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The pentapeptides methionine-enkephalin and leucine-enkephalin belong to the opioid family of peptides, and the non-opiate peptide adrenocorticotropin hormone (ACTH) to the melanocortin peptide family. Enkephalins/ACTH are derived from pro-enkephalin, pro-dynorphin or pro-opiomelanocortin precursors and, via opioid and melanocortin receptors, are responsible for many biological activities. Enkephalins exhibit the highest affinity for the δ receptor, followed by the μ and κ receptors, whereas ACTH binds to the five subtypes of melanocortin receptor, and is the only member of the melanocortin family of peptides that binds to the melanocortin-receptor 2 (ACTH receptor). Enkephalins/ACTH and their receptors exhibit a widespread anatomical distribution. Enkephalins are involved in analgesia, angiogenesis, blood pressure, embryonic development, emotional behavior, feeding, hypoxia, limbic system modulation, neuroprotection, peristalsis, and wound repair; as well as in hepatoprotective, motor, neuroendocrine and respiratory mechanisms. ACTH plays a role in acetylcholine release, aggressive behavior, blood pressure, bone maintenance, hyperalgesia, feeding, fever, grooming, learning, lipolysis, memory, nerve injury repair, neuroprotection, sexual behavior, sleep, social behavior, tissue growth and stimulates the synthesis and secretion of glucocorticoids. Enkephalins/ACTH are also involved in many pathologies. Enkephalins are implicated in alcoholism, cancer, colitis, depression, heart failure, Huntington's disease, influenza A virus infection, ischemia, multiple sclerosis, and stress. ACTH plays a role in Addison's disease, alcoholism, cancer, Cushing's disease, dermatitis, encephalitis, epilepsy, Graves' disease, Guillain-Barré syndrome, multiple sclerosis, podocytopathies, and stress. In this review, we provide an updated description of the enkephalinergic and ACTH systems., (© 2019 Elsevier Inc. All rights reserved.)

ACTH resistance is a rare disorder typified by familial glucocorticoid deficiency (FGD), a genetically heterogeneous disease. Previously, genetic defects in FGD have been identified in the ACTH receptor gene (MC2R), its accessory protein (MRAP) and the steroidogenic acute regulatory protein gene (STAR). The defective mechanisms here are failures in ACTH ligand binding and/or receptor trafficking for MC2R and MRAP and, in the case of STAR mutations, inefficient cholesterol transport to allow steroidogenesis to proceed. Novel gene defects in FGD have recently been recognised in mini-chromosome maintenance-deficient 4 homologue (MCM4) and nicotinamide nucleotide transhydrogenase (NNT). MCM4 is one part of a DNA repair complex essential for DNA replication and genome stability, whilst NNT is involved in the glutathione redox system that protects cells against reactive oxygen species. The finding of mutations in these two genes implicates new pathogenetic mechanisms at play in FGD, and implies that the adrenal cortex is exquisitely sensitive to replicative and oxidative stresses., (Copyright © 2013 S. Karger AG, Basel.)

Abnormalities in the cAMP/PKA signaling pathway have been linked to the formation of benign adrenal tumors, as well as a possible predisposition to adrenocortical cancer. Mutations in the G-protein coupled receptor are associated with McCune-Albright syndrome and ACTH-independent macronodular adrenal hyperplasia, while defects in cAMP-dependent protein kinase A can lead to the development of Carney's complex, as well as primary pigmented nodular adrenocortical disease (PPNAD), and micronodular adrenocortical hyperplasia (MAH). Defects in phosphodiesterases, which regulate cAMP levels, have also been demonstrated in PPNAD and MAH. The Wnt signaling pathway, which is involved in oncogenesis in a variety of tumors, has also been implicated in adrenocortical tumorigenesis. MicroRNA profiling has added to our understanding of the signaling pathways involved in tumor formation in the adrenal cortex. Will this all lead to the development of specific targets for pharmacologic therapies? In this article, we review the molecular genetics of adrenocortical tumors and refer to potential targets for pharmacologic therapy.

Adrenocorticotropin (ACTH) is one of several peptide hormones derived from a larger molecule, proopiomelanocortin (POMC). ACTH is a classic endocrine hormone, processed and secreted from the pituitary to stimulate cortisol production from the fasciculata cells in the adrenal gland. However, ACTH is also produced by other cells, including macrophages, at many sites in the body. ACTH binds to a specific member of the melanocortin receptor family, the MC2R. MC2R is expressed in osteoblastic cells in vivo, as shown by in situ hybridization. MC2R expression is strongest at sites of active bone deposition, and thus ACTH response probably varies with osteoblastic activity or stage of osteoblast differentiation. In vitro ACTH stimulates proliferation of osteoblasts in a dose-dependent manner. ACTH at 10 nM increases collagen I mRNA in the osteoblastic cell line SaOs2, although at lower concentrations ACTH may oppose osteoblast differentiation. ACTH is thus, at high concentrations, anabolic for the osteoblast, and it is highly likely that the hormone has concentration-dependent effects on bone metabolism in vivo.

Context: Primary adrenal Cushing's syndrome can result from the aberrant adrenal expression of several hormone receptors; this mechanism has not been explored in detail in aldosterone-producing tumors., Objective: The objective of the study was to evaluate a 56-yr-old male patient with an aldosteronoma for the regulation of aldosterone secretion by aberrant hormone receptors., Results: Renin-independent stimulation of aldosterone secretion was observed in vivo after a mixed meal, oral glucose, or administration of glucose-dependent insulinotropic peptide (GIP), vasopressin, and tegaserod. The mixed meal-mediated stimulation of aldosterone was not present in five other cases of aldosteronoma. A smaller response of aldosterone after GIP infusion was observed in a normal subject. Aldosterone secretion was stimulated by GIP in primary cultures of this patient's aldosteronoma. Increased expression of GIP receptor was found in this aldosteronoma by real-time RT-PCR and immunohistochemistry. The GIP receptor protein was also found at lower levels in zona glomerulosa cells of the normal adjacent adrenal gland. Increased expression of serotonin 4 and ACTH receptors was also present in this aldosteronoma., Conclusions: This case report provides new evidence of the implication of aberrant hormone receptors in the regulation of this aldosteronoma and suggests that further detailed studies of the role of aberrant hormone receptors in this frequent pathology should be undertaken.

The vacuolated lens (vl) mouse mutant arose spontaneously on the C3H/HeSn background and exhibits neural tube defects (NTDs), congenital cataract, and occasionally a white belly spot. We previously reported that 1) the vl phenotypes are due to a mutation in an orphan G protein-coupled receptor (GPCR), Gpr161; 2) the penetrance of the vl NTD and cataract phenotypes are affected by genetic background, allowing three unlinked quantitative trait loci (QTL) to be mapped (modifiers of vacuolated lens, Modvl1-3); and 3) phenotype-based bioinformatics followed by genetic and molecular analysis identified a lens-specific transcription factor that contributes to the cataract-modifying effect of Modvl3. We now extend this analysis in three ways. First, using the Gpr161 mutation to unequivocally identify mutant adults and embryos, we determined that approximately 50% of vl/vl NTD-affected embryos die during development. Second, the MOLF/Ei genetic background suppresses this embryonic lethality but increases the incidence of the adult belly spot phenotype. Additional QTL analysis was performed, and two novel modifiers were mapped [Modvl4, logarithm of odds ratio (LOD) 4.4; Modvl5, LOD 5.0]. Third, phenotype-based bioinformatics identified candidate genes for these modifiers including two GPCRs that cause NTD or skin/pigmentation defects (Modvl4: Frizzled homolog 6; Modvl5: Melanocortin 5 receptor). Because GPCRs form oligomeric complexes, these genes were resequenced and nonsynonymous coding variants were identified. Bioinformatics and protein modeling suggest that these variants may be functional. Our studies further establish vl as a multigenic mouse model for NTDs and identify additional QTL that interact with Gpr161 to regulate neurulation.

The genetic mechanisms underlying the regulation of adrenarche are unknown. The aim of the study was to find out whether ACTH receptor (MC2R) promoter polymorphism associates with premature adrenarche (PA) and its characteristics. DNA samples of 74 prepubertal children with PA and their age- and gender-matched 97 healthy controls were genotyped for the -2 bp T/C diallelic MC2R promoter polymorphism (MC2R -2 T>C) All children were examined clinically, and hormonal measurements after an overnight fast and a low-dose ACTH stimulation test were performed. In controls, the baseline ACTH/cortisol ratio was significantly higher (p = 0.002) in subjects with the polymorphism than in the T/T group indicating decreased ACTH sensitivity. The frequency of the MC2R -2 T>C polymorphism was significantly higher in PA children with premature pubarche than in those with milder signs of PA or in control children (p = 0.04). In children with PA, the polymorphism associated with higher baseline serum dehydroepiandrosterone (p = 0.03), androstenedione (p = 0.02), plasma ACTH (p = 0.03) levels and with lower birth weight (p = 0.02). Our study provides evidence that the MC2R promoter polymorphism modulates the hypothalamo-pituitary-adrenal axis in children and may play a role in altered regulation of adrenarche.

Vasopressin (VP) secreted from parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) stimulates pituitary adrenocorticotropic hormone (ACTH) secretion, through interaction with receptors of the V1b subtype (V1bR) in the pituitary corticotroph, mainly by potentiating the stimulatory effects of corticotrophin-releasing hormone (CRH). Chronic stress paradigms associated with corticotroph hyperresponsiveness lead to preferential expression of hypothalamic VP over CRH and upregulation of pituitary V1bR, suggesting that VP has a primary role during adaptation of the hypothalamic pituitary adrenal (HPA) axis to long-term stimulation. However, studies using pharmacological or genetic ablation of V1bR have shown that VP is required for full ACTH responses to some stressors, but not for the sensitization of ACTH responses to a novel stress observed during chronic stress. Studies using minipump infusion of a peptide V1 antagonist in long-term adrenalectomized rats have revealed that VP mediates proliferative responses in the pituitary. Nevertheless, only a minor proportion of cells undergoing mitogenesis co-express markers for differentiated corticotrophs or precursors, suggesting that new corticotrophs are recruited from yet undifferentiated cells. The overall evidence supports a limited role of VP regulating acute ACTH responses to some acute stressors and points to cell proliferation and pituitary remodelling as alternative roles for the marked increases in parvocellular vasopressinergic activity during prolonged activation of the HPA axis.

Familial glucocorticoid deficiency (FGD) and triple A syndrome belong to a rare group of autosomal recessive disorders characterized by adrenocorticotropin (ACTH) insensitivity. Unlike triple A syndrome which presents a range of clinical features, FGD is solely characterized by glucocorticoid deficiency. ACTH regulates steroid biosynthesis in the adrenal cortex by exerting its effects via the ACTH receptor (melanocortin- 2 receptor, MC2R). In FGD, mutations in the MC2R account for only approximately 25% of cases (FGD type 1). The inability to express a functional MC2R in non-adrenal cell lines had implied the presence of an adrenal specific accessory factor(s), essential for MC2R expression. More recently, this factor was identified as melanocortin receptor accessory protein (MRAP). Mutations in MRAP account for 20% of cases (FGD type 2). Like the receptor activity-modifying proteins (RAMPs) and receptor transporter proteins (RTPs), which are well-characterized accessory proteins for G-protein-coupled receptors (GPCRs), MRAP is a small single transmembrane domain protein. MRAP is essential for the functional expression of the MC2R. About 55% of FGD cases have no identifiable gene defect, implying the involvement of additional genes. This chapter briefly describes the clinical and biochemical features of ACTH resistance syndromes. However, we will focus on the recent progress made towards understanding the molecular defect underlying these conditions, in particular the interaction of MC2R and MRAP.

The melanocortin system refers to a set of hormonal, neuropeptidergic, and paracrine signaling pathways that are defined by components that include the five G protein-coupled melanocortin receptors; peptide agonists derived from the proopiomelanocortin preprohormone precursor; and the endogenous antagonists, agouti and agouti-related protein. This signaling system regulates a remarkably diverse array of physiological functions including pigmentation, adrenocortical steroidogenesis, energy homeostasis, natriuresis, erectile responses, energy homeostasis, and exocrine gland secretion. There are many complex and unique aspects of melanocortin signaling, such as the existence of endogenous antagonists, the agouti proteins, that act at three of the five melanocortin receptors. However, there is an aspect of melanocortin signaling that has facilitated highly reductionist approaches aimed at understanding the physiological functions of each receptor and peptide: in contrast to many peptides, the melanocortin agonists and antagonists are expressed in a limited number of very discrete locations. Similarly, the melanocortin receptors are also expressed in a limited number of discrete locations where they tend to be involved in rather circumscribed physiological functions. This review examines my laboratory's participation in the cloning of the melanocortin receptors and characterization of their physiological roles.

The nervous system contributes to the mechanisms of ocular immune privilege by the constitutive presence of the immunosuppressive neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) in the eye. Alpha-MSH through the melanocortin 5 receptor (MC5r) mediates induction of CD4+ regulatory T cells that suppress in an antigen specific manner autoimmune disease. We investigated whether there was a role for MC5r expression in ocular immunity and the natural induction of regulatory T cells that emerged following resolution of experimental autoimmune uveoretinitis (EAU). Unlike wild type mice, EAU in MC5r-/- mice caused severe retinal damage, did mice expressed a not induce the emergence of ocular autoantigen regulatory immunity in the spleen, and the MC5r-/- classical memory immune response when reimmunized with ocular autoantigen. There was expression of MC5r in retinal pigment epithelial cells, in the ganglion cell and neural outer plexiform layers of healthy wild type mice retinas. The recovery of the ocular microenvironment from EAU was not dependent on the expression of MC5r, nor was the recovery dependent on the induction of CD4+ regulatory T cells (Treg cells) in the spleen. However, protection of the retina from the inflammatory damage of EAU and the induction of ocular autoantigen-responsive CD4+ Treg cells in the post EAU spleen requires expression of MC5r.

The action of the peptide hormone adrenocorticotropin (ACTH) to stimulate glucocorticoid production by the adrenal gland is an essential physiologic process, yet is dependent on a single unique genetic component--the ACTH receptor or melanocortin 2 receptor. Genetic defects that cause abnormalities in this receptor or in a protein required for its expression at the cell surface result in a potentially fatal disease (familial glucocorticoid deficiency). Overexpression of this receptor or inability to desensitize it is found in adrenal adenomas or hyperplasia associated with glucocorticoid overproduction (Cushing syndrome). These disorders are uncommon, but there are considerable data to show that the hypothalamo-pituitary-adrenal axis is overactive, or in some circumstances underactive, in more common situations including depressive illness and septic shock. The origin of these latter disturbances is undoubtedly complex and multifactorial, but there is good evidence that a component of this phenomenon is an altered responsiveness of the ACTH receptor to ACTH. Understanding the basis of ACTH responsiveness might, therefore, contribute to the understanding of disorders such as these and perhaps enable the hypothalamo-pituitary-adrenal axis to be manipulated beneficially in these circumstances.

The present series of studies aimed to further our understanding of the role of melanin-concentrating hormone (MCH) neurones in the central regulation of luteinising hormone (LH) release in the female rat. LH release was stimulated when MCH was injected bilaterally into the rostral preoptic area (rPOA) or medial preoptic area (mPOA), but not when injected into the zona incerta (ZI), of oestrogen-primed ovariectomised rats. In rats that were steroid-primed to generate a surge-like release of LH, MCH administration into the ZI blocked this rise in LH release: no such effect occurred when MCH was injected into the rPOA or mPOA. In vitro, MCH stimulated gonadotrophin-releasing hormone (GnRH) release from hypothalamic explants. Double-label immunohistochemistry showed GnRH-immunoreactive neurones in the vicinity of and intermingled with immunoreactive MCH processes. MCH is the endogenous ligand of the MCH type 1 receptor (MCH1-R). Previously, we have shown a role for melanocortin-5 receptors (MC5-R) in the stimulatory action of MCH, so we next investigated the involvement of both MCH1-R and/or MC5-R in mediating the actions of MCH on GnRH and hence LH release. The stimulatory action of MCH in the rPOA was inhibited by administration of antagonists for either MCH1-R or MC5-R. However, in the mPOA, the action of MCH was blocked only by the MC5-R antagonist. LH release was stimulated by an agonist for MC5-R injected into the rPOA or mPOA; this was blocked by the MC5-R antagonist but not the MCH1-R antagonist. These results indicate that both MCH1-R and MC5-R are involved in the central control of LH release by MCH.

Expression of melanocortin-4 receptor (MC4R) mRNA in developing rat limb buds, teeth, and skull bone first indicated a possible role for MC4R in bone metabolism. We therefore investigated whether MC4R mRNA was expressed in the rat osteosarcoma UMR106.06 cell line and in primary rat osteoblast cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot analysis, and ribonuclease protection assay (RPA) were used to demonstrate MC4R mRNA expression in UMR106.06 and primary osteoblast cells. MC4R mRNA was found to be localized to the periosteum of mouse bone using in situ hybridization. We also used RT-PCR and rat specific MC2R and MC5R oligonucleotides to amplify the correct size DNA fragments for these melanocortin receptors from rat primary osteoblasts. In conclusion, melanocortin receptor expression in mouse periosteum and rat osteoblasts suggests a direct role for POMC derived peptides in bone development and bone metabolism.

Context: Cortisol secretion is usually under the control of ACTH. However, cortisol secretion occurs in response to gastric inhibitory polypeptide (GIP) in rare cases of food-dependent Cushing's syndrome (CS)., Objective: We have investigated whether chronic ACTH stimulation or activation of the ACTH signaling pathway might be associated with GIP receptor (GIPR) expression., Design: RT-PCR analysis and primary culture of hyperplastic adrenals., Patients: All patients presented with CS: 20 unilateral adrenal adenomas, five Cushing's disease, one food-dependent CS., Results: RT-PCR revealed GIPR expression in all hyperplastic adrenals studied. No RT-PCR product could be detected in two normal adrenals or 20 hyperfunctioning adrenal adenomas. Primary culture revealed a significant cAMP response to ACTH in all adrenals available for study (EC50, 8.1 x 10(-10) M in normals, 4.7 x 10(-10) M in Cushing's disease, and 4.4 x 10(-10) M in food-dependent disease). However, cultures taken from all four ACTH-dependent and the one food-dependent hyperplastic adrenals studied were also responsive to GIP (EC50 for cAMP, 1.3 x 10(-9) M in Cushing's disease and 4.1 x 10(-10) M in food-dependent disease). Fasting cortisol levels were low in the case of food-dependant Cushing's, rising postprandially as predicted. However, there was no trend toward low fasting or high postprandial cortisol in the other cases, suggesting that the presence of detectable GIPR alone, albeit with definite function in vitro, is not sufficient to cause clinically food-dependent CS., Conclusions: These data are consistent with the hypothesis that chronic ACTH stimulation or constitutive activation of the ACTH signaling pathway may be associated with aberrant GIPR expression, and suggest one mechanism for the pathogenesis of this phenomenon.

Siberian hamsters accumulate fat reserves in long photoperiods, but show a long-term decrease in food intake and body weight when exposed to a short winter photoperiod. The aim of this study was to determine the role of central melanocortin 3/4 receptors (MC3/4-R) in generating this chronic catabolic state by investigating the effects of SHU9119, a MC3/4-R antagonist, on food intake and associated behaviours. In adult male hamsters, intra-cerebroventricular infusions of SHU9119 significantly increased food intake in a dose-dependent manner. The time course of action was slow, food intake being increased between 4 and 24 h after intra-cerebroventricular administration. A similar degree of increase in food intake occurred in fat hamsters in long days and in lean hamsters chronically exposed to short days. Intra-cerebroventricular treatment with MTII (a MC3/4-R agonist) significantly decreased food intake for up to 24 h after treatment, and SHU9119 reversed these suppressive effects between 4 and 24 h after treatment, a similar time course to that observed when SHU9119 was administered alone. We conclude that endogenous melanocortin peptides acting via MC3/4-R are involved in the regulation of food intake in hamsters in both anabolic and catabolic states, but these acute studies do not provide evidence that increased activity of this hypothalamic system underlies the seasonal decrease in food intake that contributes to the long-term catabolic state in short days., (Copyright 2003 Elsevier B.V.)

The endogenous melanocortin, alpha-melanocyte-stimulating hormone (alpha-MSH), is a neurohormone secreted by the neurointermediate lobe of the pituitary. Alpha-MSH promotes intermale aggression in mice by influencing pheromone secretion, but the role of specific melanocortin receptors has not been determined. We assessed mice made deficient in the gene for the melanocortin-5 receptor (MC5R) to determine its role in pheromone-regulated behavior. In heterotypic pairs assessed in the social interaction test (SIT), MC5R-deficient mice exhibited less aggressive behavior and more defensive behavior than their wild-type opponents. By contrast, when assessed in homotypic pairs and against stimulus animals in the SIT, MC5R-deficient and wild-type mice behaved similarly. Moreover, urine from MC5R deficient mice stimulated more aggression than did urine from wild-type mice. The results suggest that MC5R deficiency disinhibits an aggression-suppressing pheromonal signal.

The melanocortins are a group of small protein hormones derived by post-translational cleavage of the proopiomelanocortin (POMC) gene product. The known melanocortin hormones include alpha-melanocyte stimulating hormone (MSH), beta-MSH, gamma-MSH and adrenocorticotropic hormone (ACTH). Five melanocortin receptors (MCIR through to MC5R) have been identified and most of these show tissue-specific expression patterns, as well as different binding affinities for each of the melanocortin hormones. The central melanocortin system consists of alpha-MSH, agouti-related protein (AGRP), MC3R and MC4R. AGRP and alpha-MSH are believed to be the natural antagonist and agonist respectively of MC3R and MC4R. This central melanocortin system is thought to play a fundamental role in the control of feeding and body weight. Knock-out mice models and genetic studies have pointed to the importance of the melanocortins in complex human pathways such as pigmentation, lipolysis, food intake, thermogenesis, sexual behaviour, memory and inflammatory response. Recently the melanocortins and their receptors have been the target for drug-based treatment of human physiological processes. MC3R and MC4R are likely targets for controlling body weight; MCIR may be used in the treatment of inflammation and MC2R for the treatment of glucocortical deficiency. A role for MCSR still remains unclear, but the evidence suggests an exocrine gland function.

Adrenal masses are a common problem affecting 3-7% of the population. The majority turn out to be benign adrenocortical adenomas, which may be functional or non-functional. Much more rarely, these masses represent a primary adrenal carcinoma. It is becoming increasingly recognized that of the benign functioning adenomas or hyperplasias, the majority will hypersecrete aldosterone and this will be more frequently detected when hypertensive populations are screened for this disease. In contrast, the incidence of primary adrenocortical carcinoma has remained steady and for this disease, surgery represents the mainstay of treatment. The advent of laparoscopic adrenal surgery has lowered the threshold size for recommending surgery for asymptomatic adrenal masses and as such, an increased proportion of adrenocortical cancers are being resected and detected at an earlier stage. Recent progress has been made in our understanding of the key genetic changes which underpin the biology of this disease. Progression from adrenal adenoma to carcinoma involves a monoclonal proliferation of cells which, among other defects, have undergone chromosomal duplication at the 11p15.5 locus leading to overexpression of the IGF2 gene and abrogation of expression of the CDKN1C and H19 genes. TP53 is involved in progression to carcinoma in a subset of patients and the frequency of ACTH receptor deletion needs to be more fully explored. Other key oncogenes and tumour suppressor genes remain to be identified although the chromosomal loci in which they lie can be identified at 17p, 1p, 2p16 and 11q13 for tumour suppressor genes and chromosomes 4, 5 and 12 for oncogenes.

The purpose of this study was to determine if leptin could induce a leptin resistance in young rats and if leptin-induced leptin resistant rats are responsive to the melanocortin agonist, melanotan II (MT II). Recombinant adeno-associated virus encoding rat leptin cDNA (rAAV-leptin) or control viral vector were administered into young, lean rats for 300 days, and food consumption, body weight, oxygen consumption, serum leptin, and leptin signal transduction were measured. In the rAAV-leptin rats, the anorexic response attenuated by day 140, and the increase in energy expenditure attenuated prior to day 223. At day 300, the rats were challenged with centrally administered recombinant leptin to test leptin responsiveness. Whilst the control responded with a decrease in food intake, the rAAV-leptin rats were unresponsive. The same rats were administered MT II subsequently, and both the leptin-resistant and leptin-responsive groups displayed reduced food intake. Notably, the anorexic response persisted longer in the leptin-resistant group. These data demonstrate that leptin induces leptin resistance in young rats and that these leptin-resistant rats have an enhanced anorexic response to exogenous melanocortin activation. We suggest that defective endogenous melanocortin activation may represent one element of leptin resistance, leading to compensatory hypersensitivity of the melanocortin pathway.

Central melanocortin signaling plays an important role in regulation of energy homeostasis by leptin and insulin. We investigated the interaction between leptin, insulin, and melanocortin-4 receptors (MC-4Rs) in the control of renal sympathetic nerve activity (RSNA) in mice. We compared the effects of intracerebroventricular (ICV) administration of leptin, insulin, MC-3/4R agonist (MTII), and corticotrophin-releasing factor (CRF) on RSNA in leptin receptor-deficient (db/db) mice, MC-4R knock-out mice, and their wild-type controls. ICV administration of leptin and MTII caused a significant and dose-dependent increase in RSNA in control mice. As expected, leptin had no significant effect on RSNA in the db/db mice. Interestingly, db/db mice exhibited markedly attenuated RSNA responses to ICV administration of MTII. However, the increase in RSNA induced by insulin and CRF was comparable between db/db and control mice. In the heterozygous and homozygous MC-4R knock-out mice, the RSNA response to MTII was attenuated and abolished, respectively. The RSNA response to ICV leptin and insulin was also attenuated and abolished in the heterozygous and homozygous MC-4R knock-out mice, respectively. In contrast, CRF induced a similar increase in RSNA in the MC-4R knock-out and wild-type mice. Our data demonstrate that in the absence of leptin receptors, the sympathoexcitatory effects of melanocortin system stimulation are attenuated. In addition, the renal sympathoexcitatory responses to leptin and insulin are dependent on the MC-4R, demonstrating an important role for the MC-4R in the regulation of renal sympathetic nerve outflow by leptin and insulin.

The melanocortins are a family of bioactive peptides derived from proopiomelanocortin. Those peptides, included among hormones and comprising ACTH, alpha-MSH, beta-MSH and gamma-MSH, are best known mainly for their physiological effects, such as the control of skin pigmentation by alpha-MSH, and ACTH effects on pigmentation and steroidogenesis. Melanocortins are released in various sites in the central nervous system and in peripheral tissues, and participate in the regulation of multiple physiological functions. They are involved in grooming behavior, food intake and thermoregulation processes, and can also modulate the response of the immune system in inflammatory states. Research of the past decade provided evidence that melanocortins could elicit their diverse biological effects by binding to a distinct family of G protein-coupled receptors with seven transmembrane domains. To date, five melanocortin receptor genes have been cloned and characterized. Those receptors differ in their tissue distribution and in their ability to recognize various melanocortins. These advances have opened up new horizons for exploring the significance of melanocortins, their ligands and their receptors for a variety of important physiological functions. We reviewed the origin of MSH peptides, the function and distribution of melanocortin receptors and their endogenous and exogenous ligands and the role of melanocortins and their receptors in inflammatory processes, nerve regeneration and nociception. Moreover, we analyzed their interaction with opioid peptides and finally, we discussed the postulated role of the melanocortin system in pain transmission at the spinal cord level.

The melanocortin-4 receptor (MC4R) is critically involved in regulating energy balance, and obesity has been observed in mice with mutations in the gene for brain-derived neurotrophic factor (BDNF). Here we report that BDNF is expressed at high levels in the ventromedial hypothalamus (VMH) where its expression is regulated by nutritional state and by MC4R signaling. In addition, similar to MC4R mutants, mouse mutants that expresses the BDNF receptor TrkB at a quarter of the normal amount showed hyperphagia and excessive weight gain on higher-fat diets. Furthermore, BDNF infusion into the brain suppressed the hyperphagia and excessive weight gain observed on higher-fat diets in mice with deficient MC4R signaling. These results show that MC4R signaling controls BDNF expression in the VMH and support the hypothesis that BDNF is an important effector through which MC4R signaling controls energy balance.

It is now well accepted that both the cortex and medulla of the mammalian adrenal gland receive a rich innervation. Many different transmitter substances have been identified in nerves supplying both cortex and medulla and, as well as catecholamines, a wide range of neuropeptides has been found in the adrenal gland. There have been several studies on the affects of age, sodium intake, stress, ACTH, and splanchnic nerve activity on the regulation of adrenal neuropeptide content. There is evidence that the abundance of each of these peptides is actively regulated. Although there have been many studies addressing the individual actions of various neurotransmitters on steroid secretion, adrenal blood flow, and adrenal growth, few have attempted to determine the nature of any interaction between neurotransmitters and the classical adrenal stimulants. There are, however, some significant interactions, particularly in the regulation of zona glomerulosa function. This review necessarily focuses on vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), as these are the most abundant transmitter peptides in the adrenal gland and the majority of studies have investigated their regulation and actions. However, substance P, calcitonin gene-related peptide (CGRP), neurotensin, and the enkephalins are included where appropriate. Finally, it has been suggested that certain neurotransmitters, particularly VIP, may interact with classical hormone receptors in the adrenal, notably the ACTH receptor. This review attempts to evaluate our current state of knowledge in each of these areas., (Copyright 2003 Wiley-Liss, Inc.)

Sex specificity of neural mechanisms modulating nociceptive information has been demonstrated in rodents, and these qualitative sex differences appear to be relevant to analgesia from kappa-opioid receptor agonists, a drug class reported to be clinically effective only in women. Via quantitative trait locus mapping followed by a candidate gene strategy using both mutant mice and pharmacological tools, we now demonstrate that the melanocortin-1 receptor (Mc1r) gene mediates kappa-opioid analgesia in female mice only. This finding suggested that individuals with variants of the human MC1R gene, associated in our species with red hair and fair skin, might also display altered kappa-opioid analgesia. We found that women with two variant MC1R alleles displayed significantly greater analgesia from the kappa-opioid, pentazocine, than all other groups. This study demonstrates an unexpected role for the MC1R gene, verifies that pain modulation in the two sexes involves neurochemically distinct substrates, and represents an example of a direct translation of a pharmacogenetic finding from mouse to human.

Sodium-water balance is causally linked to the functional expression of a number of important ocular tissues, viz. corneal deturgescence, aqueous humor secretion by the iris, hydration of the lens, retinal photoreception, and choriocapillary angiogenesis. The regulation of sodium absorption in the eye is generally believed to be under the control of Na(+),K(+)-activated adenosine triphosphatase, although evidence for this view is at best circumstantial. Contemporary work has shown widespread distribution of the mineralocorticoid hormone receptor and its colocalization with the amiloride-sensitive sodium channel in cells of diverse embryological origins. All available evidence favors the idea that the transcriptional regulation of the apical sodium channel by adrenocorticoids, and not the basolateral sodium pump, is critically important to sodium-water homeostasis in various ocular tissues, in a manner previously believed to be limited exclusively to the epithelial cell in various peripheral organs. Based upon these parameters, models are presented to help in understanding the direction of sodium absorption in a number of ocular tissues. Thus, the regulation of the sodium channel by steroid hormones seems to be a universal feature of the living cell that may have important implications in the understanding and management of normal ocular functions and their modification in human pathology.

The gamma-melanocyte-stimulating hormone (gamma-MSH) is a natriuretic peptide derived from the N-terminal region of proopiomelanocortin (POMC). Evidence suggests that it may be part of the coordinated response to a low-sodium diet (LSD). We tested the effect of the HSD (8% NaCl) compared with LSD (0.07%) on mean arterial pressure (MAP) in mice with targeted disruption of the PC2 gene (PC2(-/-)), necessary for processing of POMC into gamma-MSH, or the melanocortin receptor 3 gene (Mc3r(-/-); the receptor for MSH). In wild-type mice, HSD for 1 week did not alter MAP versus LSD mice, but plasma gamma-MSH immunoreactivity was more than double the LSD value. In contrast, in PC2(-/-) mice, MAP on the LSD was not greater than in wild-type mice, but plasma gamma-MSH was reduced to one-seventh the wild-type value. On the HSD, MAP rose to a markedly hypertensive level while plasma gamma-MSH concentration remained severely depressed. Intravenous infusion of gamma-MSH (0.2 pmol/min) for 30 min to PC2(-/-) mice after 1 week of HSD lowered MAP from hypertensive levels to normal; infusion of alpha-MSH at the same rate had no effect. Injection of 60 fmol of gamma-MSH into the lateral cerebral ventricle of hypertensive mice also lowered MAP to normal. Administration of a stable analogue of gamma-MSH intra-abdominally by microosmotic pump to PC2(-/-) mice prevented the development of hypertension when ingesting the HSD. In mice with targeted disruption of the Mc3r gene, the HSD also led to marked hypertension accompanied by elevated plasma levels of gamma-MSH; infusion of exogenous gamma-MSH to these mice had no effect on MAP. These results strongly suggest that PC2-dependent processing of POMC into gamma-MSH is necessary for the normal response to the HSD. gamma-MSH deficiency results in marked salt-sensitive hypertension that is rapidly improved with exogenous gamma-MSH through a central site of action. alpha-MSH infused at the same rate had no effect on MAP, indicating that the hypertension is a specific consequence of impaired POMC processing into gamma-MSH. Absence of Mc3r produces gamma-MSH resistance and hypertension on the HSD. These findings demonstrate a novel pathway mediating salt-sensitivity of blood pressure.

Previous studies on mice with melanocortin-4 receptor gene (MC4r) knockout have focused on obese adults. Because humans with functional MC4r mutations show early-onset obesity, we determined the onset of excessive fat deposition in 10- to 56-day-old mice, taking into account sex and litter influences. Total body fat content of MC4r-/- on day 35 and MC4r+/- on day 56 significantly exceeds that of MC4r+/+. Plasma leptin levels increase in proportion to fat mass. According to cumulative food intake and energy expenditure measurements from day 21 to 35, onset of excessive fat deposition in MC4r-/- is fueled by hyperphagia and counteracted partially by hypermetabolism. In 35- to 56-day-old mice, arcuate nucleus neuropeptide Y (NPY) mRNA decreases and pro-opiomelanocortin (POMC) mRNA increases with fat content and plasma leptin levels independently of genotype. Taking into account fat content by ANCOVA reveals, however, increases in both NPY mRNA and POMC mRNA due to melanocortin-4 receptor (MC4R) deficiency. We conclude that hyperphagia, not hypometabolism, is the primary disturbance initiating excessive fat deposition in MC4R-deficient mice at weaning and that the overall changes in NPY and POMC expression tend to antagonize the onset of excessive fat deposition.

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (M phi) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mphi incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro M phi activation, determined as release of the CXC chemokine KC and IL-1 beta, was inhibited by the more selective MC3-R agonist gamma(2)-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1 beta release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of gamma(2)-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal M phi leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.

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 distribution of fat in the body differs between the male and female sexes and is associated with the relative secretion of the two "adiposity" hormones leptin and insulin. We now report that the brains of male and female rats are differentially sensitive to the catabolic actions of small doses of these two hormones. Leptin (1 or 3.5 microg/2 microl) or saline (2 microl) was administered into the third cerebral ventricle of age- and weight-matched male and female rats. Leptin significantly reduced food intake in female and male rats over 4 h; however, leptin reduced 24-h intake in female but not in male rats. When the same rats were administered insulin (1 or 4 mU/2 microl) or saline (2 microl), male but not female rats had a robust reduction in food intake over 24 h. Previous research demonstrates the melanocortins are a central mediator of the effects of both leptin and insulin. However, we found no sex differences in sensitivity to the melanocortin agonist MTII (0.01, 0.1, 0.3, and 1.0 nmol/2 microl). These results suggest that the sex differences in sensitivity to leptin and insulin at the doses that we injected occur upstream of the melanocortin receptors. Because insulin and leptin reflect different fat beds and are differentially distributed in the male and female sexes, the implication is that the male and female sexes regulate adiposity-relevant parameters differently.

We investigated the effects of a novel melanocortin-4 (MC4) receptor antagonist,1-[(S)-2-(4-fluorophenyl)-2-(4-isopropylpiperadin-1-yl)ethyl]-4-[4-(2-methoxynaphthalen-1-yl)butyl]piperazine (MCL0129) on anxiety and depression in various rodent models. MCL0129 inhibited [(125)I][Nle(4)-D-Phe(7)]-alpha-melanocyte-stimulating hormone (alpha-MSH) binding to MC4 receptor with a K(i) value of 7.9 nM, without showing affinity for MC1 and MC3 receptors. MCL0129 at 1 microM had no apparent affinity for other receptors, transporters, and ion channels related to anxiety and depression except for a moderate affinity for the sigma(1) receptor, serotonin transporter, and alpha(1)-adrenoceptor, which means that MCL0129 is selective for the MC4 receptor. MCL0129 attenuated the alpha-MSH-increased cAMP formation in COS-1 cells expressing the MC4 receptor, whereas MCL0129 did not affect basal cAMP levels, thereby indicating that MCL0129 acts as an antagonist at the MC4 receptor. Swim stress markedly induced anxiogenic-like effects in both the light/dark exploration task in mice and the elevated plus-maze task in rats, and MCL0129 reversed the stress-induced anxiogenic-like effects. Under nonstress conditions, MCL0129 prolonged time spent in the light area in the light/dark exploration task and suppressed marble-burying behavior. MCL0129 shortened immobility time in the forced swim test and reduced the number of escape failures in inescapable shocks in the learned helplessness test, thus indicating an antidepressant potential. In contrast, MCL0129 had negligible effects on spontaneous locomotor activity, Rotarod performance, and hexobarbital-induced anesthesia. These observations indicate that MCL0129 is a potent and selective MC4 antagonist with anxiolytic- and antidepressant-like activities in various rodent models. MC4 receptor antagonists may prove effective for treating subjects with stress-related disorders such as depression and/or anxiety.

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.

Melanocortin receptors (MC-R) activated by one of several peptides derived from the pro-opiomelanocortin (POMC) precursor have become leading contenders for a pivotal role in controlling food intake. Evidence has emerged over the last decade to implicate primarily the MC4-R and, to a lesser extent, MC3-R as the key sub-types involved and both are strategically located in those regions within the hypothalamus known to be associated with feeding. The receptors are within class A of the GPCR superfamily and the key electrostatic interaction with the positively charged peptide (Arg) has been mapped to one or more Asp or Glu residues located on helices II and III of the seven helical bundle characteristic of this class of receptor. Sites for secondary interactions from which sub-type selectivity may be derived have also been located in the extracellular and helical domains. Unique amongst GPCRs is the presence of endogenous antagonist peptides, Agouti and Agouti-related peptide (AGRP), which confer an extra level of control on the system. Recently, several reports of potent and selective non-peptide ligands have been published and these are seen as prototypic molecules from which drugs may emerge to treat obesity (agonists) and cachexia (antagonists). The role played by the melanocortin system is the subject of this review and advances in our understanding of the structure of the endogenous ligand(s), non-peptide, small molecule ligands and the receptors at which they interact will be discussed.

Energy balance is a highly regulated, complex process which is modulated by central and peripheral systems. Dysregulation of energy homeostasis can result in metabolic disorders, such as obesity and type II diabetes. Obesity and type II diabetes are two of the most prevalent and challenging clinical conditions in society today. A growing body of evidence has implicated the melanocortin system as an important component in the maintenance of energy balance. alpha-MSH, a 13 amino acid peptide secreted as a product of the pro-opiomelanocortin (POMC) gene in the pituitary is a potent agonist of 4 of the 5 cloned melanocortin receptors (MCR). MC receptors are members of a G-protein-coupled receptor (GPCR) family, which signal through cAMP. Agouti and agouti-related protein (AGRP) are natural antagonists of melanocortin receptors and participate in regulation of skin/fur pigmentation, body weight, and adiposity. Stimulation of MC receptors has pleiotropic effects, which impact the nervous system as well as endocrine and immune functions. One of the most prominent effects of MC receptor stimulation is a dramatic suppression of food intake and body weight, which has led to the hypothesis that the MC receptor system plays a primary role in the maintenance of energy balance. This idea is supported by a large body of pharmacological, molecular and human genetic evidence. The following review summarizes the role of melanocortin receptors in the regulation of food intake and energy homeostasis and highlights the opportunities for MC receptors as drug development targets in treating eating disorders and diabetes.

The alpha-melanocyte-stimulating hormone (alphaMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of alphaMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent alphaMSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships.

It is now recognized that appropriate regulation of reproduction, energy intake, and energy expenditure, and thus maintenance of body weight and fertility, relies on complex hypothalamic neurocircuitry. Feeding and reproductive function are closely linked. During times of undernourishment and falling body fat the reproductive axis is down-regulated. Circulating factors and hypothalamic circuits coordinate these responses. Leptin has been described to be an important peripheral signal that indicates body fat stores to the hypothalamus and thus links nutrition and reproduction. Leptin acts by altering neuropeptide circuits in the hypothalamus, which alter gonadotropin-releasing hormone release and food intake. The importance of key neuropeptide systems identified in rodents is now being established in man. Notably mutations in the melanocortin MC4 receptor are found in up to 4% of the morbidly obese, and in a proportion of patients with anorexia nervosa mutations have been identified in the agouti-related peptide gene (AgRP), which codes for an endogenous antagonist of this receptor. Intranasal administration of a melanocortin fragment known to activate the MC4 receptor decreases adiposity in humans. The melanocortin system has been shown to influence the reproductive axis in rodents. However, the role of the melanocortin system in the control of reproduction in humans remains to be established. Since the discovery of leptin, attention has also been focused on peripheral signals that regulate reproduction, food intake, and energy expenditure, either directly or via feedback on hypothalamic circuits. Notable new discoveries in this area include the gastric hormone ghrelin. Circulating ghrelin stimulates food intake in rodents and humans, although an influence on the reproductive axis is yet to be reported.

By using a combination of genetic, pharmacological, and anatomical approaches, we show that the melanocortin 4 receptor (MC4R), implicated in the control of food intake and energy expenditure, also modulates erectile function and sexual behavior. Evidence supporting this notion is based on several findings: (i) a highly selective non-peptide MC4R agonist augments erectile activity initiated by electrical stimulation of the cavernous nerve in wild-type but not Mc4r-null mice; (ii) copulatory behavior is enhanced by administration of a selective MC4R agonist and is diminished in mice lacking Mc4r; (iii) reverse transcription (RT)-PCR and non-PCR based methods demonstrate MC4R expression in rat and human penis, and rat spinal cord, hypothalamus, brainstem, pelvic ganglion (major autonomic relay center to the penis), but not in rat primary corpus smooth muscle cavernosum cells; and (iv) in situ hybridization of glans tissue from the human and rat penis reveal MC4R expression in nerve fibers and mechanoreceptors in the glans of the penis. Collectively, these data implicate the MC4R in the modulation of penile erectile function and provide evidence that MC4R-mediated proerectile responses may be activated through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis. Our results provide a basis for the existence of MC4R-controlled neuronal pathways that control sexual function.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Melanocortin is the downstream mediator of leptin signaling and absence of leptin signaling in ob/ob and db/db mice revealed the enhancement of bone formation through the central regulation. While alpha-melanocyte-stimulating hormone (alphaMSH) inhibits the secretion of interleukin-1alpha and tumor necrosis factor-alpha from the inflammatory cells, alphaMSH can also enhance clonal expansion of pro B cells linked to stimulation of osteoclastogenesis. Therefore, we tested the effect of melanocortin on bones. alphaMSH analogues [(6)His]alphaMSH-ND and [(6)Asn]alphaMSH-ND were synthesized and the radio-ligand receptor binding- and cyclic AMP generating activity were analyzed in China Hamster Ovary cell line over- expressing melanocortin receptors. The EC(50) of [(6)His]alphaMSH-ND measured from melanocortin-1, 3, 4 and 5 receptors were 0.008 +/- 0.0045, 1.523 +/- 0.707, 0.780 +/- 0.405, and 250.320 +/- 42.234 nM, respectively, and the EC(50) of [(6)Asn]alphaMSH-ND were 16.8 +/- 6.94, 271.8 +/- 21.95, 8.0 +/- 1.21, and 1132.5 +/- 635.46 nM, respectively. Four weeks after the subcutaneous injection of the analogues, the body weights in the [(6)His]alphaMSH-ND and the [(6)Asn]alphaMSH-ND treated groups (346.0 +/- 20.63 g vs. 350.0 +/- 13.57 g) were lower than that of the vehicle treated group (375.8 +/- 17.31 g, p < 0.05). There was no difference in the total femoral BMD measured by dual x-ray absorptiometry among the three groups. Among the three groups, there were no differences in the total numbers of crystal violet positive- or alkaline phosphatase positive colonies, in the expression of Receptor Activator of Nuclear Factor Kappa-B ligand on the tibia and the total number of multinucleated osteoclast-like cells differentiated from primary cultured bone marrow cells. From the above results, no evidence of bone gain or loss was found after treatment of the alphaMSH analogues peripherally.

Myocardial ischemia/reperfusion induces ventricular tachycardia (VT), ventricular fibrillation (VF) and a high degree of lethality. Since ACTH-(1-24) (adrenocorticotropin) protects against such injuries in rats, we investigated which melanocortin MC receptor is involved. Ischemia was produced in anesthetized rats by ligature of the left anterior descending coronary artery (5 min), and reperfusion-induced VT, VF, lethality and time-course of arterial blood pressure within the 5 min following reperfusion were evaluated. I.v. administration of the selective MC(3) receptor agonist gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), as well as of an equimolar dose (162 nmol/kg) of both the non-selective agonist ACTH-(1-24) and alpha-MSH, significantly prevented VT and VF, and increased survival. Coronary reperfusion was followed by an abrupt and massive fall in mean arterial pressure and pulse pressure, in saline-treated rats. Treatment either with ACTH-(1-24) or gamma(1)-MSH completely prevented such fall. The protective effect of ACTH-(1-24) against the occurrence of VT, VF and lethality was neither affected by adrenalectomy, nor by i.v. pretreatment with the selective MC(4) receptor antagonist HS014 and the MC(4)-MC(5) antagonist HS059. On the other hand, the MC(3)-MC(4) receptor antagonist SHU 9119 prevented such protective effect. Moreover, the selective MC(1) receptor agonist MS05 (162 nmol/kg i.v.) failed to reduce the incidence of arrhythmias and lethality. These data demonstrate that MC(3) receptors mediate the protective effect of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias, in rats.

The AA (Alko, Alcohol) rats are selectively bred for their preference of alcohol to water, contrasting to ANA rats that avoid alcohol. They also exhibit a lower growth rate compared to ANA rats, as well as differences in their response to substances affecting food intake. The melanocortin (MC) system is involved in the regulation of feeding behaviour and in mechanisms underlying drug addiction and tolerance. Recently, administration of an MC receptor agonist proved to reduce alcohol intake in AA rats. We predicted that the ratio of endogenous MC receptor agonists (proopiomelanocortin, POMC) and antagonists (agouti-related protein, AgRP) would differ from ANA rats, and that subsequent differences in MC receptor levels would be detectable. We used in situ hybridization to detect an increased ratio of POMC/AgRP mRNA in the arcuate nucleus (Arc) of AA rats. Receptor autoradiography indicated that MC3 receptor binding differed in the nucleus accumbens and several hypothalamic nuclei, possibly reflecting differences in MC peptide transmission in the AA rats. Our results support the claim that AA rats have a high ratio of POMC/AgRP expression, and that this observation is accompanied by differences in MC3 receptor levels.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Obesity is associated with numerous health complications, which range from non-fatal debilitating conditions such as osteoarthritis, to life-threatening chronic diseases such as coronary heart disease, diabetes and certain cancers. The psychological consequences of obesity can range from lowered self-esteem to clinical depression. Despite the high prevalence of obesity and the many advances in our understanding of how it develops, current therapies have persistently failed to achieve long-term success. This review focuses on how fat mass can be reduced by altering the balance between energy intake and expenditure.

Identifying the role of the melanocortin system in regulating energy homeostasis has relied on both genetic and pharmacological studies. The key findings included 1) that the coat color phenotype in the lethal yellow (A(Y)/a) mouse is due to antagonism of the melanocortin-1 receptor (MC1R) by the agouti gene product; 2) the MC3R and MC4R are expressed in CNS centers involved in energy homeostasis, and 3) the combined results of pharmacological studies showing that agouti is an antagonist of the MC4R and transgenic studies showing that inhibition or loss of the MC4R recapitulate the lethal yellow phenotype. Pro-opiomelanocortin (POMC), MC3R, and MC4R knockouts are obese and are now being used to further analyze melanocortin receptor function. The obesity phenotype observed in the MC3R and MC4R knockouts (KO) differ markedly. MC4RKO mice are hyperphagic, do not regulate pathways that increase energy expenditure (diet-induced thermogenesis) and physical activity in response to hyperphagia, and can develop type 2 diabetes. In contrast, MC3R deficient mice are not hyperphagic, have a normal metabolic response to increased energy consumption, and do not develop diabetes. The mechanism underlying the increased adiposity in the MC3R knockout remains unclear, but might be related to changes in nutrient partitioning or physical activity.

The contribution of the UV component of sunlight to the development of skin cancer is widely acknowledged, although the molecular mechanisms that are disrupted by UV radiation (UVR) resulting in the loss of normal growth controls of the epidermal stem cell keratinocytes and melanocytes is still poorly understood. Alpha-melanocyte stimulating hormone (alpha-MSH), acting via its receptor MC1, has a key role in skin pigmentation and the melanizing response after exposure to UVR. The cell cycle inhibitor p16/CDKN2A also appears to have an important function in a cell cycle checkpoint response in skin after exposure to UVR. Both of these genes have been identified as risk factors in skin cancer, MC1R variants are associated with increased risk to both melanoma and nonmelanoma skin cancers, and p16/CDKN2A with increased risk of melanoma. Here we demonstrate that the increased expression of p16 after exposure to suberythemal doses of UVR is potentiated by alpha-MSH, a ligand for MC1R, and this effect is mimicked by cAMP, the intracellular mediator of alpha-MSH signaling via the MC1 receptor. This link between p16 and MC1R may provide a molecular basis for the increased skin cancer risk associated with MC1R polymorphisms.