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Recently there has been a considerable rise in the frequency of metabolic diseases, such as obesity, due to changes in lifestyle and resultant imbalances between energy intake and expenditure. Whey proteins are considered as potentially important components of a dietary solution to the obesity problem. However, the roles of individual whey proteins in energy balance remain poorly understood. This study investigated the effects of a high-fat diet (HFD) containing α-lactalbumin (LAB), a specific whey protein, or the non-whey protein casein (CAS), on energy balance, nutrient transporters expression and enteric microbial populations. C57BL/6J mice (n 8) were given an HFD containing either 20 % CAS or LAB as protein sources or a low-fat diet containing CAS for 10 weeks. HFD-LAB-fed mice showed a significant increase in cumulative energy intake (P=0·043), without differences in body weight, energy expenditure, locomotor activity, RER or subcutaneous and epididymal white adipose tissue weight. HFD-LAB intake led to a decrease in the expression of glut2 in the ileum (P=0·05) and in the fatty acid transporter cd36 (PLactobacillus, Parabacteroides and Bifidobacterium were present in significantly higher proportions in the HFD-LAB group. These data indicate a possible functional relationship between gut microbiota, intestinal nutrient transporters and energy balance, with no impact on weight gain.

Whey protein isolate (WPI) is considered a dietary solution to obesity. However, the exact mechanism of WPI action is still poorly understood but is probably connected to its beneficial effect on energy balance, adiposity, and metabolism. More recently its ability to modulate the gut microbiota has received increasing attention. Here, we used a microbiota depletion, by antibiotic cocktail (ABX) administration, to investigate if the gut microbiota mediates the physiological and metabolic changes observed during high‐fat diet (HFD)‐WPI consumption. C57BL/6J mice received a HFD containing WPI (HFD‐WPI) or the control non‐whey milk protein casein (HFD‐CAS) for 5 or 10 weeks. HFD‐fed mice supplemented with WPI showed reduced body weight gain, adiposity, Ob gene expression level in the epidydimal adipose tissue (eWAT) and plasma leptin relative to HFD‐CAS‐fed mice, after 5‐ or 10‐weeks intervention both with or without ABX treatment. Following 10‐weeks intervention, ABX and WPI had an additive effect in lowering adiposity and leptin availability. HFD‐WPI‐fed mice showed a decrease in the expression of genes encoding pro‐inflammatory markers (MCP‐1, TNFα and CD68) within the ileum and eWAT, compared to HFD‐CAS‐fed mice, without showing alterations following microbiota depletion. Additionally, WPI supplementation decreased HFD‐induced intestinal permeability disruption in the distal ileum; an effect that was reversed by chronic ABX treatment. In summary, WPI reverses the effects of HFD on metabolic and physiological functions through mainly microbiota‐independent mechanisms. Moreover, we demonstrate a protective effect of WPI on HFD‐induced inflammation and ileal permeability disruption, with the latter being reversed by gut microbiota depletion., In this study, we provided new insights on the anti‐obesity effect of whey protein consumption. In this instance, we investigated the role of the gut microbiota by using an antibiotic treatment‐based depletion approach. Moreover, we showed novel findings on the effect of a high‐fat diet containing whey protein on inflammation, intestinal permeability and metabolites profile.

We investigated how protein quantity (10%-30%) and quality (casein and whey) interact with dietary fat (20%-55%) to affect metabolic health in adult mice. Although dietary fat was the main driver of body weight gain and individual tissue weight, high (30%) casein intake accentuated and high whey intake reduced the negative metabolic aspects of high fat. Jejunum and liver transcriptomics revealed increased intestinal permeability, low-grade inflammation, altered lipid metabolism, and liver dysfunction in casein-fed but not whey-fed animals. These differential effects were accompanied by altered gut size and microbial functions related to amino acid degradation and lipid metabolism. Fecal microbiota transfer confirmed that the casein microbiota increases and the whey microbiota impedes weight gain. These data show that the effects of dietary fat on weight gain and tissue partitioning are further influenced by the quantity and quality of the associated protein, primarily via effects on the microbiota.

The growth of the intestine requires energy, which is known to be met by catabolism of ingested nutrients. Paradoxically, during whole body energy deficit including calorie restriction, the intestine grows in size. To understand how and why this happens, we reviewed data from several animal models of energetic challenge. These were bariatric surgery, cold exposure, lactation, dietary whey protein intake and calorie restriction. Notably, these challenges all reduced the adipose tissue mass, altered hypothalamic neuropeptide expression and increased intestinal size. Based on these data, we propose that the loss of energy in the adipose tissue promotes the growth of the intestine via a signalling mechanism involving the hypothalamus. We discuss possible candidates in this pathway including data showing a correlative change in intestinal (ileal) expression of the cyclin D1 gene with adipose tissue mass, adipose derived-hormone leptin and hypothalamic expression of leptin receptor and the pro-opiomelanocortin gene. The ability of the intestine to grow in size during depletion of energy stores provides a mechanism to maximize assimilation of ingested energy and in turn sustain critical functions of tissues important for survival.

Saturated fatty acid (SFA) high-fat diets (HFDs) enhance interleukin (IL)-1β–mediated adipose inflammation and insulin resistance. However, the mechanisms by which different fatty acids regulate IL-1β and the subsequent effects on adipose tissue biology and insulin sensitivity in vivo remain elusive. We hypothesized that the replacement of SFA for monounsaturated fatty acid (MUFA) in HFDs would reduce pro-IL-1β priming in adipose tissue and attenuate insulin resistance via MUFA-driven AMPK activation. MUFA-HFD–fed mice displayed improved insulin sensitivity coincident with reduced pro-IL-1β priming, attenuated adipose IL-1β secretion, and sustained adipose AMPK activation compared with SFA-HFD–fed mice. Furthermore, MUFA-HFD–fed mice displayed hyperplastic adipose tissue, with enhanced adipogenic potential of the stromal vascular fraction and improved insulin sensitivity. In vitro, we demonstrated that the MUFA oleic acid can impede ATP-induced IL-1β secretion from lipopolysaccharide- and SFA-primed cells in an AMPK-dependent manner. Conversely, in a regression study, switching from SFA- to MUFA-HFD failed to reverse insulin resistance but improved fasting plasma insulin levels. In humans, high-SFA consumers, but not high-MUFA consumers, displayed reduced insulin sensitivity with elevated pycard-1 and caspase-1 expression in adipose tissue. These novel findings suggest that dietary MUFA can attenuate IL-1β–mediated insulin resistance and adipose dysfunction despite obesity via the preservation of AMPK activity.

The present study examined the underlying mechanisms by which whey protein isolate (WPI) affects energy balance. C57BL/6J mice were fed a diet containing 10 % energy from fat, 70 % energy from carbohydrate (35 % energy from sucrose) and 20 % energy from casein or WPI for 15 weeks. Mice fed with WPI had reduced weight gain, cumulative energy intake and dark-phase VO2 compared with casein-fed mice (PPWnt5a) (PFzd4) (PRor2) and LDL receptor-related protein 5 (Lrp5). In the ileum, WPI increased the mRNA expression of Wnt5a (PFzd4 (P= 0·094), with no change in the expression of Ror2 and Lrp5. These genes were unresponsive in the duodenum. Among the nutrient-responsive genes, WPI specifically reduced ileal mRNA expression of peptide YY (PPP= 0·05), with a trend towards a decreased expression of Na–glucose co-transporter 1 (P= 0·07). The effects of WPI on gastrointestinal Wnt signalling may explain how this protein affects gastrointestinal structure and function and, in turn, energy intake and balance.

Different dietary fat and energy subtypes have an impact on both the metabolic health and the intestinal microbiota population of the host. The present study assessed the impact of dietary fat quality, with a focus on dietary fatty acid compositions of varying saturation, on the metabolic health status and the intestinal microbiota composition of the host. C57BL/6J mice (n9–10 mice per group) were fed high-fat (HF) diets containing either (1) palm oil, (2) olive oil, (3) safflower oil or (4) flaxseed/fish oil for 16 weeks and compared with mice fed low-fat (LF) diets supplemented with either high maize starch or high sucrose. Tissue fatty acid compositions were assessed by GLC, and the impact of the diet on host intestinal microbiota populations was investigated using high-throughput 16S rRNA sequencing. Compositional sequencing analysis revealed that dietary palm oil supplementation resulted in significantly lower populations of Bacteroidetes at the phylum level compared with dietary olive oil supplementation (PBacteroidaceaecompared with dietary supplementation of palm oil, flaxseed/fish oil and high sucrose (PPBifidobacteriumat the genus level compared with the LF-high-maize starch diet (P

The intake of whey protein isolate (WPI) is known to reduce high-fat diet (HFD)-induced body-weight gain and adiposity. However, the molecular mechanisms are not fully understood. To this end, we fed C57BL/6J mice for 8 weeks with diets containing 10 % energy as fat (low-fat diet, LFD) or 45 % energy as fat (HFD) enriched with either 20 % energy as casein (LFD and HFD) or WPI (high-fat WPI). Metabolic parameters and the hypothalamic and epididymal adipose tissue expression of energy balance-related genes were investigated. The HFD increased fat mass and plasma leptin levels and decreased the dark-phase energy intake, meal number, RER, and metabolic (VO2and heat) and locomotor activities compared with the LFD. The HFD increased the hypothalamic tissue mRNA expression of the leptin receptor, insulin receptor (INSR) and carnitine palmitoyltransferase 1b (CPT1b). The HFD also reduced the adipose tissue mRNA expression ofGLUT4andINSR. In contrast, WPI reduced fat mass, normalised dark-phase energy intake and increased meal size in HFD-fed mice. The dietary protein did not have an impact on plasma leptin, insulin, glucose or glucagon-like peptide 1 levels, but increased plasma TAG levels in HFD-fed mice. At a cellular level, WPI significantly reduced the HFD-associated increase in the hypothalamic tissue mRNA expression of the leptin receptor,INSRandCPT1b. Also, WPI prevented the HFD-induced reduction in the adipose tissue mRNA expression ofINSRandGLUT4. In comparison with casein, the effects of WPI on energy intake and hypothalamic and adipose tissue gene expression may thus represent a state of reduced susceptibility to weight gain on a HFD.

Obesity develops from a prolonged imbalance of energy intake and energy expenditure. However, the relatively recent discovery that the composition and function of the gut microbiota impacts on obesity has lead to an explosion of interest in what is now a distinct research field. Here, research relating to the links between the gut microbiota, diet and obesity will be reviewed under five major headings: (1) the gut microbiota of lean and obese animals, (2) the composition of the gut microbiota of lean and obese humans, (3) the impact of diet on the gut microbiota, (4) manipulating the gut microbiota and (5) the mechanisms by which the gut microbiota can impact on weight gain.

Siberian hamsters display photoperiodically regulated annual cycles in body weight, appetite, and reproduction. Previous studies have revealed a profound up-regulation of type 3 deiodinase (DIO3) mRNA in the ventral ependyma of the hypothalamus associated with hypophagia and weight loss in short-day photoperiods. DIO3 reduces the local availability of T(3), so the aim of this study was to test the hypothesis that decreased hypothalamic T(3) availability underlies the short-day-induced catabolic state. The experimental approach was to determine whether a local increase in T(3) in the hypothalamus of hamsters exposed to short days could reverse the behavioral and physiological changes induced by this photoperiod. In study 1, microimplants releasing T(3) were placed bilaterally into the hypothalamus. This treatment rapidly induced a long-day phenotype including increased appetite and body weight within 3 wk of treatment and increased fat mass and testis size by the end of the 10-wk study period. In study 2, hypothalamic T(3) implants were placed into hamsters carrying abdominal radiotelemetry implants. Again body weight increased significantly, and the occurrence of winter torpor bouts was dramatically decreased to less than one bout per week, whereas sham-implanted hamsters entered torpor up to six times a week. Our findings demonstrate that increased central T(3) induces a long-day metabolic phenotype, but in neither study was the molt cycle affected, so we infer that we had not disrupted the initial detection of photoperiod. We conclude that hypothalamic thyroid hormone availability plays a key role in seasonal regulation of appetite, body weight, and torpor.

In a previous study performed in mouse models of energetic challenge, there was evidence to suggest that the orphan G protein-coupled receptor GPCR101 may have a role in the regulation of energy balance. To further investigate this possibility, we utilised in situ hybridisation to determine the effect of energetic challenges experienced by pregnant and lactating rats on GPCR101 mRNA expression. In the rat hypothalamus, GPCR101 mRNA expression was detected in a number of hypothalamic nuclei. During pregnancy and lactation, GPCR101 mRNA level remained unchanged in most nuclei, but had increased in the supraoptic nucleus by the end of pregnancy and remained elevated during lactation. GPCR101 mRNA expression showed a similar pattern of expression in the rostral ventromedial parvocellular subdivision of the paraventricular nucleus. A common feature of these two nuclei is the production of the peptide oxytocin. Dual in situ hybridisation revealed GPCR101 and oxytocin mRNA co-expression in neurons of these two nuclei. In the supraoptic nucleus, in situ hybridisation revealed that the temporal regulation of oxytocin and GPCR101 mRNA expression were similar. In the paraventricular nucleus, although temporal changes in oxytocin mRNA expression were similar to GPCR101, the spatial expression of the two mRNA species was different; in contrast to GPCR101, oxytocin mRNA expression changed in both parvo- and magnocellular neurons during lactation. In conclusion, increased GPCR101 mRNA expression in supraoptic and paraventricular nuclei from late pregnancy to late lactation may reflect the functional importance of this receptor in the regulation of neurons of these nuclei during this period.

The Siberian hamster survives winter by decreasing food intake and catabolizing abdominal fat reserves, resulting in a sustained, profound loss of body weight. VGF gene expression is photoperiodically regulated in the hypothalamus with significantly higher expression in lean Siberian hamsters. The aim of this study was to investigate the role of VGF in regulating these seasonal cycles by determining the effects of a VGF-derived peptide (TLQP-21) on food intake and body weight. Acute intracerebroventricular administration of TLQP-21 decreased food intake, and chronic treatment caused a sustained reduction in food intake and body weight and decreased abdominal fat depots. Behavioral analysis revealed that TLQP-21 reduced meal size but not the frequency of feeding bouts, suggesting a primary action on satiety. Hamsters treated with TLQP-21 lost a similar amount of weight as a pair-fed group in which food intake was matched to that of the TLQP-21-treated group. Central or peripheral treatment with TLQP-21 did not produce a significant effect on resting metabolic rate. We conclude that the primary action of TLQP-21 is to decrease food intake rather than increase energy expenditure. TLQP-21 treatment caused a decrease in UCP-1 mRNA in brown adipose tissue, but hypothalamic expression of orexigenic and anorexigenic neuropeptide genes remained unchanged after TLQP-21 treatment, although compensatory increases in NPY and AgRP mRNA were observed in the pair-fed hamsters. The effects of TLQP-21 administration are similar to those in hamsters in short days, suggesting that increased VGF activity may contribute to the hypophagia that underlies the seasonal catabolic state.

Neuromedin U (NMU) has been associated with the regulation of food-intake and energy balance in rats. The objective of this study was to identify the sites of gene expression for NMU and the NMU receptor-2 (NMU2R) in the mouse and rat hypothalamus and ascertain the effects of nutritional status on the expression of these genes. In situ hybridization studies revealed that NMU is expressed in several regions of the mouse hypothalamus associated with the regulation of energy balance. Analysis of NMU expression in the obese ob/ob mouse revealed that NMU mRNA levels were elevated in the dorsomedial hypothalamic (DMH) nucleus of obese ob/ob mice compared to lean litter-mates. In addition, NMU mRNA levels were elevated in the DMH of mice fasted for 24 h relative to ad libitum fed controls. The pattern of expression of NMU and NMU2R were more widespread in the hypothalamus of mice than rats. These data provide the first detailed anatomical analysis of the NMU and NMU2R expression in the mouse and advance our knowledge of expression in the rat. The data from the obese rodent models supports the hypothesis that NMU is involved in the regulation of nutritional status.

The expression of precursor-protein convertase (PC)1, PC2 and paired basic amino acid cleaving enzyme four mRNA was studied by in situ hybridisation in regions of the hypothalamus involved in energy regulation in relation to obese (ob) gene mutation and energy deficit. PC1 gene was differentially expressed in hypothalamic nuclei of mice from different genetic backgrounds or energetic status, whereas no differences in expression were observed for either the PC2 or paired basic amino acid cleaving enzyme four genes. In obese ob/ob mice, PC1 mRNA levels were increased in the paraventricular nucleus, decreased in the lateral hypothalamus and unchanged in the ventromedial nucleus and arcuate nucleus relative to lean controls. In response to intraperitoneal injection of murine leptin, PC1 mRNA levels in obese ob/ob mice decreased in the arcuate nucleus, increased in the lateral hypothalamus and were unchanged in both the paraventricular nucleus and ventromedial nucleus. In mice deprived of food for 24 h, PC1 mRNA levels were reduced in the ventromedial nucleus, increased in the lateral hypothalamus and unchanged in the paraventricular nucleus and arcuate nucleus relative to ad libitum-fed controls. Overall, whilst the data show effects related to leptin and energetic status, they do not support a strong and consistent link between PC1 gene expression and energy balance. This suggests that if PC1 is important to the control of energy balance then protein expression and activity, rather than gene expression may be the more critical parameters of regulation. The relationship between PC1 and candidate energy balance-related genes in the lateral hypothalamus was investigated by dual in situ hybridisation. PC1 mRNA was localised in prepro-orexin mRNA expressing neurons in the lateral hypothalamus, which suggests a functional relationship.

The basic helix-loop-helix transcription factors, neurological basic-helix-loop-helix-2 (Nhlh-2), neurogenic differentiation-1 (NeuroD-1) and single minded-1 (Sim-1) could have roles in energy balance regulation, although supporting evidence is inconclusive. This study in mice provides further evidence that Nhlh-2 and NeuroD-1 are involved in energy balance regulation. In situ hybridization was used to study the expression of the genes in relation to physiological status and genetic background within hypothalamic nuclei that are involved in energy balance regulation. These studies show reduced expression of Nhlh-2 mRNA in the arcuate (ARC) nucleus and NeuroD-1 mRNA in the paraventricular (PVN) nucleus in obese ob/ob and 24 h food-deprived mice relative to respective controls, suggesting regulation by leptin. Interestingly, Nhlh-2 mRNA expression is reduced in obese db/db mice, whereas NeuroD-1 remains unchanged, suggesting different mechanisms of regulation by leptin of these two genes. To study the role of leptin in the regulation of these genes, leptin was injected intraperitoneally in obese ob/ob mice and mRNA expression evaluated after 1 h or 4 h, or after twice-daily injection for 7 days. None of these regimes restored Nhlh-2 or NeuroD-1 to wild-type mRNA levels. These latter data suggest either that the regulation of the Nhlh-2 and NeuroD-1 genes by leptin is indirect or that the apparent leptin insensitivity of the gene expression reflects a developmental deficit that is a consequence of the phenotype of the obese ob/ob mice. The relationship between Nhlh-2 and candidate energy balance-related genes was studied by dual in situ hybridization. Nhlh-2 mRNA was coexpressed in a subpopulation (30%) of ARC neurons expressing pro-opiomelanocortin (POMC) mRNA, suggesting a potential functional relationship.

Body weight is determined by the balance between energy intake and energy expenditure. When energy intake exceeds energy expenditure, the surplus energy is stored as fat in the adipose tissue, which causes its expansion and may even lead to the development of obesity. Thus, there is a growing interest to develop dietary interventions that could reduce the current obesity epidemic. In this regard, data from a number of in vivo and in vitro studies suggest that the branched-chain amino acid leucine influences energy balance. However, this has not been consistently reported. Here, we review the literature related to the effects of leucine on energy intake, energy expenditure and lipid metabolism as well as its effects on the cellular activity in the brain (hypothalamus) and in peripheral tissues (gastro-intestinal tract, adipose tissue, liver and muscle) regulating the above physiological processes. Moreover, we discuss how obesity may influence the actions of this amino acid.

The objective of this study is to investigate the impact of photoperiod on the temporal and spatial expression of genes involved in glucose metabolism in the brain of the seasonal mammal Phodopus sungorus (Siberian hamster). In situ hybridization was performed on brain sections obtained from male hamsters held in long photoperiod (high body weight and developed testes) or short photoperiod (reduced body weight with testicular regression). This analysis revealed upregulation in expression of genes involved in glycogen and glucose metabolism in short photoperiod and localized to the tanycyte layer of the third ventricle. On the basis of these data and a previously identified photoperiod-dependent increase in activity of neighboring hypothalamic neurons, we hypothesized that the observed expression changes may reflect alteration in either metabolic fuel or precursor neurotransmitter supply to surrounding neurons. Gene expression analysis was performed for genes involved in lactate and glutamate transport. This analysis showed that the gene for the lactate transporter MCT2 and glutamate transporter GLAST was decreased in the tanycyte layer in short photoperiod. Expression of mRNA for glutamine synthetase, the final enzyme in the synthesis of the neuronal neurotransmitter precursor, glutamine, was also decreased in short photoperiod. These data suggest a role for tanycytes in modulating glutamate concentrations and neurotransmitter supply in the hypothalamic environment.

The hormone cholecystokinin is produced by the enteroendocrine I cells in the intestine, and it plays an important role in a number of physiological processes including digestion and food intake. Recent data suggest that cholecystokinin gene expression and protein secretion are regulated by macronutrients. The mechanism involves a change in intracellular levels of cAMP and Ca(+2), brought about by the activity of a number of nutrient-responsive G protein-coupled receptors, nutrient transporters, ion channels and intracellular enzymes. How these intracellular responses could lead to gene expression and protein secretion are discussed along with new directions for future investigation.

The mechanism(s) involved in the regulation of the seasonal-appropriate body weight of the Siberian hamster are currently unknown. We have identified photoperiodically regulated genes including VGF in a sub-region of the arcuate nucleus termed the dorsomedial posterior arcuate (dmpARC). Gene expression changes in this nucleus so far account for a significant number of those reported as photoperiodically regulated and are therefore likely to contribute to seasonal physiological responses of the hamsters. The present study aimed to identify additional genes expressed in the dmpARC regulated by photoperiod that could be involved in regulating the activity of this nucleus with respect to seasonal physiology of the Siberian hamster. Using laser capture microdissection coupled with a microarray analysis and a candidate gene approach, we have identified several photoperiodically regulated genes in the dmpARC that are known to have roles in secretory and intracellular signalling pathways. These include secretogranin (sg) III and SgVI (secretory pathway), melanocortin 3 receptor (MC3-R) and serotonin (5-HT) receptors 2A and 7 (signalling pathway), all of which increase in expression under a short photoperiod. The spatial relationship between receptor signalling and potential secretory pathways was investigated by dual in situ hybridisation, which revealed that 5-HT2A and 5-HT7 receptors are expressed in neurones expressing VGF mRNA and that a sub-population (approximately 40%) of these neurones express MC3-R. These gene expression changes in dmpARC neurones may reflect the functional requirement of these neurones for seasonal physiological responses of the hamster.

GPCR101 is a recently identified orphan G protein-coupled receptor (GPCR) expressed abundantly in the human and mouse hypothalamus. In the absence of a ligand, a direct approach to determine the function(s) of this receptor is not possible. However, clues to the possible functions of GPCR101 may yield from information on the distribution of the receptor and the effect of in vivo manipulation upon the expression level of the receptor. In situ hybridisation on mouse brain sections revealed GPCR101 expression in a number of nuclei, including the amygdala, lateral parabrachial nucleus and nucleus of the solitary tract, as well as in the arcuate nucleus, posterior hypothalamus and paraventricular nucleus of the hypothalamus. Food-deprivation was found to increase GPCR101 mRNA level in the posterior hypothalamus and amygdala. In obese mice bearing the ob gene mutation, GPCR101 mRNA level decreased in the posterior hypothalamus and remained unaltered in the amygdala. By contrast, in both nuclei, GPCR101 mRNA level did not change significantly in obese ob/ob mice after intraperitoneal injection of leptin or in mice fed with a high fat diet. These data suggest that GPCR101 mRNA expression in the posterior hypothalamus and amygdala is regulated by a factor(s) other than leptin. Dual in situ hybridisation was used to establish the relationship between GPCR101 and neuropeptides expressed in the hypothalamus. In the arcuate nucleus, GPCR101 mRNA was expressed in approximately half of the population of neurones expressing the mRNA for the anorexigenic neuropeptide, pro-opiomelanocortin, which suggests a potential functional relationship.

The basic helix-loop-helix transcription factors, neurological basic-helix-loop-helix-2 (Nhlh-2), neurogenic differentiation-1 (NeuroD-1) and single minded-1 (Sim-1) could have roles in energy balance regulation, although supporting evidence is inconclusive. This study in mice provides further evidence that Nhlh-2 and NeuroD-1 are involved in energy balance regulation. In situ hybridization was used to study the expression of the genes in relation to physiological status and genetic background within hypothalamic nuclei that are involved in energy balance regulation. These studies show reduced expression of Nhlh-2 mRNA in the arcuate (ARC) nucleus and NeuroD-1 mRNA in the paraventricular (PVN) nucleus in obese ob/ob and 24 h food-deprived mice relative to respective controls, suggesting regulation by leptin. Interestingly, Nhlh-2 mRNA expression is reduced in obese db/db mice, whereas NeuroD-1 remains unchanged, suggesting different mechanisms of regulation by leptin of these two genes. To study the role of leptin in the regulation of these genes, leptin was injected intraperitoneally in obese ob/ob mice and mRNA expression evaluated after 1 h or 4 h, or after twice-daily injection for 7 days. None of these regimes restored Nhlh-2 or NeuroD-1 to wild-type mRNA levels. These latter data suggest either that the regulation of the Nhlh-2 and NeuroD-1 genes by leptin is indirect or that the apparent leptin insensitivity of the gene expression reflects a developmental deficit that is a consequence of the phenotype of the obese ob/ob mice. The relationship between Nhlh-2 and candidate energy balance-related genes was studied by dual in situ hybridization. Nhlh-2 mRNA was coexpressed in a subpopulation (30%) of ARC neurons expressing pro-opiomelanocortin (POMC) mRNA, suggesting a potential functional relationship.