maternal overweight programs insulin and adiponectin signaling in the offspring

Maternal Overweight Programs Insulin andAdiponectin Signaling in the Offspring

Kartik Shankar, Ping Kang, Amanda Harrell, Ying Zhong, John C. Marecki,Martin J. J. Ronis, and Thomas M. Badger

U.S. Department of Agriculture-Arkansas Children’s Nutrition Center (K.S., P.K., A.H., Y.Z., J.C.M.,M.J.J.R., T.M.B.), Little Rock, Arkansas 72202; and the Departments of Pediatrics (K.S., M.J.J.R., T.M.B.),Pharmacology and Toxicology (K.S., J.C.M., M.J.J.R.), and Physiology and Biophysics (T.M.B.), Universityof Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Gestational exposure to maternal overweight (OW) influences the risk of obesity in adult life. Maleoffspring from OW dams gain greater body weight and fat mass and develop insulin resistancewhen fed high-fat diets (45% fat). In this report, we identify molecular targets of maternal OW-induced programming at postnatal d 21 before challenge with the high-fat diet. We conductedglobal transcriptome profiling, gene/protein expression analyses, and characterization of down-stream signaling of insulin and adiponectin pathways in conjunction with endocrine and biochem-ical characterization. Offspring born to OW dams displayed increased serum insulin, leptin, andresistin levels (P � 0.05) at postnatal d 21 preceding changes in body composition. A lipogenictranscriptome signature in the liver, before development of obesity, was evident in OW-damoffspring. A coordinated locus of 20 sterol regulatory element-binding protein-1-regulated targetgenes was induced by maternal OW. Increased nuclear levels of sterol regulatory element-bindingprotein-1 and recruitment to the fatty acid synthase promoter were confirmed via ELISA andchromatin immunoprecipitation analyses, respectively. Higher fatty acid synthase and acetyl co-enzyme A carboxylase protein and pAKT (Thr308) and phospho-insulin receptor-� were confirmedvia immunoblotting. Maternal OW also attenuated AMP kinase/peroxisome proliferator-activatedreceptor-� signaling in the offspring liver, including transcriptional down-regulation of severalperoxisome proliferator-activated receptor-�-regulated genes. Hepatic mRNA and circulating fi-broblast growth factor-21 levels were significantly lower in OW-dam offspring. Furthermore,serum levels of high-molecular-weight adiponectin (P � 0.05) were decreased in OW-damoffspring. Phosphorylation of hepatic AMP-kinase (Thr172) was significantly decreased in OW-dam offspring, along with lower AdipoR1 mRNA. Our results strongly suggest that gestationalexposure to maternal obesity programs multiple aspects of energy-balance regulation in theoffspring. (Endocrinology 151: 2577–2589, 2010)

The global rise in the prevalence of overweight (OW)and obesity is paralleled by an alarming increase in the

incidence of OW among pregnant women (1). Being OWduring pregnancy has significant impact on the health ofboth the mother and the offspring. Pregravid OW in-creases the risk of preeclampsia, gestational diabetes, and

other labor-related complications (2, 3). For the offspring,exposure to maternal OW increases the risk of being largefor gestational age at birth, which substantially increasesthe risk of OW in adulthood (3–5). Moreover, exposure tomaternal obesity at conception and during pregnancy hasbeen hypothesized to lead to developmental programming

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2010 by The Endocrine Societydoi: 10.1210/en.2010-0017 Received January 6, 2010. Accepted March 11, 2010.First Published Online April 6, 2010

Abbreviations: AMPK, AMP-activated protein kinase; BMI, body mass index; ChIP, chro-matin immunoprecipitation; Cpt-1a, carnitine palmitoyl transferase-1a; CT, computerizedtomography; FASN, fatty acid synthase; FGF21, fibroblast growth factor-21; FPLC, fastprotein liquid chromatography; HFD, high-fat diet; HMW, high molecular weight; IPA,Ingenuity pathway analysis; IR, insulin receptor; IRS, IR substrate; NEFA, nonesterified fattyacids; NMR, nuclear magnetic resonance; OW, overweight; PND21, postnatal d 21; PPAR,peroxisome proliferator-activated receptor; SREBP-1, sterol regulatory element-bindingprotein-1; TEN, total enteral nutrition.

E N E R G Y B A L A N C E - O B E S I T Y

Endocrinology, June 2010, 151(6):2577–2589 endo.endojournals.org 2577

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of excessive weight and adiposity gain in the offspring(6, 7). Several lines of evidence from epidemiological andclinical studies support this contention. These include thestronger association of maternal body mass index (BMI)(compared with paternal BMI) with offspring BMI (8), thedecreased risk of obesity in children born to obese womenafter weight loss after bariatric surgery (9, 10), and thepositive relationship between maternal weight gain be-tween pregnancies and risk of OW in offspring comparedwith their siblings (11).

Using a model of gestational OW in the rat, we previ-ously demonstrated that maternal obesity programs in-creased sensitivity to weight gain in the offspring in theabsence of changes in birth weights (12). In this model,exposure to maternal OW is limited in utero and results inmarked increase in the weight gain in the adult offspringafter weaning to an obesogenic high-fat diet (HFD). More-over maternal obesity per se leads to hyperinsulinemia,increased percent liver weight, and adipocyte hypertrophyin the offspring, suggesting the programming of increasedlipogenic responses (12). However, the changes in off-spring endocrinology at weaning [i.e. postnatal d 21(PND21)], which may lead to increased lipogenesis andobesity risk are poorly appreciated. In this report, we focuson the effect of maternal OW on the programming of keyendocrine signaling (insulin and adiponectin) in the off-spring. Although the majority of studies examining ma-ternal obesity have used energy-dense (high fat) or highlypalatable diets to produce gestational OW, relatively littleis known about the effects of overconsumption of caloriesper se. Self-limiting consumption of diets due to satiety hasbeen the primary limitation in development of obesity inanimal models, necessitating use of energy-dense diets. Weemployed controlled feeding of liquid diets via total en-teral nutrition (TEN) as a mechanistic tool to overcomethis limitation.

The present study had three objectives. First, we exam-ined the effect of maternal OW on offspring endocrineparameters, before changes in body weight or adiposity inthe offspring. Specifically, we examined whether maternalOW in utero impacts hormones involved in energy balanceand lipogenesis. Second, we elucidate global transcrip-tome changes in the liver of the offspring at weaning usingmicroarrays to identify loci of programming. Third, weexamine cellular signaling pathways regulated by insulinand adiponectin in the liver leading to altered hepaticphysiology in the offspring of OW dams. Our datastrongly suggest that exposure to maternal OW from con-ception to birth programs systemic changes in insulin andadiponectin levels and alters a diverse suite of genes in-volved in carbohydrate metabolism, lipid biosynthesis,and fatty acid catabolism.

Materials and Methods

Animals and chemicalsFemale Sprague Dawley rats (150–175 g) were obtained from

Charles River Laboratories (Wilmington, MA). Animals werehoused in an Association for Assessment and Accreditation ofLaboratory Animal Care-approved animal facility. All experi-mental treatments were conducted in accordance with the guide-lines established and approved by the Institutional Animal Careand Use Committee at University of Arkansas for Medical Sci-ences. Unless specified otherwise, all chemicals were obtainedfrom Sigma-Aldrich Chemical Co. (St. Louis, MO).

Experimental protocolVirgin female Sprague Dawley rats were intragastrically can-

nulated and allowed to recover for 10 d as previously described(12–16). Rats were fed liquid diets at either 155 kcal/kg3/4 � d(referred to as lean dams) or at 220 kcal/kg3/4 � d (40% excesscalories, referred to as OW dams). Caloric intake for the leangroup was determined from preliminary studies and designed tomimic body weights and body composition of rats consumingstandard commercial diets ad libitum (12). TEN diets met Na-tional Research Council nutrient recommendations, includingessential fatty acids, and were 20% protein (casein), 75%carbohydrate (dextrose and maltodextrin), and 5% fat (cornoil) as percentage of total calories (12, 13, 16). These dietshave been previously used by our group in a number of studies(12–19). Infusion of diets was carried out 23 h/d using com-puter-controlled syringe pumps for 3 wk. Animals had adlibitum access to drinking water throughout. Body weightswere monitored three times per week. At the end of 3 wk, bodycomposition was noninvasively estimated using nuclear mag-netic resonance (NMR) (12).

After 3 wk of overfeeding, lean and OW rats (n � 18 pergroup) were allowed to mate for 1 wk. Each female rat washoused with one male and allowed ad libitum access to AIN-93Gdiet for this period. After mating, all female rats (lean and OW)resumed receiving diets at 220 kcal/kg3/4 � d (National ResearchCouncil-recommended caloric intake for pregnancy in rats). Allrats were allowed to give birth naturally. Numbers and sex ofpups, birth weight, and crown-to-rump and anogenital distancewere measured for each pup on PND1. On PND2, four males andfour female pups from each litter were cross-fostered to damsthat had been previously time-impregnated to give birth on thesame day as the dams receiving infusion diets. Cross-fostereddams were not cannulated and had ad libitum access to AIN-93Gpelleted diets throughout lactation. Using this experimental par-adigm, we ensured that offspring’s exposure to maternal OWwas limited to only intrauterine development (12). Female off-spring of lean and OW dams were used for separate experiments,and only data from male offspring are reported here. Male off-spring were euthanized under anesthesia at PND21 (n � 8 pergroup). Blood, liver, kidneys, and adipose tissues (retroperito-neal and gonadal depots) were weighed and collected. Sampleswere fixed in neutral-buffered formalin for histological analyses,and remaining tissues were frozen in liquid nitrogen and storedat �70 C for RNA and protein analyses. Serum was obtained bycentrifugation of blood samples and stored at �20 C for endo-crine and metabolic assessments.

2578 Shankar et al. Programming of Offspring Insulin Signaling Endocrinology, June 2010, 151(6):2577–2589


Body composition analysesBody composition of dams and offspring was assessed via

NMR (Echo Medical Systems, Houston, TX), x-ray computer-ized tomography (CT) (LaTheta LCT-100; Echo Medical Sys-tems), and postmortem dissected weights of retroperitoneal andgonadal adipose tissues. NMR was performed in conscious un-anesthetized rats in duplicate, and indices of percent fat and leanmass were derived using this technique (12, 16). For CT analyses,approximately 90 sections, 1 mm apart, were acquired encom-passing the entire visceral region of the animal under anesthesia.Indices of percent fat ratio (ratio of volume occupied by fat/volume occupied by lean tissue), percent fat mass, and percentlean mass were calculated using Aloka CT software (Tokyo,Japan) as described previously (12, 16).

Serum biochemistry, liver histology, and endocrinestatus

Serum glucose, triglycerides, fibroblast growth factor-21(FGF21), nonesterified fatty acids (NEFA), insulin, leptin, adi-ponectin, and resistin was measured as described in Supplemen-tal Materials and Methods published on The Endocrine Society’sJournals Online web site at http://endo.endojournals.org. He-patic glycogen was assessed using a commercially available gly-cogen assay kit (BioVision Inc., Mountain View, CA). Liver sec-tions were stained with hematoxylin and eosin or Oil Red O aspreviously described (15, 16). The separation of serum adiponec-tin complexes was performed using the method described bySchraw et al. (20). Serum (25 �l) was separated on a Superdex200 10/300 GL column (GE Healthcare Biosciences, Piscataway,NJ) using an AKTA fast protein liquid chromatography (FPLC)system (GE Healthcare) in a HEPES/Ca2� buffer. Thirty 0.215-mlfractions (from 7–14 ml) were collected. Aliquots of fractionswere heat denatured in loading buffer containing dithiothreitoland were separated using SDS-PAGE. Immunoblotting was car-ried out using standard procedures, and adiponectin monomers(�30 kDa) were quantitated using Western blotting. Using thistechnique, we ascertained that fractions containing high molec-ular weight (HMW) adiponectin complexes (fractions 6–13).Quantitative analyses of the percentage of HMW adiponectinrelative to total adiponectin in serum was performed usingELISA (B-Bridge International, Sunnyvale, CA). Serum from in-dividual offspring from lean and OW dams (n � 8 per group) wasseparated using FPLC as described above. After separation, adi-ponectin levels in whole serum and HMW adiponectin-contain-ing fractions (6–13) was assessed using ELISA on the same plate.Data are expressed as percentage of HMW adiponectin in serumrelative to total adiponectin.

Hepatic gene expression analyses

RNA isolation and microarray analysesTotal RNA was isolated from liver of offspring at PND21

(n � 8 per group) using the RNeasy kit (QIAGEN, Valencia,CA), including on-column deoxyribonuclease digestion. Threemicroarrays (GeneChip Rat 230 2.0; Affymetrix, Santa Clara,CA) were used for each group. Pools of equal amounts of RNAfrom two to three rats were used for analyses per microarray.Thus, eight rats per group were represented over the threemicroarrays. cRNA synthesis, labeling, hybridization, andscanning were carried out using the manufacturer’s instruc-tions (16, 18, 21).

Microarray data normalization and analysisMicroarray data analyses were carried out using GeneSpring

version 7.3X software (Agilent Technologies, Santa Clara, CA)(16, 21). The .CEL files containing probe level intensities wereprocessed using the robust multiarray analysis algorithm forbackground adjustment, normalization, and log2 transforma-tion of perfect match values (22). Subsequently, the data weresubjected to normalization by setting measurements less than0.01 to 0.01 and by per-chip and per-gene normalization usingGeneSpring. The normalized data were used to generate a list ofdifferentially expressed genes between offspring of OW and leandams at PND21. Genes were filtered based on minimum �1.8-fold change (OW vs. lean) and P value �0.05 using Student’s ttest. Corrections for multiple testing were performed using thefalse discovery rate method (23). A list of transcripts that weredifferentially expressed as a function of maternal OW was gen-erated, and correlation-based hierarchical clustering betweentreatment groups was performed. Known biological functions ofgenes were queried using Affymetrix NetAffx and gene ontologyanalyses performed using GeneSpring (16, 21). Furthermore, thelist of genes affected in offspring liver by maternal OW wasanalyzed using Ingenuity pathway analysis (IPA).

Real-time RT-PCRTotal RNA from liver and retroperitoneal adipose tissues from

offspring at PND21 (same offspring that were used for microarrayanalyses) was isolated using RNeasy columns (QIAGEN). One mi-crogram of total RNA was reverse transcribed (n � 8 per groupfor PND21) using IScript cDNA synthesis kit (Bio-Rad, Her-cules, CA). Real-time PCR analysis was performed as describedpreviously. Gene-specific primers were designed using PrimerExpress Software (Supplemental Table 1). Relative amounts ofmRNA were quantitated using a standard curve and normalizedto the expression of cyclophilin A mRNA (16, 18).

Immunoblotting, immunoprecipitation, andTransAM ELISA

Immunoblotting was carried out using standard procedures(16). A detailed description is provided in Supplemental Mate-rials and Methods. Immunoblotting was performed for AMP-activated protein kinase (AMPK), phospho-AMPK Thr172,acetyl coenzyme A carboxylase, carnitine palmitoyl trans-ferase-1a (Cpt-1a), insulin receptor (IR)-�, IR substrate (IRS)-1,phospho-AKT Ser473, phospho-AKT Thr308, AKT, ERK1/2,pERK1/2, fatty acid synthase (FASN), glyceraldehyde-3-phos-phate dehydrogenase, lamin A, and sterol regulatory element-binding protein-1 (SREBP-1) proteins in total lysates or extractsfrom nuclear or mitochondrial fractions. Immunoprecipitationwas performed in triplicate using 500 �g protein from pooledliver lysates (each pool representing two to three separate ani-mals). After overnight incubation with either anti-phosphoty-rosine antibody (Santa Cruz Biotechnology, Santa Cruz, CA) ornonspecific IgG, immune complexes were pulled down usingprotein G magnetic beads, washed, and solubilized in 2� Lae-mmli buffer. Aliquots were separated using SDS-PAGE, and im-munoblotting was carried out using anti-IR-� antibody (CellSignaling Technology, Beverly, MA). TransAM ELISA (ActiveMotif, Carlsbad, CA) was used to assess abundance of SREBP-1protein in nuclear extracts. Hepatic nuclear protein (40 �g) fromindividual animals was used and the procedures carried out ac-cording to manufacturer’s instructions. Controls such as recom-



binant SREBP-1 protein and competition with consensus oligo-nucleotides (not bound to the plate) were included in the assay.Abundance of SRE-bound SREBP-1 is represented as absorbancevalues at A450 nm.

Chromatin immunoprecipitation (ChIP)Recruitment of SREBP-1 and peroxisome proliferator activated

receptor (PPAR)-� on the promoters of respective target genes wasassessed using ChIP. The ChIP-IT enzymatic kit (Active Motif) wasused with minor modifications for in vivo samples and describedpreviously (24). Three pools of liver samples (with each pool rep-resenting two to three separate animals) were used for the analyses.Details of the procedure are described in the Supplemental Mate-rialsandMethods. Immunoprecipitationwasperformedusing5�gof either anti-SREBP-1 (Novus Biologicals, Littleton, CO), anti-PPAR-� (Abcam, Cambridge, MA), or matched nonspecific IgG.Target binding regions on the FASN and FGF21 promoters wereamplified by PCR for SREBP-1 and PPAR-�, respectively.

Statistical analysisData are expressed as means � SEM. Statistical differences be-

tween lean and OW rats before conception or dams during gesta-tion were determined using two-tailed Student’s t test. Similarly,differencesbetweenoffspringof leanandOWdamsatPND21weredetermined using two-tailed Student’s t test. Statistical significancewas set at P � 0.05. Statistical analyses were performed using Sig-maStat 3.3 software (Systat Software Inc., San Jose, CA).

Results

In utero programming of offspring metabolism bymaternal OW

Overfeeding female rats at 220 kcal/kg3/4 � d via TENfor 3 wk resulted in approximately 120% greater bodyweight gain (P � 0.001, Supplemental Fig. 1A). Body com-position assessed via NMR revealed that total body fat inOW females was 156% greater than lean controls (P �0.001) and was accompanied by a significantly lower leanmass (P � 0.001, Supplemental Fig. 1B). Body weights andadiposity of rats fed diets at 155 kcal/kg3/4 � d (lean group)were similar to ad libitum-fed controls at the start of thestudy (232 � 1.3 g ad libitum-fed rats and 235 � 1.5 g inthe lean group; n � 13 and n � 18, respectively) and at 3wk of feeding via TEN (265 � 2.4 g ad libitum-fed rats and267 � 1.9 g in the lean group; n � 13 and n � 18, re-spectively). We previously reported the development ofsignificant fasting hyperinsulinemia, hyperleptinemia,and elevated serum triglyceride and NEFA concentrationsin OW dams (12). Between wk 3 and 4 (from the beginningof diets), female rats were housed with one male breederrat and given ad libitum access to AIN-93G diets (pellets).After 1 wk of mating, both lean and OW dams resumedreceiving the TEN feeding at 220 kcal/kg3/4 � d. Duringthis period, the rate of pregnancy-related weight gain (ges-tation d 8–21) was the same for both lean and OW dams

(SupplementalFig. 1C).However, theOWdamsremainedheavier, and thebodycompositiondifferencesbetween thegroups persisted throughout gestation.

Offspring were nursed (starting PND1) by noncannu-lated surrogate dams that received ad libitum access toAIN-93G-based pellet diets. Hence, exposure to maternalOW was limited to conception and gestation. Birthweights (lean dams, 6.5 � 0.1 g, vs. OW dams, 6.2 �0.2 g), number of pups (lean dams, 11.7 � 0.3, vs. OWdams, 10.7 � 1.0), male-to-female ratio (lean dams,0.99 � 0.1, vs. OW dams, 1.0 � 0.1), crown-to-rumpdistance (lean dams, 1.5 � 0.03 in., vs. OW dams, 1.5 �0.02 in.), and anogenital distance (lean dams, 0.11 �0.005 in., vs. OW dams, 0.11 � 0.005 in.) did not differbetween offspring of lean- and OW-dam groups. To ex-amine the consequences of fetal programming due to ma-ternal OW, we assessed metabolic and endocrine param-eters at weaning (PND21) (Table 1). Body weights of maleoffspring did not differ at weaning (Table 1). However,percent liver weight (P � 0.001) in the offspring of OWdams was approximately 125% greater compared withtheir lean dam counterparts. Dissected weights of the kid-neys and visceral adipose tissues (retroperitoneal and go-nadal depots) at weaning did not differ between groups.Lack of increase in body adiposity at weaning in the off-spring of OW dams was also confirmed by quantitativeNMR(Table1) andx-rayCTanalyses.Despitenochangesin body weights or adiposity, serum insulin, leptin, andresistin concentrations were significantly elevated (140,200, and 180%, respectively) in the offspring of OW dams(P � 0.05) (Table 1). Serum triglyceride levels remained

TABLE 1. Effect of maternal OW on offspring metabolicand endocrine parameters at weaning

Parameter Lean OW P value

Body weight (g) 68 � 3.1 66 � 2.0 0.70Liver weight (g) 2.3 � 0.13 2.8 � 0.11 �0.05% Liver weight (g) 3.4 � 0.09 4.2 � 0.1 �0.0001% Visceral fat weight 1.06 � 0.06 0.85 � 0.07 0.07% Kidney weight 1.04 � 0.02 1.05 � 0.01 0.63% Lean mass 66 � 1.84 64 � 0.99 0.31% Fat mass 12.8 � 0.83 11.9 � 0.52 0.36Glucose (mg/dl) 134 � 8.9 141 � 4.9 0.55Insulin (ng/ml) 0.76 � 0.02 1.07 � 0.03 �0.05Leptin (ng/ml) 1.82 � 0.66 3.65 � 0.44 �0.05Adiponectin (�g/ml) 6.05 � 0.8 3.92 � 0.89 0.09Resistin (ng/ml) 13.9 � 0.82 25.1 � 2.1 �0.001Triglyceride (mg/dl) 227 � 32 221 � 26 0.90NEFA (�M) 249 � 26 135 � 20 �0.005Liver glycogen (�g/mg) 143 � 17 228 � 17 �0.005

Data were obtained from offspring of lean or OW dams at PND21 (n � 8per group). Lean and OW dams were fed via TEN as described inMaterials and Methods. Data are expressed as means � SEM. Indices ofpercent lean and fat mass were determined using noninvasivequantitative NMR. Weights of liver, kidney, and visceral adipose tissues(retroperitoneal plus gonadal fat depots) were assessed at the time ofeuthanasia. P values were determined using Student’s t test.



unchanged, whereas circulating NEFA levels were signif-icantly lower in OW-dam offspring (Table 1). Consistentwith increased liver weight, hepatic glycogen levels werealso increased to approximately 159% compared withlean dam offspring (Table 1).

Maternal OW alters hepatic transcriptome in theoffspring

To assess whether in utero exposure to maternal OWaltered hepatic gene expression in the offspring at wean-

ing, we performed gene expression analyses. Unsupervisedglobal condition clustering revealed clustering of expres-sion profiles between offspring based on their maternalphenotypes, suggesting significant treatment effect onglobal gene expression (Supplemental Fig. 2). After nor-malization, 147 transcripts were identified to be differen-tially expressed in offspring of OW vs. lean dams (�1.8-fold, P � 0.05, Supplemental Table 2). Correlation-basedhierarchical clustering of the genes affected by maternalOW is depicted in Fig. 1A. These transcripts were used for

FIG. 1. A, Hierarchical clustering of 147 transcripts altered by maternal OW in offspring liver. Gene expression was assessed in offspring liver atPND21 using Rat Genome 230 2.0 microarrays (Affymetrix) (n � 3 microarrays per group). Genes were filtered based on a minimum �1.8-foldchange (OW vs. lean) and P value �0.05 using Student’s t test. B, Correlation-based clustering of genes regulated by SREBP-1 with functions incarbohydrate or lipid metabolism derived from the list of genes altered by maternal OW. Heat maps were generated using GeneSpring Gx.Orange, yellow, and blue represent up-regulation, no relative effect, and down-regulation of transcripts, respectively. C, IPA gene network ofhighest significance identified using IPA software from the list of altered genes. A set of SREBP-1-regulated lipogenic genes and those involved infatty acid/cholesterol metabolism is evident. Colors green and red represent down-regulation and up-regulation respectively. D, Representativephotomicrographs of liver tissues from offspring of lean and OW dams at PND21. Top panel, H&E-stained sections; bottom panel, Oil RedO-stained sections. Magnification, �400.



gene ontology analyses based on molecular function, bi-ological function, and pathway analyses. Altered genespossessed binding or catalytic functions (46% each),transporter activity (5%), or signal transduction or regu-lated transcription (4% each) (Supplemental Fig. 3). Ofthe 147 transcripts altered, 11 were expressed sequencetag sequences with poorly defined biological functions.Approximately 26 transcripts were identified based onsequence similarity via the Rat Genome Database (Sup-plemental Table 2).

Of the genes with known biological functions, we iden-tified 33 genes involved in carbohydrate/lipid metabolismand fatty acid biosynthesis, whose expression was alteredin offspring of OW dams (Supplemental Table 2). Addi-tionally, we found suites of genes with known roles inelectron transport and cholesterol metabolism (Supple-

mental Table 2). We further used path-way analysis software (IPA) to identifycommon regulators of the altered genes.Two transcription factors, SREBP-1 andPPAR-�, were identified as critical nodesof regulation, consistent with increasedhepatic lipid accumulation (Fig. 1C).Correlation-based hierarchical cluster-ing of known SREBP-1 target genes in-volved in lipid biosynthesis is shown inFig. 1B. A uniform induction of 20SREBP-1-regulated genes, primarily in-volved in lipid biosynthesis, includingSREBP-1, was revealed. Consistent withincreased percent liver weight and lipo-genic gene expression, histological ex-amination of hematoxylin- and eosin-stained liver sections revealed enlargedhepatocytes and lipid accumulation (inOil Red O-stained sections), characteris-tic of hepatic steatosis (Fig. 1D).

We performed independent verifica-tion of 11 lipogenesis-related genesusing real-time RT-PCR (Fig. 2A). Tofurther understand the induction in li-pogenic genes, we also assessed expres-sion of PPAR-�2 (Fig. 2A). mRNA ex-pression of all transcripts identified viamicroarray analyses was confirmed viareal-time PCR. Most remarkably, ex-pression of SREBP-1 (2.1-fold), ATP-citrate lyase (3.6-fold), FASN (7-fold),adiponutrin (40-fold), and PPAR-�2(30-fold) was significantly induced (P �0.01) in livers from offspring of OWdams (Fig. 2A). Next we attempted to as-certain whether changes in SREBP-1

mRNAwere translated tohigherprotein levels. Immunoblotanalyses of hepatic nuclear extracts clearly showed �1.7-fold higher (P � 0.05) SREBP-1 protein levels in the OW-dam offspring compared with lean cohorts (Fig. 2, B andC). Greater nuclear SREBP-1 was also confirmed viaTransAM ELISA, which uses binding to an SREBP-1 bind-ing site followed by immunodetection. Consistent withimmunoblotting results, TransAM ELISA revealed ap-proximately 1.9-fold (P � 0.05) greater SREBP-1 proteinlevels in hepatic nuclear extracts from OW-dam offspring(Fig. 2D). Finally, we used ChIP assay to assess recruit-ment of SREBP-1 on binding sites on the FASN promoter.In addition to being induced transcriptionally in OW-damoffspring, FASN is recognized as a SREBP-1 target geneand has a well-characterized proximal promoter with de-

FIG. 2. A, Hepatic mRNA expression of genes from offspring of lean and OW dams at PND21(n � 8 per group). B–D, Gene expression was assessed via real-time RT-PCR. Hepatic SREBP-1in nuclear extracts using Western blots (n � 5 per group) (B and C) and TransAM ELISA(n � 8 per group) (D) from offspring lean and OW dams at PND21. E, ChIP analyses of SREBP-1 recruitment on FASN promoter. Statistical differences were determined using a Student’s ttest. *, P � 0.05. ADU, Arbitrary density units.



finedSREBP-1binding sites (25).Recruitmentof SREBP-1on the FASN promoter was increased approximately2-fold (P � 0.05) in OW-dam offspring relative to leancontrols, consistent with results from both gene expres-sion and nuclear protein assessments (Fig. 2E).

Insulin-responsive lipogenic proteins are inducedin offspring of OW dams

Because both serum insulin levels and hepatic expressionof lipogenic genes (downstream of insulin-responsiveSREBP-1) were elevated in offspring of OW dams, we ex-amined whether components of insulin signaling were al-tered. First, protein expression of key lipogenic enzymes,FASNandacetylcoenzymeAcarboxylase,weresignificantlyinduced (4- and 7-fold, respectively; Fig. 3, A and C), con-firming both gene expression and hepatic steatosis data. Nodifferences were observed in protein levels of IR-�, IRS-1, ortotal Akt levels (Fig. 3, A and C). However, tyrosine phos-phorylation of IR-� and the phosphorylation of Akt atThr308 were significantly increased (P � 0.05, Fig. 3, A–C) inliver of OW-dam offspring, consistent with modestly increasedinsulin signaling. We also observed significantly decreasedphosphorylation of ERK1/2 in OW offspring (Fig. 3, A and C).

Maternal OW decreases PPAR-�-AMPK signaling inoffspring liver

Examination of microarray data suggested that mRNAexpression of at least eight PPAR-�-regulated genes wasdecreased in livers of OW-dam offspring at PND21.Because PPAR-� is a key regulator of lipid catabolic pro-cesses, we further examined whether maternal OW pro-grammed decreased PPAR-� in offspring liver. Hierarchi-cal clustering of these genes is depicted in Fig. 4A. Usingreal-time PCR, we confirmed decreased gene expression ofat least six genes (Fig. 4B). Most genes were involved in�-hydroxylation or peroxisomal �-oxidation of fatty ac-ids. Importantly, gene expression of FGF21 and Cpt-1awas decreased approximately 5- and 2-fold, respectively(P � 0.05, Fig. 4B). Serum concentrations of FGF21 werealso significantly lower (P � 0.05) in offspring of OWdams (Fig. 4C). Mitochondrial Cpt-1a protein in OW-dam offspring was decreased to 30% of levels in lean-damoffspring, confirming gene expression results (Fig. 4D).Moreover, ChIP analyses revealed that recruitment ofPPAR-� to the FGF21 promoter was significantly lower inlivers of OW-dam offspring (P � 0.05, Fig. 4E). These datastrongly suggest that offspring of OW dams demonstratedecreased hepatic PPAR-� signaling.

AMPK, a critical regulator of fatty acid catabolic pro-cesses, also cooperates with PPAR-� signaling. We henceexamined phosphorylation status of AMPK in liver lysatesfrom lean- and OW-dam offspring. Although total AMPK

protein levels remained unchanged, phosphorylation ofAMPK (at Thr172) was decreased to 50% in OW-damoffspring (P � 0.05, Fig. 5, A and B). Circulating adi-ponectin has been implicated in regulating both AMPKand PPAR-� signaling. Moreover, recent studies have sug-gested that HMW forms of adiponectin may be importantin metabolic signaling (20, 26, 27). Serum concentrationsof total adiponectin were modestly lowered in OW-dam off-

FIG. 3. A, Expression of lipogenic enzymes and signaling proteins intotal lysates from livers of offspring from lean and OW dams at PND21by Western blotting (n � 6 per group). B, Immunoprecipitation (IP) ofphosphotyrosine in total liver lysates from offspring at PND21 (n � 3pools representing a total of eight animals per group). Immunoblotting(IB) was performed using anti-IR-� antibody. C, Densitometricquantitation of immunoblots from offspring at PND21. Statisticaldifferences were determined using a Student’s t test. *, P � 0.05.ADU, Arbitrary density units.



spring, albeit not significantly (Table 1). FPLC-based sepa-ration of serum was used to specifically quantitate HMWforms of circulating adiponectin (Fig. 5, C and D). Offspringfrom OW dams showed a 32% decrease in HMW adiponec-tin levels (P � 0.05) compared with offspring of lean dams atweaning (Fig. 5E). Adiponectin mRNA levels in retroperito-neal adipose tissues were unchanged, suggesting that mater-nal OW affects either packaging and/or secretion of adi-ponectin from adipocytes (Fig. 5F). Hepatic expression ofAdipoR1 mRNA was also significantly (P � 0.05) lower inoffspring of OW dams with modest nonsignificant decreaseobserved in AdipoR2 mRNA expression (Fig. 5F). Thesefindings collectively suggest that exposure to maternal OWdecreased adiponectin-AMPK-PPAR-� signaling along withseveral downstream targets involved in lipid catabolism.

Discussion

The risk of obesity in adulthood is subject to programmingbeginning at conception. Although several reports have

confirmed similar programming of offspring obesity risk,much remains to be known about the underlying mecha-nisms (28–35). We aimed at identifying critical endocrineand transcriptome changes before the development ofovert obesity in the offspring. Several novel findings areevident from the present studies. In the absence of changesin body weights at weaning, offspring of OW dams dem-onstrated 1) underlying endocrine abnormalities in sys-temic insulin, resistin, and adiponectin levels; 2) globalchanges in the hepatic transcriptome, revealing a repro-gramming of lipogenic and lipid degradative pathways;and 3) changes in critical signaling pathways regulatingnutrient utilization (AMPK-PPAR-�) and lipogenesis(SREBP-1) in the offspring. A schematic summarizing thechanges in OW-dam offspring is presented in Fig. 6.

We used TEN for its ability to overfeed rats in a con-trolled manner while maintaining dietary composition.This is a distinctive aspect of the present experimentaldesign. Using this model, we replicated many of the met-abolic and endocrine features of OW individuals in TEN-

FIG. 4. A, Correlation-based clustering of genes regulated by PPAR-� from the list of transcripts altered by maternal OW. B, mRNA expression ofPPAR-�-regulated genes in livers of offspring from lean and OW dams at PND21 (n � 8 per group). Gene expression was assessed via real-timeRT-PCR. C, Serum FGF21 levels determined by RIA in offspring of lean and OW dams at PND21 (n � 8 per group). D, Mitochondrial Cpt-1a proteinlevels and densitometric quantitation in liver of offspring at PND21 (n � 4 per group, each pool represents two separate animals). E, ChIP analyses showingPPAR-� recruitment on FGF21 promoter. Statistical differences were determined using a Student’s t test. *, P � 0.05. ADU, Arbitrary density units.



fed OW dams, including hyperinsulinemia, hyperleptine-mia, insulin resistance, and increased serum triglycerideand NEFA concentrations (12). Our data are consistentwith results from White et al. (31) suggesting that maternaladiposity is a key determinant of fetal programming. Al-though high-fat consumption certainly contributes to in-creased weight gain, obesity in the population at largeresults from a variety of meal and activity patterns. Theprecise control of gestational weight gain is another im-portant aspect of the experimental design. Because highgestational weight gain significantly increases the risk ofchildhood obesity (36), we matched weight gains in boththe lean and OW rats. Our data suggest that maternal OWeven in the absence of excessive weight gain has significantimpact on offspring metabolism at weaning. Finally, bylimiting the exposure of offspring to maternal OW spe-cifically to gestation, we excluded confounding variablessuch as changes in lactation efficiency and milk quality inOW dams. Hence, the differences between offspring of

lean and OW dams are direct consequences of program-ming events initiated in utero.

A central finding of our studies is the identification oftwo loci controlling fatty acid metabolism, SREBP-1 andPPAR-�, as potential targets of programming. SREBP-1 isexquisitely sensitive to circulating insulin and serves as acritical effector of the lipogenic arm of insulin (37–39).Importantly, our results highlight that not only is SREBP-1mRNA induced, but also a battery of (�20) downstreamtargets involved in lipid and cholesterol biosynthesis reg-ulated via SREBP-1 are coordinately increased. These aresupported by definitive data showing increased recruit-ment of SREBP-1 to one of its target promoters. Recently,using a mouse model of diet-induced obesity, Bruce et al.(28) also reported increased hepatic gene expression ofSREBP-1 and FASN in 15-wk-old offspring. Our resultscorroborate these findings and further suggest that lipo-genic gene expression occurs before onset of obesity in theoffspring of OW dams. Our data do not unequivocally

FIG. 5. A and B, Expression and densitometric quantitation of hepatic phosphorylated (Thr172) and total AMPK in total liver lysates from offspringof lean and OW dams at PND21. C and D, Analyses of adiponectin monomers by immunoblotting after FPLC-based separation of serum fromoffspring at PND21. Thirty fractions (0.215 ml) from over the retention volume of the complexes (7–14 ml) were collected. Aliquots of eachfraction were denatured and separated by SDS-PAGE. E, Percentage of HMW adiponectin in serum of offspring from lean or OW dams at PND21(n � 8 per group). F, mRNA expression of AdipoR1 and AdipoR2 in liver and adiponectin in retroperitoneal white adipose tissue in offspring atPND21 (n � 8 per group). Gene expression was assessed via real-time RT-PCR. Statistical differences were determined using Student’s t test.*, P � 0.05. ADU, Arbitrary density units.



address whether the observed hyperinsulinemia and in-creased hepatic insulin signaling underlie greater SREBP-1mRNA/protein and subsequent lipogenic gene expression.However, offspring of OW dams demonstrate modestlyhigher circulating insulin levels and increased postrecep-tor signaling of some aspects in the liver. Insulin signalingis orchestrated through a complex network of mediatorslinked to two main signaling pathways: the phosphatidyl-inositol 3-kinase-AKT pathway and the Ras-MAPK path-way activated via growth factor receptor-bound protein2/son of sevenless (Grb2/SOS). The former pathway is re-sponsible for most of the metabolic aspects of insulin,whereas the latter regulates mitogenic responses and dif-ferentiation (40, 41). Although generally considered dis-tinct, there is cross talk between these pathways via ERK-mediated negative regulation of phosphatidylinositol3-kinase through phosphorylation of IRS-1 at Ser636(42). Offspring of OW dams demonstrated increasedphosphorylation of AKT; however, phosphorylation ofERK1/2 was decreased, suggesting that specific aspects ofinsulin signaling may be targeted in the offspring. In-creases in serum leptin concentrations were observed inoffspring of OW dams, independent of changes in bodyadiposity. Because insulin is an important regulator ofleptin gene expression and secretion (43, 44), the increasein serum leptin may be the result of hyperinsulinemia ob-served inoffspringofOWdams.Althoughspeculativeat this

stage, it is also possible that higher leptin levels are a result ofearly leptin resistance in the offspring of OW dams.

Hepatic lipogenic signaling was enhanced in offspringof OW dams. However, we caution against an interpre-tation of systemic sensitization to insulin, because it ispossible that insulin signaling pathways in the skeletalmuscle may be inhibited in OW-dam offspring. Examplesof such divergent insulin signaling between skeletal muscleand other insulin-sensitive tissues, such as liver (and adiposetissue), have been reported. Mice with muscle-specific dele-tion of IR (MIRKO mice) develop mild hyperinsulinemiawithout hyperglycemia (45). Although insulin-mediatedglucose uptake in the muscle is decreased by 74%, uptakeof glucose in the adipose tissue is increased 3-fold in thesemice (45). Similarly, MIRKO mice demonstrate increasedhepatic glycogen synthesis and glucose uptake in the liver(46). Furthermore, studies in rats infused with insulinchronically using osmotic mini-pumps suggest that al-though insulin-induced glucose utilization is decreased inmuscles of insulinized rats, lipogenesis and glycogen syn-thesis in liver and white adipose tissues are increased (47,48). These studies demonstrate that mild hyperinsulin-emia systemically may be sufficient to drive hepatic ste-atosis and obesity if the muscle is not responsive to insulin.In fact, data from our own studies in older offspring (atPND130) clearly reveal significant insulin resistance in theoffspring of OW dams, despite increased lipogenic expres-

FIG. 6. Schematic summarizing changes in genes regulating lipid biosynthesis and oxidation in offspring liver at PND21. Genes represented inblack and gray boxes are up-regulated and down-regulated, respectively (either transcriptionally or via phosphorylation). Overall, genes involved inlipid biosynthesis and insulin signaling are up-regulated, and lipid oxidation regulating genes via PPAR-� and AMPK are down-regulated.



sion in liver and adipose tissues (12). Recent findings fromtwo models of maternal OW also suggest that skeletalmuscle insulin signaling is decreased in offspring in uteroand at weaning (33, 49). Furthermore, decreased circu-lating adiponectin and higher resistin levels are likely todecrease insulin sensitivity and glucose uptake in skeletalmuscles (26, 50). Hence, it appears that maternal OW mayselectively increase lipogenic signaling via insulin in liverand adipose tissues.

Gestational exposure to OW is likely to alter signalingin multiple tissues in the developing offspring, and changesin circulating levels of HMW adiponectin might be onesuch target. Decreases in HMW adiponectin levels oc-curred in the absence of changes in adiponectin mRNA inthe adipose tissue. The formation of higher-order com-plexes occurs in the adipocyte and is regulated mainly atthe level of secretion (51, 52). Hence, it is plausible thatgestational OW may influence mechanisms regulating adi-ponectin complex formation. Binding of adiponectin to itsreceptors (AdipoR1 or AdipoR2) increases their interac-tion with the adaptor protein APPL1 (adaptor protein, PHdomain, and leucine zipper containing 1), which promotesthe phosphorylation of AMPK (53). Activated AMPK me-diates its pleiotropic functions in target tissues, acting asan energy sensor, increasing fatty acid oxidation and in-sulin sensitivity, and inhibiting lipogenesis (26, 54). Sim-ilarly, the nuclear receptor PPAR-� plays critical roles inmobilizing energy during energy-deficient states (55, 56).AMPK cooperatively stimulates PPAR-�-dependent geneexpression especially in the liver to increase expression ofcritical components of the peroxisomal and mitochondrialfatty oxidation pathways (57, 58). Offspring of OW damsdisplayed a distinct down-regulation of both AMPK phos-phorylation and PPAR-�-dependent target genes such asCpt-1a and Cyp4A. Our results of lower hepatic AMPKphosphorylation are consistent with decreased HMW adi-ponectin levels and with previous results (33). In studiesemploying an overnourished sheep model of maternalobesity, Zhu and colleagues elegantly demonstrated thatphosphorylation of AMPK in offspring skeletal musclewas decreased in late gestation (33). The present data dem-onstrating decreased AMPK phosphorylation in the liverare in line with their findings and suggest a global decreasein AMPK signaling due to maternal obesity.

In the present studies, FGF21 was identified as a noveltarget influenced by maternal OW. FGF21 is a hepatichormone recently identified for its critical role to orches-trate integrative responses during fasting by promotinglipolysis in adipose tissue (59). Expression of FGF21 isregulated by PPAR-� (60, 61), and recent studies haveincreasingly recognized its function as a regulator of whole-body energy balance (62). Mice overexpressing FGF21 de-

velop resistance to diet-induced adiposity, and exogenousadministration of FGF21 to mice ameliorates both geneti-cally driven and diet-induced obesity, without decreasing ca-loric intake (59, 63). Taken together, it is appealing to spec-ulate that sustained decreases in FGF21 in offspring of OWdams might contribute to increased adiposity and adiposehypertrophy, as observed previously.

In conclusion, we have demonstrated that exposure tomaternal obesity, specifically during gestation, results inhepatic steatosis and extensive gene expression changes inthe liver of the offspring at weaning. Increased expressionof a number of genes regulating lipid biosynthesis appearsto be coordinated via the lipogenic transcription factorSREBP-1, associated with increased systemic insulin lev-els. Offspring born to OW dams also display lower circu-lating HMW adiponectin levels and decreased hepaticAMPK phosphorylation. Several downstream PPAR-�signaling targets including FGF21 are decreased in OW-dam offspring, consistent with hepatic steatosis and in-creased susceptibility to obesity. These results suggest tar-geting lipogenic pathways may be an effective strategy inmitigating increased adiposity.

Acknowledgments

We thank Matt Ferguson and the members of the ArkansasChildren’s Nutrition Center Animal Research Core Facility fortheir assistance with TEN. We thank Michael Blackburn, JamieBadeaux, Renee Till, Crystal Combs, and Michele Perry fortheir technical assistance. We gratefully acknowledge Dr. Vic-toria Esser (University of Texas Southwestern Medical Center,Dallas, TX) for providing the anti-Cpt-1a antibody.

Address all correspondence and requests for reprints to:Kartik Shankar, Ph.D., Arkansas Children’s Nutrition Center,15 Children’s Way, Slot 512-20B, Little Rock, Arkansas 72202.E-mail: [email protected].

This work was supported by National Institutes of HealthGrant R01-DK084225 (to K.S.) and U.S. Department of Agri-culture-ARS CRIS (Agricultural Research Service-Current Re-search Information System) 6251-51000-005-00D.

Disclosure Summary: The authors have nothing to disclose.

References

1. Yeh J, Shelton JA 2005 Increasing prepregnancy body mass index:analysis of trends and contributing variables. Am J Obstet Gynecol193:1994–1998

2. King JC 2006 Maternal obesity, metabolism, and pregnancy out-comes. Annu Rev Nutr 26:271–291

3. Castro LC, Avina RL 2002 Maternal obesity and pregnancy out-comes. Curr Opin Obstet Gynecol 14:601–606

4. Mei Z, Grummer-Strawn LM, Scanlon KS 2003 Does overweight ininfancy persist through the preschool years? An analysis of CDC



Pediatric Nutrition Surveillance System data. Soz Praventivmed 48:161–167

5. Sebire NJ, Jolly M, Harris JP, Wadsworth J, Joffe M, Beard RW,Regan L, Robinson S 2001 Maternal obesity and pregnancy out-come: a study of 287,213 pregnancies in London. Int J Obes RelatMetab Disord 25:1175–1182

6. Levin BE 2000 The obesity epidemic: metabolic imprinting on ge-netically susceptible neural circuits. Obes Res 8:342–347

7. Mingrone G, Manco M, Mora ME, Guidone C, Iaconelli A, GniuliD, Leccesi L, Chiellini C, Ghirlanda G 2008 Influence of maternalobesity on insulin sensitivity and secretion in offspring. DiabetesCare 31:1872–1876

8. Danielzik S, Langnase K, Mast M, Spethmann C, Muller MJ 2002Impact of parental BMI on the manifestation of overweight 5–7 yearold children. Eur J Nutr 41:132–138

9. Kral JG, Biron S, Simard S, Hould FS, Lebel S, Marceau S, MarceauP 2006 Large maternal weight loss from obesity surgery preventstransmission of obesity to children who were followed for 2 to 18years. Pediatrics 118:e1644–e1649

10. Smith J, Cianflone K, Biron S, Hould FS, Lebel S, Marceau S,Lescelleur O, Biertho L, Simard S, Kral JG, Marceau P 2009 Effectsof maternal surgical weight loss in mothers on intergenerationaltransmission of obesity. J Clin Endocrinol Metab 94:4275–4283

11. Villamor E, Cnattingius S 2006 Interpregnancy weight changeand risk of adverse pregnancy outcomes: a population-basedstudy. Lancet 368:1164 –1170

12. Shankar K, Harrell A, Liu X, Gilchrist JM, Ronis MJ, Badger TM2008 Maternal obesity at conception programs obesity in the off-spring. Am J Physiol Regul Integr Comp Physiol 294:R528–R538

13. Badger TM, Ronis MJ, Lumpkin CK, Valentine CR, Shahare M,Irby D, Huang J, Mercado C, Thomas P, Ingelman-Sundberg M1993 Effects of chronic ethanol on growth hormone secretion andhepatic cytochrome P450 isozymes of the rat. J Pharmacol Exp Ther264:438–447

14. Badger TM, Crouch J, Irby D, Hakkak R, Shahare M 1993 Episodicexcretion of ethanol during chronic intragastric ethanol infusion inthe male rat: continuous vs. cyclic ethanol and nutrient infusions.J Pharmacol Exp Ther 264:938–943

15. Baumgardner JN, Shankar K, Hennings L, Badger TM, Ronis MJ2008 A new model for nonalcoholic steatohepatitis in the rat uti-lizing total enteral nutrition to overfeed a high-polyunsaturated fatdiet. Am J Physiol Gastrointest Liver Physiol 294:G27–G38

16. Shankar K, Harrell A, Kang P, Singhal R, Ronis MJ, Badger TM2010 Carbohydrate-responsive gene expression in the adipose tissueof rats. Endocrinology 151:153–164

17. Korourian S, Hakkak R, Ronis MJ, Shelnutt SR, Waldron J,Ingelman-Sundberg M, Badger TM 1999 Diet and risk of ethanol-induced hepatotoxicity: carbohydrate-fat relationships in rats.Toxicol Sci 47:110–117

18. Shankar K, Hidestrand M, Liu X, Xiao R, Skinner CM, Simmen FA,Badger TM, Ronis MJ 2006 Physiologic and genomic analyses ofnutrition-ethanol interactions during gestation: Implications for fe-tal ethanol toxicity. Exp Biol Med (Maywood) 231:1379–1397

19. Shankar K, Liu X, Singhal R, Chen JR, Nagarajan S, Badger TM,Ronis MJ 2008 Chronic ethanol consumption leads to disruption ofvitamin D3 homeostasis associated with induction of renal 1,25dihydroxyvitamin D3-24-hydroxylase (CYP24A1). Endocrinology149:1748–1756

20. Schraw T, Wang ZV, Halberg N, Hawkins M, Scherer PE 2008Plasma adiponectin complexes have distinct biochemical character-istics. Endocrinology 149:2270–2282

21. Su Y, Shankar K, Simmen RC 2009 Early soy exposure via maternaldiet regulates rat mammary epithelial differentiation by paracrinesignaling from stromal adipocytes. J Nutr 139:945–951

22. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ,Scherf U, Speed TP 2003 Exploration, normalization, and summa-ries of high density oligonucleotide array probe level data. Biosta-tistics 4:249–264

23. Hochberg Y, Benjamini Y 1990 More powerful procedures for mul-tiple significance testing. Stat Med 9:811–818

24. Singhal R, Shankar K, Badger TM, Ronis MJ 2008 Estrogenic statusmodulates aryl hydrocarbon receptor-mediated hepatic gene ex-pression and carcinogenicity. Carcinogenesis 29:227–236

25. Wang Y, Jones Voy B, Urs S, Kim S, Soltani-Bejnood M, Quigley N,Heo YR, Standridge M, Andersen B, Dhar M, Joshi R, Wortman P,Taylor JW, Chun J, Leuze M, Claycombe K, Saxton AM, Moustaid-Moussa N 2004 The human fatty acid synthase gene and de novolipogenesis are coordinately regulated in human adipose tissue.J Nutr 134:1032–1038

26. Shetty S, Kusminski CM, Scherer PE 2009 Adiponectin in health anddisease: evaluation of adiponectin-targeted drug development strat-egies. Trends Pharmacol Sci 30:234–239

27. Wang Y, Lam KS, Yau MH, Xu A 2008 Post-translational modifi-cations of adiponectin: mechanisms and functional implications.Biochem J 409:623–633

28. Bruce KD, Cagampang FR, Argenton M, Zhang J, Ethirajan PL,Burdge GC, Bateman AC, Clough GF, Poston L, Hanson MA,McConnell JM, Byrne CD 2009 Maternal high-fat feeding primessteatohepatitis in adult mice offspring, involving mitochondrial dys-function and altered lipogenesis gene expression. Hepatology 50:1796–1808

29. Samuelsson AM, Matthews PA, Argenton M, Christie MR,McConnell JM, Jansen EH, Piersma AH, Ozanne SE, Twinn DF,Remacle C, Rowlerson A, Poston L, Taylor PD 2008 Diet-inducedobesity in female mice leads to offspring hyperphagia, adiposity,hypertension, and insulin resistance: a novel murine model of de-velopmental programming. Hypertension 51:383–392

30. Waterland RA, Travisano M, Tahiliani KG, Rached MT, Mirza S2008 Methyl donor supplementation prevents transgenerationalamplification of obesity. Int J Obes (Lond) 32:1373–1379

31. White CL, Purpera MN, Morrison CD 2009 Maternal obesity isnecessary for programming effect of high-fat diet on offspring. Am JPhysiol Regul Integr Comp Physiol 296:R1464–R1472

32. Du M, Yan X, Tong JF, Zhao J, Zhu MJ 2010 Maternal obesity,inflammation, and fetal skeletal muscle development. Biol Reprod82:4–12

33. Zhu MJ, Han B, Tong J, Ma C, Kimzey JM, Underwood KR, XiaoY, Hess BW, Ford SP, Nathanielsz PW, Du M 2008 AMP-activatedprotein kinase signalling pathways are down regulated and skeletalmuscle development impaired in fetuses of obese, over-nourishedsheep. J Physiol 586:2651–2664

34. Levin BE, Govek E 1998 Gestational obesity accentuates obesity inobesity-prone progeny. Am J Physiol 275:R1374–R1379

35. McCurdy CE, Bishop JM, Williams SM, Grayson BE, Smith MS,Friedman JE, Grove KL 2009 Maternal high-fat diet triggers lipo-toxicity in the fetal livers of nonhuman primates. J Clin Invest 119:323–335

36. Oken E, Taveras EM, Kleinman KP, Rich-Edwards JW, GillmanMW 2007 Gestational weight gain and child adiposity at age 3 years.Am J Obstet Gynecol 196:322–328

37. Foretz M, Guichard C, Ferre P, Foufelle F 1999 Sterol regulatoryelement binding protein-1c is a major mediator of insulin action onthe hepatic expression of glucokinase and lipogenesis-related genes.Proc Natl Acad Sci USA 96:12737–12742

38. Shimano H, Yahagi N, Amemiya-Kudo M, Hasty AH, Osuga J,Tamura Y, Shionoiri F, Iizuka Y, Ohashi K, Harada K, Gotoda T,Ishibashi S, Yamada N 1999 Sterol regulatory element-binding pro-tein-1 as a key transcription factor for nutritional induction of li-pogenic enzyme genes. J Biol Chem 274:35832–35839

39. Griffin MJ, Sul HS 2004 Insulin regulation of fatty acid synthasegene transcription: roles of USF and SREBP-1c. IUBMB Life 56:595–600

40. Taniguchi CM, Emanuelli B, Kahn CR 2006 Critical nodes in sig-nalling pathways: insights into insulin action. Nat Rev Mol Cell Biol7:85–96

41. Virkamaki A, Ueki K, Kahn CR 1999 Protein-protein interaction in



insulin signaling and the molecular mechanisms of insulin resis-tance. J Clin Invest 103:931–943

42. Bouzakri K, Roques M, Gual P, Espinosa S, Guebre-Egziabher F,Riou JP, Laville M, Le Marchand-Brustel Y, Tanti JF, Vidal H 2003Reduced activation of phosphatidylinositol-3 kinase and increasedserine 636 phosphorylation of insulin receptor substrate-1 in pri-mary culture of skeletal muscle cells from patients with type 2 dia-betes. Diabetes 52:1319–1325

43. Leroy P, Dessolin S, Villageois P, Moon BC, Friedman JM, AilhaudG, Dani C 1996 Expression of ob gene in adipose cells. Regulationby insulin. J Biol Chem 271:2365–2368

44. Bradley RL, Cheatham B 1999 Regulation of ob gene expressionand leptin secretion by insulin and dexamethasone in rat adipocytes.Diabetes 48:272–278

45. Kim JK, Michael MD, Previs SF, Peroni OD, Mauvais-Jarvis F,Neschen S, Kahn BB, Kahn CR, Shulman GI 2000 Redistribution ofsubstrates to adipose tissue promotes obesity in mice with selectiveinsulin resistance in muscle. J Clin Invest 105:1791–1797

46. Mauvais-Jarvis F, Virkamaki A, Michael MD, Winnay JN, ZismanA, Kulkarni RN, Kahn CR 2000 A model to explore the interactionbetween muscle insulin resistance and �-cell dysfunction in the de-velopment of type 2 diabetes. Diabetes 49:2126–2134

47. Cusin I, Terrettaz J, Rohner-Jeanrenaud F, Jeanrenaud B 1990 Met-abolic consequences of hyperinsulinaemia imposed on normal ratson glucose handling by white adipose tissue, muscles and liver. Bio-chem J 267:99–103

48. Cusin I, Rohner-Jeanrenaud F, Terrettaz J, Jeanrenaud B 1992 Hy-perinsulinemia and its impact on obesity and insulin resistance. IntJ Obes Relat Metab Disord 16(Suppl 4):S1–S11

49. Bayol SA, Simbi BH, Stickland NC 2005 A maternal cafeteria dietduring gestation and lactation promotes adiposity and impairs skel-etal muscle development and metabolism in rat offspring at wean-ing. J Physiol 567:951–961

50. Steppan CM, Lazar MA 2002 Resistin and obesity-associated in-sulin resistance. Trends Endocrinol Metab 13:18–23

51. Wang ZV, Schraw TD, Kim JY, Khan T, Rajala MW, Follenzi A,Scherer PE 2007 Secretion of the adipocyte-specific secretory pro-tein adiponectin critically depends on thiol-mediated protein reten-tion. Mol Cell Biol 27:3716–3731

52. Qiang L, Wang H, Farmer SR 2007 Adiponectin secretion is regu-lated by SIRT1 and the endoplasmic reticulum oxidoreductaseEro1-L�. Mol Cell Biol 27:4698–4707

53. Mao X, Kikani CK, Riojas RA, Langlais P, Wang L, Ramos FJ, Fang

Q, Christ-Roberts CY, Hong JY, Kim RY, Liu F, Dong LQ 2006APPL1 binds to adiponectin receptors and mediates adiponectinsignalling and function. Nat Cell Biol 8:516–523

54. Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S,Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K,Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y,Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T,Shimizu T, Nagai R, Kadowaki T 2003 Cloning of adiponectinreceptors that mediate antidiabetic metabolic effects. Nature 423:762–769

55. Kersten S, Seydoux J, Peters JM, Gonzalez FJ, Desvergne B, WahliW 1999 Peroxisome proliferator-activated receptor � mediates theadaptive response to fasting. J Clin Invest 103:1489–1498

56. Yoon M 2009 The role of PPAR� in lipid metabolism and obesity:focusing on the effects of estrogen on PPAR� actions. Pharmacol Res60:151–159

57. Yoon MJ, Lee GY, Chung JJ, Ahn YH, Hong SH, Kim JB 2006Adiponectin increases fatty acid oxidation in skeletal muscle cells bysequential activation of AMP-activated protein kinase, p38 mito-gen-activated protein kinase, and peroxisome proliferator-activatedreceptor �. Diabetes 55:2562–2570

58. Bronner M, Hertz R, Bar-Tana J 2004 Kinase-independent tran-scriptional co-activation of peroxisome proliferator-activated re-ceptor � by AMP-activated protein kinase. Biochem J 384:295–305

59. Kharitonenkov A, Shiyanova TL, Koester A, Ford AM, MicanovicR, Galbreath EJ, Sandusky GE, Hammond LJ, Moyers JS, OwensRA, Gromada J, Brozinick JT, Hawkins ED, Wroblewski VJ, Li DS,Mehrbod F, Jaskunas SR, Shanafelt AB 2005 FGF-21 as a novelmetabolic regulator. J Clin Invest 115:1627–1635

60. Badman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, Maratos-Flier E 2007 Hepatic fibroblast growth factor 21 is regulated byPPAR� and is a key mediator of hepatic lipid metabolism in ketoticstates. Cell Metab 5:426–437

61. Inagaki T, Dutchak P, Zhao G, Ding X, Gautron L, Parameswara V,Li Y, Goetz R, Mohammadi M, Esser V, Elmquist JK, Gerard RD,Burgess SC, Hammer RE, Mangelsdorf DJ, Kliewer SA 2007 En-docrine regulation of the fasting response by PPAR�-mediated in-duction of fibroblast growth factor 21. Cell Metab 5:415–425

62. Kliewer SA, Mangelsdorf DJ 2010 Fibroblast growth factor 21:from pharmacology to physiology. Am J Clin Nutr 91:254S–257S

63. Coskun T, Bina HA, Schneider MA, Dunbar JD, Hu CC, Chen Y,Moller DE, Kharitonenkov A 2008 Fibroblast growth factor 21corrects obesity in mice. Endocrinology 149:6018–6027



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