Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.

Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.

Mutations reducing the functional activity of leptin, the leptin receptor, alpha-melanocyte stimulating hormones (alpha-MSH) and the melanocortin-4 receptor (Mc4r) all lead to obesity in mammals. Moreover, mutant mice that ectopically express either agouti (Ay/a mice) or agouti-related protein (Agrp), antagonists of melanocortin signalling, become obese. These data suggest that alpha-MSH signalling transduced by Mc4r tonically inhibits feeding; however, it is not known to what extent this pathway mediates leptin signalling. We show here that Mc4r-deficient (Mc4r-/-) mice do not respond to the anorectic actions of MTII, an MSH-like agonist, suggesting that alpha-MSH inhibits feeding primarily by activating Mc4r. Obese Mc4r-/-mice do not respond significantly to the inhibitory effects of leptin on feeding, whereas non-obese Mc4r-/- mice do. These data demonstrate that melanocortin signalling transduced by Mc4r is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. Leptin resistance of obese Mc4r-/- mice does not prevent their response to the anorectic actions of ciliary neurotrophic factor (CNTF), corticotropin releasing factor (CRF), or urocortin; or the orexigenic actions of neuropeptide Y (NPY) or peptide YY (PYY), indicating that these neuromodulators act independently or downstream of Mc4r signalling.

Molecular genetics of adrenocortical tumor formation and potential pharmacologic targets.

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.

Studies on the physiological functions of the melanocortin system.

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.

Enkephalins and ACTH in the mammalian nervous system.

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.

Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects.

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.

Adrenocorticotropin resistance syndromes.

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.

Melanocortin-4 receptor is required for acute homeostatic responses to increased dietary fat.

In response to moderately increased dietary fat content, melanocortin-4 receptor-null mutant (MC4R-/-) mice exhibit hyperphagia and accelerated weight gain compared to wild-type mice. An increased feed efficiency (weight gain/kcal consumed) argues that mechanisms in addition to hyperphagia are instrumental in causing weight gain. We report two specific defects in coordinating energy expenditure with food intake in MC4R-/- mice. Wild-type mice respond to an increase in the fat content of the diet by rapidly increasing diet-induced thermogenesis and by increasing physical activity, neither of which are observed in MC4R-/- mice. Leptin-deficient and MC3R-/- mice regulate metabolic rate similarly to wild-type mice in this protocol. Melanocortinergic pathways involving MC4-R-regulated neurons, which rapidly respond to signals not requiring changes in leptin, thus seem to be important in regulating metabolic and behavioral responses to dietary fat.

Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptor.

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.

Inheritance and population structure of the white-phased "Kermode" black bear.

We report that a single nucleotide replacement in the melanocortin 1 receptor gene [1] (mc1r) is responsible for the white coat color of the "Kermode" bear [2], a color phase of the black bear (Ursus americanus Pallus) found in the rainforests along the north coast of British Columbia. In a sample of 220 bears, of which 22 were white, there was complete association of a recessive Tyr-to-Cys replacement at codon 298 with the white phase. This variant has not been yet been reported in other mammals, and it also is the lightest-colored variant yet found at mc1r. Also, we found that heterozygotes, which act as a hidden reservoir for the allele among black bears, were infrequent outside of the three islands where Kermodes are common and that, within these three islands, heterozygotes were less frequent than expected under random mating. Immigration of black bears into Kermode populations can depress the occurrence of the white phase, and management practices should be designed to avoid facilitating higher immigration rates.

Genetic disruption of gamma-melanocyte-stimulating hormone signaling leads to salt-sensitive hypertension in the mouse.

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.

Obesity therapy: altering the energy intake-and-expenditure balance sheet.

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.

Hypothalamic control of feeding.

Our understanding of the hypothalamic control of energy homeostasis has increased greatly since the discovery of leptin, the adipose cell derived protein. Recent studies have identified several new hypothalamic neuropeptides that affect food intake and energy balance. By studying these molecules and their neuronal systems, receptors and interactions, we are beginning to unravel the circuitry between peripheral adipogenic signals and hypothalamic effector pathways.

Evidence for a stimulatory action of melanin-concentrating hormone on luteinising hormone release involving MCH1 and melanocortin-5 receptors.

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.

Melanocortin 5 receptor and ocular immunity.

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.

Alpha-melanocyte stimulating hormone potentiates p16/CDKN2A expression in human skin after ultraviolet irradiation.

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.

Role of melanocortin-4 receptors in mediating renal sympathoactivation to leptin and insulin.

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.

A possible involvement of melanocortin 3 receptor in the regulation of adrenal gland function in the chicken.

The melanocortin 3 receptor (MC3-R) in the melanocortin receptor family has been identified as a neural receptor subtype mainly expressed in the brain in mammals. We report here the isolation of the chicken gene for MC3-R, CMC3, displaying different tissue distribution from mammalian counterparts. The CMC3 gene was found to be a single copy gene encoding a 325 amino acid protein, sharing 75.3-76.8% identity with mammalian counterparts. When assessed by RT-PCR, the CMC3 mRNA was not detected in the brain but was exclusively expressed in adrenal glands, where Agouti-related protein/Agouti-related transcript (AGRP/ART), a newly identified endogenous antagonist of MC3-R, is expressed in mammals, raising the possibility that the CMC3 plays a role in complicated regulation of the gland function by melanocortins and AGRP/ART in the chicken. Noteworthy, MC1-R gene was found to be a quite unique member of the chicken MC-R family with regard to GC content and codon usage. It may reflect as yet unidentified evolutionary pressure operating specifically on the gene.