Central proopiomelanocortin but not neuropeptide Y mediates sympathoexcitation and hypertension in fat fed conscious rabbits.

OBJECTIVE: High-fat diet (HFD)-induced hypertension in rabbits is neurogenic because of the central sympathoexcitatory actions of leptin. Hypothalamic melanocortin and neuropeptide Y (NPY) neurons are recognized as the major signalling pathways through which leptin exerts its central effects. In this study, we assessed the effects of specific antagonists and agonists to melanocortin and NPY receptors on HFD-induced sympathoexcitation and hypertension. METHODS: Rabbits were instrumented with intracerebroventricular cannula, renal sympathetic nerve activity (RSNA) electrode, and blood pressure telemetry transmitter. RESULTS: After 3 weeks HFD (13.5% fat, n = 12) conscious rabbits had higher RSNA (+3.8  nu, P = 0.02), blood pressure (+8.6  mmHg, P < 0.001) and heart rate (+15  b/min, P = 0.01), and brain-derived neurotrophic factor levels in the hypothalamus compared with rabbits fed a control diet (4.2% fat, n = 11). Intracerebroventricular administration of the melanocortin receptor antagonist SHU9119 reduced RSNA (-2.7  nu) and blood pressure (-8.5  mmHg) in HFD but not control rabbits, thus reversing 100% of the hypertension and 70% of the sympathoexcitation induced by a HFD. By contrast, blocking central NPY Y1 receptors with BVD10 increased RSNA only in HFD rabbits. Intracerebroventricular α-melanocortin stimulating hormone increased RSNA and heart rate (P < 0.001) in HFD rabbits but had no effect in control rabbits. CONCLUSION: These findings suggest that obesity-induced hypertension and increased RSNA are dependent on the balance between greater activation of melanocortin signalling through melanocortin receptors and lesser activation of NPY sympathoinhibitory signalling. The amplification of the sympathoexcitatory effects of α-melanocortin stimulating hormone also indicates that the underlying mechanism is related to facilitation of leptin-melanocortin signalling, possibly involving chronic activation of brain-derived neurotrophic factor.

The HPA axis modulates the CNS melanocortin control of liver triacylglyceride metabolism.

The central melanocortin system regulates lipid metabolism in peripheral tissues such as white adipose tissue. Alterations in the activity of sympathetic nerves connecting hypothalamic cells expressing melanocortin 3/4 receptors (MC3/4R) with white adipocytes have been shown to partly mediate these effects. Interestingly, hypothalamic neurons producing corticotropin-releasing hormone and thyrotropin-releasing hormone co-express MC4R. Therefore we hypothesized that regulation of hypothalamo-pituitary adrenal (HPA) and hypothalamo-pituitary thyroid (HPT) axes activity by the central melanocortin system could contribute to its control of peripheral lipid metabolism. To test this hypothesis, we chronically infused rats intracerebroventricularly (i.c.v.) either with an MC3/4R antagonist (SHU9119), an MC3/4R agonist (MTII) or saline. Rats had been previously adrenalectomized (ADX) and supplemented daily with 1mg/kg corticosterone (s.c.), thyroidectomized (TDX) and supplemented daily with 10 µg/kgL-thyroxin (s.c.), or sham operated (SO). Blockade of MC3/4R signaling with SHU9119 increased food intake and body mass, irrespective of gland surgery. The increase in body mass was accompanied by higher epididymal white adipose tissue (eWAT) weight and higher mRNA content of lipogenic enzymes in eWAT. SHU9119 infusion increased triglyceride content in the liver of SO and TDX rats, but not in those of ADX rats. Concomitantly, mRNA expression of lipogenic enzymes in liver was increased in SO and TDX, but not in ADX rats. We conclude that the HPA and HPT axes do not play an essential role in mediating central melanocortinergic effects on white adipose tissue and liver lipid metabolism. However, while basal hepatic lipid metabolism does not depend on a functional HPA axis, the induction of hepatic lipogenesis due to central melanocortin system blockade does require a functional HPA axis.

Alpha-melanocyte-stimulating hormone stimulates prolactin secretion through melanocortin-3 receptors expressed in mammotropes in the mouse pituitary.

The intermediate lobe of rodent pituitaries is involved in the regulation of prolactin (PRL) secretion from the anterior lobe. In a previous study, we demonstrated the stimulatory effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on PRL release and the expression of melanocortin-3 receptors (MC3-Rs) in cultured mouse pituitary cells. The aim of the present study was to clarify whether alpha-MSH directly stimulates PRL release through the MC3-Rs by determining the cell type of MC3-R-expressing cells in the mouse pituitary anterior lobe. Northern blot analysis revealed a 2.7-kb transcript for MC3-R mRNA in the anterior and neurointermediate lobes of pituitary glands of adult male and female mice. Dual cellular localization of MC3-R mRNA and PRL or growth hormone (GH) in the mouse pituitary glands was performed by in situ hybridization analysis of MC3-R mRNA followed by immunocytochemical detection of PRL or GH. MC3-R mRNA was detected in most mammotropes and some somatotropes. alpha-MSH increased PRL release and stimulated DNA replication in mammotropes, and these effects were blocked by SHU9119, an antagonist of MC3-R and MC4-R. These results indicate that alpha-MSH stimulates PRL release and proliferation of mammotropes through MC3-Rs, and suggest that alpha-MSH from intermediate lobes can regulate mammotrope functions in the mouse pituitary.

Physiologic effect of leptin on insulin secretion is mediated mainly through central mechanisms.

Leptin has been shown to decrease glucose-stimulated insulin secretion in both in vivo and in vitro studies. As some of the effects of leptin have been elicited through both peripheral and central mechanisms, we assessed whether leptin modulates insulin secretion also through the central nervous system. We infused leptin or saline through implanted intracerebro-ventricular (ICV) catheters to chronically catheterized, conscious rats (n=15), 2 h after initiation of hyperglycemic (approximately 11 mM) clamp. On ICV administration of leptin, there was a gradual and progressive decrease in plasma insulin levels by 52% with 30 ng (P<0.005) and by 28% with 20 ng (P<0.05) of leptin compared with ICV saline. The effect of 20 ng leptin ICV was replicated by intravenous (IV) leptin infusion that achieved physiological leptin levels of approximately 17 ng/ml (n=5). When the melanocortin (MC) pathway was blocked with a nonselective MC-3/4 antagonist SHU 9119 administered ICV, and either saline or leptin (n=12) was infused IV, intravenous leptin failed to produce a decrease in glucose-stimulated insulin levels. We conclude that leptin decreases insulin levels by a predominantly central mechanism, probably via the melanocortin receptors; and peripheral leptin receptors on the beta-cells do not play a major role. The physiological features of this response suggest a possible role for leptin in the evolution of diabetes in overweight individuals.

Hypothalamic melanocortin receptors and chronic regulation of arterial pressure and renal function.

This study examined control of cardiovascular and renal function during chronic melanocortin-3/4 receptor (MC3/4-R) activation or inhibition. Arterial and venous catheters were implanted in Sprague-Dawley rats for measurements of mean arterial pressure (MAP) and heart rate (HR) 24 h/d and for intravenous infusions, and the lateral ventricle was cannulated for chronic intracerebroventricular (ICV) infusions. In experiment 1, after a 5-day control period, rats were administered the MC3/4-R agonist MTII (n=7, 10 ng/h ICV) or 0.9% saline (n=6, ICV) for 14 days, followed by a 5-day recovery period. In experiment 2, after a 5-day control period, rats were administered the MC3/4-R antagonist SHU-9119 (n=7, 1 nmol/h ICV) or 0.9% saline vehicle (n=7, ICV), or pair-fed during SHU-9119 infusion (n=5, 1 nmol/h ICV) for 12 days, followed by a 5-day recovery period. MC4-R activation transiently decreased food intake from 23+/-1 to 10+/-2 g/d. Despite the hypophagia, MC3/4-R activation increased MAP by 7+/-1 mm Hg. MC3/4-R inhibition for 12 days increased food intake from 21+/-1 to 35+/-4 g/d, decreased HR by 53+/-11 bpm, and caused no change in MAP despite the marked weight gain. In rats that were pair-fed to prevent increased food intake, MC3/4-R inhibition further decreased HR (-87+/-9 bpm), whereas MAP was unchanged. Thus, chronic hypothalamic MC3/4-R activation raises arterial pressure despite decreased food intake, whereas MC3/4-R inhibition causes marked weight gain without raising arterial pressure. These observations are consistent with the hypothesis that an intact hypothalamic MC3/4-R may be necessary for excess weight gain to raise arterial pressure.

The role of the melanocortin system and the melanocortin-4 receptor in ring dove (Streptopelia risoria) feeding behavior.

The melanocortin-4 receptor (MC4-R) is an important mediator of the effects of two melanocortin system ligands, alpha melanocyte stimulating hormone (alpha-MSH) and agouti-related peptide (AGRP), on feeding behavior and energy balance in mammals. Although an avian homologue of the mammalian MC4-R has recently been identified, there is little information on the role of this receptor and the melanocortin system in avian feeding and body weight regulation. In these studies, we measured changes in feeding behavior in ring doves (Streptopelia risoria) following intracerebroventricular (i.c.v.) injection of various melanocortin receptor agonists and antagonists. The selective MC4-R antagonist HS014 elevated food intake within 4 h at all three doses tested (0.02, 0.2, and 2 nmol). A 1 nmol dose of the endogenous antagonist AGRP also stimulated feeding but only after a post-injection interval of 10 h. Surprisingly, the MC3-R and MC4-R antagonist SHU9119 not only failed to stimulate food intake at the same doses as HS014, but actually inhibited food intake at 8 h after injection. Whether this was due to toxicity effects or differences in the pharmacology of avian and mammalian melanocortin receptors remains to be determined. Food-deprived doves showed a fourfold increase in the number of AGRP-immunoreactive cells in the tuberal region of the hypothalamus and 5 ng of the MC3-R and MC4-R agonist MTII significantly attenuated the amount of food consumed by food-deprived birds that were allowed to re-feed. These data support a role for the melanocortin system and the melanocortin-4 receptor in the ring dove feeding behavior.

Hypothalamic insulin signaling is required for inhibition of glucose production.

Circulating insulin inhibits endogenous glucose production. Here we report that bidirectional changes in hypothalamic insulin signaling affect glucose production. The infusion of either insulin or a small-molecule insulin mimetic in the third cerebral ventricle suppressed glucose production independent of circulating levels of insulin and of other glucoregulatory hormones. Conversely, central antagonism of insulin signaling impaired the ability of circulating insulin to inhibit glucose production. Finally, third-cerebral-ventricle administration of inhibitors of ATP-sensitive potassium channels, but not of antagonists of the central melanocortin receptors, also blunted the effect of hyperinsulinemia on glucose production. These results reveal a new site of action of insulin on glucose production and suggest that hypothalamic insulin resistance can contribute to hyperglycemia in type 2 diabetes mellitus.

The catabolic action of insulin in the brain is mediated by melanocortins.

Like leptin, the pancreatic hormone insulin is an important adiposity signal to the brain. We report that the hypothalamic melanocortin system is an important target of the actions of insulin to regulate food intake and body weight. Hypothalamic neurons expressing insulin receptors were found to coexpress the melanocortin precursor molecule pro-opiomelanocortin (POMC), and administration of insulin into the third cerebral ventricle of fasted rats increased expression of POMC mRNA. Finally, a subthreshold dose of the melanocortin antagonist SHU-9119 prevented the reduction in food intake caused by third-ventricular insulin administration. These data suggest that the hypothalamic melanocortin system mediates the anorexic effects of central insulin, as well as of leptin.

Estradiol treatment fails to affect the feeding responses to melanocortin-3/4 receptor agonism or antagonism in ovariectomized rats.

The involvement of the hypothalamic melanocortin-3 and -4 (MC3/4) receptors system in the inhibitory actions of estradiol (E2) on feeding was investigated. Ovariectomized Long-Evans rats with lateral ventricular (LICV) injection cannulae were maintained on a near-physiological, cyclic schedule of E2 treatment. LICV injections of 0.5 nmol of the MC3/4 agonist MTII decreased feeding, and LICV injections of the MC3/4 antagonists SHU9119 (12.5-500 pmol) and AgRP (1.0 nmol) stimulated feeding. None of these effects was affected by E2 treatment. Thus, hypothalamic MC3/4 receptors play a physiological role in the control of feeding in female rats as in males but do not mediate E2's feeding effects during the ovarian cycle.

Sustained orexigenic effect of Agouti related protein may be not mediated by the melanocortin 4 receptor.

Intracerebroventricular (ICV) injection of Agouti related protein (AgRP), an endogenous melanocortin 3 and 4 receptor (MC3/4-R) antagonist, produces a prolonged increase in food intake. To clarify the roles of the MC3-R and MC4-R in AgRP-induced hyperphagia, the feeding effect of AgRP (83-132) was compared with that of the selective MC4-R antagonist, JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH11-22). Single ICV administration of AgRP (83-132) increased food intake for 48 h whilst ICV JKC-363 increased food intake for 8h. An increase in body weight at 24 and 48 h was observed following AgRP (83-132) but not JKC-363 treatment. These data suggest that the sustained orexigenic action of AgRP (83-132) may not be through MC4-R antagonism.

Central melanocortin receptors regulate insulin action.

Energy balance and insulin action are tightly coregulated. Leptin regulates energy intake and expenditure partly by modulation of the melanocortin pathway in the hypothalamus. Here we demonstrate potent effects of the melanocortin pathway on insulin action and body distribution of adiposity. Conscious rats received week-long infusions of either a melanocortin receptor agonist, alpha-melanocyte-stimulating hormone (alpha-MSH), or antagonist, SHU9119, in the third cerebral ventricle while food intake was maintained constant in each group. alpha-MSH decreased intra-abdominal fat and markedly enhanced the actions of insulin on both glucose uptake and production, while SHU9119 exerted opposite effects. Our findings elucidate a neuroendocrine network that is likely to play a central role in the coupling of energy intake and insulin action.

The role of the hypothalamic melanocortin system in behavioral appetitive processes.

Much evidence suggests that the hypothalamic melanocortin (MC) system plays an important role in the control of food intake. However, investigations of the potential behavioral mechanisms have been limited to measures of aversion. The purpose of the present experiment was to assess whether other behavioral consequences of administration of MC peptides were similar to those produced by 0- or 24-h food deprivation, respectively. Rats were first trained while food deprived that a tone predicted the delivery of peanut oil. They then received exposure to oil under food deprivation, satiation, intra-third-cerebroventricular (i3vt) infusion of MTII (a potent MC agonist) or SHU-9119 (a potent MC antagonist). All rats were then tested during extinction for levels of responding to the tone under food satiation. Previous results demonstrated that sated exposure reduces subsequent test responding to the tone. During the present extinction test, rats that received sated exposure exhibited reduced responding to the tone, relative to rats that received deprived exposure. Unlike satiation, rats that received exposure after MTII exhibited continued high levels of responding to the tone. Further, rats that received SHU-9119 exhibited a small reduction in responding. These data suggest that MTII and SHU-9119 do not influence intake via the same mechanisms as hunger and food satiation, respectively.

Hypothalamic, metabolic, and behavioral responses to pharmacological inhibition of CNS melanocortin signaling in rats.

The CNS melanocortin (MC) system is implicated as a mediator of the central effects of leptin, and reduced activity of the CNS MC system promotes obesity in both rodents and humans. Because activation of CNS MC receptors has direct effects on autonomic outflow and metabolism, we hypothesized that food intake-independent mechanisms contribute to development of obesity induced by pharmacological blockade of MC receptors in the brain and that changes in hypothalamic neuropeptidergic systems known to regulate weight gain [i.e., corticotropin-releasing hormone (CRH), cocaine-amphetamine-related transcript (CART), proopiomelanocortin (POMC), and neuropeptide Y (NPY)] would trigger this effect. Relative to vehicle-treated controls, third intracerebroventricular (i3vt) administration of the MC receptor antagonist SHU9119 to rats for 11 d doubled food and water intake (toward the end of treatment) and increased body weight ( approximately 14%) and fat content ( approximately 90%), hepatic glycogen content ( approximately 40%), and plasma levels of cholesterol ( approximately 48%), insulin ( approximately 259%), glucagon ( approximately 80%), and leptin ( approximately 490%), whereas spontaneous locomotor activity and body temperature were reduced. Pair-feeding of i3vt SHU9119-treated animals to i3vt vehicle-treated controls normalized plasma levels of insulin, glucagon, and hepatic glycogen content, but only partially reversed the elevations of plasma cholesterol ( approximately 31%) and leptin ( approximately 104%) and body fat content ( approximately 27%). Reductions in body temperature and locomotor activity induced by i3vt SHU9119 were not reversed by pair feeding, but rather were more pronounced. None of the effects found can be explained by peripheral action of the compound. The obesity effects occurred despite a lack in neuropeptide expression responses in the neuroanatomical range selected across the arcuate (i.e., CART, POMC, and NPY) and paraventricular (i.e., CRH) hypothalamus. The results indicate that reduced activity of the CNS MC pathway promotes fat deposition via both food intake-dependent and -independent mechanisms.

Central melanocortin receptors mediate changes in food intake in the rhesus macaque.

In rodents, stimulation of melanocortin-3 and -4 receptor subtypes (MC3-R and MC4-R) causes a reduction in food intake, whereas antagonism of MC3-R and MC4-R increases food intake. This report describes the effects of the stable alphaMSH analog, NDP-MSH ([Nle(4), D-Phe(7)]alphaMSH), and the endogenous alphaMSH receptor antagonist, agouti-related protein, on feeding behavior in adult male rhesus macaques. Infusion of NDP-MSH into the lateral cerebral ventricle dose dependently suppressed intake of a normally scheduled meal without affecting nonfeeding behaviors. Conversely, infusion of agouti-related protein stimulated food intake during the scheduled afternoon meal. In addition to these physiological experiments, the effect of fasting on hypothalamic POMC gene expression was assessed by in situ hybridization. Missing a single meal or fasting for 48 h caused a similar reduction in POMC gene expression in the arcuate nucleus. These results demonstrate that in the primate, central melanocortin receptors can acutely regulate food intake and suggest that the central melanocortinergic system is a physiological regulator of energy balance in primate species.

Chronic blockade of the melanocortin 4 receptor subtype leads to obesity independently of neuropeptide Y action, with no adverse effects on the gonadotropic and somatotropic axes.

Neuropeptide Y (NPY) is a powerful orexigenic factor, and alphaMSH is a melanocortin (MC) peptide that induces satiety by activating the MC4 receptor subtype. Genetic models with disruption of MC4 receptor signaling are associated with obesity. In the present study, a 7-day intracerebroventricular infusion to male rats of either the MC receptor antagonist SHU9119 or porcine NPY (10 nmol/day) was shown to strongly stimulate food and water intake and to markedly increase fat pad mass. Very high plasma leptin levels were found in NPY-treated rats (27.1 +/- 1.8 ng/ml compared with 9.9 +/- 0.9 ng/ml in SHU9119-treated animals and 2.1 +/- 0.2 ng/ml in controls). As expected, NPY infusion induced hypogonadism, characterized by an impressive decrease in seminal vesicle and prostate weights. No such effects were seen with the SHU9119 infusion. Similarly, whereas the somatotropic axis of NPY-treated rats was fully inhibited, this axis was normally activated in the obese SHU9119-treated rats. Chronic infusion of SHU9119 strikingly reduced hypothalamic gene expression for NPY (65.2 +/- 3.6% of controls), whereas gene expression for POMC was increased (170 +/- 19%). NPY infusion decreased hypothalamic gene expression for both POMC and NPY (70 +/- 9% and 75.4 +/- 9.5%, respectively). In summary, blockade of the MC4 receptor subtype by SHU9119 was able to generate an obesity syndrome with no apparent side-effects on the reproductive and somatotropic axes. In this situation, it is unlikely that hyperphagia was driven by increased NPY release, because hypothalamic NPY gene expression was markedly reduced, suggesting that hyperphagia mainly resulted from loss of the satiety signal driven by MC peptides. NPY infusion produced hypogonadism and hyposomatotropism in the face of markedly elevated plasma leptin levels and an important reduction in hypothalamic POMC synthesis. In this situation NPY probably acted both by exacerbating food intake through Y receptors and by reducing the satiety signal driven by MC peptides.

Development of a facile method for high throughput screening with reporter gene assays.

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

The effect of leptin on luteinizing hormone release is exerted in the zona incerta and mediated by melanin-concentrating hormone.

The adipose hormone, leptin, not only restrains appetite, but also influences energy expenditure. One such influence is to promote sexual maturation and fertility. The neuromodulatory circuits that mediate this effect are not well known but the present study suggests that one mediator could be melanin-concentrating hormone (MCH). We show that the long-form receptor (Ob-Rb) is expressed in the zona incerta of the rat and that administration of leptin (both 0.5 microg and 1.0 microg/side) into this area of ovariectomized, oestrogen-primed rats stimulated the release of luteinizing hormone (LH) within 1 h, the effect enduring for a further 1 h. Injections of leptin into the arcuate nucleus induced a smaller, transient rise in LH while injections into the paraventricular and ventromedial nuclei were without effect. MCH neurones are present in the zona incerta and administration of this hormone into the medial preoptic area (mPOA) stimulates LH release, therefore we investigated the possibility that MCH might mediate this effect of leptin. An injection of MCH antiserum into mPOA prevented the rise in LH normally induced by leptin injected into the zona incerta. In addition, melanocortin receptor antagonists ([D-Arg8]ACTH(4-10) and [Ala6]ACTH(4-10)), previously shown to inhibit the stimulatory effect of MCH on LH release, also inhibited the effect of leptin. We propose that one route by which leptin may promote reproductive activity is by enhancing MCH release from fibres within the mPOA. Speculative mechanisms for the action of MCH include the following possibilities: MCH may be acting on the specific MCH receptor which in turn interacts with a melanocortin or melanocortin-like receptor; MCH may bind directly to one of the melanocortin receptors; or melanocortin antagonists may interact with the MCH receptor.

The central melanocortin system affects the hypothalamo-pituitary thyroid axis and may mediate the effect of leptin.

Prolonged fasting is associated with a downregulation of the hypothalamo-pituitary thyroid (H-P-T) axis, which is reversed by administration of leptin. The hypothalamic melanocortin system regulates energy balance and mediates a number of central effects of leptin. In this study, we show that hypothalamic melanocortins can stimulate the thyroid axis and that their antagonist, agouti-related peptide (Agrp), can inhibit it. Intracerebroventricular (ICV) administration of Agrp (83-132) decreased plasma thyroid stimulating hormone (TSH) in fed male rats. Intraparaventricular nuclear administration of Agrp (83-132) produced a long-lasting suppression of plasma TSH, and plasma T4. ICV administration of a stable alpha-MSH analogue increased plasma TSH in 24-hour-fasted rats. In vitro, alpha-MSH increased thyrotropin releasing hormone (TRH) release from hypothalamic explants. Agrp (83-132) alone caused no change in TRH release but antagonized the effect of alpha-MSH on TRH release. Leptin increased TRH release from hypothalami harvested from 48-hour-fasted rats. Agrp (83-132) blocked this effect. These data suggest a role for the hypothalamic melanocortin system in the fasting-induced suppression of the H-P-T axis.

Effects of neuropeptide Y deficiency on hypothalamic agouti-related protein expression and responsiveness to melanocortin analogues.

Central administration of neuropeptide Y (NPY) potently induces feeding and its abundance in the hypothalamus increases when energy stores fall. Consequently, NPY is considered to be a physiological effector of feeding behavior. Surprisingly, NPY-deficient (NPY-/-) mice feed and grow normally with ad libitum access to food and manifest a normal hyperphagic response after fasting, suggesting that other feeding effectors may compensate for the lack of NPY. Agouti-related protein (AgRP), a melanocortin receptor antagonist, can also stimulate feeding behavior when administered centrally and is coexpressed in a majority of hypothalmamic NPY-ergic neurons, making AgRP a candidate compensatory factor. To test this possibility, we evaluated AgRP mRNA and protein expression, as well as responsiveness to centrally administered AgRP in NPY-/- mice. These studies demonstrate that hypothalamic AgRP mRNA and immunoreactivity are upregulated with fasting and that these increases are not affected by NPY deficiency. Interestingly, NPY-/- mice are hypersensitive to central administration of AgRP(83-132), yet exhibit a normal response to centrally administered MTII, a melanocortin receptor agonist. These data suggest that if AgRP compensates for the lack of NPY in NPY-/- mice, it is not at the level of AgRP synthesis and may instead involve alterations in the postsynaptic signaling efficacy of AgRP. Moreover, the effects of AgRP are not limited to its actions at the melanocortin-4 receptor (MC4R), because MC4R-deficient (MC4R-/-) mice manifest a significant response to centrally administered AgRP. These data imply that AgRP has additional targets in the hypothalamus.