Effects of AgRP Inhibition on Energy Balance and Metabolism in Rodent Models

Activation of brain melanocortin-4 receptors (MC4-R) by α-melanocyte-stimulating hormone (MSH) or inhibition by agouti-related protein (AgRP) regulates food intake and energy expenditure and can modulate neuroendocrine responses to changes in energy balance. To examine the effects of AgRP inhibition on energy balance, a small molecule, non-peptide compound, TTP2515, developed by TransTech Pharma, Inc., was studied in vitro and in rodent models in vivo. TTP2515 prevented AgRP from antagonizing α-MSH-induced increases in cAMP in HEK 293 cells overexpressing the human MC4-R. When administered to rats by oral gavage TTP2515 blocked icv AgRP-induced increases in food intake, weight gain and adiposity and suppression of T4 levels. In both diet-induced obese (DIO) and leptin-deficient mice, TTP2515 decreased food intake, weight gain, adiposity and respiratory quotient. TTP2515 potently suppressed food intake and weight gain in lean mice immediately after initiation of a high fat diet (HFD) but had no effect on these parameters in lean chow-fed mice. However, when tested in AgRP KO mice, TTP2515 also suppressed food intake and weight gain during HFD feeding. In several studies TTP2515 increased T4 but not T3 levels, however this was also observed in AgRP KO mice. TTP2515 also attenuated refeeding and weight gain after fasting, an effect not evident in AgRP KO mice when administered at moderate doses. This study shows that TTP2515 exerts many effects consistent with AgRP inhibition however experiments in AgRP KO mice indicate some off-target effects of this drug. TTP2515 was particularly effective during fasting and in mice with leptin deficiency, conditions in which AgRP is elevated, as well as during acute and chronic HFD feeding. Thus the usefulness of this drug in treating obesity deserves further exploration, to define the AgRP dependent and independent mechanisms by which TTP2515 exerts its effects on energy balance.     

Transgenic expression of syndecan-1 uncovers a physiological control of feeding behavior by syndecan-3.

Transgenic expression in the hypothalamus of syndecan-1, a cell surface heparan sulfate proteoglycan (HSPG) and modulator of ligand-receptor encounters, produces mice with hyperphagia and maturity-onset obesity resembling mice with reduced action of alpha melanocyte stimulating hormone (alphaMSH). Via their HS chains, syndecans potentiate the action of agouti-related protein and agouti signaling protein, endogenous inhibitors of alphaMSH. In wild-type mice, syndecan-3, the predominantly neural syndecan, is expressed in hypothalamic regions that control energy balance. Food deprivation increases hypothalamic syndecan-3 levels several-fold. Syndecan-3 null mice, otherwise apparently normal, respond to food deprivation with markedly reduced reflex hyperphagia. We propose that oscillation of hypothalamic syndecan-3 levels physiologically modulates feeding behavior.     

Heparanase enhances syndecan-1 shedding: a novel mechanism for stimulation of tumor growth and metastasis

When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth.     

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.     

Role of hypothalamic melanocortin system in adaptation of food intake to food protein increase in mice

The hypothalamic melanocortin system--the melanocortin receptor of type 4 (MC4R) and its ligands: α-melanin-stimulating hormone (α-MSH, agonist, inducing hypophagia), and agouti-related protein (AgRP, antagonist, inducing hyperphagia)--is considered to play a central role in the control of food intake. We tested its implication in the mediation of the hunger-curbing effects of protein-enriched diets (PED) in mice. Whereas there was a 20% decrease in food intake in mice fed on the PED, compared to mice fed on an isocaloric starch-enriched diet, there was a paradoxical decrease in expression of the hypothalamic proopiomelanocortin gene, precursor of α-MSH, and increase in expression of the gene encoding AgRP. The hypophagia effect of PED took place in mice with invalidation of either MC4R or POMC, and was even strengthened in mice with ablation of the AgRP-expressing neurons. These data strongly suggest that the hypothalamic melanocortin system does not mediate the hunger-curbing effects induced by changes in the macronutrient composition of food. Rather, the role of this system might be to defend the body against the variations in food intake generated by the nutritional environment.     

Enterostatin inhibition of dietary fat intake is modulated through the melanocortin system

Enterostatin injected into the amygdala selectively reduces dietary fat intake by an action that involves a serotonergic component in the paraventricular nucleus. We have investigated the role of melanocortin signaling in the response to enterostatin by studies in melanocortin 4 receptor (MC4R) knock out mice and by the use of the MC4R and MC3R antagonist SHU9119, and by neurochemical phenotyping of enterostatin activated cells. We also determined the effect of enterostatin in vivo on the expression of AgRP in the hypothalamus and amygdala of rats and in culture on a GT1-7 neuronal cell line. Enterostatin had no effect on food intake in MC4R knock out mice. SHU9119 i.c.v. blocked the feeding response to amygdala enterostatin in rats. Amygdala enterostatin induced fos activation in alpha-melanocyte stimulating hormone (alpha-MSH) neurons in the arcuate nucleus. Enterostatin also reduced the expression of AgRP in the hypothalamus and amygdala and in GT1-7 cells. These data suggest enterostatin inhibits dietary fat intake through a melanocortin signaling pathway.     

Inhibition of the central melanocortin system decreases brown adipose tissue activity

The melanocortin system is an important regulator of energy balance, and melanocortin 4 receptor (MC4R) deficiency is the most common monogenic cause of obesity. We investigated whether the relationship between melanocortin system activity and energy expenditure (EE) is mediated by brown adipose tissue (BAT) activity. Therefore, female APOE*3-Leiden.CETP transgenic mice were fed a Western-type diet for 4 weeks and infused intracerebroventricularly with the melanocortin 3/4 receptor (MC3/4R) antagonist SHU9119 or vehicle for 2 weeks. SHU9119 increased food intake (+30%) and body fat (+50%) and decreased EE by reduction in fat oxidation (−42%). In addition, SHU9119 impaired the uptake of VLDL-TG by BAT. In line with this, SHU9119 decreased uncoupling protein-1 levels in BAT (−60%) and induced large intracellular lipid droplets, indicative of severely disturbed BAT activity. Finally, SHU9119-treated mice pair-fed to the vehicle-treated group still exhibited these effects, indicating that MC4R inhibition impairs BAT activity independent of food intake. These effects were not specific to the APOE*3-Leiden.CETP background as SHU9119 also inhibited BAT activity in wild-type mice. We conclude that inhibition of central MC3/4R signaling impairs BAT function, which is accompanied by reduced EE, thereby promoting adiposity. We anticipate that activation of MC4R is a promising strategy to combat obesity by increasing BAT activity.     

Divergence of melanocortin pathways in the control of food intake and energy expenditure

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase. Mice homozygous for the loxTB Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. Disassociation of food intake and energy expenditure reveals unexpected divergence in melanocortin pathways controlling energy balance.     

Pregnancy-induced hyperphagia is associated with increased gene expression of hypothalamic agouti-related peptide in rats

Pregnancy is characterized by an increase in food intake that, in turn, produce a positive energy balance in order to face the considerable metabolic demands associated with the challenge of reproduction. Since hypothalamus is a key brain region involved in many peripheral signals and neuronal pathways that control energy homeostasis and food intake, we investigated if during pregnancy the increase in food intake is mediated by stimulating orexigenic and/or inhibiting anorexigenic neural pathways. We examined hypothalamic gene expressions of Ob-Rb, NPY, AgRP, POMC, MC4-R, and preproorexins in pregnant Wistar rats at day 19 of gestation. Food intake and body weight were increased progressively during the pregnancy. Visceral fat mass depots and serum leptin levels were also increased when compared with virgin animals. No differences were found in mRNA expression of Ob-Rb, POMC, MC4-R, NPY or preproorexin between virgin and pregnant animals. However, pregnancy produced a selective increase in AgRP mRNA levels. These results indicate that the positive energy balance that occurred during pregnancy can hardly be explained by changes in Ob-Rb despite hyperleptinemia associated with pregnancy. The enhanced expression of AgRP suggests the involvement of this neuropeptide in mediating pregnancy-associated hyperphagia.     

Effect of gonadectomy on AgRP-induced weight gain in rats

Agouti-related peptide (AgRP), the endogenous antagonist to the melanocortin 3 and 4 receptors, elicits robust hyperphagia and weight gain in rodents when administered directly into the central nervous system. The relative influence of AgRP to cause weight gain in rodents partially depends on the activity level of the melanocortin agonist-producing proopiomelanocortin neurons. Both proopiomelanocortin and AgRP neurons within the arcuate nucleus receive energy storage information from circulating peripheral signals such as leptin and insulin. Another modulator of AgRP activity includes the cell surface molecule syndecan-3. Because leptin and insulin affect food intake in a sexually dimorphic way in rodents and syndecan-3-deficient mice regulate adiposity levels through distinct physiological mechanisms, we hypothesized that AgRP-induced weight gain would also be sexually dimorphic in rats. In the present study, the behavioral and physiological effects of centrally-administered AgRP in male and female were investigated. In male rats, AgRP (1 nmol) induced 5 days (P < 0.0001) of significantly elevated feeding compared with vehicle-treated controls, while females displayed 3 days of hyperphagia (P < 0.05). However, 1 wk after the injection, both male and female rats gained the same percent body weight (6%). Interestingly, female rats exhibited a greater reduction in energy expenditure (Vo2) following AgRP compared with male rats (P < 0.05). Removal of the gonads did not alter cumulative food intake in male or female rats but did attenuate the dramatic reduction in Vo2 exhibited by females. Both intact and gonadectomized rats demonstrated significantly increased respiratory quotient supporting the anabolic action of AgRP (P < 0.01). These findings are novel in that they reveal sex-specific underlying physiology used to achieve weight gain following central AgRP in rats.     

Central infusion of the melanocortin receptor antagonist agouti-related peptide (AgRP(83-132)) prevents cachexia-related symptoms induced by radiation and colon-26 tumors in mice

Cachexia is a clinical wasting syndrome that occurs in multiple disease states, and is associated with anorexia and a progressive loss of body fat and lean mass. The development of new therapeutics for this disorder is needed due to poor efficacy and multiple side effects of current therapies. The pivotal role played by the central melanocortin system in regulating body weight has made this an attractive target for novel cachexia therapies. The mixed melanocortin receptor antagonist AgRP is an endogenous peptide that induces hyperphagia. Here, we used AgRP(83-132) to investigate the ability of melanocortin antagonism to protect against clinical features of cachexia in two distinct animal models. In an acute model, food intake and body weight gain were reduced in mice exposed to radiation (300 RAD), and delivery of AgRP(83-132) into the lateral cerebral ventricle prevented these effects. In a chronic tumor cachexia model, adult mice were injected subcutaneously with a cell line derived from murine colon-26 adenocarcinoma. Typical of cachexia, tumor-bearing mice progressively reduced body weight and food intake, and gained significantly less muscle mass than controls. Administration of AgRP(83-132) into the lateral ventricles significantly increased body weight and food intake, and changes in muscle mass were similar to the tumor-free control mice. These findings support the idea that antagonism of the central melanocortin system can reduce the negative impact of cachexia and radiation therapy.     

Growth hormone receptor deficiency results in blunted ghrelin feeding response, obesity, and hypolipidemia in mice

We have previously shown that growth hormone (GH) overexpression in the brain increased food intake, accompanied with increased hypothalamic agouti-related protein (AgRP) expression. Ghrelin, which stimulates both appetite and GH secretion, was injected intracerebroventricularly to GHR-/- and littermate control (+/+) mice to determine whether ghrelin's acute effects on appetite are dependent on GHR signaling. GHR-/- mice were also analyzed with respect to serum levels of lipoproteins, apolipoprotein (apo)B, leptin, glucose, and insulin as well as body composition. Central injection of ghrelin into the third dorsal ventricle increased food consumption in +/+ mice, whereas no change was observed in GHR-/- mice. After ghrelin injection, AgRP mRNA expression in the hypothalamus was higher in +/+ littermates than in GHR-/- mice, indicating a possible importance of AgRP in the GHR-mediated effect of ghrelin. Compared with controls, GHR-/- mice had increased food intake, leptin levels, and total and intra-abdominal fat mass per body weight and deceased lean mass. Moreover, serum levels of triglycerides, LDL and HDL cholesterol, and apoB, as well as glucose and insulin levels were lower in the GHR-/- mice. In summary, ghrelin's acute central action to increase food intake requires functionally intact GHR signaling. Long-term GHR deficiency in mice is associated with high plasma leptin levels, obesity, and increased food intake but a marked decrease in all lipoprotein fractions.     

The melanocortin system and energy balance

The melanocortins, a family of peptides produced from the post-translational processing of pro-opiomelanocortin (POMC), regulate ingestive behavior and energy expenditure. Loss of function mutations of genes encoding POMC, or of either of two melanocortin receptors expressed in the central nervous system (MC3R, MC4R), are associated with obesity. The analyses of MC4R knockout mice indicate that activation of this receptor is involved in the regulation of appetite, the adaptive metabolic response to excess caloric consumption, and negative energy balance associated with cachexia induced by cytokines. In contrast, MC3R knockout mice exhibit a normal, or even exaggerated, response to signals that induce a state of negative energy balance. However, loss of the MC3R also results in an increase in adiposity. This article discusses the regulation of energy balance by the melanocortins. Published and newly presented data from studies analyzing of energy balance of MC3R and MC4R knockout mice indicate that increased adiposity observed in both models involves an imbalance in fat intake and oxidation.     

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.     

Viral mediated neuropeptide Y expression in the rat paraventricular nucleus results in obesity

Objective: Chronic central administration of neuropeptide Y (NPY) has dramatic effects on energy balance; however, the exact role of the hypothalamic paraventricular nucleus (PVN) in this is unknown. The aim of this study was to further unravel the contribution of NPY signaling in the PVN to energy balance. Research Methods and Procedures: Recombinant adeno‐associated viral particles containing NPY (rAAV‐NPY) were injected in the rat brain with coordinates targeted at the PVN. For three weeks, body weight, food intake, endocrine parameters, body temperature, and locomotor activity were measured. Furthermore, effects on insulin sensitivity and expression of NPY, agouti‐related protein (AgRP), and pro‐opiomelanocortin in the arcuate nucleus were studied. Results: Food intake was increased specifically in the light period, and dark phase body temperature and locomotor activity were reduced. This resulted in obesity characterized by increased fat mass; elevated plasma insulin, leptin, and adiponectin; decreased AgRP expression in the arcuate nucleus; and decreased insulin sensitivity; whereas plasma corticosterone was unaffected. Discussion: These data suggest that increased NPY expression targeted at the PVN is sufficient to induce obesity. Interestingly, plasma concentrations of leptin and insulin were elevated before a rise in food intake, which suggests that NPY in the PVN influences leptin and insulin secretion independently from food intake. This strengthens the role of the PVN in regulation of energy balance by NPY.     

Voluntary exercise delays monogenetic obesity and overcomes reproductive dysfunction of the melanocortin-4 receptor knockout mouse

The melanocortin system is involved in hypothalamic regulation of energy homeostasis. The melanocortin-4 receptor (MC4R) has been linked to both obesity and reproductive dysfunction. Deletion of the MC4R from the mouse genome has resulted in phenotypes including adult onset obesity, hyperphagia, and difficulty in reproducing when homozygote parents are bred. Additionally, polymorphisms of the human MC4R have been identified in morbidly obese children and adults. Herein, we have identified that voluntary exercise, provided via the presence of a running wheel, impedes the monogenetic obesity (at 20 weeks of age running wheel housed body weight=31+/-1.8 g versus conventionally housed body weight=41+/-2.3 g, a 25% decrease in body weight p<0.01), hyperphagia (average cumulative food intake is not statistically different than wild type mice housed in running wheel cages), and reproductive dysfunction phenotypes associated with the MC4R knockout mice housed by conventional means. These data demonstrate the novel finding that voluntary exercise at a young age may hinder genetically induced obesity.     

Melanocortin 1 receptor regulates melanoma cell migration by controlling syndecan-2 expression

The melanocortin 1 receptor (MC1R), a key regulator of melanogenesis, is known to control inflammation, acting in concert with the MC1R ligand α-melanocyte-stimulating hormone. Although cell migration is a key event in inflammation, few studies have addressed the function of MC1R in this context. Using highly motile melanoma cells, we found that the expression level of MC1R was associated with the extent of migration of mouse melanoma cells, suggesting that MC1R plays a functional role in controlling this migration. Overexpression of MC1R enhanced melanoma cell migration, whereas the opposite was true when MC1R levels were knocked down using small inhibitory RNAs. Interestingly, MC1R expression enhanced the synthesis of syndecan-2, a cell surface heparan sulfate proteoglycan known to be involved in melanoma cell migration. Knockdown of syndecan-2 expression decreased MC1R-mediated cell migration. Further, MC1R inhibited the activation of p38 MAPK, subsequently enhancing expression of sydnecan-2, in parallel with an increase in the extent of cell migration. Consistently, activation of p38 by H(2)O(2) inhibited syndecan-2 expression and cell migration, whereas inhibition of p38 activation enhanced syndecan-2 expression and cell migration. Finally, we found that α-melanocyte-stimulating hormone inhibited MC1R-mediated cell migration via activation of p38 and inhibition of syndecan-2 expression. Together, the data strongly suggest that MC1R regulates melanoma cell migration via inhibition of syndecan-2 expression.     

EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase

The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.     

Central insulin sensitivity in male and female juvenile rats

The incidence of juvenile obesity is increasing at an alarming rate. In adults, central insulin administration decreases hypothalamic orexigenic neuropeptides, food intake and body weight more effectively in males than females. Mechanisms regulating energy balance in juvenile animals are inherently different from those in adults due to differences in growth rates and hormonal milieu. Therefore, we sought to determine if central insulin treatment in juvenile rats (4 wk) would have similar sex-dependent effects on food intake as those reported in adult rats. Twenty-four hour food intake was measured following icv saline or insulin (0.01 or 0.1 U) prior to the onset of dark phase of the light cycle. An additional set of animals was used to assess the effects of central insulin on hypothalamic orexigenic (NPY, AgRP) and anorexigenic (POMC) neuropeptide mRNA expression. In both males and females, insulin reduced meal size initially (first 4 h) and later decreased meal frequency (4-24 h) to reduce cumulative food intake. Consistent with this, central insulin decreased hypothalamic NPY and AgRP and increased POMC mRNA expression. In contrast to adult studies, there were no demonstrated sex differences. These studies indicate that juvenile females and males are equally sensitive to central insulin anorexigenic effects, perhaps due to a lack of circulating gonadal hormones. The anorexigenic responsiveness of both genders suggests a potential pharmacologic approach to childhood obesity.     

AgRP(83-132) acts as an inverse agonist on the human-melanocortin-4 receptor

The central melanocortin (MC) system has been demonstrated to act downstream of leptin in the regulation of body weight. The system comprises alpha-MSH, which acts as agonist, and agouti-related protein (AgRP), which acts as antagonist at the MC3 and MC4 receptors (MC3R and MC4R). This property suggests that MCR activity is tightly regulated and that opposing signals are integrated at the receptor level. We here propose another level of regulation within the melanocortin system by showing that the human (h) MC4R displays constitutive activity in vitro as assayed by adenylyl cyclase (AC) activity. Furthermore, human AgRP(83-132) acts as an inverse agonist for the hMC4R since it was able to suppress constitutive activity of the hMC4R both in intact B16/G4F melanoma cells and membrane preparations. The effect of AgRP(83-132) on the hMC4R was blocked by the MC4R ligand SHU9119. Also the hMC3R and the mouse(m)MC5R were shown to be constitutively active. AgRP(83-132) acted as an inverse agonist on the hMC3R but not on the mMC5R. Thus, AgRP is able to regulate MCR activity independently of alpha-MSH. These findings form a basis to further investigate the relevance of constitutive activity of the MC4R and of inverse agonism of AgRP for the regulation of body weight.