A unique melanocortin-4-receptor signaling profile for obesity-associated constitutively active variants.

The melanocortin-4 receptor (MC4R) plays a critical role in regulating energy homeostasis. Studies on obesogenic human MC4R (hMC4R) variants have not yet revealed how hMC4R maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q hMC4R variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP) response element (CRE)-driven transcription, and calcium mobilization but not phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating hormone-induced CRE-driven transcription but not impaired α-melanocyte-stimulating hormone-induced AC, calcium, or pERK1/2. This profile was not observed for transfected H158R, a constitutively active hMC4R variant associated with overweight but not obesity. We concluded that there is potential for α-melanocyte-stimulating hormone-induced CRE-driven transcription in HEK293 cells transfected with obesogenic hMC4R variants to be the key predictive tool for determining whether they exhibit loss of function. Furthermore, in vivo, α-melanocyte-stimulating hormone-induced hMC4R CRE-driven transcription may be key for maintaining body weight.

γ2-Melanocyte stimulation hormone (γ2-MSH) truncation studies results in the cautionary note that γ2-MSH is not selective for the mouse MC3R over the mouse MC5R.

The melanocortin system has been implicated in a multitude of physiological pathways including obesity, satiety, energy homeostasis, sexual behavior, pigmentation, sodium regulation, hypertension, and many others. Based upon studies of the endogenous melanocortin receptor agonists at the cloned human melanocortin receptor proteins, it was concluded that the γ-MSH related agonist ligands are selective for the MC3 versus the MC4 and MC5 receptors. In attempts to understand and identify the specific amino acids of γ2-MSH important for MC3R selectivity, we have performed N- and C-terminal truncation studies and pharmacologically characterized twenty-eight ligands at the mouse MC1 and MC3-5 melanocortin receptors. The C-terminal Trp-Asp9-Arg¹°-Phe¹¹ residues are important for nM potency at the mMC3R and the Arg7-Trp8 residues are important for mMC5R nM potency. We observed the unanticipated results that several of the C-terminal truncated analogs possessed nM agonist potency at the mMC3 and mMC5Rs which lead us to perform a comparative side-by-side study of the mouse and human MC5R. These data resulted in µM γ2-MSH analog potency at the hMC5R, consistent with previous reports, however at the mMC5R, nM γ2-MSH analog potency was observed. Thus, these data support the hypothesis of important species specific differences in γ-MSH related ligand potency at the rodent versus human MC5R subtype that is critical for the interpretation of in vivo rodent physiological studies. These results prompted us to examine the affects of a peripherally administered melanocortin agonist on hypothalamic gene expression levels of the MC3R, MC4R, and MC5R. The super potent non-selective NDP-MSH agonist was administered i.p. and resulted in significantly decreased levels of mMC3R and mMC5R hypothalamic mRNA versus saline control. These data provide for the first time data demonstrating peripherally administered NDP-MSH can modify hypothalamic melanocortin receptor expression levels.

Functions for pro-opiomelanocortin-derived peptides in obesity and diabetes.

Melanocortin peptides, derived from POMC (pro-opiomelanocortin) are produced in the ARH (arcuate nucleus of the hypothalamus) neurons and the neurons in the commissural NTS (nucleus of the solitary tract) of the brainstem, in anterior and intermediate lobes of the pituitary, skin and a wide range of peripheral tissues, including reproductive organs. A hypothetical model for functional roles of melanocortin receptors in maintaining energy balance was proposed in 1997. Since this time, there has been an extraordinary amount of knowledge gained about POMC-derived peptides in relation to energy homoeostasis. Development of a Pomc-null mouse provided definitive proof that POMC-derived peptides are critical for the regulation of energy homoeostasis. The melanocortin system consists of endogenous agonists and antagonists, five melanocortin receptor subtypes and receptor accessory proteins. The melanocortin system, as is now known, is far more complex than most of us could have imagined in 1997, and, similarly, the importance of this system for regulating energy homoeostasis in the general human population is much greater than we would have predicted. Of the known factors that can cause human obesity, or protect against it, the melanocortin system is by far the most significant. The present review is a discussion of the current understanding of the roles and mechanism of action of POMC, melanocortin receptors and AgRP (agouti-related peptide) in obesity and Type 2 diabetes and how the central and/or peripheral melanocortin systems mediate nutrient, leptin, insulin, gut hormone and cytokine regulation of energy homoeostasis.

Evidence for direct actions of melanocortin peptides on bone metabolism.

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.

alpha -melanocyte-stimulating hormone is a novel regulator of bone.

alpha-Melanocyte-stimulating hormone (alpha-MSH), a 13-amino acid peptide produced in the brain and pituitary gland, is a regulator of appetite and body weight, and its production is regulated by leptin, a factor that affects bone mass when administered centrally. alpha-MSH acts via melanocortin receptors. Humans deficient in melanocortin receptor 4 (MC4-R) have increased bone mass, and MC4-R has been identified in an osteoblast-like cell line. Thus alpha-MSH may act directly on the skeleton, a question addressed by the present studies. In primary cultures of osteoblasts and chondrocytes, alpha-MSH dose dependently (>or=10(-9) M) stimulated cell proliferation. In bone marrow cultures, alpha-MSH (>10(-9) M) stimulated osteoclastogenesis. Systemic administration of alpha-MSH to mice (20 injections of 4.5 microg/day) decreased the trabecular bone volume in the proximal tibiae from 19.5 +/- 1.8 to 15.2 +/- 1.4% (P = 0.03) and reduced trabecular number (P = 0.001). Radiographic indexes of trabecular bone, assessed by phase-contrast X-ray imaging, confirmed the bone loss. It is concluded that alpha-MSH acts directly on bone, increasing bone turnover, and, when administered systemically, it decreases bone volume. The latter result may also be contributed to by alpha-MSH effects elsewhere, such as the adipocyte, pancreatic beta-cell, or central nervous system.