Differential expression of the melanocortin-4 receptor in male and female C57BL/6J mice

The melanocortin 4 receptor is a member of melanocortin receptors of G-protein-coupled receptors. By binding to melanocortin receptor agonists or antagonists, MC4R participates in the regulating of food intake, weight, energy homeostasis and sexual behavior. By activating the protein kinase A and leptin-melanocortin signalling pathways, MC4R mediates the amplification of signals from the hypothalamo–pituitary–adrenal and hypothalamo–pituitary–thyroid axes. This process permits peripheral information about the status of energy metabolism to be transmitted to the central nervous system. The hypothalamic nuclei then integrate these signals to evoke the appropriate reaction. We found that different sexes exhibited distinct metabolic regulation abilities, likely due to differences in these signalling pathways. MC4R plays a key role in coordinating the afferent messages from the peripheral and regulatory signals by controlling food intake and energy expenditure. To probe the disparities in metabolism and weight regulation between the sexes, we analyzed the expression of MC4R in different tissues from male and female mice by qRT-PCR and immunofluorescence. The results show that the expression of MC4R in brain and kidney is higher in female mice than in male mice, but in the livers, the result is opposition. Additionally, in both sexes, the expression of MC4R is higher in the brain than in the kidneys, and its expression in the liver is lowest, in males, the expression of MC4R in the testis is higher than that in the kidneys. These data show that the expression of MC4R exist different between sexes mice.     

黑皮质素受体-4调节通路的研究进展

黑皮质素系统来自阿片-促黑素细胞皮质素原,在中枢摄食行为和能量平衡代谢中起到重要作用,此系统生理功能的发挥主要通过与下丘脑神经元细胞上特定膜受体（黑皮质素受体）结合完成。黑皮质素受体（MCR）有五种亚型（MC1R-MC5R）,其中参与体重调节的受体主要是黑皮质素受体3（MC3R）和黑皮质素受体4（MC4R）。MC4R属于G蛋白耦联受体,具有七次跨膜结构。作为一种膜受体,MC4R发挥体重调节作用,一方面受外界激动剂或拮抗剂的调节;另一方面,此受体活化后会影响到细胞内的信号调节通路。研究MC4R的功能首先要了解受体的结构,本文对G蛋白耦联受体的结构进行了较详细的叙述,MC4R经信号调节通路,激活腺苷酸环化酶,增加cAMP的浓度,最终通过影响细胞内基因的转录和翻译,来调节体重和能量的消耗。     

中枢ACTH受体研究进展

除垂体以外,中枢神经系统也含有促肾上腺皮质激素（ＡＣＴＨ）能神经元,其神经纤维在中枢具有较广泛的投射。ＡＣＴＨ相关肽类在中枢发挥着多种生理功能。近年来对于中枢ＡＣＴＨ受体的研究取得很大的进展,现已确认ＡＣＴＨ结合位点在中枢具有广泛的分布。新近克隆出的四种ＡＣＴＨ受体中,有两种是中枢神经系统占优势的受体亚型。     

Role of alpha-melanocyte stimulating hormone and melanocortin 4 receptor in brain inflammation

Inflammatory processes contribute widely to the development of neurodegenerative diseases. The expression of many inflammatory mediators was found to be increased in central nervous system (CNS) disorders suggesting that these molecules are major contributors to neuronal damage. Melanocortins are neuropeptides that have been implicated in a wide range of physiological processes. The melanocortin alpha-melanocyte stimulating hormone (alpha-MSH) has pleiotropic functions and exerts potent anti-inflammatory actions by antagonizing the effects of pro-inflammatory cytokines and by decreasing important inflammatory mediators. Five subtypes of melanocortin receptors (MC1R-MC5R) have been identified. Of these, the MC4 receptor is expressed predominantly throughout the CNS. Evidence of effectiveness of selective MC4R agonists in modulating inflammatory processes and their low toxicity suggest that these molecules may be useful in the treatment of CNS disorders with an inflammatory component. This review describes the involvement of the MC4R in central anti-inflammatory effects of melanocortins and discusses the potential value of MC4R agonists for the treatment of inflammatory-related disorders.     

Melanocortin-4 receptors, beta-MSH and leptin: key elements in the satiety pathway

This paper reviews aspects of our research, focusing on the role of the melanocortin system in the central regulation of feeding and energy balance, which was begun in 1997. It describes data from successive physiological studies, concerning the identity of the appetite-regulating melanocortin receptor, melanocortin-4 receptor (MC4R) regulation with altered nutritional status, the role of MC4R in dietary obesity and the identity of the endogenous MC4R ligand.     

Involvement of melanocortin-4 receptor in anxiety and depression

The melanocortins, which are derived from proopiomelanocortin, have a variety of physiological functions mediated membrane surface receptors. To date, five subtypes have been cloned. With the cloning of melanocortin receptors, studies with genetic models, and development of selective compounds, the physiological roles of the five melanocortin receptors have begun to be understood. The melanocortin-4 receptor (MC4R), which is predominantly expressed in the central nervous system, has in particular become the focus of much attention in recent years because of the critical roles it plays in a wide range of functions, including feeding, sexual behavior, and stress. Recent development of selective antagonists for the MC4R has provided pharmacological evidence that blockade of MC4R could be a useful way of alleviating numerous conditions such as anxiety/depression, pain, and addiction to drugs of abuse.     

Gamma-MSH/MC3R natriuretic signaling system: The possible explanation for contrasting blood pressure effects of agouti mutation and melanocortin receptor knockout

Overweight and obesity are the major risk factors of arterial hypertension. Recent studies indicate that adipose tissue hormone, leptin, is involved in the development of obesity-induced hypertension. Models of genetically determined obesity in rodents are commonly used to study the pathogenesis of obesity-associated hypertension. One of such models are agouti yellow obese (A^y/a) mice which ubiquitously overexpress agouti protein—an endogenous antagonist of melanocortin receptors normally synthesized only in the hair follicle. In A^y/a mice, agouti protein is synthesized also in the hypothalamus and blocks the anorectic effect of leptin mediated by alpha-melanocyte-stimulating hormone (α-MSH) which binds to melanocortin type 3 and 4 receptors (MC3R and MC4R). Consequently, A^y/a mice are hyperphagic, obese, hyperinsulinemic and hyperleptinemic. Blood pressure is increased in A^y/a mice due to increased serum leptin level. In contrast, blood pressure is reduced in MC4R-null mice despite obesity and hyperleptinemia, and is not increased by the administration of leptin in these animals, suggesting an essential role of the melanocortin pathway in the hypertensive effect of leptin. Herein, I propose the hypothesis which might explain why blood pressure is increased in A^y/a mice but reduced in MC4R−/− mice, although hypothalamic melanocortin signaling is impaired in both models. According to this proposal, in MC4R−/− mice the natriuretic effect of γ-MSH mediated by intrarenal MC3R is preserved and counteracts prohypertensive mechanisms triggered by leptin. In contrast, in A^y/a mice, ubiquitously expressed agouti protein blocks not only hypothalamic MC4R but also renal MC3R and thus impairs γ-MSH-induced natriuresis, leading to blood pressure elevation due to unopposed central and/or peripheral pressor effects of leptin.     

α-MSH Stimulates Glucose Uptake in Mouse Muscle and Phosphorylates Rab-GTPase-Activating Protein TBC1D1 Independently of AMPK

The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure regulation, which are mainly mediated by MC4R. The pituitary gland is the source of various POMC-derived hormones released to the circulation, which raises the possibility that there may be actions of the melanocortins on peripheral energy homeostasis. In this study, we examined the molecular signaling pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate α-MSH stimulation in both wild type and AMPK deficient mice. We found that α-MSH significantly induces phosphorylation of TBC1 domain (TBC1D) family member 1 (S237 and T596), which is independent of upstream PKA and AMPK. We find no evidence to support that α-MSH-stimulated glucose uptake involves TBC1D4 phosphorylation (T642 and S704) or GLUT4 translocation.     

Serotonergic pathways converge upon central melanocortin systems to regulate energy balance

Multiple lines of research provide compelling support for an important role for central serotonergic (5-hydroxytryptamine, 5-HT) and melanocortin pathways in the regulation of food intake and body weight. In this brief review, we outline data supporting a model in which serotonergic pathways affect energy balance, in part, by converging upon central melanocortin systems to stimulate the release of the endogenous melanocortin agonist, alpha-melanocyte stimulating hormone (alpha-MSH). Further, we review the neuroanatomical mapping of a downstream target of alpha-MSH, the melanocortin 4 receptor (MC4R), in the rodent brain. We propose that downstream activation of MC4R-expressing neurons substantially contributes to serotonin's effects on energy homeostasis.     

Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.     

Ghrelin-induced food intake and growth hormone secretion are altered in melanocortin 3 and 4 receptor knockout mice

Ghrelin stimulates food intake in part by activating hypothalamic neuropeptide Y (NPY) neurons/agouti related peptide (AGRP) neurons. We investigated the role of AGRP/melanocortin signaling in ghrelin-induced food intake by studying melanocortin 3 and 4 receptor knockout (MC3R KO and MC4R KO) mice. We also determined whether reduced ghrelin levels and/or an altered sensitivity to the GH-stimulating effects of ghrelin accompany the obesity syndromes of MC3R KO and MC4R KO mice. Compared to wild-type (WT) mice, the effects of ghrelin on food intake were reduced in MC3R KO and MC4R KO mice and circulating ghrelin levels were reduced in female MC4R KO mice. Female MC3R KO and MC4R KO mice exhibited a diminished responsiveness to the GH-releasing effects of ghrelin. Thus, deletion of the MC3R or MC4R results in a decreased sensitivity to ghrelin and verifies the involvement in the melanocortin system in ghrelin-induced food intake.     

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.     

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.     

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.     

Feeding effects of melanocortin ligands--a historical perspective

The process of energy homeostasis is a highly regulated process involving interacting signals between a variety of anorexigenic and orexigenic peptides, proteins and signaling molecules. The melanocortin system is an important component of this complex regulatory network. Involvement of the melanocortin pathway in the control of food intake and body weight regulation has been studied extensively in the past two decades. Previous studies that involve central administration of melanocortin molecules and examination of molecules that effect food intake in melanocortin knockout (KO) mice (MC3R, MC4R, POMC, AGRP and NPY) have been examined. In this review, we have summarized feeding studies that have resulted in the recognition of the melanocortin system as a major contributor to the complex neuroendocrine system regulating energy homeostasis.     

Discovery and in vivo evaluation of new melanocortin-4 receptor-selective peptides

The melanocortin-4 receptor (MC4R) is involved in several physiological processes, including body weight regulation and grooming behaviour in rats. It has also been suggested that the MC4R mediates the effects of melanocortin ligands on neuropathic pain. Selective compounds are needed to study the exact role of the MC4R in these different processes. We describe here the development and evaluation of new melanocortin compounds that are selective for the MC4R as compared with the other centrally expressed receptors, MC3R and MC5R. First, a library of 18 peptides, in which a melanocortin-based sequence was systematically point-mutated, was screened for binding to and activity on the MC3R, MC4R and MC5R. Compound Ac-Nle-Gly-Lys-D-Phe-Arg-Trp-Gly-NH(2) (JK1) appeared to be the most selective MC4R compound, based on affinity. This compound is 90- and 110-fold selective for the MC4R as compared to the MC3R and MC5R, respectively. Subsequent modification of JK1 yielded compound Ac-Nle-Gly-Lys-D-Nal(2)-Arg-Trp-Gly-NH(2) (JK7)(,) a selective MC4R antagonist with 34-fold MC4R/MC3R and 109-fold MC4R/MC5R selectivity. The compounds were active in vivo as determined in a grooming assay and a model for neuropathic pain in rats. Intravenous (i.v.) injections suggested that they were able to pass the blood-brain barrier.The compounds identified here will be useful in further research on the physiological roles of the MC4R.     

Design and bioactivities of melanotropic peptide agonists and antagonists: Design based on a conformationally constrained somatostatin template

-Melanotropin and ACTH, POMC peptides, initiate biological activity by interaction with the classical pigment cell (-MSH receptor, MC1R) and adrenal gland (ACTH receptor, MC2R) melanocortin receptors, respectively. The recently discovered MC3R, MC4R and MC5R receptors provide new targets and new biological functions for POMC peptides. We have developed conformationally constrained -melanotropin peptides that interact with all of these receptors as agonists and antagonists and are examining new approaches to obtain highly selective ligands for each of these melanocortin receptors. Previously, we had converted somatostatin-derived peptides into potent and highly selective analogues that act as antagonists at the opioid receptors. Using the reverse turn template that came out of these studies, we have designed, de novo, agonist and antagonist peptide analogues that interact with melanocortin receptors.