TXNIP in Agrp neurons regulates adiposity, energy expenditure, and central leptin sensitivity

Thioredoxin interacting protein (TXNIP) has recently been described as a key regulator of energy metabolism through pleiotropic actions that include nutrient sensing in the mediobasal hypothalamus (MBH). However, the role of TXNIP in neurochemically specific hypothalamic subpopulations and the circuits downstream from MBH TXNIP engaged to regulate energy homeostasis remain unexplored. To evaluate the metabolic role of TXNIP activity specifically within arcuate Agrp neurons, we generated Agrp-specific TXNIP gain-of-function and loss-of-function mouse models using Agrp-Ires-cre mice, TXNIP (flox/flox) mice, and a lentivector expressing the human TXNIP isoform conditionally in the presence of Cre recombinase. Overexpression of TXNIP in Agrp neurons predisposed to diet-induced obesity and adipose tissue storage by decreasing energy expenditure and spontaneous locomotion, without affecting food intake. Conversely, Agrp neuronal TXNIP deletion protected against diet-induced obesity and adipose tissue storage by increasing energy expenditure and spontaneous locomotion, also without affecting food intake. TXNIP overexpression in Agrp neurons did not primarily affect glycemic control, whereas deletion of TXNIP in Agrp neurons improved fasting glucose levels and glucose tolerance independently of its effects on body weight and adiposity. Bidirectional manipulation of TXNIP expression induced reciprocal changes in central leptin sensitivity and the neural regulation of lipolysis. Together, these results identify a critical role for TXNIP in Agrp neurons in mediating diet-induced obesity through the regulation of energy expenditure and adipose tissue metabolism, independently of food intake. They also reveal a previously unidentified role for Agrp neurons in the brain-adipose axis.     

Effect of opioid receptor ligands injected into the rostral lateral hypothalamus on c-fos and feeding behavior

The lateral hypothalamic area (LHa) is an important brain site for the regulation of food intake. Central injection of opioids increases food intake, and the LHa contains mu and kappa opioid receptors, both of which are involved in feeding behavior. It is unclear whether opioids impact feeding when injected directly into the rostral portion of the LHa (rLHa) in rats. We performed a series of studies in which free-feeding rLHa-cannulated rats were injected with opioid agonists (DAMGO, morphine, dynorphin, U-50488H) followed by the measurement of food intake at 1, 2, and 4 h postinjection. To determine whether opioid receptor ligands administered into the rLHa affect neuronal activation in this brain site, we studied cFos immunoreactivity (cFos IR) in response to rLHa stimulation with naltrexone. We found that the only compound that stimulated feeding behavior was morphine. The other agonists had no effect on food consumption. Naltrexone injection into the rLHa increased neural activation in the LHa, indicating the presence of functional opioid receptors in this region. These data suggest that although neuronal activity is affected by opioid agents acting in the rLHa, administration of selective mu and kappa opioid ligands in this subdivision of the LHa does not have a reliable effect on feeding behavior.     

Receptor-selective changes in mu-, delta- and kappa-opioid receptors after chronic naltrexone treatment in mice

Chronic treatment with the opioid antagonist naltrexone induces functional supersensitivity to opioid agonists, which may be explained by receptor up-regulation induced by opioid receptor blockade. In the present study, the levels of opioid receptor subtypes through the brain of mice were determined after chronic naltrexone treatment using quantitative in vitro autoradiography. This is the first complete mapping study in mice for micro-, delta- and kappa-opioid receptors after chronic naltrexone exposure. Treatment with naltrexone clearly induced up-regulation of micro- (mean 80%) and, to a lesser extent, delta-opioid receptors (mean 39%). The up-regulation of micro- and delta-opioid receptors was evident throughout the brain, although there was variation in the percentage change across brain regions. In contrast, consistent up-regulation of kappa-opioid receptors was observed in cortical structures only and was not so marked as for micro- and delta-opioid receptors. In noncortical regions kappa-opioid receptor expression was unchanged. Taken together, the present findings suggest opioid receptor subtype-selective regulation by chronic naltrexone treatment in mice.     

Effect of Agouti-related protein delivered to the dorsomedial nucleus of the hypothalamus on intake of a preferred versus a non-preferred diet

Agouti-related protein (Agrp), a high-affinity antagonist of the melanocortin-3/4 receptors, increases feeding when administered centrally. Previous studies have shown that this increase is long-lasting (at least 24 h) and delayed, unless the animal is first stimulated to feed by fasting or onset of the dark phase. The present studies first demonstrate that long-lasting and delayed increases in food intake are also evident when Agrp is microinjected into the dorsomedial nucleus of the hypothalamus (DMH). Next, the effects of DMH-administered Agrp were assessed on intake of two foods, isocaloric but differing in flavor (with or without sucrose). Following exposure to the two diets, rats were injected via cannula aimed at the DMH with 100 pmol Agrp at 10:00 h and allowed ad libitum access to either: (1) a choice of both diets or (2) one of the diets alone. Food intake was determined at 2, 4, and 24 h post-injection. In the first (choice) paradigm, Agrp only increased intake of the sucrose-containing diet. In the second (no-choice) paradigm, animals on either diet showed an Agrp-induced increase in intake 24 h following injection; only animals on the sucrose-containing diet showed an increase in intake 4 h post-injection. The results are discussed in the context of the possible involvement of Agrp/MC4-R in the rewarding characteristics of food intake.     

Naloxone benzoylhydrazone, a kappa 3 opioid agonist, stimulates food intake in rats.

Naloxone benzoylhydrazone (NalBzoH) is a selective, short-acting agonist at the kappa 3 opioid receptor and a slowly dissociating potent antagonist at the mu opioid receptor. Given the important role of kappa receptors in the opioid control of food intake, the present study examined the central and peripheral effects of NalBzoH upon food intake. Central administration of NalBzoH (1-20 micrograms, i.c.v.) significantly increased food intake for up to 12 h, but failed to alter intake or body weight after 24 or 48 h. The 12 h duration of NalBzoH-mediated effects may be due to either persistent kappa 3 receptor occupancy, and/or activation of an ingestive system which maintains its activity. Peripheral administration of NalBzoH (20 mg/kg, s.c.) significantly increased food intake for up to 1 h. To distinguish kappa 1 (U50,488H) and kappa 3 (NalBzoH) hyperphagic effects, these agonist effects were compared following pretreatment with either naltrexone or the kappa 1 antagonist, nor-binaltorphamine (Nor-BNI). Whereas naltrexone significantly reduced both U50,488H and NalBzoH hyperphagia, Nor-BNI blocked U50,448H, but not NalBzoH hyperphagia. These data indicate a distinct role for the kappa 3 receptor in ingestive behavior separable from that of kappa 1 effects.     

Effects of agouti-related protein on metabolism and hypothalamic neuropeptide gene expression

The hypothalamic melanocortin system regulates feeding in part through interaction of the appetite stimulating peptide, agouti-related protein (AGRP), and the anorectic peptide, alpha-melanocyte stimulating hormone, a peptide derived from the pro-opiomelanocortin (POMC) polyprotein. Central administration of AGRP induces hyperphagia and increased gain in body weight in rodents, but may also exert metabolic effects even when hyperphagia is prevented. In the present studies, the effects of AGRP on hypothalamic neuropeptide gene expression and metabolism were examined in the rat. Central administration of AGRP for 3- and 7-day periods resulted in hyperphagia, increased body weight and increased plasma leptin and insulin concentrations compared to saline-injected controls. Hypothalamic concentrations of Pomc mRNA were also increased by 27% and 44% (in 3- and 7-day experiments, respectively). The hypothalamic concentration of Agrp mRNA was unchanged after 3 days, but was significantly decreased by 33% after 7 days of AGRP infusion. To determine if these changes were dependent upon AGRP-induced hyperphagia, pair-fed rats with restricted food intake receiving central administration of AGRP were also studied. In the absence of hyperphagia, intracerebralventricular administration of AGRP caused significant increases in plasma leptin and insulin concentrations (two-fold and 1.5-fold, respectively) and fat pad mass. A significant increase in hypothalamic Pomc mRNA concentrations was not detected in pair-fed rats. In contrast, Agrp mRNA concentrations remained suppressed by 45% in the pair-fed group after 7 days of AGRP infusion despite equal body weight compared to saline controls. The ratio of hypothalamic Pomc to Agrp mRNA was elevated two-fold in ad libitum and pair-fed AGRP-injected rats, which is consistent with increased stimulation of central melanocortin signalling pathways. Thus, central administration of AGRP exerts changes in hypothalamic neuropeptide gene expression and metabolic effects that are independent of the effects on food intake and body weight.     

Agouti-related protein prevents self-starvation

Food restriction leads to a paradoxical increase in physical activity and further suppression of food intake, such as observed in anorexia nervosa.(1,2) To understand this pathophysiological process, we induced physical hyperactivity and self-starvation in rats by restricting food in the presence of running wheels. Normally, decreased melanocortin receptor activity will prevent starvation.(3,4) However, we found that self-starvation increased melanocortin receptors in the ventral medial hypothalamus, a brain region involved in eating behavior.(5) Suppression of melanocortin receptor activity, via central infusion of Agouti-related protein (AgRP), increased survival rate in these rats by counteracting physical hyperactivity, food intake suppression as well as deregulated body temperature. We conclude that self-starvation may result from insufficient suppression of central melanocortin receptor activity.     

Effect of Agouti-related protein on development of conditioned taste aversion and oxytocin neuronal activation

Agouti-related protein (Agrp) is an orexigenic peptide that acts as an antagonist of the melanocortin-3 and -4 receptors. Initial studies suggest similarities between the effects of Agrp and opioid peptides on ingestive behavior. Given these observations, we examined whether Agrp, similarly to opioids, alleviates conditioned taste aversion (CTA) generated by peripheral injection of LiCl. Agrp (1 nmol) delivered to the lateral cerebral ventricle, a dose known to cause orexigenic effects, was shown to partially block acquisition of LiCl-induced CTA. Agrp also decreased the percentage of c-Fos-positive oxytocin neurons induced by LiCl in the hypothalamic paraventricular and supraoptic nuclei. Inhibitory effects of Agrp on acquisition of CTA and aversion-associated activation of oxytocin neurons parallel what has previously been shown with opioid receptor agonists.     

The cannabinoid anticonvulsant effect on pentylenetetrazole-induced seizure is potentiated by ultra-low dose naltrexone in mice

Cannabinoid compounds are anticonvulsant since they have inhibitory effects at micromolar doses, which are mediated by activated receptors coupling to G(i/o) proteins. Surprisingly, both the analgesic and anticonvulsant effects of opioids are enhanced by ultra-low doses (nanomolar to picomolar) of the opioid antagonist naltrexone and as opioid and cannabinoid systems interact, it has been shown that ultra-low dose naltrexone also enhances cannabinoid-induced antinociception. Thus, concerning the seizure modulating properties of both classes of receptors this study investigated whether the ultra-low dose opioid antagonist naltrexone influences cannabinoid anticonvulsant effects. The clonic seizure threshold was tested in separate groups of male NMRI mice following injection of vehicle, the cannabinoid selective agonist arachidonyl-2-chloroethylamide (ACEA) and ultra-low doses of the opioid receptor antagonist naltrexone and a combination of ACEA and naltrexone doses in a model of clonic seizure induced by pentylenetetrazole (PTZ). Systemic injection of ultra-low doses of naltrexone (1pg/kg to 1ng/kg, i.p.) significantly potentiated the anticonvulsant effect of ACEA (1mg/kg, i.p.). Moreover, the very low dose of naltrexone (500pg/kg) unmasked a strong anticonvulsant effect for very low doses of ACEA (10 and 100mug/kg). A similar potentiation by naltrexone (500pg/kg) of anticonvulsant effects of non-effective dose of ACEA (1mg/kg) was also observed in the generalized tonic-clonic model of seizure. The present data indicate that the interaction between opioid and cannabinoid systems extends to ultra-low dose levels and ultra-low doses of opioid receptor antagonist in conjunction with very low doses of cannabinoids may provide a potent strategy to modulate seizure susceptibility.     

Melanocortin mediated inhibition of feeding behavior in rats

Melanocortinergic neurons are believed to play a role in the control of food intake. Melanocortin receptor agonists and antagonists modulate feeding in several mouse models of chemically and genetically induced hyperphagia. To date, little information is available describing the role of this neurological system in the control of the natural feeding cycle in genetically intact rats.To evaluate the involvement of melanocortins in spontaneous nocturnal feeding, the synthetic melanocortin receptor agonist, MTII and the antagonist, SHU9119 were administered ICV (third ventricle) alone and in combination. Dose-dependent inhibition or stimulation of food intake was observed with MTII or SHU9119, respectively. Co-injections containing equal concentrations of MTII and SHU9119 resulted in food intake that was indistinguishable from controls. Food intake patterns observed in studies in which various dose combinations of MTII and SHU9119 were co-injected are consistent with the concept that both affect feeding by acting on similar melanocortin receptors.The hypothesis that effects of melanocortins on feeding may be mediated via an NPY related pathway was tested by co-injecting MTII and NPY in a 2-h satiated food intake paradigm. MTII inhibited food intake induced by 5.0 μg hNPY in a dose dependent manner with the highest dose tested abolishing the NPY feeding response.The studies suggest that melanocortins act via specific receptors to control food intake in rats, possibly via an NPY related pathway. If similar neurochemical processes operate in humans, selectively modulating specific melanocortin receptor signaling may be an approach to the treatment of human obesity.     

Feeding and behavioural effects of central administration of the melanocortin 3/4-R antagonist SHU9119 in obese and lean Siberian hamsters

Siberian hamsters accumulate fat reserves in long photoperiods, but show a long-term decrease in food intake and body weight when exposed to a short winter photoperiod. The aim of this study was to determine the role of central melanocortin 3/4 receptors (MC3/4-R) in generating this chronic catabolic state by investigating the effects of SHU9119, a MC3/4-R antagonist, on food intake and associated behaviours. In adult male hamsters, intra-cerebroventricular infusions of SHU9119 significantly increased food intake in a dose-dependent manner. The time course of action was slow, food intake being increased between 4 and 24 h after intra-cerebroventricular administration. A similar degree of increase in food intake occurred in fat hamsters in long days and in lean hamsters chronically exposed to short days. Intra-cerebroventricular treatment with MTII (a MC3/4-R agonist) significantly decreased food intake for up to 24 h after treatment, and SHU9119 reversed these suppressive effects between 4 and 24 h after treatment, a similar time course to that observed when SHU9119 was administered alone. We conclude that endogenous melanocortin peptides acting via MC3/4-R are involved in the regulation of food intake in hamsters in both anabolic and catabolic states, but these acute studies do not provide evidence that increased activity of this hypothalamic system underlies the seasonal decrease in food intake that contributes to the long-term catabolic state in short days.     

Beta-melanocyte-stimulating hormone potently reduces appetite via the hypothalamus in chicks

The melanocortin system together with other appetite-related systems plays a significant role in appetite regulation. The appetite-related effects of one such melanocortin, β-melanocyte-stimulating hormone (MSH), are well documented in rodents; however, its effects in the avian class are not thoroughly understood. Thus, we designed a study to determine the effects of i.c.v. β-MSH injection on food and water intake, plasma corticosterone concentration, ingestive and non-ingestive behaviours, and hypothalamic neuronal activation using Cobb-500 chicks. Chicks responded to β-MSH-treatment with a reduction in food and water intake; however when water intake was measured independently of food intake, it was not affected. β-MSH-treated chicks also had increased plasma corticosterone concentrations and increased c-Fos reactivity in the periventricular, paraventricular and infundibular nuclei, and the ventromedial hypothalamus; however, the lateral hypothalamus was not affected. The effect on food intake is primary because behaviours that may be competitive with food intake were not increased in β-MSH-treated chicks. Based on these results, we conclude that β-MSH causes anorexigenic effects that are likely primarily mediated via stimulation of satiety-related hypothalamic nuclei in broiler-type chicks.     

Investigation of the melanocyte stimulating hormones on food intake. Lack Of evidence to support a role for the melanocortin-3-receptor

The melanocortin receptors, melanocortin-3-receptor (MC3-R) and melanocortin-4-receptor (MC4-R), are expressed in many discrete medial hypothalamic nuclei implicated in feeding regulation. The pro-opiomelanocortin product alpha-melanocyte stimulating hormone (alpha-MSH), an MC3/4-R agonist, decreases food intake following intracerebroventricular (ICV) injection in rats. MC4-R's involvement in feeding has been established although a function for the MC3-R is unclear. We investigated endogenous melanocortin ligand binding and activation at the MC3-R and MC4-R and their effects on feeding. We have shown that alpha-MSH, desacetyl-alpha-MSH and beta-MSH bound to the MC3-R and MC4-R with similar affinity and stimulated cAMP with similar potency in HEK 293 cells transfected with MC3-R and MC4-R. In contrast gamma(2)-MSH showed selectivity for the MC3-R over the MC4-R both in binding affinity and cAMP stimulation. alpha-MSH and beta-MSH injected ICV into fasted rats at doses of 1, 3 and 6 nmol resulted in a decrease in food intake, (2 h food intake: alpha-MSH 6 nmol, 1.7+/-0.3 g; beta-MSH 6 nmol, 1.5+/-0.3 g vs. saline 6.0+/-0.5 g, P<0.001). Desacetyl alpha-MSH did not reduce food intake at low doses but was significant at 25 nmol (2 h food intake: desacetyl-alpha-MSH 6.1+/-1.0 g vs. saline 9.5+/-1.4 g, P<0.05). In contrast, gamma(2)-MSH had no effect on food intake when administered ICV to fasted rats. We were unable to establish a role for the MC3-R in feeding regulation. Our evidence, however, strengthens the hypothesis that the melanocortin's effects on food intake are mediated via the MC4-R.     

Endogenous opioids encode relative taste preference

Endogenous opioid signaling contributes to the neural control of food intake. Opioid signaling is thought to regulate palatability, the reward value of a food item as determined by orosensory cues such as taste and texture. The reward value of a food reflects not only these sensory properties but also the relative value of competing food choices. In the present experiment, we used a consummatory contrast paradigm to manipulate the relative value of a sucrose solution for two groups of rats. Systemic injection of the nonspecific opioid antagonist naltrexone suppressed sucrose intake; for both groups, however, this suppression was selective, occurring only for the relatively more valuable sucrose solution. Our results indicate that endogenous opioid signaling contributes to the encoding of relative reward value.     

Leptin Regulation of Agrp and Npy mRNA in the Rat Hypothalamus

Agouti-related protein (AGRP) is synthesized in the same neurones in the arcuate nucleus as neuropeptide Y (NPY), another potent orexigenic peptide. AGRP antagonizes the action of alpha-melanocyte stimulating hormone, a derivative of pro-opiomelanocortin (POMC) at the hypothalamic MC4 receptor to increase food intake. Although leptin has been shown to regulate Agrp/Npy and Pomc-expressing neurones, there are differences with respect to Agrp regulation in leptin receptor-deficient mice and rats. Unlike the obese leptin receptor-deficient db/db mouse, which exhibits upregulation of Agrp mRNA expression in the medial basal hypothalamus (MBH) compared to lean controls, the obese leptin receptor-deficient (faf; Koletsky) rat does not exhibit upregulation of Agrp expression. To determine whether this represents a general difference between leptin receptor-deficient mice and rats, neuropeptide gene expression was analysed in the MBH of lean and obese rats segregating for a different leptin receptor mutation, Leprfa (Zucker). Fasting in lean rats (+/fa) for 72 h significantly increased Agrp and Npy mRNA expression, and decreased Pomc mRNA expression as detected by a sensitive solution hybridization/S1 nuclease protection assay. Npy mRNA levels were significantly increased in fed obese fa/fa compared to lean rats, and further increased in the obese animals after fasting. In contrast, Agrp mRNA levels did not differ between fed lean and fed obese rats, and fasting did not significantly change Agrp levels in obese rats. To determine whether the change in Agrp expression that occurs with food deprivation in lean rats could be prevented by leptin replacement, Sprague-Dawley rats were fasted and infused via subcutaneous osmotic micropumps for 48 h with either saline or recombinant mouse leptin. Fasting significantly increased Agrp and Npy, and decreased Pomc mRNA levels. Leptin infusion almost completely reversed these changes such that there was no significant difference between the levels in the fasted rats and those that were fed ad libitum. Thus, in fasted lean rats, Agrp and Npy are upregulated in parallel when leptin levels fall and are downregulated by leptin infusion. By contrast, the absence of a functional leptin receptor results in the upregulation of Npy but not Agrp mRNA.     

Effects of u50,488, a selective kappa agonist, on atypical mouse opiate systems

We have proposed that endogenous opioids play a critical role in the etiology of anorexia nervosa, mediating an auto-addiction, and that atypical opioid systems in mice may be representative of those in anorexia nervosa patients, in contrast to normal humans and rats. A biological predisposition to eating disorders may result from these atypical opioid systems. Definition of these systems as atypical is based on their responses to morphine, which are preferential for the mu receptor subtype. Three patterns have been described in four strains of mice: anorexia with hyperactivity (BALB/C and C57BL/J), anorexia without hyperactivity (DBA/J), and a biphasic curve (CF-1). The latter showed anorexia and hyperactivity at high doses but increased food intake without a change in motor activity at low doses. These patterns contrast to the increase in food intake and sedation in typical species, including rats and normal humans. In the present study, U50,488, a selective kappa agonist, increases food intake in all four mouse strains, as previously reported in rats. Thus, these two agonists have opposite effects on the atypical mouse systems, but similar effects on the typical rat system. The typical and atypical opioid systems respond oppositely to morphine but similarly to U50,488.     

Mercaptoacetate induces feeding through central opioid-mediated mechanisms in rats

The endogenous opioid system has been implicated in the mediation of food intake elicited by such regulatory challenges as glucoprivation induced by 2-deoxy-D-glucose (2DG) or food deprivation in rodents. Administration of the free fatty acid oxidation inhibitor, mercaptoacetate (MA), produces a potent short-term increase in feeding in rats, the mechanisms of which have been dissociated from that elicited by 2DG. The present study evaluated whether MA-induced feeding in rats was mediated by the endogenous opioid system through systemic administration of the general opioid antagonist, naltrexone, through central administration of either general, mu, mu(1), kappa(1) or delta opioid antagonists, and through central administration of antisense oligodeoxynucleotide (AS ODN) probes directed against specific exons of either the mu (MOR-1), kappa (KOR-1), kappa(3) (KOR-3/ORL-1) or delta (DOR-1) opioid receptor clones. MA-induced feeding was significantly and dose-dependently reduced by systemic naltrexone (0.005-5 mg/kg); these ingestive effects were quite selective since neither total, ambulatory nor stereotypic activity was affected by either MA itself or MA paired with naltrexone. MA-induced feeding was significantly reduced by central pretreatment with either naltrexone (0.1-20 microgram) or mu-selective (beta-funaltrexamine, 0.1-20 microgram), mu(1)-selective (naloxonazine, 1-20 microgram), kappa(1)-selective (nor-binaltorphamine, 0.1-20 microgram), or delta-selective (naltrindole, 1-20 microgram) opioid receptor antagonists. MA-induced feeding was significantly reduced by AS ODN probes directed against either exons 1, 2 or 3, but not exon 4 of the MOR-1 clone, exon 3, but not exons 1 or 2 of the KOR-1 clone, exons 1 or 2, but not exon 3 of the KOR-3/ORL-1 clone, and exon 1, but not exons 2 or 3 of the DOR-1 clone. These data are discussed in terms of opioid mediation of ingestive responses related to fat, and in terms of potential central sites of action at which lipoprivic ingestive responses might act.     

Anorexigenic melanocortin signaling in the hypothalamus is augmented in association with failure-to-thrive in a transgenic mouse model for Prader-Willi syndrome

As in Prader-Willi syndrome (PWS) infants, mouse models of PWS display failure-to-thrive during the neonatal period. In rodents, the hypothalamic neuropeptide, Neuropeptide Y (NPY) and Agouti-related peptide (AgrP) stimulate while alpha-melanocyte stimulating hormone (alpha-MSH) inhibits appetite. We hypothesized that altered expression of these neuropeptides in the hypothalamus may underlie the failure-to-thrive in PWS neonatal mice. To test this hypothesis we evaluated mRNA expression of Npy, Agrp, and Pomc by in situ hybridization in the hypothalamic arcuate nucleus (ARC) of 3-day-old female and male PWS neonates. The results showed that Agrp mRNA expression was decreased relative to wild-type (WT) controls in neonates of both sexes, while mRNA expression of Pomc was upregulated in PWS neonates. Since AgrP and the Pomc-derived peptide, alpha-MSH, are functional antagonists at melanocortin 4 receptors in the hypothalamic regulation of appetitive behavior, these results show that robust anorexigenic melanocortin signaling, may contribute to the failure-to-thrive in PWS neonatal mice.     

Food deprivation decreases responsiveness of ventromedial hypothalamic neurons to melanocortins

The melanocortin system is involved in regulation of food intake and energy balance. Melanocyte-stimulating hormone (alpha-MSH) is an endogenous melanocortin receptor (MC-R) agonist. It acts on MC3/4 receptors to reduce appetite and to increase energy expenditure. The production of alpha-MSH is reduced during food deprivation, but MC4-R density is increased. The net effect of reduced alpha-MSH production and increased receptor level is not clear. To address this question, responses of ventromedial hypothalamic (VMH) neurons to melanotan II (MTII; a synthetic analogue of alpha-MSH) were recorded in brain slices from fed and food-deprived rats. Responses to the highest dose MTII were observed in 61% of VMH neurons from fed rats but only 33% of VMH neurons from food-deprived rats. To assess a possible mechanism by which responsiveness to melanocortins is diminished even though receptor number is augmented during fasting, we examined the effect of agouti gene-related peptide (AGRP), an endogenous MC-R antagonist that stimulates food intake. The synthesis of AGRP increases during fasting. AGRP significantly reduced VMH responsiveness to MTII. Additionally, AGRP by itself evoked neuronal responses, in contrast to synthetic MC-R antagonists. AGRP (1 nM) induced a predominant inhibitory effect on VMH neurons in food-deprived rats but not in fed rats. In the presence of AGRP, MTII induced a significant inhibition of neuronal activity in deprived rats, but not in fed rats. Inhibition of VMH neurons reduces energy expenditure and the satiety signal. These findings suggest that although food deprivation increases MC4-R density, it nevertheless reduces the effectiveness of melanocortins on VMH neurons, possibly by the involvement of AGRP.