Melanocortin-4 Receptor: a Novel Signalling Pathway Involved in Body Weight Regulation

Home , Melanocortin, Melanocortin 1 receptor, Melanocortin 4 receptor, Melanocortin receptor

International Journal of Obesity (1999) 23, Suppl 1, 54±58 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo Melanocortin-4 receptor: A novel signalling pathway involved in body weight regulation

SL Fisher1, KA Yagaloff1 and P Burn1*

1Department of Metabolic Diseases, Hoffmann LaRoche, Nutley, NJ 07110, USA

For many years, genetically obese mouse strains have provided models for human obesity. The Avy=-agouti mouse, one of the oldest obese mouse models, is characterized by maturity-onset obesity and diabetes as a result of ectopic expression of the secreted protein hormone, agouti protein. Agouti protein is normally expressed in hair follicles to regulate pigmentation through antagonism of the melanocortin-1 receptor, but in-vitro studies have demonstrated that the hormone also has potent antagonist activity for the melanocortin-4 receptor (MC4-R). Subsequent development of the MC4-R knockout mouse model demonstrated that MC4-R plays a role in weight homeostasis as these mice recapitulated the metabolic defects of the agouti mouse. Further evidence for this hypothesis was obtained from pharmacological studies utilizing peptides with MC4-R agonist activity, that inhibitied food intake (when administered intracerebrally). Additional studies with peptide antagonists have now implicated the MC4-R in the leptin signalling pathway. Finally, evidence that the MC4-R may play a role in human obesity has been obtained from the identi®cation of a dis-functional variant of the receptor in genetically obese subjects.

Keywords: obesity; diabetes; agouti; melanocortin; POMC; leptin; ob

Introduction There has been an explosion in obesity research and with this has come an understanding of the molecular mechanisms that underly the disease. This explosion Obesity is reaching epidemic status in the developed was sparked by the cloning of the ob gene from the world and is rapidly threatening the near-developed obese mouse model ob=ob5 and the subsequent regions of the globe. The clinically obese comprise demonstration that the protein product is a centrally nearly 30% of the total population in the US alone1 active hormone.6 These initial observations have led and it is estimated over $60 billion are spent annually to an increasingly complex view of the factors and in treatments for this disease,2 which is characterized regulatory processes that govern weight homeostasis by increased storage of energy input as adipose tissue in which the OB protein (or leptin) plays a central role as a consequence of unbalanced energy intake and coordinating a number of metabolic and reproductive energy expenditure. Obese patients suffer from an functions.7±10 Use of mouse genetics as a predictor increased risk of a number of severe and debilitating and model of human disease has provided the frame- diseases including non-insulin dependent diabetes work for the identi®cation of novel drug discovery mellitus (NIDDM), hypertension, hyperlipidemia, targets. Our goal in this report is to describe the sleep apnea, and certain cancers.3 While previous current understanding of the weight regulation pro- generations regarded obesity as a social disease that cesses underlying another genetically obese mouse could be remedied with appropriate diet and exercise model, the agouti viable yellow Avy=-mouse. regimens, it is now clear that human obesity is a multifactorial disease with both environmental and genetic components. Studies in human populations4 suggest that at least 35% (and potentially as high as 50%) of the obese population is genetically predis- Agouti mouse model posed to become overweight. This segment of the population is most likely to bene®t from therapeutic vy intervention and it is hoped that with proper diet and The A =-mouse is the oldest known mouse model of obesity and was initially prized by mouse fanciers for exercise, therapeutic treatment will allow for long- 11 term weight reduction and weight maintenance. its golden yellow coat color (Figure 1). In addition to the change in coat color, these mice display an age- onset (10 ± 20 weeks) metabolic defect characterized by hyperphagia, increased adiposity, hyperglycemia, hyperinsulinemia and increased somatic growth. *Correspondence: Paul Burn, Department of Metabolic Dieases, Unlike other mouse models, the increased adipocyte Hoffmann LaRoche, Nutley, NJ 07110, USA; 12 Stewart Fisher, Biochemistry Dept, Astra Research Center, mass results from adipocyte hypertropy Ðan attri- Boston, Cambridge MA 02139, USA. bute that, along with the age-onset of development, Melanocortin-4 receptor SL Fisher et al 55 puri®ed protein product con®rmed this assumption. The agouti protein was found to be a potent ( < 2 nM) antagonist for the MC1-R,16 but interestingly was also found to be a potent anatagonist of the MC4-R. Agouti does not bind to MC2, MC3 or MC 5 receptors. The activity at the MC4-R was puzzling due to the selective tissue expression patterns of agouti and the MC4-R, but careful analysis of the Avy=-mouse identi®ed a mutation in the promoter region of the agouti gene that results in high-level, ectopic expression of the gene.17 This suggested that the characteristic phenotypes of the Avy=-mouse arise from separable antagonist activities at the MC1-R and MC4-R as shown in Figure 2; the yellow coat color from MC1- Figure 1 Agouti mice (right) display a yellow coat color and age- R antagonism and the observed metabolic defects onset obesity compared to wild-type mice (left). from MC4-R antagonism. This hypothesis was the foundation for the development of an MC4-R knock- closely resembles the characteristics most commonly out mouse model to directly assess the role of MC4-R observed in human obesity. in the regulation of weight homeostasis. The underlying cause of the Avy=-mouse metabolic defect remained an enigma until the early 1990s. The yellow coat color strongly suggested that the pigmentation regulatory pathways governed by the melano- Studies of the MC4-R cortin-1 receptor (MC1-R) were affected. Primarily expressed in the skin, the MC1-R is a G-protein coupled receptor which regulates pigmentation in The phenotype of the MC4-R knockout mouse pro- response to the melanocyte stimulating hormone (a- vided con®rmation of the model depicted in Figure 2. 13 These mice completely recapitulated the metabolic MSH), a potent agonist of MC1-R. Agonism of the vy 18 MC1-R results in eumelanin production (and therefore defect of the A =-mice. The MC4-R knockout mice black pigmentation) whereas antagonism produces have a normal coat color, but develop age-onset phaeomelanin (resulting in bleaching of the skin obesity (12 ± 20 weeks) that results from hyperphagia color). There are ®ve known melanocortin receptors and adipocyte hypertrophy. In addition, the mice based on sequence homology that ranges from 35 ± become hyperglycemic and hyperinsulinemic in a 60% homology between family members.13 The similar age-dependent manner. Furthermore, these MC2-R, also known as the adrenocorticotropin hor- effects are attenuated approx 2-fold in the hetero- mone (ACTH) receptor, is expressed in the adrenal zygote mice ± a ®nding consistent with the expected 2- cortex and regulates corticosteroid synthesis in fold increase in receptor density relative to the knock- response to ACTH levels. The MC3-R and MC4-R out mice. The mice have normal corticosteriod con- are both expressed in the central nervous system centrations, demonstrating an intact hypothalamic ± (CNS), and little was known about the function of pituitary ± adrenal (HPA) axis, but also show an these receptors when the agouti gene was cloned. The increase in the somatic growth as observed in the MC5-R is expressed in speci®c exocrine glands agouti mice. The extent of hyperglycemia and hyper- throughout the body (for example, Harderian and insulinemia observed in the knockout mice is gender perputial glands) and has been implicated in the dependent, with male offspring exhibiting more control of lipid and pheromone production in these severe elevation than females in both cases. The sites.14 Melanocyte stimulating hormones are pro- overall phenotype clearly demostrates the separate duced from pro-opiomelanocortin (POMC), a pro- activities of the agouti protein on the melanocortin hormone of 131 amino acids that is processed into three classes of hormones; the melanocortins (a, b, and g), ACTH and various endorphins (for example, lipotropin).13 POMC is produced primarily in the pituitary, but also in the arcuate nucleus of the hypothalamus. The cloning of the agouti gene in 1992 represented a breakthrough for understanding the Avy=-mouse.15 The agouti gene codes for small (131 residues), soluble, secreted protein, that is normally expressed only in the skin, consistent with the premise that this gene plays a role in coat color regulation through Figure 2 Agouti protein antagonism of MC1-R and MC4-R leads MC1-R interactions. Pharmacological studies on the to yellow pigmentation and obesity. Melanocortin-4 receptor SL Fisher et al 56 receptors in vivo (Figure 2) and implicates the MC4-R concentrations of POMC expression were observed in as a regulator of energy balance. fasted mice (low leptin concentrations), but POMC Additional proof of the concept for this model expression was elevated in response to rising leptin (Figure 2) was provided by studies that relied on concentrations after feeding.24,26 Acute measurements two cyclic heptapeptide analogs of aMSH, MT-II of POMC levels in Long ± Evans rats were found to be and SHU-9119, which display potent ( < 1 nM) directly in¯uenced by the administration of leptin; MC4-R agonist and antagonist activities, respec- after leptin administration, the POMC concentrations tively.19 The authors found that injection of MT-II rose by nearly 39%.26 In addition, repeated dosing of into the third ventricle of the brain caused a dose- ob mice with leptin (single dose 5 d) showed elevated dependent inhibition of food intake that was long POMC concentrations, when compared to control lasting ( > 8 h) and reversible when co-administered mice.25,26 Similar changes were not observed in the with the antagonist SHU-9119. Intraperitoneal admin- db=db mouse, demonstrating the requirement for an istration of higher doses of MT-II had similar effects intact ob-signalling pathway (in this case functional on food intake, clearly demonstrating that these mole- ob-R) for POMC modulation. cules, although relatively large (MW > 1300), were Additional evidence for melanocortin effects on capable of crossing the blood ± brain barrier in con- leptin signalling was obtained from studies using the centrations high enough to result in ef®cacy. To sum antagonist, SHU-9119, in Long-Evans rats.27 The up, these studies provided the ®rst demonstration that authors ®rst identi®ed a dose that was suf®cient to MC4-R agonists can inhibit food intake and provide a interact with the receptor, but below the threshold foundation for the development of small molecule required for modulation of food intake when adminis- anti-obesity agents. tered by intracerebral injection into the thrid ventricle (iv3t, dose 0.5 nmol). Under these conditions, both the control animals and those treated with the antagonist demonstrate normal feeding behavior after injec- Evidence for MC4-R involvement in tion, while those treated with leptin (3.5 mg) show a leptin signalling signi®cant reduction in food intake after 4 h ( 7 56%). Interestingly, animals subjected to co-administration of the antagonist with leptin exhibit normal feeding There were several lines of evidence suggesting the behavior, clearly showing a pharmacological blockade MC4-R played a role in the control of metabolic of leptin signalling by the antagonist. Similar results pathways regulated by leptin. First, agouti mice were obtained upon assessment of cFos activation in were highly resistant to leptin effects on food intake the paraventricular nucleus (PVN) ± a region known to inhibition.20 Second, functional (long-form) ob-R be activated by leptin and the MC4-R receptor. As receptors were found in the neurons of the arcuate expected from previous work,22 animals treated with nucleus that produce POMC, providing a basis for the leptin exhibited a signi®cant activation of cFos nuclei hypothesis that activation of the leptin signalling when the mice were pre-treated with vehicle (254% pathway in these neurons could result in elevated increase), but this effect was completely abolished POMC production and processing to yield MC4-R when the mice were pretreated with SHU-9119. These agonists.21 In addition, comparative analysis (using studies indicate that the MC4-R is downstream of ob- cFos immunohistochemistry analysis of brain sections R in the leptin pathway; consistent with the working of mice treated with leptin or MC4-R agonist) demon- model shown in Figure 3. This simple model has strated that overlapping activation patterns were support from known mouse models of obesity. At observed in a number of sites in the brain, for each of the steps in the signalling cascade, there is a example, paraventricular nucleus (PVN), dorsomedial hypothalamus (DMH) and the central nucleus of the Amygdala (CeA).22; 23 Furthermore, the long duration of action observed in food intake inhibition upon administration of MC4-R agonists, was consistent with similar results observed with leptin dosing ± in both cases, the effects extend well beyond the measured half-life of the administered agent. These observations, while largely circumstantial, led to detailed mechanistic studies of leptin and MC4-R signalling activation. The ®rst direct evidence that leptin in¯uenced the melanocortin signalling pathway was obtained from in-situ analysis of POMC neurons in the arcuate nucleus of a number of mouse and rat models.24 ± 26 Expression of POMC was found to be proportional to Figure 3 Proposed model for MC4-R in the leptin signaling circulating leptin concentrations in the serum; low pathway. Melanocortin-4 receptor SL Fisher et al 57 known genetic mutation that would inhibit the path- activities at other melanocortin receptors.33 In addi- way and result in the expected obesity phenotype. tion, there is evidence to suggest that there are addi- Whilte this model is appealing and correlates well tional melanocortin receptors that remain to be to the data mentioned above, there is a report that identi®ed.32 As a result, con®rmation of additional challenges its underlying tenets. A genetic cross of the MC4-R speci®c regulatory pathways will await the Ay=a and the ob=ob mice was prepared and the generation of more speci®c compounds and the poten- phenotype of these mice was examined for leptin tial discovery of new melanocortin receptors. sensitivity.28 Unexpectedly, these mice exhibited a near normal response to leptin administration, when measured as a function of body weight reduction, demonstrating that the leptin signalling pathway Evidence for melanocortin remained intact in these animals despite the expected regulation of human obesity antagonism of the MC4-R by the agouti protein. In addition, deletion of the leptin gene in agouti mice restored the senstivity to leptin administration, sug- While the data supporting a role for MC4-R in weight gesting that the development of obesity in the Ay=a regulation in rodent models of weight homeostasis is animals is achieved through a pathway that is inde- quite strong, there is growing evidence that the pendent of leptin signalling. Resolution of the appar- melanocortins also play a similar role in human ent con¯ict between the conclusions of this paper and obesity. A quantitative trait locus (QTL) associated with obesity in a human population was recently the data supporting the model in Figure 3 awaits 34 further investigation, but it should be noted that the identi®ed near the POMC gene locus, suggesting two studies differ considerably in their design (acute that mutations in POMC production=processing may administration vs chronic antagonism by agouti) and play a role in human obesity. Furthermore, a novel in their measurement of leptin ef®cacy (food intake agouti-related transcript (ART, also known as agouti- inhibition and cFos activation vs body weight reduc- related protein or AGRP) has been identi®ed in both tion). One possibility is that the amount of endogen- rodents and man. This protein is produced in the ous agouti protein present in the hypothalamus is not hypothalamus and has potent antagonist activity for both MC3-R and MC4-R, despite sharing consider- able to inhibit ob signalling through the MC4-R 35,36 receptor. In the end, the differing results from the able sequence homology to agouti protein. This two studies may represent the likely possibility that homology, along with the expression pattern and the MC4-R and the leptin signalling pathways, while pharmacological pro®le, suggests that AGRP plays a clearly connected under some conditions, may not be role in allowing speci®c melanocortin subtype (MC3- completely overlapping under all situations. R and MC4-R) regulation similar to that observed with the agouti protein and MC1-R in pigmentaion control. Consistent with this hypothesis, expression levels of ART have recently been shown to be Other melanocortin effects modulated by leptin in an inverse relationship to POMC.37 The current model therefore suggests that attributed to MC4-R regulation the MC4-R is speci®cally switched between activation and inactivation by the action of two secreted hor- Recent studies have suggested that the MC4-R may be mones, MSH and AGRP. involved in the control of additional physiological processes such as grooming behaviour29,30 and blood 31 pressure regulation. The conclusions from these Conclusion studies were bases on selective administration of various MSH analogs (a, b, and g) and the antagonist (SHU-9119) into regions of the brain known to The rodent data supporting the role of MC4-R regula- express MC4-R. For example, administration of a- tion in weight homeostasis is compelling and the MSH into the dorsal motor nucleus of the vagus nerve recent data supporting a similar function in humans (a site where MC4-R is expressed32) results in lower will no doubt be expanded in the near future. This blood pressure and bradycardia. This effect is blocked strong proof-of-concept, along with the rich history of by administration of SHU-9119. Additional side therapeutic agents derived from G-protein coupled effects have been noted at high doses of both MT-II receptors, augurs well for the development of novel and SHU-9119, which includes barrel-rolling in rats therapeutic anti-obesity agents targeted to the MC4-R. and the development of conditioned-taste aversions.23 These side effects are inconsistent with agonism of the MC4-R. It should be noted that although these com- References pounds were originally reported as speci®c for the 1 Parker S. Obesity: Trends and Treatments. 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