Download - Maternal obesity impairs brain glucose metabolism and neural response to hyperglycemia in male rat offspring

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*School of Medical and Molecular Biosciences, Faculty of Science, Centre for Health Technology,

University of Technology, Sydney, NSW, Australia

†Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney,

NSW, Australia

‡Inflammation and Infection Research, School of Medical Sciences, University of New South Wales,

Sydney, NSW, Australia

AbstractHypothalamic appetite regulators neuropeptide Y (NPY) andpro-opiomelanocortin (POMC) are modulated by glucose.This study investigated how maternal obesity disturbs glu-cose regulation of NPY and POMC, and whether thisderegulation is linked to abnormal hypothalamic glucoseuptake-lactate conversion. As post-natal high-fat diet (HFD)can exaggerate the effects of maternal obesity, its additionalimpact was also investigated. Female Sprague Dawley ratswere fed a HFD (20 kJ/g) to model maternal obesity. Atweaning, male pups were fed chow or HFD. At 9 weeks, invivo hypothalamic NPY and POMC mRNA responses toacute hyperglycemia were measured; while hypothalami wereglucose challenged in vitro to assess glucose uptake-lactaterelease and related gene expression. Maternal obesitydampened in vivo hypothalamic NPY response to acute

hyperglycemia, and lowered in vitro hypothalamic glucoseuptake and lactate release. When challenged with 20 mMglucose, hypothalamic glucose transporter 1, monocarboxy-late transporters, lactate dehydrogenase-b, NPY and POMCmRNA expression were down-regulated in offspring exposedto maternal obesity. Post-natal HFD consumption reducedin vitro lactate release and monocarboxylate transporter 2mRNA, but increased POMC mRNA levels when challengedwith 20 mM glucose. Overall, maternal obesity producedstronger effects than post-natal HFD consumption to impairhypothalamic glucose metabolism. However, they both dis-turbed NPY response to hyperglycemia, potentially leading tohyperphagia.Keywords: GLUT1, lactate production, MCTs, mTOR, NPY,POMC.J. Neurochem. (2014) 129, 297–303.

Traditionally, glucose-responsive neurons are defined as thosethat increase their action potential frequency when ambientglucose is increased above resting levels (up to 10–20 mmol/L), whereas glucose-sensitive neurons decrease their actionpotential frequency under the same conditions (Song et al.2001). In this regard, in the hypothalamic arcuate nucleus(Arc), neurons expressing orexigenic neuropeptide Y (NPY)are inhibited by rising glucose levels, while neurons express-ing anorexigenic pro-opiomelanocortin (POMC) are excitedby glucose abundance (Muroya et al. 1999; Ibrahim et al.2003; Stefater and Seeley 2010). Mammalian target ofrapamycin (mTOR) can sense changes in ambient glucoseand amino acid levels; a function that is critical for regulationof cellular activity. mTOR is colocalized in NPY- and POMC-expressing cells (Cota et al. 2006), suggesting mTOR may

play an important role in NPY and POMC neural glucosesensing and changes in their mRNA expression. Indeed, theshort-term activation of mTOR can inhibit NPY expression to

Received April 3, 2013; revised manuscript received November 6, 2013;accepted November 21, 2013.Address correspondence and reprint requests to Hui Chen, School of

Medical and Molecular Biosciences, Faculty of Science, University ofTechnology, Sydney, NSW 2007, Australia. E-mail: [email protected]; Margaret J. Morris, Department of Pharmacology, School ofMedical Sciences, University of New South Wales, Sydney, NSW 2052,Australia. E-mail: [email protected] used: Arc, arcuate nucleus; GLUT, glucose transporter;

HFD, high-fat diet; IPGTT, intraperitoneal glucose tolerance test; LDH,lactate dehydrogenase; MCT, monocarboxylate transporter; mTOR,mammalian target of rapamycin; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; PVN, paraventricular nucleus; TG, triglyceride.

© 2013 International Society for Neurochemistry, J. Neurochem. (2014) 129, 297--303 297

JOURNAL OF NEUROCHEMISTRY | 2014 | 129 | 297–303 doi: 10.1111/jnc.12623

reduce energy intake and body weight (Cota et al. 2006).High-fat diet (HFD) consumption can impair brain mTORactivation, leading to overeating in rodents (Um et al. 2006;Cota et al. 2006).We previously showed that maternal obesityis also linked to deregulation of both hypothalamic mTOR andNPY expression, contributing to hyperphagia and obesity inoffspring (Chen and Morris 2009; Chen et al. 2012).It has been suggested that brain glucose sensing activity by

mTOR relies on the ATP generated from glucose–lactate–pyruvate conversion (Stefater and Seeley 2010). In astro-cytes, glucose is taken up and can be further metabolizedanaerobically into lactate, which is transferred to neurons tosupply fuel (Magistretti 2006), in addition to the directglucose uptake by neurons from the extracellular space. Inneurons, lactate is further metabolized into pyruvate throughlactate dehydrogenase (LDH)b (Lam et al. 2007). Thisprocess can not only induce appetite suppression, but canalso lower blood lipid and glucose levels through amechanism involving the liver (Lam et al. 2008; Lam2007; Lam et al. 2005). Therefore, altered central glucosemetabolism may potentially lead to abnormal glucoseregulation of NPY and POMC-expressing neurons, and thusappetite deregulation. We have previously shown thathypothalamic mTOR expression and activity are significantlydown-regulated by maternal obesity (Chen et al. 2008,2012). It is not known whether this is linked to abnormalhypothalamic glucose sensing and metabolic ability. Thisformed one of the rationales of this study.We have observed that a change in blood glucose level

(hypoglycemia) led to hyper-activation of hypothalamicNPY expression in offspring from obese dams compared tooffspring from lean mothers (Chen and Morris 2009). Studieshave also reported impaired glucose sensing under lowglucose conditions in both genetic and dietary obese ratmodels, as well as in rat models of maternal overnutrition(Fuente-Martin et al. 2012; Song et al. 2001; Parton et al.2007; Canabal et al. 2007). However, it is unknown whethermaternal obesity leads to impaired hyperglycemia-inducedregulation of NPY and POMC (Sarbassov et al. 2005). Wehypothesized that hypothalamic glucose metabolism wasimpaired by maternal obesity, which further leads toderegulated hypothalamic NPY and POMC expression inresponse to hyperglycemia. In addition, these alterations canbe further exacerbated by post-natal HFD consumption.Therefore, we aimed to determine the effect of maternalobesity and post-natal HFD on the mRNA expression ofhypothalamic NPY and POMC during hyperglycemia in vivoand under increased ambient glucose level in vitro.

Materials and method

Animals

All procedures were approved by the Animal Care and EthicsCommittee of the University of New South Wales. Female

Sprague–Dawley rats (8 weeks, Animal Resource Centre, Yanderra,WA, Australia) were assigned to two groups, one fed a pelleted HFD(n = 15, 20 kJ/g, 43% fat; SF03-020, Specialty Feeds, Glen Forrest,WA, Australia) to induce obesity, and a control group fed standardrodent chow (n = 10, 11 kJ/g, 14% fat, Gordon’s Specialty Stock-feeds, NSW, Australia) for 6 weeks prior to mating, throughoutgestation and lactation (Chen et al. 2012). At post-natal day 1, littersizes were adjusted to 10–12 pups/litter (sex 1 : 1). At 20 days,within each litter half of the male pups were fed chow and the otherhalf were fed HFD. This yielded four groups, male offspring fromchow-fed dams consuming chow (CC) or HFD (CH), and maleoffspring from HFD-fed dams consuming chow (HC) or HFD (HH).There were 2–3 pups from the same litter within each group.Intraperitoneal glucose tolerance test (IPGTT) was performed at8 weeks (n = 6–8 in each group) (Chen et al. 2009). After 5 hfasting, baseline glucose (T0) was measured using tail tip blood(Accu-Chek� glucose meter, Roche Diagnostics, Nutley, NJ, USA).Blood glucose levels were measured at 15, 30, 60, and 90-min post-glucose injection (2 g/kg, i.p.). Area under the curve (AUC) wascalculated for each rat using the equation 0.5 9 (GT0 + GT15)9 15 min + 0.5 9 (GT15 + GT30) 9 15 min + 0.5 9 (GT30 +GT60) 9 30 min + 0.5 9 (GT60 + GT90) 9 30 min.

Sample collection and analysis

At 9 weeks, offspring were fasted for 5 h then anesthetized withPentothal� (0.1 mg/g, i.p., Abbott Australasia Pty. Ltd., Lane Cove,NSW, Australia). Half of the rats within each group were injectedwith glucose (0.5 g/kg, i.v.), while the other half received saline ascontrol (n = 5–6 under each condition). Cardiac blood was collected10 min after injection and glucose levels were measured immedi-ately. The hypothalamic areas containing Arc and paraventricularnucleus (PVN) were isolated (Chen et al. 2009) and stored at�80°C. Abdominal fat pads (retroperitoneal, mesenteric, andepididymal fat)) and liver were weighed.

Another cohort (9-week old, n = 11–12) was anesthetized as aboveand killed in a non-fasting state. Thewhole hypothalamuswas quicklysliced into 440 lm prisms (McIlwain tissue chopper, Mickle Labo-ratory Engineering Co. Ltd. Guildford, Surrey, UK.), then carefullyplaced into individual wells containing 1 mL of modified Krebssolution (containing 5 mM glucose and no lactate as previouslydescribed (Parton et al. 2007; Bertrand et al. 2011). The culture platewas incubated at 37°C/5% CO2 in an orbital incubator at 55 rpm(oscillation amplitude 20 mm) and the Krebs solution was replacedevery 20 min. After 1 h equilibration, a basal 20 min samplecollection was performed. Then, the tissues were challenged with20 mM glucose for 20 min. Superfusates were collected, centrifugedand stored at �20°C. Brain tissue was kept for later determination ofmRNA expression of NPY, POMC, mTOR, Glucose transporter(GLUT)1, monocarboxylate transporter (MCT)2 and 4, and LDHb.

Plasma triglyceride (TG) was measured using glycerol standard(Sigma, St. Louis, MO, USA) and TG reagent (Roche Diagnostics)(Chen et al. 2008); plasma insulin was measured using radioim-munoassay (Linco, St. Charles, MI, USA). Glucose and lactateconcentrations in the hypothalamus superfusate were measuredusing an electrode based glucose analyzer (EML105-analyzer,Radiometer Medical A/S, Copenhagen, Denmark) (Simar et al.2012). The difference in glucose between Krebs solution and thesuperfusate after 20 min incubation was calculated as hypothalamic

© 2013 International Society for Neurochemistry, J. Neurochem. (2014) 129, 297--303

298 H. Chen et al.

glucose consumption in vitro. As the Krebs solution was lactate free,the lactate in the superfusate represented hypothalamic lactaterelease.

Tissue total RNA was extracted using TRIZOL reagent (Invitro-gen Australia Pty Ltd, VIC, Australia). First-strand cDNA wassynthesized from total RNA using M-MLV Reverse Transcriptase,RNase H-, Point Mutant Kit (Promega, Madison, WI, USA).Manufacturer optimized and validated TaqMan� probe/primers(Applied Biosystem, Foster City, CA, USA) were used forquantitative real-time PCR (Eppendorf Realplex2, Hamburg, Ger-many). The target gene and housekeeping 18s rRNA probes werelabeled with FAM and VIC, respectively. Gene expression wasquantified in a single multiplexing reaction, by standardizing thegene of interest to 18s rRNA. The average value of the CC groupwas assigned as the calibrator, against which all other samples areexpressed as fold difference. In both Arc and whole hypothalamusmRNA expression of NPY, POMC was measured. In addition,mRNA expression of mTOR, GLUT1, MCT2, MCT4, LDHB wasmeasured in the whole hypothalamus. In the PVN, mRNAexpression of NPY, POMC, and GLUT1 was measured.

The results were expressed as mean � SE. The differencebetween groups was analyzed using two-way analysis of variance(Statistica 10, StatSoft Inc., Tulsa, OK, USA). If there wassignificant interaction between maternal obesity and post-natalHFD consumption, the data were further analyzed by post hocFisher’s Least Significance Difference tests. Pearson correlationcoefficient was used to assess the correlations between lactateconcentration and mRNA expression of hypothalamic markers(Statistica 10). Differences between saline and glucose-injected ratswithin the same group were analyzed using Student’s t-test(Statistica 10). p < 0.05 was considered as significantly differentbetween groups.

Results

Growth and adiposity

At 9 weeks of age, pups from obese mothers had significantlygreater body weight, liver weight and fat mass (p < 0.05,maternal effect, n = 11–15, Table 1). Post-weaning HFDconsumption also significantly increased body and liverweights, with fat mass more than doubled in rats comparedwith their chow-fed litter mates (p < 0.05, post-natal HFD

effect, Table 1). There were significant interactions betweenmaternal obesity and post-weaning HFD consumption onretroperitoneal, mesenteric, and epididymal fat mass (post hoctest p < 0.05, HH vs. CH, Table 1).

In vivo response to glucose injectionAt 8 weeks, baseline (T0) glucose levels during IPGTT weresimilar between groups (Fig. 1a). Maternal obesity exerted amore pronounced effect than post-weaning HFD consump-tion to increase blood glucose levels during IPGTT. Maternalobesity led to increased glucose levels from 15 to 90-minpost-glucose injection, whereas post-natal HFD consumptionincreased blood glucose level only at 15 min and 30-minpost-glucose injection (p < 0.05, n = 6–8, Fig. 1a). Signif-icant maternal and post-natal HFD effects, as well as asignificant interaction between these two factors were shownon AUC (p < 0.05, Fig. 1a), with the AUC of HH nearlydoubled compared to HC (p < 0.05 post hoc test); however,it was not different between the two groups from the leanmothers (Fig. 1a).At 9 weeks, saline-injected rats showed similar glucose

levels between groups; while both plasma insulin and TGlevels were significantly higher in post-natal HFD-fed rats(p < 0.05, post-natal HFD effect, n = 5–6, Fig. 1c, d).Glucose injection significantly increased blood glucoseconcentrations in all groups (p < 0.05, n = 5–6, Fig. 1b),but only significantly increased plasma insulin levels in theHC rats (p < 0.05, glucose effect, Fig. 1c). In saline-injectedrats, Arc NPY mRNA was significantly down-regulated byboth maternal obesity and post-weaning HFD consumption(p < 0.05, maternal and post-weaning HFD effects, Fig. 1e).There was a significant interaction between maternal obesityand post-natal HFD on Arc NPY mRNA expression(p < 0.05), with the level in CH and HC significantly lowerthan CC (p < 0.05, post hoc tests, n = 5–6, Fig. 1e). POMCmRNA was only up-regulated by post-natal HFD consump-tion (Fig. 1f). There was no difference in Arc GLUT1mRNA expression (data not shown), or in PVN NPY, POMCand GLUT1 mRNA expression (data not shown).

Table 1 Parameters of male offspring from chow and HFD-fed dams at 9 weeks

CCn = 11

CHn = 11

HCn = 15

HHn = 12

Body Weight (g)*# 303 � 12 408 � 11 349 � 10 493 � 12Liver (g)*# 12.6 � 0.8 19.3 � 0.7 14.7 � 0.6 25.0 � 1.4Retroperitoneal fat (g)*#a 1.53 � 0.18 6.28 � 0.47# 2.19 � 0.16 9.40 � 0.43*#

Mesenteric fat (g)*#a 2.14 � 0.17 5.62 � 0.37# 2.71 � 0.27 9.26 � 0.63*#

Epididymal fat (g)*#a 2.62 � 0.19 8.72 � 0.57# 3.28 � 0.20 13.64 � 0.80*#

Data are expressed as mean � SEM. ap < 0.05, significant interaction between the maternal and post-natal HFD effect. *p < 0.05, maternal effect.#p < 0.05, post-natal HFD effect. * and # next to the numbers indicate post hoc test significance p < 0.05.Rp: retroperitoneal.


Brain glucose metabolism and neural response 299

In glucose-injected rats, circulating glucose and TG levelswere higher in HFD-fed rats regardless of maternal group(p < 0.05, post-natal HFD effect, n = 5–6, Fig. 1d), whereasplasma insulin was higher in rats from obese dams (p < 0.05,n = 5–6, Fig. 1c). In CC, Arc NPY mRNA was reduced bymore than 50% after glucose injection (glucose effect,p < 0.05, n = 6), whereas in the other groups NPY mRNAwas not significantly different after glucose injection(Fig. 1e). Arc POMC (Fig. 1f, n = 5–6) and GLUT1 (notshown) mRNA levels were not different between saline andglucose injection in any group. mRNA expression of NPY,POMC, and GLUT1 were not different between saline andglucose injection in the PVN either (data not shown).

In vitro hypothalamic glucose metabolism and response to

increased glucose

When incubated in Krebs solution containing 5 mM glucose,hypothalamic glucose uptake was similar between groups(Fig. 2a, n = 11–12); however, lactate release was signifi-cantly reduced by maternal obesity (p < 0.05, Fig. 2b). At20 mM glucose, hypothalamic glucose uptake and lactaterelease were significantly reduced by maternal obesity(p < 0.05, Fig. 2a, b). There was a significant interactionbetween maternal and post-natal HFD consumption onlactate release (p < 0.05), which was significantly lower in

CH compared with CC group, and in HC compared to CCgroups (p < 0.05, post hoc, n = 11–12, Fig. 2b).To further investigate the underlying mechanisms, the

expression of fuel sensor mTOR, fuel transporters, andmetabolic enzyme was examined. At 20 mM glucose,hypothalamic mRNA levels of NPY, POMC, mTOR,GLUT1, MCT2, MCT4, and LDHb were significantly lowerin offspring from obese dams compared to those from leandams (p < 0.05 maternal effect, n = 11–12, Fig. 2c, d).POMC mRNA levels were higher, whereas mTOR mRNAexpression was lower in post-natal HFD-fed rats (p < 0.05post-natal HFD effect, n = 11–12, Fig. 2c, d). There was asignificant interaction between maternal obesity and post-natal HFD on MCT2 mRNA expression (p < 0.05), resultingin lower levels in HC versus CC according to post hoc test(p < 0.05, Fig. 2d). There was a significant positive corre-lation between hypothalamic mTOR mRNA expression andlactate release at the 20 mM glucose concentration (r = 0.37,p < 0.05, n = 37) and LDHb (r = 0.42, p < 0.05, n = 38).

Discussion

The major finding of this study is that maternal obesityresulted in reduced hypothalamic glucose metabolism; whilehypothalamic NPY response to hyperglycemia was also

(a) (b) (c)

(d) (e) (f)

Fig. 1 (a) IP glucose tolerance test (IPGTT) and Area under the curve(AUC) at 8 weeks (n = 6-8). Plasma glucose (b), insulin (c), andtriglyceride (TG) (d) levels, and mRNA levels of neuropeptide Y (NPY,e) and proopiomelanocortin (POMC, f) in the arcuate nucleus (Arc)

10 min after saline (open bars) and glucose injection (solid bars)(n = 5–6) in vivo. Glucose levels in (a) were analyzed by ANOVA withrepeated measures followed by a post hoc LSD test. Two-way ANOVA

followed by a post hoc LSD test was used to analyze data in (A AUG,

b–e). Data on the same group after saline and glucose injection wereanalyzed by Student’s t-test. * overall maternal effect, p < 0.05. #overall post-natal high fat diet (HFD) effect, p < 0.05. c post hoc testsignificance, p < 0.05. t glucose injected versus saline-injected rats in

the same group, p < 0.05. CC: offspring from chow-fed damsconsuming chow; CH: offspring from chow-fed dams consumingHFD; HC: offspring from HFD-fed dams consuming chow; HH:

offspring from HFD-fed dams consuming HFD.


300 H. Chen et al.

dampened by maternal obesity, likely to be driven byimpaired glucose sensing. However, post-natal HFD con-sumption was not as potent as maternal obesity to impair theglucose regulation of hypothalamic appetite regulators.These findings may reveal additional hypothalamic mecha-nisms underlying the phenotype of early onset of obesity,hyperlipidemia, and hyperglycemia in offspring from obesedams (Chen et al. 2008, 2009; Chen and Morris 2009).The firing of Arc neurons including POMC and NPY

neurons is known to respond to changes in ambient glucoseconcentrations (Belgardt et al. 2009). Previous research onthe electrophysiological characterization of these neuronsmainly focused on hypoglycaemia. Problems with offspringfrom obese mothers include hyperresponsiveness of NPY tofasting despite being obese which could lead to an inabilityto stop eating during nutrient abundance (Chen and Morris2009; Chen et al. 2009). In this study, we measured thechanges of NPY/POMC expression in response to hypergly-cemia, which may directly impact feeding behavior. Asexpected, in control rats, increased glucose levels reducedNPY expression, which would be expected to decreasefeeding during conditions of nutrient abundance. Thisresponse was impaired by both maternal obesity and post-natal HFD consumption, but only in the Arc where the

majority of NPY-expressing cells related to feeding reside.This lack of responsiveness to hyperglycemia could resultfrom down-regulated fuel sensor mTOR in response to bothmaternal obesity and post-natal HFD consumption. Thisimpairment could be because of reduced fuel supply fromhypothalamic glucose-lactate metabolism (Stefater and See-ley 2010). POMC-expressing neurons are glucose-excitedneurons and inhibit feeding upon satiation (Stefater andSeeley 2010). In a previous study, in vitro release of POMCderived a-melanocyte-stimulating hormone in hypothalamifrom normal mice was increased when ambient glucose wasraised from 8 to 15 mM (Parton et al. 2007). This responsewas impaired by post-natal HFD, potentially contributing tohyperphagia (Parton et al. 2007). Here, we failed to observeany change in POMC mRNA in response to glucose injectionin vivo. Timing might be a factor as previously a significantchange was observed after 45 min incubation with highconcentration of glucose (Parton et al. 2007). On the otherhand, nervous activity reflected by firing rate may changewell ahead of mRNA expression changes measured here(Song et al. 2001; Parton et al. 2007). Increased hypotha-lamic POMC in post-natal HFD-fed rats at baseline couldrepresent an adaptation to counteract long-term overnutrition;whereas it was diminished post-glucose injection suggesting

(a)

(d)

(b) (c)

Fig. 2 In vitro hypothalamic glucose consumption (a) and lactate

release (b) under 5 mM (open bars) and 20 mM (checked bars)glucose concentrations. At 20 mM glucose in vitro hypothalamicmRNA expression of neuropeptide Y (NPY) and pro-opiomelanocortin(POMC, c), mammalian target of rapamycin (mTOR), glucose trans-

porter 1 (GLUT1), Monocarboxylate transporter 2 (MCT2), MCT4 andlactic dehydrogenase b (LDHb) (D, n = 12). Results are expressed as

mean � SEM. Two-way ANOVA followed by a post hoc LSD test was

used for data analysis. * overall maternal effect, p < 0.05. # overallpost-natal high-fat diet (HFD) effect, p < 0.05. c post hoc test,p < 0.05, compared to CC. CC: offspring from chow-fed damsconsuming chow; CH: offspring from chow-fed dams consuming

HFD; HC: offspring from HFD-fed dams consuming chow; HH:offspring from HFD-fed dams consuming HFD.



an abnormal response to hyperglycemia. In the absence ofconcurrent NPY down-regulation under the same condition,this could promote overfeeding in post-natal HFD-fed ratseven upon satiation (Chen et al. 2009).Both glucose and lactate are energy sources for neurons

(Song and Routh 2005). It has been suggested that brainextracellular glucose level is ~ 30% of the blood level (Cremeret al. 1981; Silver and Erecinska 1994; Burdakov et al. 2005;Routh 2010). Under anesthesia, blood glucose level was about15 mM in saline-injected rats because of the inhibition ofinsulin by Pentothal, so the brain extracellular glucose levelwould be expected to have reached 5 mM in control rathypothalamus (Brown et al. 2004; Silver andErecinska 1994).Therefore, the 5 mM of glucose used as baseline level in vitrowas considered to be close to the in vivo condition. Indeed,in vitro glucose levels higher than 5 mM have been suggestedto be a physiologically relevant stimulus to study hypergly-cemic conditions (Fioramonti et al. 2004). Our study directlymeasured for the first time hypothalamic glucose uptake andlactate release. Here, maternal obesity reduced hypothalamicglucose uptake at high glucose levels, which could beattributed to down-regulated hypothalamic GLUT1 (Vannucci1994). The change in GLUT1 contradicts the observation byFuente-Martin et al. where GLUT1 level was increased inweaning offspring from obese dams (Fuente-Martin et al.2012). We can only postulate that this discrepancy may be agerelated and that after a transient over-expression of GLUT1 atweaning, levels drop during adulthood. GLUT1 is insulin-independent and the main glucose transporter in the brain(Carruthers et al. 2009). While there are also insulin-depen-dent glucose transporters expressed in the hypothalamus, theirrole seem to be less important in regulating glucose influx tothe hypothalamus and maternal obesity has been shown to notresult in hypothalamic insulin resistance (Chen et al. 2011).At 20 mM glucose level in vitro, hypothalami from post-

natal HFD-fed rats showed reduced lactate release withoutchanging glucose uptake, suggesting impaired glucose-lactate conversion. As a fuel, lactate can also determine theactivity of glucose-sensing neurons (Mobbs et al. 2001;Song and Routh 2005; Yang et al. 1999). It is of interest thatNPY expression mirrored lactate release at 20 mM glucose,suggesting a possible link between NPY expression and locallactate levels. POMC followed the same pattern as glucoseuptake, consistent with previous observation where ArcPOMC activity is positively correlated with plasma glucoseconcentrations (Adam et al. 2008). Therefore, it is postulatedhere that low hypothalamic glucose uptake in offspring fromobese dams may potentially limit the fuel supply and thus theactivity of POMC-expressing neurons. Decreased NPY andPOMC expression in offspring from obese dams wasconsistent with low hypothalamic glucose uptake and lactaterelease, suggesting that their expression is glucose-lactatedependent.

Under high ambient glucose conditions, hypothalamicglucose uptake was reduced by maternal obesity, which if itwere recapitulated in vivo, would potentially result indecreased lactate production. Low lactate release could alsobe linked to reduced MCT2 and MCT4 levels, the predom-inant lactate transporters in neurons and astrocytes, respec-tively (Bergersen 2007). Astrocytic MCT4 exports lactateinto the extracellular space, where MCT2 transports it intoneurons (Bergersen 2007). Hypothalamic MCT expressionhas been shown to be positively correlated with glucosesupply (Simpson et al. 2007); therefore, low MCTs inoffspring of obese dams may result from local glucoseshortage because of reduced uptake suggested by the in vitroexperimental data here. Lactate is further converted intopyruvate by LDH (Lam et al. 2007). In this study, reducedLDHb expression because of maternal obesity may followreduced hypothalamic glucose uptake and consequent lactaterelease. We acknowledge that further work is needed toexamine whether hyperglycemia changes the protein levelsand the translocation of these glucose and monocarboxylatetransporters on different cell types in the hypothalamus.Glucose infusion was shown to lead to reduced blood TG

levels within 60 min in normal rats, whereas impaired brainglucose-lactate-pyruvate conversion leads to hyperlipidemia(Lam et al. 2007). Here, we failed to observe TG changes inthe control rats, potentially because of the short duration ofhyperglycemia (10 min). Hypothalamic glucose metabolismwas impaired by both maternal obesity and post-natal HFD,whereas hyperlipidemia was only observed in post-natalHFD-fed rats. We postulate that increased fat in the dietinduces hyperlipidemia more rapidly than maternal obesity.As such, hyperlipidemia because of maternal obesity wasseen in 18-week-old offspring (Chen et al. 2009), but not atthe 9 week time point used in this study.In conclusion, maternal obesity posed stronger effects than

post-natal HFD consumption to impair hypothalamic glucosemetabolism in offspring. However, they both disturbed NPYresponse to hyperglycemia, potentially leading to hyperpha-gia. Down-regulated glucose and monocarboxylate trans-porters and LDH may contribute to the deregulatedhypothalamic glucose metabolism seen in response tomaternal obesity, with the mechanism underlying post-natalHFD consumption yet to be determined.

Acknowledgements and Conflict of interestdisclosure

HC received Early Career Researcher grant (2008) from the Facultyof Medicine, University of New South Wales, and the Faculty ofScience, University of Technology, Sydney. MJM received fundingsupport from NH & MRC. We thank Dr Paul Bertrand (Departmentof Physiology, UNSW) for his facility and reagents to perform therelease experiment. All experiments were conducted in compliance


302 H. Chen et al.

with the ARRIVE guidelines. There is no conflict of interest for allthe authors.

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