JOURNAL OF NEUROCHEMISTRY
| 2014 | 129 | 297–303
doi: 10.1111/jnc.12623
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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
Abstract Hypothalamic appetite regulators neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) are modulated by glucose. This study investigated how maternal obesity disturbs glucose regulation of NPY and POMC, and whether this deregulation is linked to abnormal hypothalamic glucose uptake-lactate conversion. As post-natal high-fat diet (HFD) can exaggerate the effects of maternal obesity, its additional impact was also investigated. Female Sprague Dawley rats were fed a HFD (20 kJ/g) to model maternal obesity. At weaning, male pups were fed chow or HFD. At 9 weeks, in vivo hypothalamic NPY and POMC mRNA responses to acute hyperglycemia were measured; while hypothalami were glucose challenged in vitro to assess glucose uptake-lactate release and related gene expression. Maternal obesity dampened in vivo hypothalamic NPY response to acute
hyperglycemia, and lowered in vitro hypothalamic glucose uptake and lactate release. When challenged with 20 mM glucose, hypothalamic glucose transporter 1, monocarboxylate transporters, lactate dehydrogenase-b, NPY and POMC mRNA expression were down-regulated in offspring exposed to maternal obesity. Post-natal HFD consumption reduced in vitro lactate release and monocarboxylate transporter 2 mRNA, but increased POMC mRNA levels when challenged with 20 mM glucose. Overall, maternal obesity produced stronger effects than post-natal HFD consumption to impair hypothalamic glucose metabolism. However, they both disturbed NPY response to hyperglycemia, potentially leading to hyperphagia. Keywords: GLUT1, lactate production, MCTs, mTOR, NPY, POMC. J. Neurochem. (2014) 129, 297–303.
Traditionally, glucose-responsive neurons are defined as those that increase their action potential frequency when ambient glucose is increased above resting levels (up to 10–20 mmol/ L), whereas glucose-sensitive neurons decrease their action potential 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 expressing anorexigenic pro-opiomelanocortin (POMC) are excited by glucose abundance (Muroya et al. 1999; Ibrahim et al. 2003; Stefater and Seeley 2010). Mammalian target of rapamycin (mTOR) can sense changes in ambient glucose and amino acid levels; a function that is critical for regulation of cellular activity. mTOR is colocalized in NPY- and POMCexpressing cells (Cota et al. 2006), suggesting mTOR may
play an important role in NPY and POMC neural glucose sensing and changes in their mRNA expression. Indeed, the short-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 of Technology, Sydney, NSW 2007, Australia. E-mail: hui.chen-1@uts. edu.au; Margaret J. Morris, Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia. E-mail: [email protected] Abbreviations 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, proopiomelanocortin; PVN, paraventricular nucleus; TG, triglyceride.
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reduce energy intake and body weight (Cota et al. 2006). High-fat diet (HFD) consumption can impair brain mTOR activation, leading to overeating in rodents (Um et al. 2006; Cota et al. 2006). We previously showed that maternal obesity is also linked to deregulation of both hypothalamic mTOR and NPY expression, contributing to hyperphagia and obesity in offspring (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 astrocytes, glucose is taken up and can be further metabolized anaerobically into lactate, which is transferred to neurons to supply fuel (Magistretti 2006), in addition to the direct glucose uptake by neurons from the extracellular space. In neurons, lactate is further metabolized into pyruvate through lactate dehydrogenase (LDH)b (Lam et al. 2007). This process can not only induce appetite suppression, but can also lower blood lipid and glucose levels through a mechanism involving the liver (Lam et al. 2008; Lam 2007; Lam et al. 2005). Therefore, altered central glucose metabolism may potentially lead to abnormal glucose regulation of NPY and POMC-expressing neurons, and thus appetite deregulation. We have previously shown that hypothalamic mTOR expression and activity are significantly down-regulated by maternal obesity (Chen et al. 2008, 2012). It is not known whether this is linked to abnormal hypothalamic glucose sensing and metabolic ability. This formed one of the rationales of this study. We have observed that a change in blood glucose level (hypoglycemia) led to hyper-activation of hypothalamic NPY expression in offspring from obese dams compared to offspring from lean mothers (Chen and Morris 2009). Studies have also reported impaired glucose sensing under low glucose conditions in both genetic and dietary obese rat models, 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 whether maternal obesity leads to impaired hyperglycemia-induced regulation of NPY and POMC (Sarbassov et al. 2005). We hypothesized that hypothalamic glucose metabolism was impaired by maternal obesity, which further leads to deregulated hypothalamic NPY and POMC expression in response to hyperglycemia. In addition, these alterations can be further exacerbated by post-natal HFD consumption. Therefore, we aimed to determine the effect of maternal obesity and post-natal HFD on the mRNA expression of hypothalamic NPY and POMC during hyperglycemia in vivo and under increased ambient glucose level in vitro.
Materials and method Animals All procedures were approved by the Animal Care and Ethics Committee 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 standard rodent chow (n = 10, 11 kJ/g, 14% fat, Gordon’s Specialty Stockfeeds, NSW, Australia) for 6 weeks prior to mating, throughout gestation and lactation (Chen et al. 2012). At post-natal day 1, litter sizes 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 other half were fed HFD. This yielded four groups, male offspring from chow-fed dams consuming chow (CC) or HFD (CH), and male offspring 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 at 8 weeks (n = 6–8 in each group) (Chen et al. 2009). After 5 h fasting, baseline glucose (T0) was measured using tail tip blood (Accu-Chekâ glucose meter, Roche Diagnostics, Nutley, NJ, USA). Blood glucose levels were measured at 15, 30, 60, and 90-min postglucose injection (2 g/kg, i.p.). Area under the curve (AUC) was calculated 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 with Pentothalâ (0.1 mg/g, i.p., Abbott Australasia Pty. Ltd., Lane Cove, NSW, Australia). Half of the rats within each group were injected with glucose (0.5 g/kg, i.v.), while the other half received saline as control (n = 5–6 under each condition). Cardiac blood was collected 10 min after injection and glucose levels were measured immediately. The hypothalamic areas containing Arc and paraventricular nucleus (PVN) were isolated (Chen et al. 2009) and stored at 80°C. Abdominal fat pads (retroperitoneal, mesenteric, and epididymal fat)) and liver were weighed. Another cohort (9-week old, n = 11–12) was anesthetized as above and killed in a non-fasting state. The whole hypothalamus was quickly sliced into 440 lm prisms (McIlwain tissue chopper, Mickle Laboratory Engineering Co. Ltd. Guildford, Surrey, UK.), then carefully placed into individual wells containing 1 mL of modified Krebs solution (containing 5 mM glucose and no lactate as previously described (Parton et al. 2007; Bertrand et al. 2011). The culture plate was incubated at 37°C/5% CO2 in an orbital incubator at 55 rpm (oscillation amplitude 20 mm) and the Krebs solution was replaced every 20 min. After 1 h equilibration, a basal 20 min sample collection was performed. Then, the tissues were challenged with 20 mM glucose for 20 min. Superfusates were collected, centrifuged and stored at 20°C. Brain tissue was kept for later determination of mRNA 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 radioimmunoassay (Linco, St. Charles, MI, USA). Glucose and lactate concentrations in the hypothalamus superfusate were measured using 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 the superfusate after 20 min incubation was calculated as hypothalamic
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glucose consumption in vitro. As the Krebs solution was lactate free, the lactate in the superfusate represented hypothalamic lactate release. Tissue total RNA was extracted using TRIZOL reagent (Invitrogen Australia Pty Ltd, VIC, Australia). First-strand cDNA was synthesized from total RNA using M-MLV Reverse Transcriptase, RNase H-, Point Mutant Kit (Promega, Madison, WI, USA). Manufacturer optimized and validated TaqManâ probe/primers (Applied Biosystem, Foster City, CA, USA) were used for quantitative real-time PCR (Eppendorf Realplex2, Hamburg, Germany). The target gene and housekeeping 18s rRNA probes were labeled with FAM and VIC, respectively. Gene expression was quantified in a single multiplexing reaction, by standardizing the gene of interest to 18s rRNA. The average value of the CC group was assigned as the calibrator, against which all other samples are expressed as fold difference. In both Arc and whole hypothalamus mRNA expression of NPY, POMC was measured. In addition, mRNA expression of mTOR, GLUT1, MCT2, MCT4, LDHB was measured in the whole hypothalamus. In the PVN, mRNA expression of NPY, POMC, and GLUT1 was measured. The results were expressed as mean SE. The difference between groups was analyzed using two-way analysis of variance (Statistica 10, StatSoft Inc., Tulsa, OK, USA). If there was significant interaction between maternal obesity and post-natal HFD consumption, the data were further analyzed by post hoc Fisher’s Least Significance Difference tests. Pearson correlation coefficient was used to assess the correlations between lactate concentration and mRNA expression of hypothalamic markers (Statistica 10). Differences between saline and glucose-injected rats within the same group were analyzed using Student’s t-test (Statistica 10). p < 0.05 was considered as significantly different between groups.
Results Growth and adiposity At 9 weeks of age, pups from obese mothers had significantly greater body weight, liver weight and fat mass (p < 0.05, maternal effect, n = 11–15, Table 1). Post-weaning HFD consumption also significantly increased body and liver weights, with fat mass more than doubled in rats compared with their chow-fed litter mates (p < 0.05, post-natal HFD
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effect, Table 1). There were significant interactions between maternal obesity and post-weaning HFD consumption on retroperitoneal, mesenteric, and epididymal fat mass (post hoc test p < 0.05, HH vs. CH, Table 1).
In vivo response to glucose injection At 8 weeks, baseline (T0) glucose levels during IPGTT were similar between groups (Fig. 1a). Maternal obesity exerted a more pronounced effect than post-weaning HFD consumption to increase blood glucose levels during IPGTT. Maternal obesity led to increased glucose levels from 15 to 90-min post-glucose injection, whereas post-natal HFD consumption increased blood glucose level only at 15 min and 30-min post-glucose injection (p < 0.05, n = 6–8, Fig. 1a). Significant maternal and post-natal HFD effects, as well as a significant interaction between these two factors were shown on AUC (p < 0.05, Fig. 1a), with the AUC of HH nearly doubled compared to HC (p < 0.05 post hoc test); however, it was not different between the two groups from the lean mothers (Fig. 1a). At 9 weeks, saline-injected rats showed similar glucose levels between groups; while both plasma insulin and TG levels 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 glucose concentrations in all groups (p < 0.05, n = 5–6, Fig. 1b), but only significantly increased plasma insulin levels in the HC rats (p < 0.05, glucose effect, Fig. 1c). In saline-injected rats, Arc NPY mRNA was significantly down-regulated by both 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 obesity and post-natal HFD on Arc NPY mRNA expression (p < 0.05), with the level in CH and HC significantly lower than CC (p < 0.05, post hoc tests, n = 5–6, Fig. 1e). POMC mRNA was only up-regulated by post-natal HFD consumption (Fig. 1f). There was no difference in Arc GLUT1 mRNA expression (data not shown), or in PVN NPY, POMC and GLUT1 mRNA expression (data not shown).
Table 1 Parameters of male offspring from chow and HFD-fed dams at 9 weeks CC n = 11 Body Weight (g)*# Liver (g)*# Retroperitoneal fat (g)*#a Mesenteric fat (g)*#a Epididymal fat (g)*#a
303 12.6 1.53 2.14 2.62
CH n = 11 12 0.8 0.18 0.17 0.19
408 19.3 6.28 5.62 8.72
HC n = 15 11 0.7 0.47# 0.37# 0.57#
349 14.7 2.19 2.71 3.28
HH n = 12 10 0.6 0.16 0.27 0.20
493 25.0 9.40 9.26 13.64
12 1.4 0.43*# 0.63*# 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.
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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), and triglyceride (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 with repeated 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 were analyzed 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 test significance, p < 0.05. t glucose injected versus saline-injected rats in the same group, p < 0.05. CC: offspring from chow-fed dams consuming 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.
In glucose-injected rats, circulating glucose and TG levels were higher in HFD-fed rats regardless of maternal group (p < 0.05, post-natal HFD effect, n = 5–6, Fig. 1d), whereas plasma insulin was higher in rats from obese dams (p < 0.05, n = 5–6, Fig. 1c). In CC, Arc NPY mRNA was reduced by more than 50% after glucose injection (glucose effect, p < 0.05, n = 6), whereas in the other groups NPY mRNA was not significantly different after glucose injection (Fig. 1e). Arc POMC (Fig. 1f, n = 5–6) and GLUT1 (not shown) mRNA levels were not different between saline and glucose injection in any group. mRNA expression of NPY, POMC, and GLUT1 were not different between saline and glucose injection in the PVN either (data not shown).
CH compared with CC group, and in HC compared to CC groups (p < 0.05, post hoc, n = 11–12, Fig. 2b). To further investigate the underlying mechanisms, the expression of fuel sensor mTOR, fuel transporters, and metabolic enzyme was examined. At 20 mM glucose, hypothalamic mRNA levels of NPY, POMC, mTOR, GLUT1, MCT2, MCT4, and LDHb were significantly lower in offspring from obese dams compared to those from lean dams (p < 0.05 maternal effect, n = 11–12, Fig. 2c, d). POMC mRNA levels were higher, whereas mTOR mRNA expression was lower in post-natal HFD-fed rats (p < 0.05 post-natal HFD effect, n = 11–12, Fig. 2c, d). There was a significant interaction between maternal obesity and postnatal HFD on MCT2 mRNA expression (p < 0.05), resulting in lower levels in HC versus CC according to post hoc test (p < 0.05, Fig. 2d). There was a significant positive correlation between hypothalamic mTOR mRNA expression and lactate 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).
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 significantly reduced by maternal obesity (p < 0.05, Fig. 2b). At 20 mM glucose, hypothalamic glucose uptake and lactate release were significantly reduced by maternal obesity (p < 0.05, Fig. 2a, b). There was a significant interaction between maternal and post-natal HFD consumption on lactate release (p < 0.05), which was significantly lower in
Discussion The major finding of this study is that maternal obesity resulted in reduced hypothalamic glucose metabolism; while hypothalamic NPY response to hyperglycemia was also
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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 hypothalamic mRNA expression of neuropeptide Y (NPY) and pro-opiomelanocortin (POMC, c), mammalian target of rapamycin (mTOR), glucose transporter 1 (GLUT1), Monocarboxylate transporter 2 (MCT2), MCT4 and lactic 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. # overall post-natal high-fat diet (HFD) effect, p < 0.05. c post hoc test, p < 0.05, compared to CC. CC: offspring from chow-fed dams consuming 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.
dampened by maternal obesity, likely to be driven by impaired glucose sensing. However, post-natal HFD consumption was not as potent as maternal obesity to impair the glucose regulation of hypothalamic appetite regulators. These findings may reveal additional hypothalamic mechanisms underlying the phenotype of early onset of obesity, hyperlipidemia, and hyperglycemia in offspring from obese dams (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 glucose concentrations (Belgardt et al. 2009). Previous research on the electrophysiological characterization of these neurons mainly focused on hypoglycaemia. Problems with offspring from obese mothers include hyperresponsiveness of NPY to fasting despite being obese which could lead to an inability to stop eating during nutrient abundance (Chen and Morris 2009; Chen et al. 2009). In this study, we measured the changes of NPY/POMC expression in response to hyperglycemia, which may directly impact feeding behavior. As expected, in control rats, increased glucose levels reduced NPY expression, which would be expected to decrease feeding during conditions of nutrient abundance. This response was impaired by both maternal obesity and postnatal 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 result from down-regulated fuel sensor mTOR in response to both maternal obesity and post-natal HFD consumption. This impairment could be because of reduced fuel supply from hypothalamic glucose-lactate metabolism (Stefater and Seeley 2010). POMC-expressing neurons are glucose-excited neurons and inhibit feeding upon satiation (Stefater and Seeley 2010). In a previous study, in vitro release of POMC derived a-melanocyte-stimulating hormone in hypothalami from normal mice was increased when ambient glucose was raised from 8 to 15 mM (Parton et al. 2007). This response was impaired by post-natal HFD, potentially contributing to hyperphagia (Parton et al. 2007). Here, we failed to observe any change in POMC mRNA in response to glucose injection in vivo. Timing might be a factor as previously a significant change was observed after 45 min incubation with high concentration of glucose (Parton et al. 2007). On the other hand, nervous activity reflected by firing rate may change well ahead of mRNA expression changes measured here (Song et al. 2001; Parton et al. 2007). Increased hypothalamic POMC in post-natal HFD-fed rats at baseline could represent an adaptation to counteract long-term overnutrition; whereas it was diminished post-glucose injection suggesting
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an abnormal response to hyperglycemia. In the absence of concurrent NPY down-regulation under the same condition, this could promote overfeeding in post-natal HFD-fed rats even upon satiation (Chen et al. 2009). Both glucose and lactate are energy sources for neurons (Song and Routh 2005). It has been suggested that brain extracellular glucose level is ~ 30% of the blood level (Cremer et al. 1981; Silver and Erecinska 1994; Burdakov et al. 2005; Routh 2010). Under anesthesia, blood glucose level was about 15 mM in saline-injected rats because of the inhibition of insulin by Pentothal, so the brain extracellular glucose level would be expected to have reached 5 mM in control rat hypothalamus (Brown et al. 2004; Silver and Erecinska 1994). Therefore, the 5 mM of glucose used as baseline level in vitro was considered to be close to the in vivo condition. Indeed, in vitro glucose levels higher than 5 mM have been suggested to be a physiologically relevant stimulus to study hyperglycemic conditions (Fioramonti et al. 2004). Our study directly measured for the first time hypothalamic glucose uptake and lactate release. Here, maternal obesity reduced hypothalamic glucose uptake at high glucose levels, which could be attributed to down-regulated hypothalamic GLUT1 (Vannucci 1994). The change in GLUT1 contradicts the observation by Fuente-Martin et al. where GLUT1 level was increased in weaning offspring from obese dams (Fuente-Martin et al. 2012). We can only postulate that this discrepancy may be age related and that after a transient over-expression of GLUT1 at weaning, levels drop during adulthood. GLUT1 is insulinindependent and the main glucose transporter in the brain (Carruthers et al. 2009). While there are also insulin-dependent glucose transporters expressed in the hypothalamus, their role seem to be less important in regulating glucose influx to the hypothalamus and maternal obesity has been shown to not result in hypothalamic insulin resistance (Chen et al. 2011). At 20 mM glucose level in vitro, hypothalami from postnatal HFD-fed rats showed reduced lactate release without changing glucose uptake, suggesting impaired glucoselactate conversion. As a fuel, lactate can also determine the activity of glucose-sensing neurons (Mobbs et al. 2001; Song and Routh 2005; Yang et al. 1999). It is of interest that NPY expression mirrored lactate release at 20 mM glucose, suggesting a possible link between NPY expression and local lactate levels. POMC followed the same pattern as glucose uptake, consistent with previous observation where Arc POMC activity is positively correlated with plasma glucose concentrations (Adam et al. 2008). Therefore, it is postulated here that low hypothalamic glucose uptake in offspring from obese dams may potentially limit the fuel supply and thus the activity of POMC-expressing neurons. Decreased NPY and POMC expression in offspring from obese dams was consistent with low hypothalamic glucose uptake and lactate release, suggesting that their expression is glucose-lactate dependent.
Under high ambient glucose conditions, hypothalamic glucose uptake was reduced by maternal obesity, which if it were recapitulated in vivo, would potentially result in decreased lactate production. Low lactate release could also be linked to reduced MCT2 and MCT4 levels, the predominant lactate transporters in neurons and astrocytes, respectively (Bergersen 2007). Astrocytic MCT4 exports lactate into the extracellular space, where MCT2 transports it into neurons (Bergersen 2007). Hypothalamic MCT expression has been shown to be positively correlated with glucose supply (Simpson et al. 2007); therefore, low MCTs in offspring of obese dams may result from local glucose shortage because of reduced uptake suggested by the in vitro experimental data here. Lactate is further converted into pyruvate by LDH (Lam et al. 2007). In this study, reduced LDHb expression because of maternal obesity may follow reduced hypothalamic glucose uptake and consequent lactate release. We acknowledge that further work is needed to examine whether hyperglycemia changes the protein levels and the translocation of these glucose and monocarboxylate transporters 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 brain glucose-lactate-pyruvate conversion leads to hyperlipidemia (Lam et al. 2007). Here, we failed to observe TG changes in the control rats, potentially because of the short duration of hyperglycemia (10 min). Hypothalamic glucose metabolism was impaired by both maternal obesity and post-natal HFD, whereas hyperlipidemia was only observed in post-natal HFD-fed rats. We postulate that increased fat in the diet induces hyperlipidemia more rapidly than maternal obesity. As such, hyperlipidemia because of maternal obesity was seen in 18-week-old offspring (Chen et al. 2009), but not at the 9 week time point used in this study. In conclusion, maternal obesity posed stronger effects than post-natal HFD consumption to impair hypothalamic glucose metabolism in offspring. However, they both disturbed NPY response to hyperglycemia, potentially leading to hyperphagia. Down-regulated glucose and monocarboxylate transporters and LDH may contribute to the deregulated hypothalamic glucose metabolism seen in response to maternal obesity, with the mechanism underlying post-natal HFD consumption yet to be determined.
Acknowledgements and Conflict of interest disclosure HC received Early Career Researcher grant (2008) from the Faculty of Medicine, University of New South Wales, and the Faculty of Science, University of Technology, Sydney. MJM received funding support from NH & MRC. We thank Dr Paul Bertrand (Department of Physiology, UNSW) for his facility and reagents to perform the release experiment. All experiments were conducted in compliance
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with the ARRIVE guidelines. There is no conflict of interest for all the authors.
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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
Abstract Hypothalamic appetite regulators neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) are modulated by glucose. This study investigated how maternal obesity disturbs glucose regulation of NPY and POMC, and whether this deregulation is linked to abnormal hypothalamic glucose uptake-lactate conversion. As post-natal high-fat diet (HFD) can exaggerate the effects of maternal obesity, its additional impact was also investigated. Female Sprague Dawley rats were fed a HFD (20 kJ/g) to model maternal obesity. At weaning, male pups were fed chow or HFD. At 9 weeks, in vivo hypothalamic NPY and POMC mRNA responses to acute hyperglycemia were measured; while hypothalami were glucose challenged in vitro to assess glucose uptake-lactate release and related gene expression. Maternal obesity dampened in vivo hypothalamic NPY response to acute
hyperglycemia, and lowered in vitro hypothalamic glucose uptake and lactate release. When challenged with 20 mM glucose, hypothalamic glucose transporter 1, monocarboxylate transporters, lactate dehydrogenase-b, NPY and POMC mRNA expression were down-regulated in offspring exposed to maternal obesity. Post-natal HFD consumption reduced in vitro lactate release and monocarboxylate transporter 2 mRNA, but increased POMC mRNA levels when challenged with 20 mM glucose. Overall, maternal obesity produced stronger effects than post-natal HFD consumption to impair hypothalamic glucose metabolism. However, they both disturbed NPY response to hyperglycemia, potentially leading to hyperphagia. Keywords: GLUT1, lactate production, MCTs, mTOR, NPY, POMC. J. Neurochem. (2014) 129, 297–303.
Traditionally, glucose-responsive neurons are defined as those that increase their action potential frequency when ambient glucose is increased above resting levels (up to 10–20 mmol/ L), whereas glucose-sensitive neurons decrease their action potential 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 expressing anorexigenic pro-opiomelanocortin (POMC) are excited by glucose abundance (Muroya et al. 1999; Ibrahim et al. 2003; Stefater and Seeley 2010). Mammalian target of rapamycin (mTOR) can sense changes in ambient glucose and amino acid levels; a function that is critical for regulation of cellular activity. mTOR is colocalized in NPY- and POMCexpressing cells (Cota et al. 2006), suggesting mTOR may
play an important role in NPY and POMC neural glucose sensing and changes in their mRNA expression. Indeed, the short-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 of Technology, Sydney, NSW 2007, Australia. E-mail: hui.chen-1@uts. edu.au; Margaret J. Morris, Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia. E-mail: [email protected] Abbreviations 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, proopiomelanocortin; PVN, paraventricular nucleus; TG, triglyceride.
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reduce energy intake and body weight (Cota et al. 2006). High-fat diet (HFD) consumption can impair brain mTOR activation, leading to overeating in rodents (Um et al. 2006; Cota et al. 2006). We previously showed that maternal obesity is also linked to deregulation of both hypothalamic mTOR and NPY expression, contributing to hyperphagia and obesity in offspring (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 astrocytes, glucose is taken up and can be further metabolized anaerobically into lactate, which is transferred to neurons to supply fuel (Magistretti 2006), in addition to the direct glucose uptake by neurons from the extracellular space. In neurons, lactate is further metabolized into pyruvate through lactate dehydrogenase (LDH)b (Lam et al. 2007). This process can not only induce appetite suppression, but can also lower blood lipid and glucose levels through a mechanism involving the liver (Lam et al. 2008; Lam 2007; Lam et al. 2005). Therefore, altered central glucose metabolism may potentially lead to abnormal glucose regulation of NPY and POMC-expressing neurons, and thus appetite deregulation. We have previously shown that hypothalamic mTOR expression and activity are significantly down-regulated by maternal obesity (Chen et al. 2008, 2012). It is not known whether this is linked to abnormal hypothalamic glucose sensing and metabolic ability. This formed one of the rationales of this study. We have observed that a change in blood glucose level (hypoglycemia) led to hyper-activation of hypothalamic NPY expression in offspring from obese dams compared to offspring from lean mothers (Chen and Morris 2009). Studies have also reported impaired glucose sensing under low glucose conditions in both genetic and dietary obese rat models, 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 whether maternal obesity leads to impaired hyperglycemia-induced regulation of NPY and POMC (Sarbassov et al. 2005). We hypothesized that hypothalamic glucose metabolism was impaired by maternal obesity, which further leads to deregulated hypothalamic NPY and POMC expression in response to hyperglycemia. In addition, these alterations can be further exacerbated by post-natal HFD consumption. Therefore, we aimed to determine the effect of maternal obesity and post-natal HFD on the mRNA expression of hypothalamic NPY and POMC during hyperglycemia in vivo and under increased ambient glucose level in vitro.
Materials and method Animals All procedures were approved by the Animal Care and Ethics Committee 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 standard rodent chow (n = 10, 11 kJ/g, 14% fat, Gordon’s Specialty Stockfeeds, NSW, Australia) for 6 weeks prior to mating, throughout gestation and lactation (Chen et al. 2012). At post-natal day 1, litter sizes 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 other half were fed HFD. This yielded four groups, male offspring from chow-fed dams consuming chow (CC) or HFD (CH), and male offspring 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 at 8 weeks (n = 6–8 in each group) (Chen et al. 2009). After 5 h fasting, baseline glucose (T0) was measured using tail tip blood (Accu-Chekâ glucose meter, Roche Diagnostics, Nutley, NJ, USA). Blood glucose levels were measured at 15, 30, 60, and 90-min postglucose injection (2 g/kg, i.p.). Area under the curve (AUC) was calculated 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 with Pentothalâ (0.1 mg/g, i.p., Abbott Australasia Pty. Ltd., Lane Cove, NSW, Australia). Half of the rats within each group were injected with glucose (0.5 g/kg, i.v.), while the other half received saline as control (n = 5–6 under each condition). Cardiac blood was collected 10 min after injection and glucose levels were measured immediately. The hypothalamic areas containing Arc and paraventricular nucleus (PVN) were isolated (Chen et al. 2009) and stored at 80°C. Abdominal fat pads (retroperitoneal, mesenteric, and epididymal fat)) and liver were weighed. Another cohort (9-week old, n = 11–12) was anesthetized as above and killed in a non-fasting state. The whole hypothalamus was quickly sliced into 440 lm prisms (McIlwain tissue chopper, Mickle Laboratory Engineering Co. Ltd. Guildford, Surrey, UK.), then carefully placed into individual wells containing 1 mL of modified Krebs solution (containing 5 mM glucose and no lactate as previously described (Parton et al. 2007; Bertrand et al. 2011). The culture plate was incubated at 37°C/5% CO2 in an orbital incubator at 55 rpm (oscillation amplitude 20 mm) and the Krebs solution was replaced every 20 min. After 1 h equilibration, a basal 20 min sample collection was performed. Then, the tissues were challenged with 20 mM glucose for 20 min. Superfusates were collected, centrifuged and stored at 20°C. Brain tissue was kept for later determination of mRNA 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 radioimmunoassay (Linco, St. Charles, MI, USA). Glucose and lactate concentrations in the hypothalamus superfusate were measured using 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 the superfusate after 20 min incubation was calculated as hypothalamic
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glucose consumption in vitro. As the Krebs solution was lactate free, the lactate in the superfusate represented hypothalamic lactate release. Tissue total RNA was extracted using TRIZOL reagent (Invitrogen Australia Pty Ltd, VIC, Australia). First-strand cDNA was synthesized from total RNA using M-MLV Reverse Transcriptase, RNase H-, Point Mutant Kit (Promega, Madison, WI, USA). Manufacturer optimized and validated TaqManâ probe/primers (Applied Biosystem, Foster City, CA, USA) were used for quantitative real-time PCR (Eppendorf Realplex2, Hamburg, Germany). The target gene and housekeeping 18s rRNA probes were labeled with FAM and VIC, respectively. Gene expression was quantified in a single multiplexing reaction, by standardizing the gene of interest to 18s rRNA. The average value of the CC group was assigned as the calibrator, against which all other samples are expressed as fold difference. In both Arc and whole hypothalamus mRNA expression of NPY, POMC was measured. In addition, mRNA expression of mTOR, GLUT1, MCT2, MCT4, LDHB was measured in the whole hypothalamus. In the PVN, mRNA expression of NPY, POMC, and GLUT1 was measured. The results were expressed as mean SE. The difference between groups was analyzed using two-way analysis of variance (Statistica 10, StatSoft Inc., Tulsa, OK, USA). If there was significant interaction between maternal obesity and post-natal HFD consumption, the data were further analyzed by post hoc Fisher’s Least Significance Difference tests. Pearson correlation coefficient was used to assess the correlations between lactate concentration and mRNA expression of hypothalamic markers (Statistica 10). Differences between saline and glucose-injected rats within the same group were analyzed using Student’s t-test (Statistica 10). p < 0.05 was considered as significantly different between groups.
Results Growth and adiposity At 9 weeks of age, pups from obese mothers had significantly greater body weight, liver weight and fat mass (p < 0.05, maternal effect, n = 11–15, Table 1). Post-weaning HFD consumption also significantly increased body and liver weights, with fat mass more than doubled in rats compared with their chow-fed litter mates (p < 0.05, post-natal HFD
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effect, Table 1). There were significant interactions between maternal obesity and post-weaning HFD consumption on retroperitoneal, mesenteric, and epididymal fat mass (post hoc test p < 0.05, HH vs. CH, Table 1).
In vivo response to glucose injection At 8 weeks, baseline (T0) glucose levels during IPGTT were similar between groups (Fig. 1a). Maternal obesity exerted a more pronounced effect than post-weaning HFD consumption to increase blood glucose levels during IPGTT. Maternal obesity led to increased glucose levels from 15 to 90-min post-glucose injection, whereas post-natal HFD consumption increased blood glucose level only at 15 min and 30-min post-glucose injection (p < 0.05, n = 6–8, Fig. 1a). Significant maternal and post-natal HFD effects, as well as a significant interaction between these two factors were shown on AUC (p < 0.05, Fig. 1a), with the AUC of HH nearly doubled compared to HC (p < 0.05 post hoc test); however, it was not different between the two groups from the lean mothers (Fig. 1a). At 9 weeks, saline-injected rats showed similar glucose levels between groups; while both plasma insulin and TG levels 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 glucose concentrations in all groups (p < 0.05, n = 5–6, Fig. 1b), but only significantly increased plasma insulin levels in the HC rats (p < 0.05, glucose effect, Fig. 1c). In saline-injected rats, Arc NPY mRNA was significantly down-regulated by both 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 obesity and post-natal HFD on Arc NPY mRNA expression (p < 0.05), with the level in CH and HC significantly lower than CC (p < 0.05, post hoc tests, n = 5–6, Fig. 1e). POMC mRNA was only up-regulated by post-natal HFD consumption (Fig. 1f). There was no difference in Arc GLUT1 mRNA expression (data not shown), or in PVN NPY, POMC and GLUT1 mRNA expression (data not shown).
Table 1 Parameters of male offspring from chow and HFD-fed dams at 9 weeks CC n = 11 Body Weight (g)*# Liver (g)*# Retroperitoneal fat (g)*#a Mesenteric fat (g)*#a Epididymal fat (g)*#a
303 12.6 1.53 2.14 2.62
CH n = 11 12 0.8 0.18 0.17 0.19
408 19.3 6.28 5.62 8.72
HC n = 15 11 0.7 0.47# 0.37# 0.57#
349 14.7 2.19 2.71 3.28
HH n = 12 10 0.6 0.16 0.27 0.20
493 25.0 9.40 9.26 13.64
12 1.4 0.43*# 0.63*# 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.
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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), and triglyceride (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 with repeated 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 were analyzed 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 test significance, p < 0.05. t glucose injected versus saline-injected rats in the same group, p < 0.05. CC: offspring from chow-fed dams consuming 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.
In glucose-injected rats, circulating glucose and TG levels were higher in HFD-fed rats regardless of maternal group (p < 0.05, post-natal HFD effect, n = 5–6, Fig. 1d), whereas plasma insulin was higher in rats from obese dams (p < 0.05, n = 5–6, Fig. 1c). In CC, Arc NPY mRNA was reduced by more than 50% after glucose injection (glucose effect, p < 0.05, n = 6), whereas in the other groups NPY mRNA was not significantly different after glucose injection (Fig. 1e). Arc POMC (Fig. 1f, n = 5–6) and GLUT1 (not shown) mRNA levels were not different between saline and glucose injection in any group. mRNA expression of NPY, POMC, and GLUT1 were not different between saline and glucose injection in the PVN either (data not shown).
CH compared with CC group, and in HC compared to CC groups (p < 0.05, post hoc, n = 11–12, Fig. 2b). To further investigate the underlying mechanisms, the expression of fuel sensor mTOR, fuel transporters, and metabolic enzyme was examined. At 20 mM glucose, hypothalamic mRNA levels of NPY, POMC, mTOR, GLUT1, MCT2, MCT4, and LDHb were significantly lower in offspring from obese dams compared to those from lean dams (p < 0.05 maternal effect, n = 11–12, Fig. 2c, d). POMC mRNA levels were higher, whereas mTOR mRNA expression was lower in post-natal HFD-fed rats (p < 0.05 post-natal HFD effect, n = 11–12, Fig. 2c, d). There was a significant interaction between maternal obesity and postnatal HFD on MCT2 mRNA expression (p < 0.05), resulting in lower levels in HC versus CC according to post hoc test (p < 0.05, Fig. 2d). There was a significant positive correlation between hypothalamic mTOR mRNA expression and lactate 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).
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 significantly reduced by maternal obesity (p < 0.05, Fig. 2b). At 20 mM glucose, hypothalamic glucose uptake and lactate release were significantly reduced by maternal obesity (p < 0.05, Fig. 2a, b). There was a significant interaction between maternal and post-natal HFD consumption on lactate release (p < 0.05), which was significantly lower in
Discussion The major finding of this study is that maternal obesity resulted in reduced hypothalamic glucose metabolism; while hypothalamic NPY response to hyperglycemia was also
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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 hypothalamic mRNA expression of neuropeptide Y (NPY) and pro-opiomelanocortin (POMC, c), mammalian target of rapamycin (mTOR), glucose transporter 1 (GLUT1), Monocarboxylate transporter 2 (MCT2), MCT4 and lactic 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. # overall post-natal high-fat diet (HFD) effect, p < 0.05. c post hoc test, p < 0.05, compared to CC. CC: offspring from chow-fed dams consuming 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.
dampened by maternal obesity, likely to be driven by impaired glucose sensing. However, post-natal HFD consumption was not as potent as maternal obesity to impair the glucose regulation of hypothalamic appetite regulators. These findings may reveal additional hypothalamic mechanisms underlying the phenotype of early onset of obesity, hyperlipidemia, and hyperglycemia in offspring from obese dams (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 glucose concentrations (Belgardt et al. 2009). Previous research on the electrophysiological characterization of these neurons mainly focused on hypoglycaemia. Problems with offspring from obese mothers include hyperresponsiveness of NPY to fasting despite being obese which could lead to an inability to stop eating during nutrient abundance (Chen and Morris 2009; Chen et al. 2009). In this study, we measured the changes of NPY/POMC expression in response to hyperglycemia, which may directly impact feeding behavior. As expected, in control rats, increased glucose levels reduced NPY expression, which would be expected to decrease feeding during conditions of nutrient abundance. This response was impaired by both maternal obesity and postnatal 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 result from down-regulated fuel sensor mTOR in response to both maternal obesity and post-natal HFD consumption. This impairment could be because of reduced fuel supply from hypothalamic glucose-lactate metabolism (Stefater and Seeley 2010). POMC-expressing neurons are glucose-excited neurons and inhibit feeding upon satiation (Stefater and Seeley 2010). In a previous study, in vitro release of POMC derived a-melanocyte-stimulating hormone in hypothalami from normal mice was increased when ambient glucose was raised from 8 to 15 mM (Parton et al. 2007). This response was impaired by post-natal HFD, potentially contributing to hyperphagia (Parton et al. 2007). Here, we failed to observe any change in POMC mRNA in response to glucose injection in vivo. Timing might be a factor as previously a significant change was observed after 45 min incubation with high concentration of glucose (Parton et al. 2007). On the other hand, nervous activity reflected by firing rate may change well ahead of mRNA expression changes measured here (Song et al. 2001; Parton et al. 2007). Increased hypothalamic POMC in post-natal HFD-fed rats at baseline could represent an adaptation to counteract long-term overnutrition; whereas it was diminished post-glucose injection suggesting
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an abnormal response to hyperglycemia. In the absence of concurrent NPY down-regulation under the same condition, this could promote overfeeding in post-natal HFD-fed rats even upon satiation (Chen et al. 2009). Both glucose and lactate are energy sources for neurons (Song and Routh 2005). It has been suggested that brain extracellular glucose level is ~ 30% of the blood level (Cremer et al. 1981; Silver and Erecinska 1994; Burdakov et al. 2005; Routh 2010). Under anesthesia, blood glucose level was about 15 mM in saline-injected rats because of the inhibition of insulin by Pentothal, so the brain extracellular glucose level would be expected to have reached 5 mM in control rat hypothalamus (Brown et al. 2004; Silver and Erecinska 1994). Therefore, the 5 mM of glucose used as baseline level in vitro was considered to be close to the in vivo condition. Indeed, in vitro glucose levels higher than 5 mM have been suggested to be a physiologically relevant stimulus to study hyperglycemic conditions (Fioramonti et al. 2004). Our study directly measured for the first time hypothalamic glucose uptake and lactate release. Here, maternal obesity reduced hypothalamic glucose uptake at high glucose levels, which could be attributed to down-regulated hypothalamic GLUT1 (Vannucci 1994). The change in GLUT1 contradicts the observation by Fuente-Martin et al. where GLUT1 level was increased in weaning offspring from obese dams (Fuente-Martin et al. 2012). We can only postulate that this discrepancy may be age related and that after a transient over-expression of GLUT1 at weaning, levels drop during adulthood. GLUT1 is insulinindependent and the main glucose transporter in the brain (Carruthers et al. 2009). While there are also insulin-dependent glucose transporters expressed in the hypothalamus, their role seem to be less important in regulating glucose influx to the hypothalamus and maternal obesity has been shown to not result in hypothalamic insulin resistance (Chen et al. 2011). At 20 mM glucose level in vitro, hypothalami from postnatal HFD-fed rats showed reduced lactate release without changing glucose uptake, suggesting impaired glucoselactate conversion. As a fuel, lactate can also determine the activity of glucose-sensing neurons (Mobbs et al. 2001; Song and Routh 2005; Yang et al. 1999). It is of interest that NPY expression mirrored lactate release at 20 mM glucose, suggesting a possible link between NPY expression and local lactate levels. POMC followed the same pattern as glucose uptake, consistent with previous observation where Arc POMC activity is positively correlated with plasma glucose concentrations (Adam et al. 2008). Therefore, it is postulated here that low hypothalamic glucose uptake in offspring from obese dams may potentially limit the fuel supply and thus the activity of POMC-expressing neurons. Decreased NPY and POMC expression in offspring from obese dams was consistent with low hypothalamic glucose uptake and lactate release, suggesting that their expression is glucose-lactate dependent.
Under high ambient glucose conditions, hypothalamic glucose uptake was reduced by maternal obesity, which if it were recapitulated in vivo, would potentially result in decreased lactate production. Low lactate release could also be linked to reduced MCT2 and MCT4 levels, the predominant lactate transporters in neurons and astrocytes, respectively (Bergersen 2007). Astrocytic MCT4 exports lactate into the extracellular space, where MCT2 transports it into neurons (Bergersen 2007). Hypothalamic MCT expression has been shown to be positively correlated with glucose supply (Simpson et al. 2007); therefore, low MCTs in offspring of obese dams may result from local glucose shortage because of reduced uptake suggested by the in vitro experimental data here. Lactate is further converted into pyruvate by LDH (Lam et al. 2007). In this study, reduced LDHb expression because of maternal obesity may follow reduced hypothalamic glucose uptake and consequent lactate release. We acknowledge that further work is needed to examine whether hyperglycemia changes the protein levels and the translocation of these glucose and monocarboxylate transporters 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 brain glucose-lactate-pyruvate conversion leads to hyperlipidemia (Lam et al. 2007). Here, we failed to observe TG changes in the control rats, potentially because of the short duration of hyperglycemia (10 min). Hypothalamic glucose metabolism was impaired by both maternal obesity and post-natal HFD, whereas hyperlipidemia was only observed in post-natal HFD-fed rats. We postulate that increased fat in the diet induces hyperlipidemia more rapidly than maternal obesity. As such, hyperlipidemia because of maternal obesity was seen in 18-week-old offspring (Chen et al. 2009), but not at the 9 week time point used in this study. In conclusion, maternal obesity posed stronger effects than post-natal HFD consumption to impair hypothalamic glucose metabolism in offspring. However, they both disturbed NPY response to hyperglycemia, potentially leading to hyperphagia. Down-regulated glucose and monocarboxylate transporters and LDH may contribute to the deregulated hypothalamic glucose metabolism seen in response to maternal obesity, with the mechanism underlying post-natal HFD consumption yet to be determined.
Acknowledgements and Conflict of interest disclosure HC received Early Career Researcher grant (2008) from the Faculty of Medicine, University of New South Wales, and the Faculty of Science, University of Technology, Sydney. MJM received funding support from NH & MRC. We thank Dr Paul Bertrand (Department of Physiology, UNSW) for his facility and reagents to perform the release experiment. All experiments were conducted in compliance
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with the ARRIVE guidelines. There is no conflict of interest for all the authors.
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