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.     

Paraventricular hypothalamic alpha-melanocyte-stimulating hormone and MTII reduce feeding without causing aversive effects

alpha-Melanocyte-stimulating hormone (alpha-MSH) appears to play a tonic inhibitory role in feeding and energy storage. MTII, a specific synthetic MC3-R/MC4-R agonist, has similar effects on feeding in rats. The current studies demonstrate that PVN administration of alpha-MSH or MTII decreases nocturnal and NPY-stimulated food intake without causing aversive effects. Co-administration with NPY of 600 pmol alpha-MSH or 1 pmol MTII into the PVN caused a significant decrease in NPY-induced feeding. PVN administration of MTII or alpha-MSH at doses effective to suppress feeding did not cause conditioned taste aversion (CTA). ICV administration of alpha-MSH, however, did cause weak CTA. These results indicate that the potent effects on feeding of MC3-R and MC4-R agonists when injected into the PVN are not due to aversive effects.     

MTII attenuates ghrelin- and food deprivation-induced increases in food hoarding and food intake

Food deprivation triggers a constellation of physiological and behavioral changes including increases in peripherally-produced ghrelin and centrally-produced agouti-related protein (AgRP). Upon refeeding, food intake is increased in most species, however hamsters primarily increase food hoarding. Food deprivation-induced increases in food hoarding by Siberian hamsters are mimicked by peripheral ghrelin and central AgRP injections. Because food deprivation stimulates ghrelin as well as AgRP synthesis/release, food deprivation-induced increases in hoarding may be mediated by melanocortin 3 or 4 receptor (MC3/4-R) antagonism via AgRP, the MC3/4-R inverse agonist. Therefore, we asked: Can a MC3/4-R agonist block food deprivation- or ghrelin-induced increases in foraging, food hoarding and food intake? This was accomplished by injecting melanotan II (MTII), a synthetic MC3/4-R agonist, into the 3rd ventricle in food deprived, fed or peripheral ghrelin injected hamsters and housed in a running wheel-based food delivery foraging system. Three foraging conditions were used: a) no running wheel access, non-contingent food, b) running wheel access, non-contingent food or c) a foraging requirement for food (10 revolutions/pellet). Food deprivation was a more potent stimulator of foraging and hoarding than ghrelin. Concurrent injections of MTII completely blocked food deprivation- and ghrelin-induced increases in food intake and attenuated, but did not always completely block, food deprivation- and ghrelin-induced increases in food hoarding. Collectively, these data suggest that the MC3/4-R are involved in ghrelin- and food deprivation-induced increases in food intake, but other neurochemical systems, such as previously demonstrated with neuropeptide Y, also are involved in increases in food hoarding as well as foraging.     

Effect of the melanocortin receptor stimulation or inhibition on ethanol intake in alcohol-preferring rats

It has been recently reported that acute intracerebroventricular injection of 1 nmol/rat of the non-selective melanocortin 3 and 4 receptor (MC3/4) agonist MTII reduces ethanol intake in female AA alcohol-preferring rats and alters opioid peptide levels in the ventral tegmental area of rats. To better understand the role of the MC system in the control of ethanol intake, we tested the acute and chronic effects of lateral ventricular (LV) injections of 0.01-1 nmol MTII, of 0.1-1 nmol of the MC3/4R receptor antagonist agouti related peptide (AgRP), and 0.1-0.5 nmol of the MC3/4R receptor antagonist SHU9119 on food, water, and 10% ethanol intake in Marchigian-Sardinian alcohol-preferring (msP) rats, which spontaneously ingest pharmacologically relevant quantities of ethanol both under short and long term access conditions. The data showed that with 2h/day ethanol access, LV MTII injections reduced intake of food and ethanol intakes. When food, water, and ethanol were available ad libitum and 0.01 nmol MTII was given by daily LV injection, however, ethanol intake was reduced for only the first 2 days, whereas food intake was reduced for all 5 days of treatment. Finally, acute LV injection of neither AgRP nor SHU9119 affected ethanol intake under ad libitum conditions, although both antagonists significantly increased food and water intake. In conclusion, these data fail to support a role for endogenous MC3/4R in the control of spontaneous ethanol intake in the msP rat. MC3/4R agonism, however, reduced ethanol intake in association with reduced food intake, suggesting that MTII might reduce nutrient-related controls of ethanol intake rather than, or in addition to, reward-related controls of ethanol intake.     

MTII attenuates ghrelin- and food deprivation-induced increases in food hoarding and food intake

Food deprivation triggers a constellation of physiological and behavioral changes including increases in peripherally-produced ghrelin and centrally-produced agouti-related protein (AgRP). Upon refeeding, food intake is increased in most species, however hamsters primarily increase food hoarding. Food deprivation-induced increases in food hoarding by Siberian hamsters are mimicked by peripheral ghrelin and central AgRP injections. Because food deprivation stimulates ghrelin as well as AgRP synthesis/release, food deprivation-induced increases in hoarding may be mediated by melanocortin 3 or 4 receptor (MC3/4-R) antagonism via AgRP, the MC3/4-R inverse agonist. Therefore, we asked: Can a MC3/4-R agonist block food deprivation- or ghrelin-induced increases in foraging, food hoarding and food intake? This was accomplished by injecting melanotan II (MTII), a synthetic MC3/4-R agonist, into the 3rd ventricle in food deprived, fed or peripheral ghrelin injected hamsters and housed in a running wheel-based food delivery foraging system. Three foraging conditions were used: a) no running wheel access, non-contingent food, b) running wheel access, non-contingent food or c) a foraging requirement for food (10 revolutions/pellet). Food deprivation was a more potent stimulator of foraging and hoarding than ghrelin. Concurrent injections of MTII completely blocked food deprivation- and ghrelin-induced increases in food intake and attenuated, but did not always completely block, food deprivation- and ghrelin-induced increases in food hoarding. Collectively, these data suggest that the MC3/4-R are involved in ghrelin- and food deprivation-induced increases in food intake, but other neurochemical systems, such as previously demonstrated with neuropeptide Y, also are involved in increases in food hoarding as well as foraging.     

Structure activity studies of the melanocortin-4 receptor by in vitro mutagenesis: identification of agouti-related protein (AGRP), melanocortin agonist and synthetic peptide antagonist interaction determinants

In vitro mutagenesis of the mouse melanocortin-4 receptor (mMC4R) has been performed, based upon homology molecular modeling and previous melanocortin receptor mutagenesis studies that identified putative ligand-receptor interactions. Twenty-three mMC4 receptor mutants were generated and pharmacologically characterized using several melanocortin-based ligands [alpha-MSH, NDP-MSH, MTII, DNal (1')(7)-MTII, Nal(2')(7)-MTII, SHU9119, and SHU9005]. Selected mutant receptors possessing significant differences in the melanocortin-based peptide agonist and/or antagonist pharmacology were further evaluated using the endogenous antagonist agouti-related protein fragment hAGRP(83-132) and hAGRP(109-118) molecules. These studies of the mouse MC4R provide further experimental data suggesting that the conserved melanocortin receptor residues Glu92 (TM2), Asp114 (TM3), and Asp118 (TM3) (mouse MC4R numbering) are important for melanocortin-based peptide molecular recognition. Additionally, the Glu92 and Asp118 mMC4R residues are important for molecular recognition and binding of AGRP(83-132). We have identified the Phe176 (TM4), Tyr179 (TM4), Phe254 (TM6), and Phe259 (TM6) receptor residues as putatively interacting with the melanocortin-based ligand Phe(7) by differences between alpha-MSH and NDP-MSH agonist potencies. The Glu92, Asp118, and Phe253 mMC4R receptor residues appear to be critical for hAGRP(83-132) molecular recognition and binding while Phe176 appears to be important for functional antagonism of AGRP(83-132) and AGRP(109-118) but not molecular recognition. The Phe253 mMC4R residue appears to be important for AGRP(83-132) molecular recognition and general mMC4 receptor stimulation. The Phe254 and Phe259 mMC4R amino acids may participate in the differentiation of agonist versus antagonist activity of the melanocortin-based peptide antagonists SHU9119 and SHU9005, but not AGRP(83-132) or AGRP(109-118). The Met192 side chain when mutated to a Phe results in a constitutively active mMC4R that does not effect agonist ligand binding or potency. Melanocortin-based peptides modified at the 7 position of MTII with DPhe, DNal(1'), Nal(2'), and DNal(2') have been pharmacologically characterized at these mutant mouse MC4Rs. These data suggest a revised hypothesis for the mechanism of SHU9119 antagonism at the MC4R which may be attributed to the presence of a "bulky" naphthyl moiety at the 7 position (original hypothesis), and additionally that both the stereochemistry and naphthyl ring position (2' versus 1') are important for positioning of the ligand Arg(8) residue with the corresponding mMC4R amino acids.     

Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression

Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with ICV infusion of the specific MC3-R agonist, [D-Trp(8)]-gamma-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that ICV infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake.     

Leptin-like effects of MTII are augmented in MSG-obese rats

To evaluate whether MTII, a melanocortin receptor 3/4 agonist, is working in hypophagic and hypothermogenic obese model, we measured food intake, body weight, oxygen consumption, and fat mass following intracerebroventricular (i.c.v.) infusion of MTII in monosodium glutamate (MSG)-induced obese rats. MTII, or artificial cerebrospinal fluid (aCSF), was infused into i.c.v. with an osmotic minipump for 1 week. MSG-obese rats were induced by neonatal injection of MSG. Five-month-old MSG rats were characterized by hypophagia, lower oxygen consumption, hyperleptinemia, and obesity compared to age-matched control rats. The infusion of MTII decreased their food intake, visceral fat, and body weight in MSG-obese rats compared with aCSF-infused rats. The oxygen consumption was increased by MTII treatment in MSG-obese rats compared with aCSF as well as pair fed (PF) rats. Interestingly, these leptin-like effects of MTII were greater in MSG-obese rats than in controls, which might be related to the increased expression of melanocortin receptor 4 (MC4R) in the hypothalamus of MSG-obese rats. Our results suggested that both anorexic and thermogenic mechanisms were activated by MTII in the MSG-obese rats and contributed to the decrease in body weight and fat mass. Moreover, there was a sensitization to MTII caused by upregulation of the melanocortin receptor in the MSG-obese rats.     

Changes in hypothalamic agouti-related protein (AGRP), but not alpha-MSH or pro-opiomelanocortin concentrations in dietary-obese and food-restricted rats.

Melanocortin-4 receptor (MC4-R) density is thought to be regulated by synaptic availability of endogenous agonist, alpha-melanocyte-stimulating hormone (alpha-MSH), and also by agouti-related protein (AGRP), which acts as a competitive antagonist. As hypothalamic MC4-R have been implicated in the regulation of energy balance, we examined concentrations of alpha-MSH and AGRP in hypothalami of dietary-obese and food-restricted rats. In dietary-obese rats, AGRP concentrations were significantly increased by 43% (p < 0.01) above lean controls, whereas a 91% (p < 0.01) reduction was observed in food-restricted rats. Surprisingly, hypothalamic concentrations of alpha-MSH and its precursor peptide, pro-opiomelanocortin (POMC), did not differ significantly from controls in either model. In conclusion, we suggest that MC4-R activity may not be regulated by changes in agonist (alpha-MSH) but by changes in the antagonist (AGRP) availability, which may modulate background activation of the receptor by tonic alpha-MSH release. AGRP may be an important modulator of feeding behaviour.     

Melanocortin receptors mediate the excitatory effects of blood-borne murine leptin on hypothalamic paraventricular neurons in rat

The central pathways and mediators involved in sympathoexcitatory responses to circulating leptin are not well understood, although the arcuate-paraventricular nucleus (ARC-PVN) pathway likely plays a critical role. In urethane-anesthetized rats, ipsilateral intracarotid artery (ICA) injection of murine leptin (100 microg/kg) activated most PVN neurons tested. These responses were reduced by intracerebroventricular injection of the melanocortin subtype 3 and 4 receptor (MC3/4-R) antagonist SHU-9119 (0.6 nmol). The MC3/4-R agonist MTII (0.6 nmol icv) activated PVN neurons. Some PVN neurons that were excited by ICA leptin were inhibited by local application of neuropeptide Y (NPY, 2.5 ng). ICA leptin (100 microg/kg) excited presympathetic rostral ventrolateral medulla neurons and renal sympathetic nerve activity without significant change in blood pressure or heart rate; these effects were mimicked by intracerebroventricular injection of MTII (0.6 nmol). These data provide in vivo electrophysiological evidence to support the hypothesis that circulating leptin activates the sympathetic nervous system by stimulating the release of alpha-melanocyte-stimulating hormone in the vicinity of PVN neurons that are inhibited by the orexogenic peptide NPY.     

The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity

The yellow mouse obesity syndrome is due to dominant mutations at the Agouti locus, which is characterized by obesity, hyperinsulinemia, insulin resistance, hyperglycemia, hyperleptinemia, increased linear growth, and yellow coat color. This syndrome is caused by ectopic expression of Agouti in multiple tissues. Mechanisms of Agouti action in obesity seem to involve, at least in part, competitive melanocortin antagonism. Both central and peripheral effects have been implicated in Agouti-induced obesity. An Agouti-Related Protein (AGRP) has been described recently. It has been shown to be expressed in mice hypothalamus and to act similarly to agouti as a potent antagonist to central melanocortin receptor MC4-R, suggesting that AGRP is an endogenous MC4-R ligand. Mice lacking MC4-R become hyperphagic and develop obesity, implying that agouti may lead to obesity by interfering with MC4-R signaling in the brain and consequently regulating food intake. Furthermore, food intake is inhibited by intracerebro-ventricular injection of a potent melanocortin agonist and was reversed by administration of an MC4-R antagonist. The direct cellular actions of Agouti include stimulation of fatty acid and triglyceride synthesis via a Ca²⁺-dependent mechanism. Agouti and insulin act in an additive manner to increase lipogenesis. This additive effect of agouti and insulin is demonstrated by the necessity of insulin in eliciting weight gain in transgenic mice expressing agouti specifically in adipose tissue. This suggests that agouti expression in adipose tissue combined with hyperinsulinemia may lead to increased adiposity. The roles of melanocortin receptors or agouti-specific receptor(s) in agouti regulation of adipocyte metabolism and other peripheral effects remain to be determined. In conclusion, both central and peripheral actions of agouti contribute to the yellow mouse obesity syndrome and this action is mediated at least in part by antagonism with melanocortin receptors and/or regulation of intracellular calcium.     

Parathyroid hormone-related protein and lung injury after pulmonary thromboendarterectomy

Agouti-related protein (AGRP) is one of two naturally occurring antagonists of G-Protein coupled receptors (GPCRs) identified to date, and has been physiologically implicated in regulating food intake, body weight, and energy homeostasis. AGRP has been identified in vitro, as competitively antagonizing the brain melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors, and when over expressed in transgenic mice, results in an obese phenotype. Emerging data propose that AGRP has additional targets in the hypothalamus and/or physiologically functions via a mechanism in addition to competitive antagonism of alpha-MSH at the brain melanocortin receptors. We report data herein supporting an alternative mechanism for AGRP involvement in feeding behavior. A constitutively active MC4R has been generated which possess EC(50) values for melanocortin agonists (alpha-MSH, NDP-MSH, and MTII) and a pA2 value for the synthetic peptide antagonist SHU9119 identical to the wildtype receptor, but increases basal activity to 50% maximal response. AGRP possesses inverse agonist activity at this constitutively active MC4R. These data support the hypothesis for an additional physiological mechanism for AGRP action in feeding behavior and energy homeostasis.     

Agouti-related protein: more than a melanocortin-4 receptor antagonist?

It is well established that agouti-related protein (AGRP) can act as a competitive antagonist to proopiomelanocortin (POMC)-derived peptides at the melanocortin-4 receptor (MC4R), and that this homeostatic mechanism is important as a means of coordinating appetite with perceived metabolic requirement. However, there are clearly additional facets to the physiological role of AGRP, given that it is active in MC4R knockout mice and it has strikingly long-lasting effects on food intake, compared with MC4R agonists. In this review we focus on: (i) evidence that AGRP is more sensitive to perturbations in energy balance than POMC and is therefore the primary basis of melanocortinergic regulation. (ii) Evidence that the bioactive peptide AGRP83-132, acts by alternate mechanism(s) to elicit its long-term effects on food intake. (iii) Evidence that AGRP is post-translationally cleaved to generate AGRP83-132 and one or more N terminal peptides, which may have an important physiological role(s) that are independent of the melanocortin system. A clear understanding of how proAGRP processing is regulated, and the role of resultant peptides, may define additional therapeutic targets in the treatment of obesity.     

Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo

Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.     

Melanocortin receptor 4 deficiency affects body weight regulation, grooming behavior, and substrate preference in the rat

Obesity is caused by an imbalance between energy intake and expenditure and has become a major health-care problem in western society. The central melanocortin system plays a crucial role in the regulation of feeding and energy expenditure, and functional loss of melanocortin receptor 4 (MC4R) is the most common genetic cause of human obesity. In this study, we present the first functional Mc4r knockout model in the rat, resulting from an N-ethyl-N-nitrosourea mutagenesis-induced point mutation. In vitro observations revealed impaired membrane-binding and subsequent nonfunctionality of the receptor, whereas in vivo observations showed that functional loss of MC4R increased body weight, food intake, white adipose mass, and changed substrate preference. In addition, intracerebroventricular (ICV) administration of Agouti-Related Protein(79-129) (AgRP(79-129)), an MC4R inverse agonist, or Melanotan-II (MTII), an MC4R agonist, did affect feeding behavior in wild-type rats but not in homozygous mutant rats, confirming complete loss of MC4R function in vivo. Finally, ICV administration of MTII induced excessive grooming behavior in wild-type rats, whereas this effect was absent in homozygous mutant rats, indicating that MTII-induced grooming behavior is exclusively regulated via MC4R pathways. Taken together, we expect that the MC4R rat model described here will be a valuable tool for studying monogenic obesity in humans. More specifically, the relative big size and increased cognitive capacity of rats as compared to mice will facilitate complex behavioral studies and detailed mechanistic studies regarding central function of MC4R, both of which ultimately may help to further understand the specific mechanisms that induce obesity during loss of MC4R function.     

Brain stem melanocortinergic modulation of meal size and identification of hypothalamic POMC projections

Metabolic, cognitive, and environmental factors processed in the forebrain modulate food intake by changing the potency of direct controls of meal ingestion in the brain stem. Here, we behaviorally and anatomically test the role of the hypothalamic proopiomelanocortin (POMC) system in mediating some of these descending, indirect controls. Melanotan II (MTII), a stable melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC3R) agonist injected into the fourth ventricle near the dorsal vagal complex, potently inhibited 14-h food intake by decreasing meal size but not meal frequency; SHU9119, an antagonist, increased food intake by selectively increasing meal size. Furthermore, MTII injected into the fourth ventricle increased and SHU9119 tended to decrease heart rate and body temperature measured telemetrically in freely moving rats. Numerous alpha-melanocyte-stimulating hormone-immunoreactive axons were in close anatomical apposition to nucleus tractus solitarius neurons showing c-Fos in response to gastric distension, expressing neurochemical phenotypes implicated in ingestive control, and projecting to brown adipose tissue. In retrograde tracing experiments, a small percentage of arcuate nucleus POMC neurons was found to project to the dorsal vagal complex. Thus melanocortin signaling in the brain stem is sufficient to alter food intake via changing the potency of satiety signals and to alter sympathetic outflow. Although the anatomical findings support the involvement of hypothalamomedullary POMC projections in mediating part of the descending, indirect signal, they do not rule out involvement of POMC neurons in the nucleus tractus solitarius in mediating part of the direct signal.     

Effect of i.c.v. infusion of the alpha-MSH agonist MTII on meal patterns in male rats following nicotine withdrawal

The present study explored the role of endogenous alpha-MSH in the alteration of meal patterns induced by nicotine (NIC) withdrawal. Male Sprague Dawley rats bearing third ventricle cannulas were placed in computerized food intake monitors. On days 1-21, the rats were given 4 mg/kg/day of NIC or saline (SAL) in four equal i.p. doses during the dark period. NIC suppressed (P < 0.05) food intake only during the first week. The normalization of food intake occurred when the reduced meal size of the NIC injected rats was countered by an increase in meal number. Despite the normalization of 24-h food intake, body weight in NIC rats was decreased (P < 0.05) for 21 days. On day 22, the rats were divided into 4 groups (n's = 7-8 each) and injected into the third ventricle with various doses of the alpha-MSH agonist MTII or artificial cerebrospinal fluid (aCSF): SAL + aCSF, SAL + MTII, NIC + aCSF, NIC + MTII. Infusion of MTII (30 ng/rat) suppressed (P < 0.01) dark phase food intake in both groups, but the NIC + MTII group ate (P < 0.05) more than the SAL + MTII group. Meal number during the dark phase was suppressed by MTII, but the NIC + MTII group took significantly more meals that the SAL + MTII group. Infusion of MTII suppressed meal size in SAL and NIC treated rats, but this effect was attenuated in NIC treated rats. All meal parameters normalized by the day after i.c.v. infusion. These data indicate that NIC treatment differentially affects the neural controls of meal number and meal size and attenuates the suppression by MTII of meal number and meal size.     

Agouti-related protein increases food hoarding more than food intake in Siberian hamsters

Agouti-related protein (AgRP), an endogenous melanocortin 3/4 receptor antagonist, appears to play an important role in the control of food intake and energy balance because exogenous administration in rats and overexpression in mice result in hyperphagia and body mass gain. Furthermore, arcuate nucleus AgRP mRNA is increased with fasting in laboratory rats and mice and is decreased with refeeding. In Siberian hamsters, fasting also increases arcuate nucleus AgRP mRNA, but these animals increase food hoarding, rather than food intake with refeeding. Therefore, we tested whether exogenous AgRP increased food hoarding in this species. Hamsters were trained in a hoarding/foraging apparatus to run a programmed number of wheel revolutions to earn food pellets. Four doses of AgRP-(83-132) or vehicle were injected into the third ventricle at the beginning of the dark phase, and food hoarding, food intake, and foraging were measured at various time points subsequently. Overall, food hoarding was stimulated as much as 10 times more than food intake, and both responses occurred as early as 1 h after injection. Food hoarding was increased the greatest at the lowest dose (0.1 nmol), whereas food intake was increased the greatest at the second lowest dose (1 nmol). Food intake and especially food hoarding were increased up to seven days after the AgRP injections. Foraging was increased at all AgRP doses except the highest dose (100 nmol). These results suggest that AgRP triggers the search for food in this species, and once they find it, hoarding predominates over eating.     

Cardiovascular responses to melanocortin 4-receptor stimulation in conscious unrestrained normotensive rats

In the present studies, we used a non-selective melanocortin MC3/4 receptor agonist (HP228) and a novel selective melanocortin MC4 receptor (MC4-R) agonist (MK-cpd1) to study the cardiovascular, temperature, locomotor and feeding responses to melanocortin receptor stimulation in comparison to sibutramine in rats instrumented with a telemetry transmitter. Moreover, norepinephrine turnover rates in heart and brown adipose tissue were determined. HP228 (1, 3 and 10mg/kg, i.p.) reduced 24h food intake dose-dependently and increased heart rate and mean arterial pressure (maximal differences: +60+/-8beats/min and +8+/-1mmHg, means+/-S.E.M., p<0.001 and p<0.01, respectively). After 10mg/kg HP228 showed a three-fold increase in norepinephrine turnover in the heart. The selective MC4-R agonist MK-cpd1 tended to decrease 24h food intake only at the highest dose tested (10mg/kg, i.p., p=0.06) and increased both heart rate (+17+/-4 and +22+/-5beats/min at 3 and 10mg/kg, p<0.01) and mean arterial pressure (+4+/-1mmHg at 10mg/kg, p<0.05). Sibutramine reduced food intake at all doses tested (1, 3 and 10mg/kg, i.p.). It did not change mean arterial pressure significantly, and increased heart rate only at the highest dose tested (+36+/-6beats/min, p<0.05). If also observed in humans, the pharmacological profile of MC4-R agonists would not offer a significant therapeutic advantage over currently used appetite suppressants such as sibutramine.