A Novel Selective Melanocortin-4 Receptor Agonist Reduces Food Intake in Rats and Mice Without Producing Aversive Consequences
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The Journal of Neuroscience, May 1, 2000, 20(9):3442–3448 A Novel Selective Melanocortin-4 Receptor Agonist Reduces Food Intake in Rats and Mice without Producing Aversive Consequences Stephen C. Benoit,1 Michael W. Schwartz,2 Jennifer L. Lachey,1 Mary M. Hagan,1 Paul A. Rushing,1 Kathleen A. Blake1 Keith A. Yagaloff,3 Grazyna Kurylko,3 Lucia Franco,3 Waleed Danhoo,3 and Randy J. Seeley1 1Department of Psychiatry, University of Cincinnati, Cincinnati, Ohio 45267, 2University of Washington, Department of Veterans Affairs, Puget Sound Health Care System, Seattle, Washington 98108-1597, and 3Department of Metabolic Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 Studies using nonselective agonists and antagonists of mice that lack functional leptin receptors via a mechanism that melanocortin-3 receptor (MC3R) and MC4R point to the impor- is not accompanied by illness or other nonspecific effects. tance of the CNS melanocortin system in the control of food Conversely, a related compound that is a selective MC4R an- intake. We describe here a novel compound that is highly tagonist potently increased food intake when administered cen- selective as an agonist at the MC4 receptor but has minimal trally in rats. These results support the hypothesis that the brain activity at the MC3 receptor. When administered centrally to MC4R is intimately involved in the control of food intake and rats, this selective agonist increased Fos-like immunoreactivity body weight and provide evidence that selective activation of in the paraventricular nucleus, central nucleus of the amygdala, MC4R causes anorexia that is not secondary to aversive nucleus of the solitary tract, and area postrema, a pattern of effects. neuronal activation that is similar to that induced by a nonse- lective MC3/4R agonist. Additionally, it suppresses food intake Key words: melanocortins; MC4-R; hypothalamus; food in- when administered centrally to rats or peripherally to db/db take; taste aversion; c-Fos Recent evidence points to the hypothalamic melanocortin (MC) Ectopic overexpression of the agouti protein in A Y mice there- peptide system as critical in the control of energy balance. Neu- fore results in both yellow coat color because of antagonism of rons that express mRNA for the MC precursor molecule, pro- MC1R and increased food intake and body weight because of opiomelanocortin (POMC), are found in the arcuate nucleus abnormal antagonism of MC4R (Lu et al., 1994). Agouti-related (ARC) and project to other hypothalamic sites. Evidence sug- protein (AgRP) is homologous to agouti, which is synthesized gests that these POMC neurons express leptin receptors and that exclusively in a population of ARC neurons that are distinct from central MC signaling contributes to the effects of the adipocyte those expressing POMC. AgRP antagonizes both MC3R and hormone leptin (Cheung et al., 1997; Mountjoy and Wong, 1997; MC4R but not MC1R (Fong et al., 1997). Consequently, genetic Seeley et al., 1997). Within the brain, two receptor subtypes for overexpression of AgRP or exogenous administration of an AgRP melanocortins are expressed, MC3R and MC4R (Mountjoy et al., fragment increases food intake and causes obesity but does not 1994). Several nonselective ligands of these receptors have been alter pigmentation (Ollmann et al., 1997; Rossi et al., 1998). identified and assessed for their effect on food intake (Fan et al., Furthermore, central administration of a nonselective MC3/4R 1997; Grill et al., 1998). ␣-Melanocyte-stimulating hormone (␣- antagonist (SHU-9119) increases food intake in normal rats and MSH), synthesized within ARC POMC neurons, is an agonist of mice (Fan et al., 1997; Hagan et al., 1999). Collectively, these data MC3R and MC4R (Adan et al., 1994) that, after exogenous suggest that central MC3/4 receptors participate in energy central administration, robustly decreases food intake in rats homeostasis. (Tsujii and Bray, 1989). MTII is a synthetic analog of ␣-MSH that An important unanswered question is whether the two cen- also has agonistic properties at both MC3R and MC4R and trally expressed MC receptors have different roles in the control decreases food intake and body weight of mice and rats (Fan et of energy balance. We report here the use of two new compounds al., 1997; Thiele et al., 1998). that have specific affinity for the MC1R and MC4R. Because Interestingly, MC receptors are one of the few signaling sys- MC1R has only been found in periaquiductal gray matter of tems for which endogenous antagonists, as well as agonists, have humans (Xia et al., 1995), we expect central administration of been identified. Agouti is a protein that is normally expressed in these compounds results in selective activation at MC4R. We the skin and is an antagonist of the cutaneous MC receptor therefore measured the effects of these compounds on food intake involved in pigmentation, MC1R (Ollmann et al., 1997, 1998). in rats and db/db mice, which develop obesity because of autoso- mal recessive mutation of the long-form leptin receptor. In addi- Received Oct. 25, 1999; revised Feb. 3, 2000; accepted Feb. 17, 2000. tion, we report results of behavioral tests for aversive conse- This work was supported by National Institutes of Health Grants DK54890, DK54080, DK17844, and NS32273. quences of the selective MC4R agonist. Correspondence should be addressed to Stephen C. Benoit, Department of Psychiatry, University of Cincinnati, College of Medicine, Box 670559, Cincinnati, MATERIALS AND METHODS OH 45267-0559. E-mail: [email protected]. Subjects and materials. All animal procedures were approved by the Copyright © 2000 Society for Neuroscience 0270-6474/00/203442-07$15.00/0 institutional animal care and use committee of the University of Cincin- Benoit et al. • Selective MC4 Agonist and Food Intake J. Neurosci., May 1, 2000, 20(9):3442–3448 3443 nati. Male Long–Evans rats were individually housed in stainless steel cages and maintained on a 12 hr light/dark cycle. Laboratory chow (Purina 5001) and water were provided ad libitum (except where noted) during the experiments. Seven days after arrival in the laboratory, rats were anesthetized with 3.3 ml/kg equithesin and implanted with a 21-gauge stainless steel cannula (Plastics One, Roanoke, VA) aimed at the third ventricle (i3vt) 2.2 mm posterior to bregma and 7.4 mm ventral to dura (for review, see Seeley et al., 1996). Placement and patency of cannulas were confirmed by administration of 10 ng of angiotensin II in saline while the animals were water replete. Animals that did not drink at least 5 ml of water within 60 min were considered to have failed cannula placement and were not used in the experiments. Rats that received taste aversion training were also implanted unilaterally with intraoral cannulas constructed of polyethelene-100 tubing, which allows liquid stimuli to be infused directly into the oral cavity. These were placed anterolateral to the first maxillary molar and were threaded subcutaneously to exit the top of the head (for review, see Thiele et al., 1998). Obese C57/B6 db/db (n ϭ 10) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were individually housed in plastic tubs and maintained on a 12 hr light/dark cycle. They were fed and watered ad libitum, except where noted. Each mouse received one intraperitoneal injection of saline (0.9%, 200 l) and one injection of Ro27–3225 (200 g, 200 l). For all experiments with rats, i3vt infusions were administered in volumes of 2 l. Intraperitoneal injections were all isotonic and delivered in NaCl vehicle volumes of 20 ml/kg (rat) or 200 l (mouse). In all experiments on food intake, subjects received manipulations immedi- ately before onset of the dark phase. Experiment 1: MCR-based assays. All assays were performed in dupli- cate. Binding assays were performed using membranes prepared from Figure 1. Two-dimensional structure of the selective MC4R agonist HEK293 cells stably transfected with human MC1R, MC3R, MC4R, and (Ro27–3225) and the selective antagonist (Ro27–4680). 125 4 7 7 MC5R. Binding of [ I] [Nle , D-Phe ] -MSH (NDP-MSH) (labeled via chloramines T method, 2000 Ci/nmol specific activity) (catalog #IM316; 10 (MC1R and MC4R) or 30 (MC3R and MC5R) l of ethanol extract, Amersham Pharmacia Biotech, Arlington Heights, IL) to the membrane 50 lof[125I]cAMP, 50 l of antiserum, and 50 l of SPA beads were homogenates was done as follows: 20 mg of wheat germ agglutinin added to a 96 well plate. The total well volume after addition was 200 l. scintillation proximity assay (SPA) beads (Amersham Pharmacia Bio- The plates were sealed and incubated for 15–20 hr at room temperature. tech) was resuspended with 10 ml of 50 mM HEPES, 10 mM MgCl2, and [ 125I]cAMP binding to the SPA beads was determined by counting for 2 0.1% BSA, pH 7.4. Concentrated membrane homogenate was added to min. Each plate contained controls for unstimulated cells and NDP- give a final concentration of 0.04 mg/ml (MC1R, MC3R, and MC5R) and ␣MSH for stimulated cells. 0.032 mg/ml (MC4R). The mixture of SPA beads plus homogenate was Experiment 2: food intake in rats. After recovery from i3vt cannulation, incubated for 1 hr at 4°C with constant and vigorous mixing. Assays were food intake was measured for at least3dtoestablish baseline. On test initiated by distributing 50 l of SPA membrane into 96 well plates days, rats were weighed and food hoppers were removed from cages 2 hr (Optiplate polystyrene microplate; Packard, Meridian, CT), and 2 lof before the beginning of the dark phase. Rats (n ϭ 8 per group) then compounds at various concentrations or DMSO was added. Nonspecific received i3vt infusion of 0.0, 0.1, 1.0, 3.0, 5.0, or 10.0 nmol of either the binding was determined by adding an excess of unlabeled NDP-MSH (2 MC4R antagonist (Ro27–4680) or MC4R agonist (Ro27–3225) in saline M).