ≥10% weight loss at 12 months.

Reported AEs were mild (event rate 85.0%) or moderate (event rate 14.5%) in severity and mostly GI in nature. Hypoglycemia was observed in patients on insulin or sulfonylurea, but no severe hypoglycemia occurred. EBT did not induce hypoglycemia in non-diabetic subjects, suggesting no innate hypo-glycemia potential. Early device removal was due to anchor migration, GI distress, liner obstruction, or GI bleed; all resolved w/o permanent sequelae. SAEs were reported infrequently at a rate of 3%.

EBT elicits metabolic benefi ts, with an acceptable safety profi le, in obese subjects with comorbidities.

Supported By: GI Dynamics, Inc.

2218-PSubcutaneous Adipose Tissue Circadian Genes NR1D2 and DBP Ex-pression Is Associated with Obesity, Insulin Action and Increases after Weight Loss in HumansMONIKA KARCZEWSKA-KUPCZEWSKA, NATALIA MATULEWICZ, MAGDALENA STEFANOWICZ, RADOSLAW MAJEWSKI, REMIGIUSZ FILARSKI, AGNIESZKA NIKOLAJUK, MAREK STRACZKOWSKI, Białystok, Poland, Olsztyn, Poland

Nuclear receptor subfamily 1, group D, member 2 (NR1D2) and D site of albumin promoter binding protein (DBP) are proteins regulating circadian rhythms, which have also been linked to obesity and metabolism. Data on the expression of genes encoding these proteins in human adipose tissue are very limited. The aim of the present study was to assess the mRNA ex-pression of NR1D2 and DBP in human subcutaneous adipose tissue in rela-tion to obesity and insulin action and to examine their response to weight loss. We examined 20 normal-weight individuals (8 males and 12 females, control for the baseline conditions) and 40 subjects with marked overweight and obesity (BMI between 28 and 40 kg/m2, 18 males and 22 females) with normal glucose tolerance. Euglycemic hyperinsulinemic clamp and a biopsy of subcutaneous adipose tissue were performed. Then overweight/obese subjects underwent 12-weeks moderate weight loss program, whit indi-vidually planned diet of 20 kcal per kg of due weight. All the initial analyses were repeated at the end of the program. Tissue NR1D2 and DBP mRNA expression was measured with Real Time PCR. Baseline adipose tissue NR1D2 and DBP expression was lower in the obese subjects (both p<0.05) and was positively related to insulin sensitivity in the entire study group (r=0.31, p=0.016 and r=0.26, p=0.046, respectively). Dietary intervention re-sulted in a signifi cant reduction in body weight (-11%, p<0.001), which was accompanied by an increase in insulin sensitivity (+27%, p=0.001). We also observed an increase in adipose tissue NR1D2 (+19%, p=0.009) ,and DBP (+21%, p=0.032) after weight loss. After the intervention, both NR1D2 and DBP were related to post-intervention insulin sensitivity (r=0.35, p=0.031 and r=0.32, p=0.049, respectively). Our data show that weight loss is associ-ated with up-regulation of circadian genes in adipose tissue, in proportion with an increased insulin sensitivity.

Supported By: National Science Center of Poland (2001/03/B/NZ7/04980)

2219-PLiraglutide 3.0 mg Improves Health Utilities in Weight Management Compared with PlaceboJAKOB B. BJØRNER, KENNETH FUJIOKA, AGATHE LE LAY, JASON BRETT, RONETTE KOLOTKIN, Copenhagen, Denmark, La Jolla, CA, Søborg, Denmark, Plainsboro, NJ, Durham, NC

Health-related quality of life (HRQoL) is important as a measure for cost-effectiveness analyses of new therapeutic entities. Health utility scores summarize HRQoL as a single value where 0 corresponds to death and 1 is equal to perfect health. Health utility scores can be derived from health questionnaires such as the Impact of Weight on Quality of Life (IWQOL)-Lite and Short Form-36 v2 (SF-36). This study assessed whether liraglutide 3.0 mg improved health utility compared with placebo, as adjunct to diet and exercise, in patients who were overweight or had obesity.

IWQOL-Lite and SF-36 data were collected from 3,731 patients during the 1-year, Phase 3a, randomized, double-blind, SCALE Obesity and Pre-diabetes trial comparing liraglutide 3.0 mg with placebo. HRQoL was assessed in subjects from countries with validated translations (82% of subjects). Body weight data were observed means with LOCF ± SD. IWQOL-Lite scores were mapped to the Short-Form-6D (SF-6D) index using a validated algorithm. SF-6D health utilities were also scored directly from the SF-36. As a sensitivity analysis, SF-36 scores were mapped to the EuroQoL-5D (EQ-5D) index.

Individuals on liraglutide 3.0 mg had greater weight loss from baseline (8.0±6.7%) compared with placebo (2.6±5.7%); Estimated treatment differ-ence (ED) -5.4% [95% CI -5.8;-5.0], p<0.0001. At end of trial, SF-6D scores

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derived from the IWQOL-Lite or from the SF-36 were signifi cantly higher for patients treated with liraglutide 3.0 mg vs. placebo. For SF-6D/IWQOL-Lite, change from baseline was 0.80 vs. 0.78 and ED was 0.017 [95% CI: 0.012; 0.021], p<0.0001. For SF-6D/SF-36, ED was 0.018 [95% CI: 0.010; 0.026], p<0.0001. EQ-5D scores derived from the SF-36 supported these fi ndings, with a higher score for liraglutide 3.0 mg vs. placebo; ED 0.014 [95% CI: 0.010; 0.018], p<0.0001.

Liraglutide 3.0 mg for weight management signifi cantly improved health utility scores compared with placebo in patients who are overweight or who have obesity.


2220-PObesity-related Neuropathy and the Effects of Bariatric SurgerySHAZLI AZMI, MARYAM FERDOUSI, GEORGIOS PONIRAKIS, IOANNIS N. PETRO-POULOS, UAZMAN ALAM, MITRA TAVAKOLI, HASSAN FADAVI, JONATHAN SCHOFIELD, TARZA SIAHMANSUR, ANDREW MARSHALL, BASIL AMMORI, HANDREAN SORAN, RAYAZ A. MALIK, Manchester, United Kingdom, Doha, Qatar

Introduction: Obese subjects have risk factors for the development of neu-ropathy. Bariatric surgery can normalise these risk factors and could benefi t neuropathy.

Methods: 60 morbidly obese subjects and 30 age-matched controls un-derwent assessment of: sural nerve conduction velocity (SNCV) and ampli-tude (SNAP), peroneal nerve conduction velocity (PMNCV) and amplitude (PNA), vibration perception threshold (VPT), cold (CT) and warm threshold (WT), cold (CIP) and warm induced pain (WIP), and assessment of corneal nerve fi bre density (CNFD), branch density (CNBD) and fi bre length (CNFL) by Corneal Confocal Microscopy. 16 subjects were re-assessed 6 months after bariatric surgery.

Results: Obese subjects had a higher baseline BMI (49.0±1.2 vs. 26.9±0.8, p<0.0001) and HbA1c (51.8±4.1 vs. 37.5±0.7 mmol/mol, p=0.002). They had a signifi cantly lower SNAP (11.4±1.4 vs. 20.5±1.9mV, p=0.02), CT (24.4±0.9 vs. 28.7±0.40C, p=0.02) and CIP (7.0±1.2 vs. 11.2±1.70C, p=0.02 and greater WT (40.0±0.5 vs. 36.4±0.4, p=0.02), WIP (45.9±0.8 vs. 44.5±0.50C, p=0.003) and VPT (12.5±1.0 vs. 5.0±0.7 V, p<0.0001). CNFD (23.7±1.1 vs. 33.4±1.3 no/mm2, p<0.0001), CNBD (30.9±2.6 vs. 50.7±3.1 no/mm2, p<0.0001) and CNFL (14.2±0.5 vs. 20.0±0.48 mm/mm2, p<0.0001) were signifi cantly reduced. Paradoxically, CNBD (38.3±5.7 vs. 22.9±2.8, p=0.03) and CNFL (15.1±1.2 vs. 12.9±0.5, p=0.05) were signifi cantly lower in the non-diabetic (n=35) com-pared to the diabetic (n=28) group. In 16 subjects assessed post-operatively at 6 months, BMI (51.8±3.8 vs. 34.7±1.9, p=0<0.0001), Systolic BP (142±4.6 vs. 119.9±4.5 mmHg, p=0.008), CIP (7.3±2.6 vs. 17.3±3.6, p=0.05), CNFD (23.7±1.7 vs. 25.9±1.4, p=0.05) and CNFL 4.1±0.8 vs. 15.6±0.9, p=0.005) im-proved, with no change in other parameters.

Conclusion: Obese subjects have evidence of a small fi bre neuropathy but paradoxically those with diabetes have less severe small fi bre damage, presumably driven by factors other than hyperglycaemia. Bariatric surgery improves small fi bre neuropathy.

2221-PAssociation of Genetic Variants in NUDT3, SEC16B, and MC4R with Visceral and Subcutaneous Fat Distribution in Chinese PopulationFENG JIANG, RONG ZHANG, TAO WANG, CHENG HU, WEIPING JIA, Shanghai, China

Central obesity, especially abdominal fat accumulation were primarily responsible for obesity related metabolic abnormalities. Numerous genetic variants associated with simple but less precise anthropometric measure-ments of obesity including body mass index (BMI), waist circumference, waist-hip ratio have been uncovered using genome-wide association stud-ies (GWAS). However, little is known regarding how the genetic variants is linked to distribution of fat between visceral and subcutaneous com-partments. We aim to defi ne the hypothesis that precise measurement of visceral and subcutaneous fat could provide signifi cant information above and beyond less precise measurements for fi nding variants in Chinese indi-viduals. A total of 2959 subjects (1352 men and 1607 women) enrolled from four community-based populations in Chinese Han ancestry were geno-typed for 19 single-nucleotide polymorphisms (SNP), who underwent Mag-netic Resonance Imaging (MRI) for measurement of visceral fat area (VFA) and subcutaneous fat area (SFA). We replicated the association of SEC16B rs574367 BDNF rs6265 MC4R rs6567160 KCTD15 rs29941 with both BMI and waist circumference (P=0.04~9.7×10-7). NUDT3 rs206936 and other SNPs in SEC16B, MC4R and KCTD15 exhibited signifi cant associations with SFA (P=0.001149~0.01337) and variants of SEC16B and MC4R were signifi cantly correlated with VFA (P=0.0182 and 0.03096, respectively) in all subjects.

The association of NUDT3 with SFA remained after adjustment for BMI (P=0.02831), in contrast to no survival correlation with VFA adjusted for BMI. In subgroup analysis, signifi cant associations were found between NUDT3 and SFA in men (P=0.025) and between RBJ and SFA in women (P=0.024) after adjustment for BMI. Our results suggest that rs206936 in NUDT3 is associated with subcutaneous fat accumulation, especially in Chinese men. The contribution of two SNPs in SEC16B and MC4R to visceral fat depends on overall adiposity.

Supported By: Shanghai Rising Star Program (12QH1401700)

2222-PUnexpected Higher Prevalence of Nonalcoholic Fatty Liver Disease in Metabolically Healthy Obese Patients with Massive Obesity—Relation with Sympathetic OveractivitySABRINA CHIHEB, BADREDDINE MERIOUD, NADA HELMY, AMEL REZKI, MARIANNE ZIOL, CHRISTOPHE BARRAT, CORINNE VONS, PAUL VALENSI, Paris, France

Obesity and metabolic syndrome (MS) were reported to be associated with sympathetic nervous system (SNS) overactivity. The role of infl amma-tion in steatohepatitis (NASH) development and the relation between proin-fl ammatory state, SNS and metabolic disorders have been suggested. We aimed to determine whether in metabolically healthy obese (MHO) patients infl ammatory markers, insulin resistance, hypercortisolemia and sympathet-ic activity are associated with non alcoholic fatty liver disease (NAFLD).

In a prospective study of patients undergoing bariatric surgery, peropera-tive liver biopsies were obtained (study approved by the Ethical Committee). We included 39 patients free of other liver disease, aged 36.4±9.6 years with body mass index 44.7±0.9 kg/m². Liver changes were classifi ed as: nor-mal liver, simple steatosis and NASH. Biological investigations included liver tests, Lipids, glucose, cortisol, infl ammatory markers (CRP, fi brinogen) and HOMA-IR. Sympathetic activity was assessed by plasma epinephrine and norepinephrine at fasting and 2 hours after an oral glucose challenge. MHO phenotype was defi ned as no or only one feature of the metabolic syndrome (IDF criteria).

Of the 39 patients 21 had defi nite MHO status. Seven of them had histo-logically normal liver, 11 had steatosis and 3 NASH. Among MHO patients, those with steatosis had higher post-glucose norepinephrine, afternoon cor-tisol and CRP levels (p=0.04, 0.01 and 0.05 respectively). There was a trend (NS) to higher Liver enzymes, fasting plasma glucose and insulin levels, and higher HOMA-IR in patients with NAFLD.

In conclusion the prevalence of liver disease is high in MHO patients. Those with NAFLD exhibit a more marked cluster of infl ammatory, higher cortisol levels and enhanced sympathetic activity.

2223-PMMP-9 to TIMP-1 Ratio Was Independently Positively Correlated with BMI at 36 Months after a 12-Week Low-Calorie Diet in Obese MenCHIA-LIN LEE, JUN-SING WANG, I-TE LEE, CHIA-PO FU, WEN-JANE LEE, KAE-WOEI LIANG, SHIH-YI LIN, WAYNE HUEY-HERNG SHEU, Taichung, Taiwan

Dietary interventions for overweight/obesity are often associated with weight regain. Matrix metalloprotease 9 (MMP-9) and the endogenous tis-sue inhibitor of matrix metalloprotease 1 (TIMP-1) have been associated with adipose differentiation and obesity. We investigated whether the MMP-9 to TIMP-1 ratio could predict long term body mass index (BMI) after dietary intervention for obesity.

This study was a 36-month follow up after a 12-week weight-reduction program which enrolled men with central obesity (waist circumference > 90 cm). Participants were instructed by a registered dietician for how to maintain a low-calorie diet (1200 Kcal/day). A magnetic resonance imaging (MRI) was conducted for the quantifi cation of participants’ subcutaneous fat and abdominal visceral fat areas. Serum MMP-9 and TIMP-1 were measured at baseline. Participants’ BMI and waist were measured before and at 3, 6, 12, and 36 months after the beginning of the program.

A total of 40 men were recruited (mean age 43±11 years, mean body mass index 33.9±3.9 kg/m2, and mean waist 110±9.5 cm). BMI at 3, 6, 12, and 36 months after the beginning of the weight-reduction program were 31.0, 30.6, 31.1, and 31.1, respectively (all p<0.05 compared with data at baseline). Waist circumference at 3, 6, 12, and 36 months were 101.1, 99.1, 101.1, and 100.7 cm, respectively (all p<0.05 compared with data at baseline). After adjusting for baseline BMI, leptin, adiponectin, subcutaneous fat and ab-dominal visceral fat areas, MMP-9 to TIMP-1 ratio was positively correlated with BMI (beta coeffi cient=1.623, p=0.038) and waist circumference (beta coeffi cient=5.106, p=0.038) at 36 months. In conclusion, we demonstrated

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that MMP-9 to TIMP-1 ratio was independently associated with long term BMI in obese men after a 12-week low-calorie-diet.

2224-PComparison of the Polymorphisms of the Endocannabinoid System between Obese and Nonobese SubjectsTINA K. THETHI, ASTER SIGEL, BONNIE L. KATALENICH, SHANKER JAPA, SHUQ-IAN LIU, TUYEN NGUYEN, JOSHUA LARRAZOLO, GANDAHARI CARPIO, DRA-GANA LOVRE, WYNN HTUN, STEPHANIE SYU, ESTHER CAREFOOT, CAROL A. STELL, CYNTHIA MOREAU, USHA JAPA, ROBERTA MCDUFFIE, VIVIAN FONSECA, New Orleans, LA, Henderson, NV

Endocannabinoid system (EC) plays a role in obesity. Our hypothesis is that genetic variants of the cannabinoid type 1 receptor (CNR1) and fatty acid amide hydrolase (FAAH) are associated with obesity. This was a cross sectional study with Caucasian (C) and African American (AA) subjects with a body mass index (BMI) < 27 ([control group]; C=76; AA=126) or > 30 ([obese group]; C=259; AA=216). Data were analyzed with Fisher’s exact test by SAS 9.1. Whole genomic DNA was isolated from buffy coat. For CNRI, poly-morphisms, 3813 A/G (rs12720071) and 4895A/G (rs806368); and for FAAH polymorphism, 385 C/A (rs324420); genotyping was performed using allelic discrimination assay and analyzed by real time polymerase chain reaction (PCR). For CNR1, there was no signifi cant difference in prevalence of poly-morphisms of 3813 A/G (P=0.08) and 4895A/G (P=0.45) between the groups. For FAAH 385 C/A, there was a signifi cant (74% in the obese group vs. 26% in the control group P=0.03) difference in prevalence of the polymorphism. Presence of polymorphisms was not associated with prevalence of diabetes (DM) [(3813 A/G; P=0.8456); (4895A/G; P=0.4981); (FAAH 385 C/A; P=0.89)]. There were no signifi cant differences in the prevalence of these polymor-phisms between ethnic groups. Our data suggests that the FAAH 385 A/C genotype is signifi cantly associated with obesity in Caucasian and AA sub-jects in the U.S., but not with DM.

Table. Characteristics of the Subjects.Obese Subjects

(n=475)Control Subjects

(n=202)P value

CNR1 3813, count (%) 131 (75.3) 43 (24.7) 0.08CNR1 4895, count (%) 132 (68.4) 61 (31.6) 0.54FAAH 385, count (%) 239 (74.0) 84 (26.0) 0.03Body Mass Index, mean (SD) 37.5 (6.5) 23.3 (2.4) <.0001Waist Hip Ratio, mean (SD) 0.9 (0.1) 0.8 (0.1) <.0001Glucose (mg/dL), mean (SD) 99.8 (39.2) 76.5 (23.5) <.0001Type 2 Diabetes, count (%) 121 (91.6) 11 (8.3) <.0001

Supported By: Tulane University

2225-PEffect of Smartphone Application on Weight Loss: A Study Based on Global Big DataSANG YOUL RHEE, CHANGWON KEUM, JUNG HOON WOO, JEHWAN PARK, JOO YOUNG KIM, JEONG-TAEK WOO, YOUNG SEOL KIM, Seoul, Republic of Korea, New York, NY, Suwon, Republic of Korea

Many smartphone applications for weight management have been intro-duced, but their clinical usefulness was not established, because most previ-ous studies were conducted on limited study design.

This study was conducted to evaluate clinical effectiveness of smart-phone based application on weight loss. We utilized de-identifi ed weight logs and the self-monitoring information about diet and exercise of all users who installed the Noom™ Coach application from October 2012 to April 2014. And we selected data of 48,095 users from 58 countries who used the appli-cation at least once in the fi rst six consecutive months. We evaluated long-term effectiveness of application on weight loss, and analyzed independent variables on the weight change.

We could analyzed the weight changes and relevant self-monitoring vari-ables from baseline to 267 median days (IQR = 182 day) of follow-up, and we found that 44.7% of participants lost weight (≥5.0% from baseline BMI). The weight loss success rate was higher in male participants (53.9%) than female participants (46.7%). In multivariate analyses, age, gender, baseline BMI, calorie intake, weight input frequency, exercise input frequency, and diet input frequency were independent for weight loss (p<0.001). Among these, effect size of dinner input frequency was the strongest (β=2.562, 95% CI 2.271-2.852, p<0.001).

In conclusion, smartphone application for weight management may be useful for persons who are ready to monitor their clinical parameters for a

certain period of time. And dinner input frequency may be the most impor-tant determinant for their weight loss.

Supported By: Korean Medical Institute; Korean Society for the Study of Obe-sity; Ministry of Health and Welfare of the Republic of Korea (HI10C2020)

2226-PValidated Metabolomics Study Shows Preoperative and Postop-erative Metabolic Indicators of the Early Remission of T2DM after Bariatric SurgeryMICHAL CIBOROWSKI, PAULINA SAMCZUK, MAGDALENA LUBA, ANNA CITKO, JOANNA GODZIEN, HADY RAZAK HADY, JACEK DADAN, CORAL BARBAS, MA-RIA GORSKA, ADAM KRETOWSKI, Białystok, Poland, Madrid, Spain

Laparoscopic sleeve gastrectomy (LSG) and Roux-en-Y gastric bypass (RYGB) bariatric procedures are effective treatments for obesity and in 80-90% of subjects for T2DM. It is suggested, that altered secretion of gut hormones, modulation of neurohumoral pathways or changes in bile acids and lipids concentration contribute to improvement of glycemic control. The aim of this study was to search for metabolic biomarkers which could de-termine the effi cacy of the bariatric surgery in the treatment of T2DM. A group of 40 obese patients with T2DM, who underwent LSG or RYGB bar-iatric procedures were evaluated for an early remission (1 month after the surgery) of T2DM. Patients were divided on two groups: primary set (n=23) and validation set (n=17). The fasting serum samples were fi ngerprinted by LC-QTOF-MS. Primary set was analyzed in Spain, while validation set in Poland. Statistical analysis was performed independently on both sets and only metabolites signifi cant (p-value < 0.05) in both sets were selected to draw conclusions. Depending on data distribution t-test or Mann-Whitney test were used. Identifi cation of signifi cant metabolites was confi rmed by MS/MS analysis. Patients with early T2DM remission had higher levels of acylcarnitines (+ 34-72%, p-value 0.05-0.04) and lysophospholipids (LPLs) (+ 47-63%, 0.03-0.01) before the surgery. One month after the surgery de-crease in choline (- 32%, 0.04), phenylalanine (- 27%, 0.04), phospholipids (PLs) (- 42-60%, 0.02-0.01), LPLs (- 27-59%, 0.05-0.009), sphingomyelins (SMs) (- 37-55%, 0.02-0.01) and increase in acylcarnitines (+ 30-135%, 0.05-0.0008) was observed. Early recovery was accompanied with signifi cantly higher decrease of lipids and increase of acylcarnitines. Obtained results indicate that preoperative status and postoperative modulation of PLs, LPLs, SMs and acylcarnitines might substantially contribute to early improve-ments of glycemic control after bariatric surgery.

Supported By: Polish Ministry of Science and Higher Education (KNOW2012-2017)

2227-PRelationships between Markers of Total and Visceral Fat Mass and Gold Standard Body Composition Assessments in the EMPA-REG H2H SU TrialIAN J. NEELAND, DARREN K. MCGUIRE, BJORN ELIASSON, MARTIN RIDDER-STRÅLE, CORDULA ZELLER, HANS-JUERGEN WOERLE, ULI C. BROEDL, ODD ERIK JOHANSEN, Dallas, TX, Gothenburg, Sweden, Gentofte, Denmark, Biberach, Ger-many, Ingelheim, Germany, Asker, Norway

The SGLT-2 inhibitor empaglifl ozin reduces weight, total body fat (TBF) and visceral adiposity (VAT) in patients with type 2 diabetes (T2D). Since use of gold-standard methodology to quantify adipose tissue depots is limited, we explored the relationship between markers and direct imaging assessments of adipose tissue mass in ~100 T2D patients enrolled in a body composition sub-study. We assessed Spearman correlations of total body fat/fat-free mass (%) by dual energy X-ray absorptiometry (DXA) and VAT/subcutane-ous adipose tissue (SAT) mass by MRI with waist circumference (WC), index of central obesity (ICO, WC/height), estimated (e)TBF (Males: 100*(-98.42 + [4.15*WC] - [0.082*weight])/ weight; Females: 100*(-76.76 + [4.15* WC] - [0.082* weight])/weight) and visceral adiposity index (VAI; Males: WC/(39.68 + 1.88 x BMI) x TG/1.03 x 1.31/HDL, Females; WC/(36.58 + 1.89 x BMI x TG/0.81 x 1.52/HDL). eTBF positively correlated with total fat mass and SAT but not with VAT (Table). WC had the strongest correlation with VAT and ICO correlated with all fat depots. VAI had a negligible correlation with VAT. Our fi ndings support use of WC, ICO and eTBF, but not VAI, for quantifi cation of fat depots when gold standard methods are not available. The EMPA-REG OUTCOME trial (NCT01131676) due to report in 2015 will evaluate if changes in such markers are associated with clinical benefi ts.

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Supported By: Boehringer Ingelheim

2228-PIn Obese Patients, Cardiac Output Is Higher in Those with Glucose Intolerance—Role of Sympathetic HyperactivityAMEL REZKI, MARINOS FYSEKIDIS, BADREDDINE MERIOUD, SABRINA CHIHEB, ISABELA BANU, EMMANUEL COSSON, PAUL VALENSI, Bondy, France

An increase in cardiac output and alterations in cardiovascular autonomic activity were demonstrated in obese patients. Glucose intolerance is associ-ated with more marked alterations of cardiac autonomic function. The aim was to compare these parameters in obese patients with normal glucose tolerance (NGT) or glucose intolerance (IGT).

Sixty-six obese patients were included and classifi ed according to an oral glucose tolerance test. They were 38 NGT and 28 IGT, with similar sex ratio, age and BMI (BMI=38.4±4.1 and 37.4±4.3kg/m², respectively). They were nor-motensive or had well-controlled hypertension and were free of cardiovascu-lar history. Arterial stiffness (carotid to femoral pulse wave velocity), central and peripheral blood pressure and left ventricular ejection time (LVET) were assessed by applanation tonometry (SphygmoCor®). Cardiac sympathetic activity (LF-HR) and sympatho-vagal balance (LF/HF-HR) were determined by spectral analysis of heart rate variations (Task Force Monitor® digital plethys-mography). Cardiac output (CO), stroke volume (SV), cardiac index (CI) and tho-racic fl uid content (TFC) were measured by thoracic impedance.

Blood pressures and arterial stiffness were similar in both groups. Com-pared with NGT, IGT patients had longer LVET (317±25 vs. 304±25 ms), higher CI (2.3±0.5 vs. 1.8±0.7 l/min*m2), SV (68±19 vs. 55±17 ml), and TFC, and lower total peripheral resistance index (p<0.04 to p<0.002). A trend to an increase in CO was observed in IGT obese patients (p=0.06). For all patients, plasma glucose at fasting and 2 hours after oral glucose challenge correlated with LF/HF-HR (r= 0.27, p=0.02; r=0.26, p=0.03), even after adjusting for age and BMI.

In obese patients, glucose intolerance is accompanied by an increase in left ventricular output and ejection duration and hypervolemia, all probably favoured by higher sympathetic activity.

2229-PEarly Weight Loss Responders to Liraglutide 3.0 mg in the SCALE Obesity and Prediabetes Trial Achieve Greater Reversal of Predia-betes than PlaceboJOHN P.H. WILDING, MATTHIAS BLÜHER, KJELD HERMANSEN, FRANK GREEN-WAY, KENNETH FUJIOKA, BIRGITTE CLAUDIUS, AGATHE LE LAY, DAVID C.W. LAU, Liverpool, United Kingdom, Leipzig, Germany, Aarhus, Denmark, Baton Rouge, LA, La Jolla, CA, Søborg, Denmark, Calgary, AB, Canada

The SCALE Obesity and Prediabetes trial investigated weight loss (WL; primary endpoint) with liraglutide 3.0 mg (N=2487) vs. placebo (N=1244; 2:1 randomization) and prediabetes status after 56 weeks. This post-hoc analy-

sis investigated whether individuals with prediabetes (ADA 2010 criteria) at screening (liraglutide: N=1528 [61.4%] vs. placebo: N=757 [60.9%]) who were early responders (≥4% WL after 16 wks) and achieved ≥5% or >10% WL at 56 wks also had greater rates of prediabetes reversal. Baseline characteristics of the completer populations were similar. More completers achieved ≥5% and >10% WL at wk 56 with liraglutide 3.0 mg (765 [50.1%] and 431 [28.2%]) vs. placebo (114 [15.1%] and 55 [7.3%]), and consistently greater mean WL was seen with liraglutide 3.0 mg in both WL categories (Table). At 56 wks reversal of prediabetes was greater in early responders on liraglutide 3.0 mg vs. placebo: 76.9% vs. 46.5% for ≥5% WL and 82.8% vs. 58.2% for >10% WL. Overall adverse event rates were similar, except gastrointestinal events were higher with liraglutide 3.0 mg (Table). In conclusion, more individuals treated with liraglutide 3.0 mg vs. placebo were early responders, lost ≥5% or >10% weight at 56 wks, and achieved greater WL after 56 wks. Compared with placebo early responders treated with liraglutide 3.0 mg had greater rates of prediabetes reversal.


2230-PmiR-100 Is Lower in Visceral Adipose Tissues of Patients with Type 2 Diabetes and Targets IGFR and mTORSHARON L.T. PEK, ANTON K.S. CHENG, SU CHI LIM, CHEE FANG SUM, SUBRA-MANIAM TAVINTHARAN, Singapore, Singapore

Obesity confers substantial risk for type 2 diabetes (T2D) and understand-ing their molecular basis is critical. MicroRNAs are small non-coding RNAs that bind to regulatory sites of target mRNA, usually resulting in decreased protein production. We previously reported mir-100 to be lower in visceral fat of those with T2D compared to those without T2D. To understand down-stream effects of miR-100, we modulated miR-100 levels by over-expression or inhibition in 3T3-L1 adipocytes.

Obese patients referred to bariatric surgeons at our institution were re-cruited. Patient demographics, anthropometric data and visceral adipose samples were collected. Primary cultures were established from visceral adipose tissues (n=4) and adipogenesis was initiated. miR-100 expression was assessed by realtime PCR. 3T3-L1 pre-adipocyte was transfected with miR-100 mimics or inhibitors (48hrs) and gene expression analyzed by mRNA array. Validation was performed by realtime PCR and western blot.

miR-100 expression was lower in human matured adipocytes compared to pre-adipocytes (p=0.01). With pathway enrichment of lipid catabolic pathways, we identifi ed genes (n=69) signifi cantly down-regulated in 3T3-L1 cells over-expressing miR-100 and 11 genes up-regulated in 3T3-L1 with miR-100 inhibition. Validation of candidate genes by real time PCR showed that Very low-density lipoprotein receptor (VLDLR), Insulin growth factor re-ceptor (IGFR) and mammalian target of rapamycin (mTOR) were up-regulated (1.79-fold, 4.81-fold, 2.21-fold, respectively) after miR-100 inhibition and suppressed (0.44-fold, 0.92-fold, 0.83-fold), respectively after miR-100 over-expression in 3T3-L1.

Bioinformatic analyses showed that VLDLR, IGFR and mTOR are potential targets of miR-100, with each playing important roles in adipogenesis and lipid metabolism Down-regulation of miR-100 in human adipogenesis and adipose tissues from patients with T2D may suggest a link between adipo-cyte dysfunction and T2D.

Supported By: Alexandra Health Pte. Ltd. of Singapore (SIGII/13002)

2231-PMonocyte Phenotype in Obese Humans with Metabolic SyndromeHUAIZHU WU, ILVIRA KHAN, YASHASHWI POKHAREL, RAZVAN DADU, DORO-THY LEWIS, RON HOOGEVEEN, CHRISTIE M. BALLANTYNE, Houston, TX

Monocytes are heterogeneous with 3 subsets in humans, which are iden-tifi ed by CD14 and CD16 and differ in numerous other markers and functions. We used multi-color fl ow cytometry to examine effect of obesity on human monocyte phenotype. Eleven obese subjects with metabolic syndrome (MS) (BMI: 34.8 ± 2.2 kg/m2; fasting triglyceride [TG]: 217 ± 42 mg/dl) and 11 gen-

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der- and age-matched lean controls (BMI: 24.0 ± 0.7 kg/m2; fasting TG: 89 ± 10 mg/dl, P<0.01 vs. obese group) were recruited. Blood was taken after an overnight fast. Compared to leans, obese subjects had higher counts of CD14dim/CD16+ nonclassical monocytes (ncM), but not CD14+/CD16- classical (cM) and CD14+/CD16+ intermediate monocytes (iM). CD11c, a β2 integrin, CCR5, HLA-DR, TNFα and IL-1β were higher on iM and ncM than on cM of the same groups, supporting infl ammatory phenotype of iM and ncM. Compared to lean, obese subjects had higher CD11c on iM and cM, CCR5 and TNFα on/in cM, and a trend to be higher in CCR5 and HLA-DR on iM and ncM, and IL-1β in cM (Table). The proportion of nile-red+ lipid-laden foamy monocytes was higher in obese subjects than leans, and positively correlated with TG levels (P=0.02, r=0.53). In summary, in obese and lean subjects, iM and ncM had proinfl ammatory phenotype. Compared to those in leans, monocyte sub-sets in obese subjects with MS had enhanced infl ammatory phenotype and increased lipid accumulation, which correlated with TG levels.

Table. Counts and Infl ammatory Markers of Monocyte Subsets.Markers Unit Subset Obese group Control group P values#

Counts cells/mm3 Total 748.7 ± 90.0 704.2 ± 122.9 0.77Classical 681.0 ± 89.9 653.0 ± 118.7 0.85Intermediate 24.01 ± 2.7 24.66 ± 4.7 0.90Nonclassical 43.70 ± 4.9 26.51 ± 2.4 0.005

CD11c MFI Classical 13.8 ± 2.9 7.8 ± 0.6 0.06Intermediate 27.7 ± 4.4*** 18.1 ± 1.4*** 0.05Nonclassical 23.3 ± 2.9*** 21.0 ± 1.0*** 0.30

CCR5 MFI Classical 12.8 ± 2.8 6.7± 0.9 0.05Intermediate 16.4 ± 2.5 11.2 ± 1.4*** 0.10Nonclassical 11.5 ± 1.2 9.99 ± 1.3*** 0.39

HLA-DR MFI Classical 64.1 ± 7.4 66.4 ± 5.0 0.80Intermediate 219.6 ± 42.1** 151.0 ± 28.7* 0.21Nonclassical 152.4 ± 20.1 106.7 ± 27.5 0.19

TNFα % of the subsets Classical 4.1 ± 0.5 1.6 ± 0.5 0.003Intermediate 51.8 ± 5.7*** 53.6 ± 7.3*** 0.85Nonclassical 45.3 ± 6.8*** 48.3 ± 7.5*** 0.77

IL-1β % of the subsets Classical 4.0 ± 0.8 2.5 ± 0.4 0.15Intermediate 40.3 ± 3.8*** 51.8 ± 6.4*** 0.14Nonclassical 23.3 ± 4.4*** 35.4 ± 6.4*** 0.13

# indicates P values for comparisons between obese and control groups;*indicates P<0.05, ***indicates P<0.001 compared to classical monocytes of the same group.

Supported By: National Institutes of Health (R01HL098839, R01DK078847)

2232-PTelemedical Coaching for Weight Loss in Overweight Employees—A 3-Armed Randomized Controlled TrialKERSTIN KEMPF, STEPHAN MARTIN, MICHAEL SCHNEIDER, Düsseldorf, Ger-many, Ingelheim, Germany

We examined the effect of a telemedical coaching (TMC) program on weight loss in comparison to two control groups. Overweight employees (n=100; 86% men, mean age 55 ± 18 years) were randomized into 3 groups. The TMC group and one control (C1) group were equipped with telemoni-toring devices (scale and pedometer) at baseline, and the 2nd control (C2) group primal after 6 months. Data were automatically transferred into a personalized online portal, which could be monitored from the participant and the coaches. Telemedical coaching included lifestyle information, data discussion, and target agreements. The TMC group were intensive coached with weekly care calls in months 3-6 and monthly calls until study end. The C2 group got a short coaching including 9 care calls in months 6-9. Weight difference within and between groups was analyzed using Wilcoxon signed rank and Mann-Whitney test. After 6 months weight loss was signifi cantly higher in the TMC and C1 group (p<0.05 vs. C2). After 12 months weight loss was signifi cant in all groups (8 ± 11 kg (TMC); 4 ± 6 kg (C1); 3 ± 6 kg (C2); p<0.001 each for within group differences) but weight loss in the TMC group was signifi cantly higher (p<0.05 vs. C1 and C2). Cardiovascular risk factors, e.g. BMI, waist circumference, blood pressure, cholesterol, and triglycerides improved in addition. Telemedical coaching is effective in reducing weight and cardiometabolic risk long-term.

Supported By: Boehringer Ingelheim Pharma GmbH & Co. KG

2233-PGLP-1-analog Treatment in the Failed Gastric Bypass PatientNICLAS ABRAHAMSSON, ARVO HÄNNI, Uppsala, Sweden

Bariatric surgery is shown to be the most effective weight loss therapy presently available, with the Gastric ByPass (GBP) surgery being the method most commonly used worldwide. All patients however do not lose weight suffi ciently (defi ned as Excess BMI Loss (EBL) <50%), and there are currently no other treatment than intensifi ed diet and exercise counseling to offer these patients.

The GLP-1-analogs are a new option in the treatment of diabetes, shown to decrease both HbA1c and weight. They have further been shown to lead to weight loss in the non-diabetic patient. Their effect in the post-GBP pa-tient is largely unknown. It is well-known that the levels of endogenous postprandial GLP-1 increases about 8-10-fold post-GBP, and the potential effects and possible side-effects of adding exogenous GLP-1-analog treat-ment to post-GBP patients are largely unknown.

We have, in a clinical setting at the Obesity Unit at Uppsala University Hospital, Sweden, used the GLP-1-analog Liraglutide as off label treatment in 13 post-GBP-patients (BMI 41.6(30.5-50.1)kg/m2, age 44(28-67)years) with insuffi cient weight loss (<50% EBL) for treatment-periods of at least 15-20 months. All patients were at least 2 years post-surgery, and the doses were individualized and between 1.8-3.0 mg/day. All patients fi rst met with specialized dieticians to optimize food intake and meal frequency, and all patients were in a weight gaining trend when initiating treatment.

No patient experienced severe side effects, motivating withdrawal of treatment. The excess weight loss at 15-20 months was 10.4% (2.3 (-0.3-5.8) BMI-units or 4.6 (-1.0-14.7) kg).

We believe that this indicates that long-acting GLP-1-analogs might be used in the failed GBP-patient with an effect of breaking the weight gaining trend and initiating weight loss, without more severe side effects than in the diabetic patient. This effect is evident even though postprandial endog-enous GLP-1-levels are already considerably raised in this patient group.

2234-PAdipose Tissue Splicing in Human Obesity LociDOROTA KAMINSKA, SARI VENESMAA, PIRJO KÄKELÄ, PEKKA MIETTINEN, IMRE ILVES, MARJUKKA KOLEHMAINEN, LEILA KARHUNEN, JOHANNA KUU-SISTO, HELENA GYLLING, MARKKU LAAKSO, JUSSI PIHLAJAMÄKI, Kuopio, Finland

Alternative splicing results in generation of transcriptomic and proteomic diversity. Genome-wide association studies (GWAS) have identifi ed multiple obesity susceptibility loci, yet the mechanisms of action by which these loci infl uence obesity remain unclear. We hypothesized that alternative splicing of genes in these loci contributes to obesity. We applied database search for alternative splicing in these loci followed by splicing analysis of human adipose tissue in one population study (METSIM) and 2 independent weight loss intervention (surgical and very low calorie diet). Seventy-two of the 136 genes in the 11 obesity loci encoded different protein isoforms. We con-fi rmed alternative splicing of 11 genes with PCR capillary electrophoresis in human adipose tissue. Interestingly, TRA2B, BAG6 and MSH5 exhibited differential splicing between lean normoglycaemic and overweight type 2 diabetic individuals. Of these genes, we detected fat depot dependent splicing of TRA2B and BAG6 and weight loss induced regulation of MSH5 splicing in two independent studies. Finally, an association between MSH5 splicing and fasting insulin levels was observed. Concluding, our study pro-vides evidence on alternative splicing in obesity loci, suggesting that the

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obesity-associated SNPs might act through regulation of splicing leading to increased obesity risk.

2235-PObese African-American Adolescents Have Increased Free Fatty Acid Levels Before and After High-Fat MealROBERT P. HOFFMAN, RACHEL CAZEAU, LINDSEY B. RAUCH, HONG HUANG, JOHN A. BAUER, Columbus, OH, Fort Wayne, IN, Lexington, KY

AA have increased metabolic syndrome diseases yet have decreased metabolic syndrome frequency and fasting triglyceride (TG) levels compared to Caucasians (C) thus presenting a conundrum as to why. We hypothesized that although AA have lower fasting TG differences in post-prandial re-sponses between AA and C might be present. We studied TG, FFA, endothe-lial function (reactive hyperemia), and infl ammatory markers in 9 AA obese adolescents (age 12.7±1.0 yrs, BMI 34.5±7.4 kg/m2, mean±SD) and 7 C (age 12.8±0.8, BMI 33.2±6.4) before and 3 hours after McDonalds Big Break-fast®. Insulin sensitivity (IS) and secretion (ISEC) were measured using the oral glucose tolerance test. TG levels increased following the meal (p<0.001) and tended to be lower in AA (64±32 mg/dl) than in C (110±80, p=0.064) at baseline and were lower after meal (112±62 vs. 188±111, p=0.039). FFA levels decreased after meal (p<0.002) and were higher (p=0.02) in AA than in C at both times (AA: 0.58±0.15 mmol/L and 0.39±0.18; C:0.44±0.14 and 0.26±0.06). Adiponectin (ADIP) was higher (p=0.007) in AA but did change post-prandially. Interleuken-6 levels but not c-reactive protein (CRP) in-creased post-prandially (p=0.022). No racial differences were present. EF did not differ between races or post-prandially. IS was not different between the races while ISEC tended to be increased in AA (p=0.08) and the oral disposition index (DI) was signifi cantly higher in AA (11.6±5.7 vs. 4.3±1.4, p=0.006). Higher baseline FFA were associated with increased ISEC (r=0.73, p=0.002) and accounted for the racial difference in ISEC. Increased CRP at both times was associated with lower DI (r=-0.65, p=0.008). Baseline ADIP positively correlated (p=0.54, p=0.036) with DI though not when race was included. Higher FFA with lower TG in AA indicate differences in fat metabo-lism are present between obese AA and C adolescents. It remains unclear how these differences explain higher frequency of type 2 diabetes and car-diovascular disease in AA.

Supported By: The Ohio State University Center for Clinical and Translational Studies (UL1TR001070)

2236-P

2237-PENPP-1 and Lipid Metabolism in Different Human Adipose Tissue CompartmentsWENTONG PAN, MANISHA CHANDALIA, EDWARD LIVINGSTON, NICOLA ABATE, Galveston, TX, Dallas, TX

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a trans-membrane glycoprotein involved in defective adipocyte maturation and sys-temic insulin resistance. Despite the existing evidence from animal studies, the effect of ENPP1 in humans remains controversial. Further more, no stud-ies in humans have thus far evaluated relationship of ENPP1 expression level and adipose tissue (AT) function in different AT compartments. The aim of this study was to assess differences of adipocyte size and expression profi le between subcutaneous abdominal and Intra-abdominal AT.

Human subcutaneous abdominal and omental AT were obtained in 40 bar-iatric surgery subjects ( 30% males ) with BMI 47.9±10.7. Image J software was used to measure adipocyte size, which is represented as the average adipocyte area (in µm2). Quantitative RT-PCR was performed to determine the level of genes expression.

ENPP1 expression was lower (0.6 vs. 1.0; p=0.001) in omental as compared to subcutaneous abdominal AT. Adipocytes were smaller (4605 vs. 3268; p=1.07E-07) and leptin receptor OB-Rb expression was higher in omental AT (150.9 vs. 28.6; p=0.02). On the other hand, expression of genes involved in lipolysis and infl ammation were lower in omental than in subcutaneous abdominal AT (92.7 vs. 133.7 ; p=0.04 for CD36; 3.3 vs. 5.8; p=0.004 for HSL; and 3.2 vs. 6.2; p=0.002 for RBP4).

Based on our results, obesity is characterized by down-regulation of ENPP1 (which promotes pre-adipocyte differentiation into mature adipo-cytes), and reduction of both lipolysis and fatty acid uptake in visceral adipo-cytes. The increase in leptin receptor expression would suggest possible in-crease in fatty acid oxidation which could explain smaller adipocyte size and decreased expression of infl ammatory genes. Thus, signifi cantly decreased ENPP1 in intra-abdominal AT may act as compensatory mechanism to high storage demands in patients with obesity and positive caloric balance thus limiting excessive expansion of an anatomically confi ned AT depot.

2238-PSerum Fibroblast Growth Factor 21 Is Elevated in Overweight/Obese Women with Polycystic Ovary Syndrome and Regulated by Insulin InfusionIRINA KOWALSKA, MONIKA KARCZEWSKA-KUPCZEWSKA, AGNIESZKA NIKO-LAJUK, AGNIESZKA ADAMSKA, ELZBIETA OTZIOMEK, SLAWOMIR WOLCZYN-SKI, MAREK STRACZKOWSKI, Białystok, Poland

Fibroblast growth factor 21 (FGF21) exerts diverse benefi cial effects on energy balance and insulin sensitivity. Animal data indicate that exogenous FGF21 administration improves plasma glucose and insulin levels. Studies in obese subjects and in patients type 2 diabetes have demonstrated an in-crease in plasma FGF21 and its association with insulin resistance. Polycys-tic ovary syndrome (PCOS) is a common endocrine disorder associated with insulin resistance. The aim of the present study was to determine serum FGF21 concentration in patients with PCOS in comparison to healthy women and its response to insulin infusion during the clamp study.

We studied 48 women with PCOS (BMI-27.34±6.78kg/m2) and 30 healthy, regularly menstruating women (BMI-26.06±5.94kg/m2). PCOS was diag-nosed with Rotterdam criteria. Clinical examination, an anthropometric mea-surements, hyperinsulinemic, euglycemic clamp and estimation of plasma concentrations of sex hormones, sex hormone binding globulin (SHBG), lipid profi le were performed in all study participants. Serum FGF21 was measured at baseline and after the clamp.

Serum FGF21 concentration was higher in PCOS group vs. controls at baseline (p=0.004) and after clamp (p=0.004). In response to insulin infusion during the clamp study we observed an increase of serum FGF21 in PCOS (p=0.019), but not in the control group (p=0.36). In entire group baseline se-rum FGF21 correlated inversely with LDL-cholesterol concentration (r=-0.24, p=0.039).

Our data indicate that serum FGF21 is elevated in PCOS and regulated by hyperinsulinemia.

Supported By: Medical University of Bialystok (123-50724L)

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2239-PDiabetes Remission after Nonsurgical Intensive Lifestyle Interven-tion in Obese Patients with Type 2 DiabetesMAHMOUD SAKR, ADHAM MOTTALIB, MOHAMED SHEHABELDIN, OSAMA HAMDY, Boston, MA

Partial or complete remission from type 2 diabetes is a new and intriguing concept that was recently observed after bariatric surgeries. However, lim-ited data are available about the possibility of inducing diabetes remission through intensive and medically supervised weight reduction. We retrospec-tively evaluated diabetes remissions after one year of the Weight Achieve-ment and Intensive Treatment (Why WAIT) program, a 12 week intensive multidisciplinary program for diabetes weight management in real-world clinical practice. Among the 120 obese patients with type 2 diabetes who completed the program, 88 patients returned for follow up at one year and have their medications and A1C evaluated. Nineteen participants (21.6%) had major improvement in their glycemic control, defi ned as achieving an A1C <6.5 after one year. Four participants (4.5%) achieved either partial or complete diabetes remission defi ned as A1C <6.5 and <5.7% respectively on no antihyperglycemic medications for one year; 2 achieved partial remission (2.3%) and 2 achieved complete remission (2.3%). At time of intervention, patients who achieved diabetes remission had shorter diabetes duration <5 years, lower A1C than <8% and were treated with fewer than 2 oral medica-tions. They achieved weight reduction >7% after 12 weeks. These results in-dicate that a subset of obese patients with type 2 diabetes who are already on established antihyperglycemic medications are appropriate for intensive lifestyle intervention with the aim of inducing diabetes remission and stop-ping antihyperglycemic medications. Long-term follow up is needed to evalu-ate durability of diabetes remission.

Supported By: Joslin Diabetes Center

2240-PWeight Loss-independent Improvement in Type 2 Diabetes with EndoBarrier TherapyLEE M. KAPLAN, AURORA LIAO, DAVID MAGGS, ELAINE CHIQUETTE, Boston, MA, Lexington, MA

EndoBarrier therapy (EBT) involves the reversible endoscopic placement of an impermeable liner that anchors in the duodenum and bypasses the fi rst 2 feet of small intestine, replicating selected features of gastric bypass. The metabolic impact of EBT in type 2 diabetes (T2D) has been speculated to include weight dependent and independent components. We studied T2D subjects with obesity (n=161, BMI 38 kg/m2, hemoglobin A1C 8.4%, average T2DM duration 5 yrs) who had EBT for an intended 12 mo., and assessed: (i) metabolic changes in subjects with minimal weight loss (≤2 kg) early after implantation, and (ii) the estimated effect of weight on A1C by multivariate linear regression analysis, with baseline A1C and weight as the independent variables.

In all subjects, signifi cant reductions in fasting blood glucose (FBG) (−1.9 mmol/L; 95% CI −2.5, −1.3), weight (−3.7 kg; 95% CI −4.2, −3.2) and triglyc-erides (−0.7 mmol/L; 95% CI −1.0, −0.4) were observed by week 1. After resuming a regular diet, signifi cant improvements in the metabolic profi le continued and extended to reduction of blood pressure and LDL cholesterol.

In the subset of subjects exhibiting minimal weight loss at week 1 (n=28, BMI 33.8 kg/m2, A1C 8.8%), we observed signifi cant reduction in FBG (−2.7 mmol/L; 95% CI −3.6, −1.7). Within this group, subjects assessed 1 month after implantation (n=15, BMI 35.6 kg/m2, A1C 8.4%) exhibited signifi cant reduction in FBG (−2.8 mmol/L; 95% CI −4.7, −1.0), despite a weight loss of only 0.4 kg.

The derived regression equation for A1C change was: A1C Δ = 4.71 + (−0.64 x baseline A1C) + (0.05 x weight Δ), predicting that in subjects with baseline A1C of 9%, a 1% reduction would be expected independent of weight loss.

These data provide evidence for both weight loss dependent and indepen-dent metabolic effects of EBT on glycemic state in T2DM. The contribution of a weight-independent effect appears clinically signifi cant. Understanding its mechanism will provide valuable insight into GI regulation of metabolic function.

Supported By: GI Dynamics, Inc.

2241-PThe Comparison of the Prevalence Rate of Metabolic Syndrome be-tween the Last Five and Ten Years in Okinawa, JapanKENICHIRO WAKUTA, YOKO MAEKAWA, KURANDO WATANABE, NORIHUMI KAMIYA, TOMOHIRO YARA, ISAO SHIROMA, Okinawa, Japan

Metabolic syndrome (Mets) is known as an independent risk-factor of a blood vessel event. In Okinawa Prefecture, Japan, compared with the na-tional average, the prevalence rate of Mets is high, and it is considered that the prevalence rate of Mets in Okinawa at the present stage refl ects that of Japan in future stage. Since the Okinawans started consuming western food almost ten years earlier than that of main land Japan after World War II. About ten years ago, Tanaka et al. did a cross-sectional research to check the prevalence rate of Mets in Okinawa and the result was that in men, the prevalence rate of Mets was 30.2% and 10.3% in women. This time we stud-ied 6,752 men and 8,048 women, a total of 14,800 in 2009, and in 2013, 7,978 men and 8,949 women, a total of 16,887 people ages 30 to 79 years old. The subjects came to our clinic for an annual checkup. The comparison was made to check the latest prevalence rate of Mets in the last fi ve years with the result from ten years ago, which was conducted by Tanaka et al. This time we used modifi ed NCEP ATP III criteria,which is the same as that of Tanaka et al. (Hypertriglyceridemia (triglyceride ≥150 mg/dl), low high-density lipopro-tein (HDL) cholesterol (HDL cholesterol <40 mg/dl for men and <50 mg/dl for women), hypertension (systolic blood pressure ≥130 mmHg and/or diastolic blood pressure ≥ 85 mmHg), and impaired fasting glucose (fasting blood glu-cose ≥110 mg/dl), abdominal circumference (abdominal circumference ≥85 cm in men, ≥90 cm in women). Those who met 3 or more of the 5 risk factors listed above were diagnosed with Mets). As a result, the prevalence rate of Mets was 32.0% in men, and 17.2% in women in 2009, and in 2013, 31.9% in men, and 16.7% in women. In both sexes, compared with the study which was done by Tanaka et al. ten years ago, the prevalence rate of Mets in the last fi ve years was high, but the prevalence rate of Mets of 2013 was lower than 2009, so it may be concluded that the increase of the prevalence rate of Mets had already peaked out and it may decrease in future.

2242-PDirect Assessment of β-Cell Amount, Function, and Turnover in Nondiabetic Obese SubjectsLORELLA MARSELLI, MARA SULEIMAN, VERONICA DE GREGORIO, FAROOQ SYED, FABRIZIO SCATENA, FRANCO FILIPPONI, UGO BOGGI, MARCO BUGLIANI, PIERO MARCHETTI, Pisa, Italy

In non-diabetic (ND) obese subjects (OB), hyperinsulinemia counterbal-ances insulin resistance to maintain glucose homeostasis. Understanding the relative contribution of β cell amount and function in this regard could help to promote better β cell performance. We compared β cell features in 13 OB (age 66±13 yrs; 4M/9F; BMI: 32.0±3.3 kg/m2) and 14 lean [LEAN; 64±15 yrs; 4M/10F; 22.5±1.8 kg/m2 (p<0.01 vs. OB)] ND organ donors. Immunofl uo-rescence staining was performed with pancreatic sections; insulin secretion was assessed from isolated islets; laser capture microdissection (LCM) was used to acquire β cell enriched samples from frozen specimens. Pancreatic insulin (INS) and glucagon (GLUC) areas were 0.71±0.34% and 0.27±0.25% in OB, and 0.85±0.50% and 0.22±0.15% in LEAN, with the respective INS/GLUC values of 3.0±1.1 and 4.1±1.3 (p=0.06). Cells positive for both INS and GLUC were 10.5% in OB and 7.0% in LEAN islets. β cell clusters (<4 cells) per mm2 were 7.8±4.9 in OB and 7.6±7.0 in LEAN (>90% in acinar tissue, the remaining in/close to ducts); similar proportions of OB and LEAN clusters contained only INS+ (around 60%) or GLUC+ (around 35%) cells; costained INS+ and GLUC+ cells were seen in 7.6 and 3.9% of clusters in OB and LEAN, respectively. TUNEL or Ki67 positive β cells were similarly rare in the two groups. Glucose stimulated (16.7 mM glucose) INS release (µU/islet/min) was 0.093±0.048 from OB and 0.058±0.031 from LEAN (both p<0.01 vs. basal) islets; INS stimulation index was higher with OB islets (3.4±1.2 vs. 2.3±1.1, p=0.03), with a similar effect also seen with glyburide. LCM was applied to islets and acinar or duct clusters. RIN values were >6 for islet β cells and >4 for cluster β cells, with respective concentrations of >4 ng/µl and approximately 100 pg/µl. In conclusion, β cells in ND OB show greater insulin secretion; the subtle morphometric differences of OB vs. LEAN β cells requires further studies; LCM use could allow detailed molecular investiga-tion of OB β cell properties.

Supported By: JDRF (99-2012-12); Innovative Medicine Initiative Joint Undertak-ing (155005); European Commission (FP7/2007-2013)

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2243-PSilencing of Collagen VI Alpha 3 (COL6A3) Gene Is Associated with Resistance to Adipocyte Infl ammation—Implications in Metabolic DisordersKALYANI V. GUNTUR, ISHITA DEB MAJUMDAR, STEPHANE GESTA, VIVEK K. VISHNUDAS, RANGAPRASAD SARANGARAJAN, NIVEN R. NARAIN, Framing-ham, MA

COL6A3 was identifi ed as a key node in a disease-normal differential human obesity model by the Berg Interrogative BiologyTM discovery plat-form. Type VI collagen consists of an assembly of 3 alpha chains (COL6A1, COL6A2 & COL6A3) and is a major component of the extracellular matrix of adipose tissue. In humans, elevated expression of COL6A3 is associated with increased body mass index, fat mass, adipose tissue fi brosis. To further investigate the role of COL6A3 in adipocyte functions, immortalized human preadipocyte cell lines stably expressing either a control or COL6A3 shRNA were generated.

Depletion of COL6A3 enhanced adipogenic capacity that was refl ected by an increase in PPARγ (~50%) and FABP4 (4 fold) mRNA expression, and a 30% increase in triglyceride content. In addition, basal and stimulated lipolytic activities as well as insulin stimulated Akt phosphorylation were signifi cantly enhanced in COL6A3 knockdown adipocytes. Importantly, CO-L6A3 gene silencing led to a decrease in basal adipocyte chemokine (C-C motif) ligand 2 (CCL2, MCP1) mRNA expression (~80%) and secreted protein levels (>90%). Furthermore, TNFα and LPS stimulated MCP1 mRNA expres-sion was inhibited in COL6A3 knockdown adipocytes. Co-culture of THP1 macrophages with untreated normal adipocytes led to 3.5-fold increase in adipocyte MCP1 mRNA expression. However, MCP1 mRNA expression was unchanged when COL6A3 knockdown adipocytes were co-cultured with THP1 macrophages, demonstrating that these cells with COL6A3 knock-down are completely resistant to infl ammatory cues.

These results demonstrate that Col6a3 partakes directly or indirectly in the adipocyte MCP1 secretion - infl ammation axis. Therefore COL6A3 knock-down or disruption in adipose tissue could serve as a therapeutic strategy for the treatment of obesity associated infl ammation & insulin resistance.

2244-PCD163+ and CD206+ Adipose Tissue Macrophage Subpopulations in Subjects with Obesity and Type 2 Diabetes Mellitus: The Effect of Gastric Plication and Duodenal-Jejunal Bypass Liner ImplantationANNA CINKAJZLOVA, ZDENKA LACINOVA, JANA KLOUCKOVA, PETRA KAVALK-OVA, PAVEL TRACHTA, MILOS MRAZ, MARTIN HALUZIK, Prague, Czech Republic

CD163 and CD206 are considered the main markers of alternatively acti-vated (M2) macrophages which play a role in the resolution of adipose tissue low-grade infl ammation. The aim of our study was to examine changes in CD163+ and CD206+ cells in subcutaneous adipose tissue (SCAT) of obese subjects with type 2 diabetes mellitus (T2DM) in the context of global meta-bolic improvements after selected bariatric procedures.

Twenty-one obese subjects with T2DM undergoing either GP or DJBL were included into the study. Anthropometric and biochemical parameters were measured and SCAT samples from the abdominal region were taken at baseline, 1 and 6 (GP) or 10 months (DJBL) after intervention. M2 mac-rophage subpopulations were identifi ed using fl ow cytometry and a combi-nation of antigens including CD14, HLA-DR, CD163 and CD206.

At baseline 2 different subsets of CD163+ (CD163+HLA-DR+CD14+ and CD163+HLA-DR+CD14-) and CD206+ cells (CD206+HLA-DR+CD14+ and CD206+HLA-DR+CD14-) were identifi ed in SCAT according to the presence of CD14. Both interventions resulted in decreased body weight (BMI 43.2±1.8 vs. 36.0±2.4 kg/m2, p<0.001 for GP and 42.6±1.2 vs. 39.3±1.4 kg/m2, p<0.001 for DJBL) and improved glucose control (HbA1C 64.8±6.3 vs. 45.0±3.4 mmol/mol, p=0.002 for GP and 74.2±5.6 vs. 56.1±4.9 mmol/mol, p<0.001 for DJBL). In SCAT, GP signifi cantly decreased CD163+HLA-DR+CD14+ (20.5±2.4 vs. 12.6±1.1 %, p=0.017) and increased CD163+HLA-DR+CD14- cells (3.7±0.4 vs. 9.5±1.8 %, p=0.004). CD206+HLA-DR+CD14+ cells also decreased (21.7±3.2 vs. 11.8±0.7 %, p=0.009), while the CD206+HLA-DR+CD14- subset remained unchanged. DJBL yielded similar results.

We conclude that human adipose M2 macrophages consist of several subpopulations which react differently to weight reduction. These changes might contribute to positive effects of GP and DJBL on metabolic control in obese T2DM subjects.

Supported By: RVOVFN64165, IGANT13299-4, SVV260019/2014

2245-PQuercetin Counteracts Hypoxia-mediated Modulation of Gene Ex-pression in SGBS AdipocytesANDREAS LEIHERER, KATHRIN GEIGER, AXEL MUENDLEIN, KATHRIN STÖM-MER, CHRISTOPH H. SAELY, PETER FRAUNBERGER, HEINZ DREXEL, Dornbirn, Austria, Feldkirch, Austria, Philadelphia, PA

Adipose tissue of obese subjects is characterized by a hypoxic environ-ment which affects gene expression and appears to play a central role in the development of type 2 diabetes. The phytochemical quercetin like hypoxia acts at the mitochondrial membrane. We therefore aimed at investigating quercetin-triggered effects on hypoxic adipocytes. To elucidate the impact of hypoxia on adipose tissue, we used mature adipocytes derived from a Simpson-Golabi-Behmel syndrome patient and applied microarray analysis and advanced computational methods for data analysis. We found a striking association between the genome-wide gene expression pattern of hypoxia-treated adipocytes and diabetes-related biomarkers. Specifi cally, as is sum-marized in the fi gure, we could demonstrate that hypoxic cultivation signifi -cantly increases transcription of genes involved in energy metabolism (Eno-lase, PFKP, and PFKFB4) and the development of diabetes (Adipsin, ANGPTL4, and PAI). However, supplementation with quercetin signifi cantly decreased gene expression of these genes despite the hypoxic environment. We con-clude that quercetin appears to be a natural opponent of the deleterious effects on gene expression induced by obesity and mediated by hypoxia.

2246-PGreater Myosteatosis Is Associated with Higher All-Cause Mortal-ity among Men of African AncestryQIAN ZHAO, JOSEPH M. ZMUDA, ALLISON L. KUIPERS, PALLAVI JONNALA-GADDA, CLAREANN H. BUNKER, ALAN L. PATRICK, IVA MILJKOVIC, Pittsburgh, PA, Scarborough, Trinidad and Tobago

Myosteatosis (adipose tissue (AT) infi ltration within skeletal muscle fi -bers (intra-muscular AT) and within the fascia surrounding skeletal muscle (inter-muscular, IMAT, visible AT)), increases with aging, is greater in African compared with European ancestry men, and may play an important role in the development of type 2 diabetes (T2D). Studies examining this important fat depot and mortality are lacking, particularly among high-risk African an-cestry populations.

We evaluated the association of all-cause mortality with quantitative com-puted tomography (QCT) measured lower leg myosteatosis (IMAT and muscle density (lower muscle density refl ects greater intra-muscular AT)), in 1657 Af-rican ancestry men (mean age, 57.7 years; range, 40 to 91). Date of death from death certifi cates was reviewed by a central physician adjudicator. Cox propor-tional hazards ratio (HR) models were used to estimate the risk of mortality.

112 deaths occurred during a mean of 6.6 years. Greater IMAT (HR (95% CI) per SD increase: 1.30 (1.07-1.58)) and lower muscle density (per SD decrease: 1.34 (1.06-1.71)) were associated with increased risk of all-cause mortality, independent of smoking habits, alcohol consumption, sedentary lifestyle, physical activity, health status, body mass index, muscle area, cancer, T2D, renal disease, stroke, myocardial infarction and hypertension.

Our study reveals an independent association between myosteatosis and all-cause mortality among middle-aged and older African ancestry men. Further studies are needed to establish if the association of myosteatosis with mortality is independent of other ectopic fat depots, to identify possible biological mecha-nisms underlying this relationship, and to assess potential mitigating factors.

Supported By: K01-DK083029, R01AR049747

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2247-PPeripheral Blood Gene Expression Profi les in Obese Subjects with-out Metabolic SyndromeDANIEL DE LUIS, ROCIO ALLER, OLATZ IZAOLA, ENRIQUE ROMERO, Simancas, Spain, Valladolid, Spain

The purpose of our study was to assess if obese patients without meta-bolic syndrome possess unique peripheral blood gene expression profi les, in a pilot study. A sample of 17 obese subjects without metabolic syndrome and 15 health control subjects was enrolled in a prospective way. Follow-ing ‘One-Color Microarray-Based Gene Expression Analysis’ protocol Ver-sion 5.7 (Agilent p/n 4140-90040), 3 g of labeled cRNA was hybridized with Whole Human Genome Oligo Microarray Kit (Agilent p/n G2519F-014850) containing 41,000+ unique human genes and transcripts.

The mean age of the study group was 43.6+19.7 years with a sex distribu-tion of 64.7% females and 35.3% males. These dates didn´t show statistical differences with healthy controls 41.9+12.3 years with a sex distribution of 70% females and 30% males. We identifi ed 1436 differentially expressed genes: 1,248 up-regulated and 168 down-regulated. Inginuity canonical path-way analysis detected 49 biochemical pathways. These canonical pathways have been analyzed with the molecular and cell function algorithm of Gene Spring and a total of 13 categories has been detected and these categories have been joined in two groups; “cellular function” and “metabolism.” In the gene set of cellular function, the most important genes were C-terminal region of Nel-like molecule 1 protein (NELL1) and Pigment epithelium-derived factor (SPEDF), both genes were over-expressed. In the gene set of metabolism, in-sulin growth factor type 1 (IGF1), ApoA5 (apolipoprotein subtype 5), PIKFYVE (PtdIns(3) P 5-kinase), ADIPOR1 (receptor of adiponectin type 1) and AQP7 (aquaporin channel proteins7) were over expressed. Moreover, Foxo4 (Fork-head transcription factor 4) and ROCK-2 (rho-kinase II) were under expressed.

In conclusion, we showed that PBMCs from obese patients without metabolic syndrome presented signifi cant changes in gene expression, exhibiting 1,436 dif-ferentially expressed genes compared to PBMCs from non-obese subjects.

2248-PMetabolomic Profi les of Diabetic and Nondiabetic Individuals with Morbid Obesity in a Multiethnic Asian CohortPHONG CHING LEE, MATTHEW TAN, OI FAH LAI, KWANG WEI THAM, ALVIN NG, SHANKER PASUPATHY, ALVIN ENG, SONALI GANGULY, SU-YEN GOH, HONG CHANG TAN, Singapore, Singapore

Abnormalities in the metabolomic profi les branched-chain amino acids (BCAA) and their related metabolites are related to insulin resistance (IR) and glycemic control. It is unclear whether these metabolomics signatures are different between diabetic and non-diabetic individuals with morbid obesity (BMI ≥ 37.5 kg/m2 or ≥ 32.5 kg/m2 with co-morbidities).

Metabolomic profi les of amino acids (AA) were compared between individu-als with morbid obesity with type 2 diabetes (T2DM) (n=10) to those who were morbidly obese but non-DM individuals (n=14), and lean healthy controls (n=5). Quantitative metabolomics were performed using liquid-chromatography tan-dem mass spectrometry. IR was estimated by Homeostasis Model Assess-ment (HOMA-IR) and body composition with bioimpedence analysis.

Twenty nine subjects were recruited (41.4% female, ethnicity: Chinese 45%, Malay 24%, Indian 24%). BCAA correlated signifi cantly with BMI (r= 0.429, p =0.02) and IR (r = 0.426; p =0.027).

Compared to lean controls, HOMA-IR, total AA, BCAA and aromatic AA were higher in obese individuals with and without T2DM. These values were however similar between individuals of both groups (DM vs. non-DM) with morbid obesity (Table 1).

In conclusion, abnormalities in BCAA metabolism in morbidly obese individu-als are primarily a refl ection of insulin resistance and not beta-cell dysfunction.

Table 1. Clinical and Metabolomic Profi le of Study Cohort.Obese

Non-DM (n=10)

Obese T2DM (n=14)

Lean controls

(n=5)

p-value† (between groups)

p-value (within groups) Obese DM vs Obese non-DM

Obese DM vs Lean control

Obese Non-DM vs Lean control

Age, years 34.5 ± 7.4 45.0 ± 8.5 24.5 ± 2.4 <0.001 0.004 <0.001 0.003BMI, kg/m2 41.3 ± 8.1 39.0 ± 4.1 23.2 ± 3.2 <0.001 0.423 <0.001 <0.001Body fat, % 51.9 ± 10.9 48.9 ± 14.4 29.1 ± 9.3 0.007 0.565 0.005 0.002HOMA-IR 40.2 ± 27.6 40.0 ± 28.6 7.2 ± 4.7 0.013 0.986 0.001 0.004Total AA, µmol/L 2461 ± 386 2770 ± 495 1751 ± 326 0.001 0.101 <0.001 0.004BCAA, µmol/L 494 ± 98 535 ± 73 331 ± 102 0.001 0.282 0.007 0.018Aromatic AA, µmol/L 175 ± 39 182 ± 47 99.4 ± 14.8 0.002 0.672 <0.001 <0.001† using one-way ANOVA.

2249-PIncreased Risk for Coronary Artery Calcifi cation in Apparently Healthy Korean Adults with Hypertriglyceridemic Waist PhenotypeBYUNG-SUB MOON, EUN-JUNG RHEE, SE EUN PARK, CHEOL-YOUNG PARK, WON-YOUNG LEE, KI-WON OH, SUNG-WOO PARK, Seoul, Republic of Korea

Objective: Hypertriglyceridemic waist phenotype is a simple and inex-pensive screening parameter to identify people at increased risk for cardio-vascular disease. We evaluated whether hypertriglyceridemic waist pheno-type increases the risk for coronary artery calcifi cation (CAC) in apparently healthy Korean adults. A total of 32,186 participants (mean age 41.3, 80.2% men) in a health screening program, in whom the coronary artery calcium score (CACS) was measured, was analyzed. Subjects were divided into four groups: 1) normal waist circumference (WC)-normal triglyceride (TG) (NWNT), 2) normal WC-high TG (NWHT), 3) enlarged WC-normal TG (EWNT), and 4) enlarged WC-high TG (EWHT). Enlarged WC was defi ned as WC ≥90 cm for men and ≥85 cm for women; high serum TG was defi ned as TG ≥150 mg/dL. The presence of CAC was defi ned by CACS >0, and CACS was ana-lyzed in a logarithmized form of CACS plus 1 {ln(CACS+1)}. A total of 14.9% of the study population had CAC. The EWHT group showed the highest mean value for ln(CACS+1) among the four groups (0.63±1.42, p<0.05 in post-hoc analysis). When logistic regression analysis was performed with CAC, the EWHT group showed the highest odds ratio for CAC, with the NWHT group second, and the EWNT group showing a lower odds ratio for CAC compared with the EWNT group after adjusting for confounding variables (1.579, 1.302, and 1.266 vs. NWNT as the reference group). The EWHT group showed the highest risk for CAC, and the NWHT group showed a higher risk for CAC than the EWNT group, suggesting that hypertriglyceridemia could affect CAC more than an enlarged WC in apparently healthy Korean adults.

2250-PMetabolic Benefi ts of LCZ696: A Randomized, Double-Blind, Active-Controlled, Parallel-Group Study in Obese Hypertensive PatientsJENS JORDAN, RUDI STINKENS, THOMAS JAX, STEFAN ENGELI, ELLEN BLAAK, MARCUS MAY, BASTIAAN HAVEKES, CHRISTOPH SCHINDLER, DIEGO AL-BRECHT, PARASAR PAL, TIM HEISE, GIJS GOOSSENS, THOMAS LANGENICKEL, Hannover, Germany, Maastricht, Netherlands, Neuss, Germany, Basel, Switzerland, Hyderabad, India

LCZ696 simultaneously inhibits neprilysin, thereby augmenting natriuretic peptide (NP) availability, and blocks the AT1-receptor. Since NPs promote lip-id mobilization and oxidation and NP defi ciency predisposes to type-2 diabe-tes, this study investigated the effects of LCZ696 on insulin sensitivity, lipid mobilization and oxidation. Ninety-eight (98) patients with mild-to-moderate hypertension and abdominal adiposity were randomized to LCZ696 400 mg or amlodipine (AML) 10 mg once daily for 8 weeks in a double-blind, double-dummy fashion. At baseline and week 8, hyperinsulinemic euglycemic glu-cose clamp, abdominal subcutaneous adipose tissue microdialysis, glycerol tracer kinetics and indirect calorimetry were performed. LCZ696 and AML groups were matched for age, and baseline blood pressure and body mass index (LCZ696 32.6±4.6 kg/m2, AML 33.3±4.4 kg/m2). At week 8, the insulin sensitivity index trended towards improvement with LCZ696 (LCZ696 1.88 µg/kg*min/(mmol/L*pmol/L); AML 1.76 µg/kg*min/(mmol/L*pmol/L); p=ns), and glucose infusion rate/body weight was signifi cantly larger with LCZ696 (LCZ696 4.49 mg/min*kg; AML 3.88; p=0.0156). Subcutaneous adipose tis-sue lipolysis increased with LCZ696, but decreased with AML (interstitial glycerol levels at week 8; LCZ696 80.53 µmol/L; amlodipine 63.99 µmol/L; p=0.003), while there were no relevant changes in ethanol ratio, and glucose and lactate levels. Whole-body lipolysis as measured by systemic glycerol concentrations and the rate of appearance of glycerol were not different between groups. Resting energy expenditure and respiratory quotient did not change with either treatment. In conclusion, 8 weeks of treatment with LCZ696 improved insulin sensitivity and abdominal lipid mobilization in pa-tients with hypertension and abdominal adiposity, thereby supporting for the fi rst time the relevance of sustained elevation of NPs to human glucose and lipid metabolism.

2251-PIncreased Risk of Diabetes Development in Subjects with the Hy-pertriglyceridemic Waist Phenotype: A 4-Year Longitudinal StudyEUN-JUNG RHEE, SE EUN PARK, CHEOL-YOUNG PARK, WON-YOUNG LEE, KI-WON OH, EUN-GYOUNG HONG, SUNG-WOO PARK, Seoul, Republic of Korea

The hypertriglyceridemic waist (HTGW) phenotype is a simple and inex-pensive screening parameter to identify people at increased risk of cardio-vascular disease. We evaluated whether the HTGW phenotype predicts dia-betes in urban Korean adults. A total of 2,900 non-diabetic subjects (mean

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ISLET BIOLOGY—APOPTOSIS

age 44.3 years), comprising 2,078 males (71.7%) and 822 females (28.3%) who underwent annual medical check-ups at our center between January 2005 and December 2009, were recruited. The subjects were divided into four groups according to baseline serum triglyceride (TG) level and waist cir-cumference (WC): normal WC-normal TG level (NWNT), normal WC-high TG level (NWHT), enlarged WC-normal TG level (EWNT) and enlarged WC-high TG level (EWHT). High serum TG level was defi ned as ≥150 mg/dL and en-larged WC was defi ned as ≥90 cm for men and ≥85cm for women. New cases of diabetes were determined according to questionnaires fi lled in by partici-pants and the diagnostic criteria of the American Diabetes Association. Cox proportional hazards model analysis was used to assess the association of HTGW phenotype with the incidence of diabetes. A total of 101 (3.5%) new diabetes cases were diagnosed during the study period. The EWHT group had a higher incidence of diabetes (8.3%) compared with the NWNT group (2.2%). The adjusted hazard ratio (aHR) for diabetes for subjects with the EWHT phenotype at baseline was 4.113 (95% confi dence interval [CI] 2.397-7.059) after adjustment for age, and 2.429 (95% CI 1.370-4.307) after ad-justment for age, sex, total cholesterol, systolic blood pressure and alcohol drinking history. It was attenuated by inclusion of baseline fasting glucose level in the model. Subjects with the HTGW phenotype showed the highest risk of incident diabetes. This tool could be useful for identifying individuals at high risk of diabetes.

2252-PLongevity of the Metabolic Impact of Laparoscopic Sleeve Gastrec-tomy: Results in Morbidly Obese Indian Diabetic Patients at the End of Seven YearsJAYASHREE S. TODKAR, SHASHANK S. SHAH, Pune, India

Laparoscopic sleeve gastrectomy (LSG) is a standard bariatric operation. It also shows a metabolic impact in terms of improvement in diabetes type 2 (T2DM) in morbidly obese patients. Sparse reports exist about the longivity of its metabolic impact. This is the study to present the results of LSG in Indi-an obese patients with T2DM at the end of seven years. From 2006 till 2010, 124 patients of Indian origin with morbid obesity and T2DM have undergone a LSG at our center by a single surgical team. The standard operation of LSG and the multidisciplinary care with regular follow ups was provided to all of them. At the end of seven years we could collect information of 81 patients. N =81. M:F: 29: 62. Age range 22 - 65 yrs. Duration of T2DM: 6 mths to 21 yrs. BMI range: 35 - 68 kg/m2. On OHA only: 56. OHA + Insulin: 25. Average glycosylated Haemoglobin was 8.5%. At the end of seven years average BMI loss was found to be 18 kg/m2. The average glycosylated hemoglobin was 6.8%. The insulin usage (in redced doses) was needed in only 3 patients. 68 patients out of 81 did not need any anti diabetic medication. 10 patients were on a single OHA. 76/81 had other medical comorbidities related to obe-sity and all showed an improvement even at the end of seven years. 21/81 showed some weight gain (average 5 kg) at the end of 7 years but could re-tain their metabolic improvements as compared to the baseline. LSG seems to be a good surgical and metabolic tool to improve the diabetic status in morbidly obese Indian T2DM patients even at the end of seven years.

2253-PSerum Adiponectin Determines Circulating Vitamin D Concentra-tion Independently of Other AdipokinesRAM P. NARAYANAN, LUKE D. BOYLE, SARAH NG, AMANDEEP KAHLON, AN-DREW CROSS, CHERLYN DING, CHEN BING, CONOR MAGEE, HASAN Z. MALIK, STEPHEN W. FENWICK, JOHN P.H. WILDING, Warrington, United Kingdom, Liver-pool, United Kingdom

Circulating vitamin D concentrations have been linked to adiponectin and leptin, but not studied in the context of other adipokines. Our study aimed to assess the effects of adipokines on serum vitamin D levels in a predomi-nantly obese middle aged Caucasian cohort in north west England.

We recruited 53 patients (11 male), the majority (79.2%) of whom were due to undergo bariatric surgery. The remainder underwent elective laparo-scopic cholecystectomy. Mean age was 48 years (95% CI 45.3 - 50.9). The majority were obese (BMI>30: 88.6%; BMI>40: 77.3%).

Fasting serum samples were obtained from patients on the morning of the day of surgery. Serum total 25 hydroxyvitamin D (D2+D3), fasting insulin, calcium, parathyroid hormone (PTH) were measured along with serum leptin, adiponectin, resistin, ASP and C reactive protein (hs-CRP). Body weight was measured using TANITA scales.

Details of measured proteins expressed as (Mean, SD, 95% CI, units): vi-tamin D (60, 28, 16-149, nmol/L); leptin (21, 8.5, 18.7-23.4, ng/ml); resistin (0.2, 0.01, 0.19-0.2, ng/ml); adiponectin (59.6, 61.3, 42-76, ng/ml); ASP (19.2, 7, 17.3-21, nm/L).

Multiple regression analysis(STATA 10SE) with leptin, adiponectin, resis-tin and ASP as determinants of serum total 25 hydroxy vitamin D used a model adjusted for age, gender, adjusted calcium, PTH , BMI, fasting insulin, smoking status, season of sampling and statin prescription. hs-CRP was re-moved due to collinearity with leptin and resistin.

Adiponectin determined circulating vitamin D concentration independent of other measured adipokines (β 0.39, 95% CI 0.14-0.63, p=0.003). Leptin, resistin and ASP did not infl uence vitamin D concentration (r2 of model 0.51, p=0.04).

Serum adiponectin infl uences circulating vitamin D concentration inde-pendent of other adipokines. Resistin and ASP do not appear to be related to circulating vitamin D in our obese middle aged cohort.

ISLET BIOLOGY—APOPTOSIS

Guided Audio Tour: Islet Biology—Apoptosis (Posters: 2254-P to 2261-P), see page 15.

& 2254-PAutophagy Induction Improves Function and Survival of Human Type 2 Diabetic β-CellsMARCO BUGLIANI, MASINI MATILDE, FAROOQ SYED, SANDRA MOSSUTO, MARA SULEIMAN, LORELLA MARSELLI, UGO BOGGI, FRANCO FILIPPONI, DECIO L. EIZIRIK, MIRIAM CNOP, PELLEGRINO MASIELLO, PIERO MARCHETTI, Pisa, Italy, Brussels, Belgium

Autophagy is the major mechanism involved in degradation and recycling of intracellular components, and its alterations have been proposed to cause β cell dysfunction. In the present study we explored the effects of autophagy modulation in islets prepared from type 2 diabetic (T2D) and non-diabetic (ND) human donors, studied under several different conditions. Islets were isolated from 5 T2D (age: 77±7yrs; gender: 3M/2F; BMI: 23.9±3.7 Kg/m2) and 10 non-diabetic (ND; age: 69±19 yrs; gender: 3M/7F; BMI: 23.6±2.9 Kg/m2) organ donors. T2D and/or ND islets were then cultured for 1-5 days with 10 ng/ml rapamycin (autophagy inducer), or 5 mM 3-methyl-adenine (3MA) and 1.0 nM concanamycin-A (ConcA) (autophagy blockers), either in the presence or absence of metabolic (0.5 mM palmitate) or chemical (0.1 ug/ml brefeldin A) endoplasmic reticulum (ER) stressors. Compared to untreated T2D islets, rapamycin exposed (24h) diabetic islets showed improved insulin secretion (glucose-induced insulin stimulation index from 1.6±0.8 to 2.1±1.1, p=0.05), reduced amount of β cells with signs of apoptosis (from 6.6±1.7 to 2.0±0.8% by electron microscopy, p<0.05), and better insulin granules, mitochondria and ER ultrastructure. This was associated with signifi cant reduction of PERK, CHOP and Bip gene expression (qRT-PCR). As expected, in ND islets palmitate exposure (5 days) induced a 4-5 fold increase of β cell apoptosis (from 0.4±0.2 to 2.0±0.8%, p<0.01); this deleterious action was completely prevented by rapamycin (0.3±0.3%) and signifi cantly exacerbated by 3-MA (7.0±1.1%) and ConcA (3.1±1.1%). Substantially similar results were observed with brefeldin treatment (24h). Both palmitate and brefeldin induced PERK, CHOP and Bip gene expression, which was signifi cantly, although partially, prevented by rapamycin. This study emphasizes the importance of autophagy in human β cell function and survival, likely in association with ER activity. Modulation of autophagy could be instrumental to β cell protection.

& 2255-PHeat Shock Factor 1 Protects Beta Cells against Glucolipotoxicity-induced Endoplasmic Reticulum Stress and ApoptosisINDRI PURWANA, JEAN BUTEAU, Edmonton, AB, Canada

Excess exposure to glucose and fatty acid causes endoplasmic reticulum (ER) stress, protein misfolding, and apoptosis, a condition termed “glucolipo-toxicity”. Heat Shock Factor 1 (HSF1) is a transcription factor that regulates cell response to diverse stressors via the induction of the molecular chaper-ones heat shock proteins 70 and 90 (HSP70 and HSP90). Upon stress, HSF1 binds to the heat shock response element (HSE) to initiate the transcription of HSPs, whose role is to keep proteins from being misfolded thereby al-leviating ER stress.

We herein sought to test the hypothesis that HSF1 would protect beta-cells against glucolipotoxicity and to identify the posttranslational modifi ca-tions involved in its activation.

Thus, INS1 cells and human islets were treated with or without 25 mM glucose and 0.4 mM palmitate to induce glucolipotoxicity. Glucolipotoxicity caused ER stress as indicated by a rise in CHOP protein levels. This was accompanied by an increase in HSE-luciferase promoter activity and the con-sistent up-regulation of both chaperones hsp70 and hsp90. These effects of

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