Antiobesity Effect of a Melanin-Concentrating Hormone 1 Receptor Antagonist in Diet-Induced Obese Mice
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内分泌学杂志 2005年第7期
Tsukuba Research Institute (S.M., A.I., A.G., R.M., M.I., H.I., M.M., A.K.), Banyu Pharmaceutical Co., Ltd., Tsukuba 300-2611, Japan; and Departments of Metabolic Disorders (Y.F., Z.S., D.J.Mar., D.J.Mac.) and Medicinal Chemistry (M.A.B.), Merck Research Laboratories, Rahway, New Jersey 07065
Address all correspondence and requests for reprints to: Dr. Akane Ishihara, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-2611, Japan. E-mail: akane_ishihara@merck.com.
Abstract
Melanin-concentrating hormone (MCH) is a cyclic orexigenic peptide expressed in the lateral hypothalamus, which plays an important role in regulating energy balance. To elucidate the physiological role of MCH in obesity development, the present study examined the effect of a selective MCH1 receptor (MCH1R) antagonist in the diet-induced obesity mouse model. The MCH1R antagonist has high affinity and selectivity for MCH-1R and potently inhibits intracerebroventricularly injected MCH-induced food intake in Sprague Dawley rats. Chronic intracerebroventricular infusion of the MCH1R antagonist (7.5 μg/d) completely suppressed body weight gain in diet-induced obese mice during the treatment periods and significantly decreased cumulative food intake, by 14%. Carcass analysis showed that the MCH1R antagonist resulted in a selective decrease of body fat in the diet-induced obese mice. In addition, the MCH1R antagonist ameliorated the obesity-related hypercholesterolemia, hyperinsulinemia, hyperglycemia, and hyperleptinemia. These results indicate that MCH has a major role in the development of diet-induced obesity in mice and that a MCH1R antagonist might be a useful candidate as an antiobesity agent.
Introduction
MELANIN-CONCENTRATING HORMONE (MCH) is a cyclic 19-amino-acid polypeptide that is expressed predominantly in the lateral hypothalamus (LH). The LH is a region of the brain involved in the regulation of feeding, the neuroendocrine axis, and thermogenesis. Several lines of investigation suggest that MCH is an important mediator of energy homeostasis. Mice lacking prepro-MCH are lean and hypophagic and have an elevated metabolic rate (1). Conversely, prepro-MCH overexpression in mice results in a greater susceptibility to obesity (2). Furthermore, overexpression of MCH mRNA has been found in obese rodents, such as ob/ob, db/db, and Ay/a mice (3, 4, 5). Exogenous administration of MCH stimulates food intake (3, 6), and chronic intracerebroventricular (ICV) infusion of MCH (7, 8) or a related MCH1 receptor (MCH1R) agonist (9) produces obesity with hyperphagia. Even when pair-feeding is employed to prevent hyperphagia, ICV infusion of MCH still produces anabolic changes (10). The effects of MCH are mediated through G protein-coupled receptors located in the central nervous system; and thus far, two receptor subtypes, MCH1R and MCH2R, have been identified (11, 12, 13, 14). Because rodents possess only MCH1R, all pharmacological effects of MCH in rodents are likely mediated via MCH1R (15). Recently, peptide and nonpeptidic MCH1R antagonist have been developed, and both antagonists produced antiobese effects in diet-induced obese (DIO) rats (9, 16). Collectively, these data indicate that MCH1R is an important regulator of energy homeostasis and suggest that it may play an important role in the development of obesity. However, because these data were obtained using MCH1R antagonists for which selectivity has not been validated in MCH1R-deficient mice, it is unclear whether the actions of these agents on energy homeostasis are mediated by MCH1R. In the present study, we evaluated the effect of the selective MCH1R antagonist (9, 17) in a diet-induced obesity model, which is considered to be a relevant model of human obesity, and also evaluated the selectivity of the MCH1R antagonist in MCH1R-deficient mice.
Materials and Methods
Materials
MCH was purchased from the Peptide Institute (Osaka, Japan). All other chemicals were analytical grade. The peptidic MCH1R antagonist (9, 17) was synthesized in Banyu Pharmaceutical Co., Ltd.
Binding and functional assays
CHO-K1-derived cell lines (CCL-61; American Type Culture Collection, Manassas, VA) that stably express human MCH1R from pEF/myc/cyto (Invitrogen, Carlsbad, CA) and human MCH2R from pEF1/V5-HisB (Invitrogen) were cultured in D-MEM/F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mg/ml geneticin at 37 C with 5% CO2 in a humidified atmosphere. For preparation of membranes used for the binding assays, the cells were washed with 10 mM 3[N-morholino]propanesulfonic acid buffer (pH 7.4) containing 20% sucrose, 154 mM NaCl, 10 mM KCl, and 0.8 mM CaCl2, homogenized, and centrifuged at 1000 x g for 15 min. The supernatant was centrifuged at 100,000 x g for 60 min. The pellet was resuspended in 5 mM HEPES buffer (pH 7.4) and centrifuged again. The precipitated membrane fraction was resuspended in the same buffer and used for the assays. The process of the binding of [125I]MCH (PerkinElmer, Wellesley, MA) to the MCH1R membrane preparations was performed in 250 μl of 50 mM Tris buffer (pH 7.4) containing 10 mM MgCl2, 2 mM EDTA, 0.1% bacitracin, and 0.2% BSA. The membranes (5 μg/ml) were incubated at 25 C for 60 min with 50 pM [125I]MCH in the presence of several concentrations of the test peptides. The MCH-2R binding assay was conducted with [Phe13,[125I]Tyr19]-MCH (PerkinElmer) in 250 μl of 20 mM HEPES buffer (pH 7.4) supplemented as above. The MCH2R membranes (75 μg/ml) were incubated at 25 C for 30 min with 50 pM [Phe13,[125I]Tyr19]-MCH. Bound and free radio ligands were separated by filtration using a GF/C glass filter presoaked with 0.3% polyethylenimine. The remaining radioactivity on the filter was quantitated using a Topcount-HTS (PerkinElmer). Specific binding of radio ligands was defined as the difference between total binding and nonspecific binding in the presence of 1 μM of the corresponding cold peptides. For the MCH1R functional assay, 1 d before the assay, the MCH1R/CHO-K1 cells were seeded into a black-wall, clear-bottom 96-well plate at 5 x 104 cells/well. The cells were loaded with Fluo-4-AM (Molecular Probes, Inc., Eugene, OR) for 1 h in D-MEM/F-12 containing 10% FBS and 2.5 mM probenecid in a CO2 incubator. After washing with the assay buffer (HBSS containing 20 mM HEPES, 0.5% BSA, and 2.5 mM probenecid), the cell plate was set into a FLIPR (Molecular Devices, Sunnyvale, CA), the assay started, and fluorescence output measured. The maximum fluorescence change induced by 5 nM MCH was determined in the presence of several concentrations of the test peptides. The IC50 and equilibrium dissociation constant (Ki) values for both the binding and functional assays were calculated by using Prism Version 3.00 software (GraphPad Software, Inc., San Diego, CA).
Animals
Male Sprague Dawley (SD) rats (20 wk old; Charles River Japan, Tokyo, Japan), male C57BL/6J mice (27 wk old; CLEA Japan, Inc., Tokyo, Japan), and male F2 50% C57BL/6J x 50% 129SvEv Mch1r wild-type and littermate Mch1r knockout mice derived from F1 heterozygous crosses were employed (21–24 wk old). Generations of MCH1R-deficient mice were described previously (7). The animals were housed individually in plastic cages kept at 23 ± 2 C, 55 ± 15% relative humidity, and maintained on a light-dark cycle with the lights on from 0700–1900 h. Water and regular chow (CE-2; CLEA Japan, Inc.) were available ad libitum. All experimental procedures followed the Japanese Pharmacological Society Guideline for Animal Use.
Surgical procedure and experimental design
Rat acute ICV injection.
SD rats were anesthetized with sodium pentobarbital (50 mg/kg, ip; Dinabot, Tokyo, Japan), and a sterile 26-gauge guide cannula (Plastics One Inc., Roanoke, VA) was stereotaxically implanted into the third ventricle. The stereotaxic coordinates used were 2.2 mm posterior to the bregma and 8.0 mm from the surface of the skull, using a flat skull position. Cannulae were attached to the skull with dental cement, and an antibiotic (Cefamedin , 50 mg/kg; Fujisawa Pharmaceutical Co., Ltd., Tokyo, Japan) was injected sc. The experiments were conducted at least 1 wk after the surgery. The placement of the cannula was confirmed before the experiment by injection of angiotensin II. Rats were injected with 150 ng angiotensin II, and water intake was monitored for 1 h. Only rats that drank more than 5 ml during the 1 h observation period were used for subsequent studies. The day before the experiment, the food was changed to a moderately high-fat diet (MHF diet; Oriental Bioservice Kanto Inc., Ibaraki, Japan) to satiate rats. The MHF diet provides 52.4% energy as carbohydrate, 15.0% as protein, and 32.6% as fat (4.41 kcal/g). Rats were injected with either MCH (5 μg) or MCH + MCH1R antagonist (1 μg) dissolved in artificial cerebrospinal fluid (aCSF), and food intake was monitored for 2 h. The injection was given 1 h before the onset of the dark cycle. To examine the effect on baseline food intake, rats were injected either with the MCH1R antagonist or aCSF under the same conditions.
Mouse chronic ICV infusion.
Experiment 1. ICV infusion of MCH1R antagonist in diet-induced obesity of C57BL/6J mice.
Mice were anesthetized with sodium pentobarbital (80 mg/kg, ip; Dinabot), and a sterile brain infusion cannula (28 gauge; Durect Corp., Cupertino, CA) was stereotaxically implanted into the right lateral ventricle. The stereotaxic coordinates used were 0.4 mm posterior to the bregma, 0.8 mm lateral to the midline, and 2.0 mm from the surface of the skull, using a flat skull position. Cannulae were attached to the skull with dental cement. The infusion cannula was connected via polyvinylchloride tubing to an osmotic minipump (model 2004; Durect Corp.) filled with 30% propylene glycol in water. The pump was implanted under the skin of the mouse’s back, and antibiotic (Cefamedin , 50 mg/kg; Fujisawa Pharmaceutical Co., Ltd., Tokyo, Japan) was injected sc. The placement of the cannula was confirmed at the end of the experiment by injection of 0.5% Evans blue dye. After a 2-wk recovery period, mice were fed a MHF diet. Five mice were kept on regular chow as a control. After 2 wk of exposure to the MHF diet, the mice were divided into two groups to match average values of basal food intake, body weight, and diet-induced body weight gain. The infusion pump was replaced with a new pump filled with the MCH1R antagonist (7.5 μg/d; n = 14) or its vehicle (30% propylene glycol; n = 14) under isoflurane anesthesia. The infusion pumps in the regular chow-fed mice were also replaced with new ones filled with vehicle (n = 5). Food intake and body weight were measured. After a 4-wk ICV infusion, the mice were fasted for 2 h, and blood samples were collected from the infraorbital vein under conscious condition, for measurement of plasma glucose, insulin, and leptin levels. Under isoflurane anesthesia, the mice were then killed via the collection of whole blood from the heart. Epididymal, retroperitoneal, and mesenteric adipose tissue and the liver were excised and weighed. All tissue was kept at –20 C for carcass analysis.
Experiment 2. ICV infusion of MCH1R antagonist in diet-induced obesity of wild-type and MCH1R-deficient mice.
Mice were anesthetized with ketamine (100 mg/kg, im) and domitor (0.75 mg/kg, im), and a sterile osmotic pump connector cannula (28 gauge; Plastics One, Inc.) was stereotaxically implanted into the dorsal third ventricle. The stereotaxic coordinates used were 0.22 mm posterior, 0.3 mm lateral, and 3.3 mm ventral to the bregma. Cannulae were attached to the skull with cyanoacrylate adhesive followed by dental cement. The infusion cannula was connected via polyvinylchloride tubing to an osmotic minipump (model 1007D; Durect Corp.) filled with aCSF (Harvard Apparatus, Inc., Holliston, MA). The pump was implanted under the skin of the mouse’s back, and antisedan (5 mg/kg) was injected im. After a 5-d recovery period, mice were anesthetized with ketamine (100 mg/kg) and domitor (0.75 mg/kg), and a dual-energy x-ray absorptiometry (DEXA) measurement was performed to evaluate body composition. After the DEXA measurements, the infusion pump was replaced with an osmotic minipump (model 2002; Durect Corp.) filled with the MCH1R antagonist (7.5 μg/d; n = 9–10) or its vehicle (aCSF; n = 10–11), and antisedan (5 mg/kg) was injected im. On the day of pump replacement, all mice were switched to a MHF diet, and food intake and body weight were measured. After a 2-wk ICV infusion, all mice were once again subjected to DEXA measurements under ketamine and domitor anesthesia.
Carcass analysis.
Carcasses were homogenized and dried at 135 C for 2 h. The carcass powder was extracted with diethyl ether to determine body fat (18). After using the Kjeldahl method to measure nitrogen content, protein content was then calculated by multiplying the nitrogen content by 6.25 (19).
Measurement of hormone and blood chemistry.
Plasma glucose, triglyceride, total cholesterol, high-density lipoprotein (HDL)-cholesterol, and low-density lipoprotein (LDL)-cholesterol levels were measured using commercial kits [Determiner GL-E, L TGII, L TCII, L HDL-C, and L LDL-C (Kyowa Medex, Tokyo, Japan)]. Plasma free fatty acids were determined using commercial kits [NEFA-HA Testwako (II) (Wako Pure Chemical Industries, Ltd., Osaka, Japan)]. Insulin and leptin levels were measured by ELISA (Morinaga, Yokohama, Japan).
Measurement of motor activity.
Another set of mice, which were ICV infused with the MCH1R antagonist, were prepared for measurement of spontaneous motor activity. The MCH1R antagonist was ICV infused for 4 wk under regular chow-fed conditions. Motor activity was measured in home cages during the last 3 d of the 4-wk treatment using an activity monitoring system (NS-AS01; Neuroscience, Tokyo, Japan). In brief, the activity monitor was composed of an infrared ray sensor placed over each home cage, a signal amplification circuit, and a control unit. The sensor detected the movement of the animal on the basis of the released infrared radiation associated with its body temperature (20, 21). The motor activity data were collected at 10-min intervals and analyzed with a computer-associated analyzing system (AB System-24A; Neuroscience).
Statistical analysis
Data are expressed as mean ± SEM. Body weight change was compared between groups using the repeated-measures ANOVA. For food intake, blood parameters, and tissue weights, a two-way ANOVA coupled to a post hoc Bonferroni test was performed. P values < 0.05 were considered significant.
Results
Affinity of the MCH1R antagonist
The binding affinities of MCH and the MCH1R antagonist were evaluated in CHO-K1 cells that stably expressed human MCH1R and MCH2R (Table 1). The MCH1R antagonist exhibited high affinity for MCH1R, with a Ki value of 9.9 nM and about 1000-fold selectivity over MCH2R, although the affinity for MCH1R was 140-fold less than that of MCH. The MCH1R antagonist showed antagonistic activity in a functional assay, with an IC50 value of 15 nM.
TABLE 1. In vitro profile of the MCH1R antagonist
Effect of MCH1R antagonist on ICV MCH-induced food intake in SD rats
ICV injection of MCH (5 μg) produced approximately a 2-fold increase of food intake in satiated SD rats (Fig. 1). Coadministration of the MCH1R antagonist at 1 μg completely suppressed the MCH-induced food intake, compared with the basal level of food intake in the aCSF-injected group. The MCH1R antagonist alone had no significant effect but tended to decrease short-term spontaneous food intake, as compared with the aCSF-injected group (Fig. 1), and did not produce any changes in gross behavior (data not shown).
FIG. 1. Effect of the MCH1R antagonist on MCH-induced food intake and on spontaneous food intake in SD rats. MCH (5 μg) and the MCH1R antagonist (1 μg) were injected simultaneously or the MCH1R antagonist (1 μg) was injected alone into the third ventricle at 1 h before the dark cycle. Food intake was measured 2 h after injection. Values are the mean ± SEM of eight to 16 rats per group. #, P < 0.05 vs. vehicle-treated (Veh.) group; *, P < 0.05 vs. MCH-treated group.
Effect of chronic ICV infusion of MCH1R antagonist in DIO mice
To investigate the antiobese effect of the MCH1R antagonist, the MCH1R antagonist was chronically ICV infused in DIO mice for 4 wk. Chronic treatment of the MCH1R antagonist significantly prevented diet-induced body weight gain and further decreased body weight in DIO mice (–1.1 ± 0.6 g) (Fig. 2A), whereas the vehicle-treated control group gained 5.5 ± 0.5 g (Fig. 2A). Treatment with the MCH1R antagonist significantly decreased food intake, and significant feeding suppression was still observed at the end of the treatment (3.58 ± 0.11 g vs. 4.02 ± 0.10 g at d 27; P < 0.01), although the feeding suppression tended to wane gradually. The MCH1R antagonist significantly decreased cumulative food intake (88.5 ± 2.8 g vs. 103.2 ± 2.1 g; P < 0.01).
FIG. 2. Effect of chronically ICV-infused MCH1R antagonist on body weight (B.W.) (A) and food intake (B) in C57BL/6J DIO mice. The MCH1R antagonist, at a dose of 7.5 μg/d, was chronically administered to the left lateral ventricle of the brain for 4 wk. Values are the mean ± SEM of 14 mice per group. #, P < 0.05; *, P < 0.01 vs. vehicle-treated group.
After the 4-wk treatment, DIO mice treated with vehicle or the MCH1R antagonist weighed 44.0 ± 0.8 g and 38.1 ± 0.8 g, respectively (P < 0.01), and regular chow-fed mice weighed 32.9 ± 0.8 g. DIO mice had a high percentage of body fat compared with the regular chow-fed mice (31.1 ± 1.0% vs. 13.6 ± 0.9%) (Fig. 3) and a relatively low protein content (15.6 ± 0.3% vs. 20.1 ± 0.6%) (Fig. 3). Treatment with the MCH1R antagonist significantly decreased the fat mass in DIO mice (22.5 ± 1.2%) (Fig. 3) and increased the relative protein content (17.5 ± 0.4%) (Fig. 3). Treatment with the MCH1R antagonist also significantly decreased the visceral adipose tissue weights, epidydimal, retroperitoneal, and mesenteric fat weight (Table 2), which confirms carcass analysis results. In addition, the MCH1R antagonist decreased liver weight to a level similar to that of the regular chow-fed mice.
FIG. 3. Effect of chronically ICV-infused MCH1R antagonist on body composition in DIO mice. The MCH1R antagonist, at a dose of 7.5 μg/d, was chronically administered to the left lateral ventricle of the brain for 4 wk. Body composition was quantified by chemical extraction methods. Values are the mean ± SEM of five to 14 mice per group. ##, P < 0.01 vs. vehicle-treated regular chow-fed group; **, P < 0.01 vs. vehicle-treated MHF diet-fed group.
TABLE 2. Effect of MCH1R antagonist on blood parameters and tissue weight in DIO mice
DIO mice exhibited obesity-related abnormalities of blood parameters, hyperinsulinemia, hyperglycemia, hyperleptinemia, and hypercholesterolemia, compared with the regular chow-fed mice (Table 2). The MCH1R antagonist significantly decreased all these parameters, plasma glucose, insulin, leptin, total cholesterol, and LDL-cholesterol.
Effect of chronic ICV infusion of the MCH1R antagonist in MCH1R-deficient and wild-type mice
To validate the selectivity of the MCH1R antagonist, it was chronically ICV infused at a dose of 7.5 μg/d in MCH1R-deficient and wild-type mice for 2 wk. After 2 wk of exposure to a MHF diet, wild-type mice gained 4.0 ± 0.6 g, and MCH1R deficient mice gained 1.8 g, respectively. The MCH1R antagonist significantly attenuated body weight gain only in wild-type mice (0.7 ± 0.4 g) but did not affect the body weight gain of MCH1R-deficient mice (Fig. 4A). Treatment with the MCH1R antagonist entirely prevented the gain in fat mass observed in wild-type mice treated with aCSF (Fig. 4C). In contrast, treatment with the MCH1R antagonist had no effect on fat mass gains in MCH1R-deficient mice. MCH1R antagonist treatment tended to decrease cumulative food intake only in wild-type mice, although this effect did not achieve statistical significance (P = 0.098) (Fig. 4B).
FIG. 4. Effect of chronically ICV-infused MCH1R antagonist on body weight (A), cumulative food intake (B), and fat mass change (C) in MCH1R-deficient and wild-type mice. The MCH1R antagonist, at a dose of 7.5 μg/d, was chronically administered to the dorsal third ventricle of the brain for 2 wk. Fat mass change was measured before and after treatment using DEXA. Values are the mean ± SEM of nine to 11 mice per group. ##, P < 0.01 vs. vehicle-treated group.
Effect of chronic ICV infusion of MCH1R antagonist on motor activity in mice
Because it has been reported that MCH1R-deficient mice showed increased motor activity (7, 22), we measured the motor activity to evaluate whether the antiobese effect of the MCH1R antagonist was due to the increased motor activity or not. In another set of regular chow-fed mice, the MCH1R antagonist was ICV infused for 4 wk at a dose of 7.5 μg/d, and the spontaneous motor activity was measured at the end of the treatment periods. Chronic treatment of the MCH1R antagonist slightly, but significantly, reduced body weight in the regular chow-fed mice. The MCH1R antagonist did not affect food intake throughout the treatment period (data not shown) and did not produce any change in motor activity during either the light or the dark cycle (Fig. 5).
FIG. 5. Effect of chronically ICV-infused MCH1R antagonist on body weight (A) and motor activity (B) in C57BL/6J lean mice. The MCH1R antagonist, at a dose of 7.5 μg/d, was chronically administered to the left lateral ventricle of the brain for 4 wk. Motor activity was measured at the end of the treatment period and expressed as cumulative activity during light and dark cycles. Values are the mean ± SEM of eight mice per group.
Discussion
To elucidate the physiological role of MCH1R in the development of obesity, we evaluated a selective, peptidic MCH1R antagonist in a diet-induced obesity model. The peptidic MCH1R antagonist was highly selective for the MCH1R, with more than a 1000-fold selectivity over human MCH2R, and did not show any significant cross-reactivity with 120 other binding assays and enzyme assays for targets that are considered to be involved in feeding regulation (data not shown). The MCH1R antagonist significantly decreased food intake and completely suppressed body weight gain in DIO mice. Similar results were previously reported with nonpeptidic MCH1R antagonists that produced antiobese effects in obese rodents (16). However, small molecules often cause nonmechanism-based feeding suppression and body weight reductions. Indeed, recently it was reported that a potent and selective Y5 antagonist reduced food intake in neuropeptide Y (NPY) Y5 receptor-deficient mice (23). Consequently, validation of an agent’s selectivity in an appropriate target receptor-deficient mouse was essential to prove the concept. In the present study, we further conducted validation of the compound’s selectivity in MCH1R-deficient mice. In the MCH1R-deficient mice, the antiobese effect of the peptidic MCH1R antagonist was completely abolished. As reported previously, the MCH1R-deficient mice are DIO resistant (7, 22); the diet-induced body weight gain was about half that of wild-type mice in present study. Thus, we cannot exclude the possibility that a relatively small increase in body weight of MCH1R-deficient mice may mask nonmechanism based body weight reductions caused by the MCH1R antagonist. However, the MCH1R antagonist had no effect on body weight gain, food intake, and fat mass gain in MCH1R-deficient mice, whereas it significantly inhibited body weight gain, even in lean mice (Fig. 5). Taken together, the antiobese effect of the MCH1R antagonist, which was observed in DIO mice, was not due to a nonselective toxic effect. Therefore, the current observations represent the first mechanism-based demonstration that MCH1R antagonism produces antiobese effects in DIO mice.
The MCH1R antagonist produced sustained reductions of food intake throughout the treatment periods, indicating that feeding suppression is one of the mechanisms of the MCH1R antagonist. Additionally, acute ICV administration of the MCH1R antagonist tended to decrease short-term spontaneous food intake in MHF diet-fed rats (Fig. 1). These data are in agreement with the observation that the exogenous administration of MCH stimulates food intake in rats (3, 6). Thus, MCH1R plays a key role in feeding regulation.
The MCH1R antagonist produced fat-selective reduction in DIO mice (Fig. 3). In general, body weight reduction that is derived merely through food restriction reduces both the fat and lean masses. Thus, the fat-selective reduction in the current experiment indicates that the MCH1R antagonist may have additional effects on the metabolic pathway beyond feeding suppression. We have demonstrated that chronic ICV infusion of MCH produced significant obesity; and even when hyperphagia was prevented, MCH still produced an increase in the fat mass (10). These data suggest that MCH regulates both energy intake and expenditure and makes it likely that the MCH1R antagonist produces an antiobesity effect by both reducing food intake and increasing energy expenditure. Further studies to elucidate the effect of the MCH1R antagonist on energy expenditure by measuring oxygen consumption or through the conduction of pair-feeding studies are needed to confirm this hypothesis.
Because the MCH1R-deficient mice showed increased motor activity (7, 22), the MCH1R antagonist might produce an antiobesity effect by increasing motor activity. However, after 4 wk of treatment with the MCH1R antagonist, no changes in spontaneous motor activity were recorded relative to the controls. Therefore, the antiobesity effect of the MCH1R antagonist in the present study is not due to hyperactivity.
We also observed that the efficacy of the MCH1R antagonist is clearly different between lean and DIO mice. The MCH1R antagonist remarkably reduced body weight in the DIO mice, whereas it reduced it only slightly in the lean mice. Because we administered the MCH1R antagonist via the ICV route, the exposure level of the MCH1R antagonist should be the same in both the DIO and lean mice. Therefore, these data indicate that the MCH pathway may be activated in obesity. In support of this idea, it has been reported that, in some obesity models, there are increased levels of hypothalamic MCH mRNA (3, 4, 5). Also, we have previously reported that the body weight gain produced by chronic ICV infusion of MCH is more pronounced under a high-fat diet-fed condition than a regular chow-fed condition (8). These results suggest that MCH may play a dominant role in energy homeostasis during obesity or high-fat intake.
Thus far, two types of MCH receptors have been identified. The rodent possesses only MCH1R, whereas humans possess both types of MCH receptors (11, 12, 13, 14, 15). The physiological role of MCH1R in species like humans, who have both receptors, is still unclear. In addition, the mechanism of MCH-mediated feeding regulation is unclear in rodents. Several studies have reported that there might be some interaction between MCH and other feeding-regulated neuropeptides. MCH neurons in the LH are innervated by NPY-, agouti-related protein (AgRP)-, and proopiomelanocortin (POMC)-containing fibers originating in the arcuate nucleus (24). ICV administration of MC4R antagonists, AgRP or SHU9119, increases MCH expression (25). However, MCH1R-deficient mice showed normal brain neuropeptides expression, such as NPY, AgRP, orexin, POMC, galanin, and cocaine- and amphetamine-regulated transcript (CART) (7, 22), and 2 wk treatment with the peptidic-MCH1R antagonist did not affect NPY and POMC mRNA levels in the arcuate nucleus (9). Further investigations using this MCH1R antagonist may clarify the pathway of MCH-mediated feeding regulation, and investigations in species with both MCH receptors will provide guidance in the elucidation of the role of MCH in human obesity.
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Address all correspondence and requests for reprints to: Dr. Akane Ishihara, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-2611, Japan. E-mail: akane_ishihara@merck.com.
Abstract
Melanin-concentrating hormone (MCH) is a cyclic orexigenic peptide expressed in the lateral hypothalamus, which plays an important role in regulating energy balance. To elucidate the physiological role of MCH in obesity development, the present study examined the effect of a selective MCH1 receptor (MCH1R) antagonist in the diet-induced obesity mouse model. The MCH1R antagonist has high affinity and selectivity for MCH-1R and potently inhibits intracerebroventricularly injected MCH-induced food intake in Sprague Dawley rats. Chronic intracerebroventricular infusion of the MCH1R antagonist (7.5 μg/d) completely suppressed body weight gain in diet-induced obese mice during the treatment periods and significantly decreased cumulative food intake, by 14%. Carcass analysis showed that the MCH1R antagonist resulted in a selective decrease of body fat in the diet-induced obese mice. In addition, the MCH1R antagonist ameliorated the obesity-related hypercholesterolemia, hyperinsulinemia, hyperglycemia, and hyperleptinemia. These results indicate that MCH has a major role in the development of diet-induced obesity in mice and that a MCH1R antagonist might be a useful candidate as an antiobesity agent.
Introduction
MELANIN-CONCENTRATING HORMONE (MCH) is a cyclic 19-amino-acid polypeptide that is expressed predominantly in the lateral hypothalamus (LH). The LH is a region of the brain involved in the regulation of feeding, the neuroendocrine axis, and thermogenesis. Several lines of investigation suggest that MCH is an important mediator of energy homeostasis. Mice lacking prepro-MCH are lean and hypophagic and have an elevated metabolic rate (1). Conversely, prepro-MCH overexpression in mice results in a greater susceptibility to obesity (2). Furthermore, overexpression of MCH mRNA has been found in obese rodents, such as ob/ob, db/db, and Ay/a mice (3, 4, 5). Exogenous administration of MCH stimulates food intake (3, 6), and chronic intracerebroventricular (ICV) infusion of MCH (7, 8) or a related MCH1 receptor (MCH1R) agonist (9) produces obesity with hyperphagia. Even when pair-feeding is employed to prevent hyperphagia, ICV infusion of MCH still produces anabolic changes (10). The effects of MCH are mediated through G protein-coupled receptors located in the central nervous system; and thus far, two receptor subtypes, MCH1R and MCH2R, have been identified (11, 12, 13, 14). Because rodents possess only MCH1R, all pharmacological effects of MCH in rodents are likely mediated via MCH1R (15). Recently, peptide and nonpeptidic MCH1R antagonist have been developed, and both antagonists produced antiobese effects in diet-induced obese (DIO) rats (9, 16). Collectively, these data indicate that MCH1R is an important regulator of energy homeostasis and suggest that it may play an important role in the development of obesity. However, because these data were obtained using MCH1R antagonists for which selectivity has not been validated in MCH1R-deficient mice, it is unclear whether the actions of these agents on energy homeostasis are mediated by MCH1R. In the present study, we evaluated the effect of the selective MCH1R antagonist (9, 17) in a diet-induced obesity model, which is considered to be a relevant model of human obesity, and also evaluated the selectivity of the MCH1R antagonist in MCH1R-deficient mice.
Materials and Methods
Materials
MCH was purchased from the Peptide Institute (Osaka, Japan). All other chemicals were analytical grade. The peptidic MCH1R antagonist (9, 17) was synthesized in Banyu Pharmaceutical Co., Ltd.
Binding and functional assays
CHO-K1-derived cell lines (CCL-61; American Type Culture Collection, Manassas, VA) that stably express human MCH1R from pEF/myc/cyto (Invitrogen, Carlsbad, CA) and human MCH2R from pEF1/V5-HisB (Invitrogen) were cultured in D-MEM/F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mg/ml geneticin at 37 C with 5% CO2 in a humidified atmosphere. For preparation of membranes used for the binding assays, the cells were washed with 10 mM 3[N-morholino]propanesulfonic acid buffer (pH 7.4) containing 20% sucrose, 154 mM NaCl, 10 mM KCl, and 0.8 mM CaCl2, homogenized, and centrifuged at 1000 x g for 15 min. The supernatant was centrifuged at 100,000 x g for 60 min. The pellet was resuspended in 5 mM HEPES buffer (pH 7.4) and centrifuged again. The precipitated membrane fraction was resuspended in the same buffer and used for the assays. The process of the binding of [125I]MCH (PerkinElmer, Wellesley, MA) to the MCH1R membrane preparations was performed in 250 μl of 50 mM Tris buffer (pH 7.4) containing 10 mM MgCl2, 2 mM EDTA, 0.1% bacitracin, and 0.2% BSA. The membranes (5 μg/ml) were incubated at 25 C for 60 min with 50 pM [125I]MCH in the presence of several concentrations of the test peptides. The MCH-2R binding assay was conducted with [Phe13,[125I]Tyr19]-MCH (PerkinElmer) in 250 μl of 20 mM HEPES buffer (pH 7.4) supplemented as above. The MCH2R membranes (75 μg/ml) were incubated at 25 C for 30 min with 50 pM [Phe13,[125I]Tyr19]-MCH. Bound and free radio ligands were separated by filtration using a GF/C glass filter presoaked with 0.3% polyethylenimine. The remaining radioactivity on the filter was quantitated using a Topcount-HTS (PerkinElmer). Specific binding of radio ligands was defined as the difference between total binding and nonspecific binding in the presence of 1 μM of the corresponding cold peptides. For the MCH1R functional assay, 1 d before the assay, the MCH1R/CHO-K1 cells were seeded into a black-wall, clear-bottom 96-well plate at 5 x 104 cells/well. The cells were loaded with Fluo-4-AM (Molecular Probes, Inc., Eugene, OR) for 1 h in D-MEM/F-12 containing 10% FBS and 2.5 mM probenecid in a CO2 incubator. After washing with the assay buffer (HBSS containing 20 mM HEPES, 0.5% BSA, and 2.5 mM probenecid), the cell plate was set into a FLIPR (Molecular Devices, Sunnyvale, CA), the assay started, and fluorescence output measured. The maximum fluorescence change induced by 5 nM MCH was determined in the presence of several concentrations of the test peptides. The IC50 and equilibrium dissociation constant (Ki) values for both the binding and functional assays were calculated by using Prism Version 3.00 software (GraphPad Software, Inc., San Diego, CA).
Animals
Male Sprague Dawley (SD) rats (20 wk old; Charles River Japan, Tokyo, Japan), male C57BL/6J mice (27 wk old; CLEA Japan, Inc., Tokyo, Japan), and male F2 50% C57BL/6J x 50% 129SvEv Mch1r wild-type and littermate Mch1r knockout mice derived from F1 heterozygous crosses were employed (21–24 wk old). Generations of MCH1R-deficient mice were described previously (7). The animals were housed individually in plastic cages kept at 23 ± 2 C, 55 ± 15% relative humidity, and maintained on a light-dark cycle with the lights on from 0700–1900 h. Water and regular chow (CE-2; CLEA Japan, Inc.) were available ad libitum. All experimental procedures followed the Japanese Pharmacological Society Guideline for Animal Use.
Surgical procedure and experimental design
Rat acute ICV injection.
SD rats were anesthetized with sodium pentobarbital (50 mg/kg, ip; Dinabot, Tokyo, Japan), and a sterile 26-gauge guide cannula (Plastics One Inc., Roanoke, VA) was stereotaxically implanted into the third ventricle. The stereotaxic coordinates used were 2.2 mm posterior to the bregma and 8.0 mm from the surface of the skull, using a flat skull position. Cannulae were attached to the skull with dental cement, and an antibiotic (Cefamedin , 50 mg/kg; Fujisawa Pharmaceutical Co., Ltd., Tokyo, Japan) was injected sc. The experiments were conducted at least 1 wk after the surgery. The placement of the cannula was confirmed before the experiment by injection of angiotensin II. Rats were injected with 150 ng angiotensin II, and water intake was monitored for 1 h. Only rats that drank more than 5 ml during the 1 h observation period were used for subsequent studies. The day before the experiment, the food was changed to a moderately high-fat diet (MHF diet; Oriental Bioservice Kanto Inc., Ibaraki, Japan) to satiate rats. The MHF diet provides 52.4% energy as carbohydrate, 15.0% as protein, and 32.6% as fat (4.41 kcal/g). Rats were injected with either MCH (5 μg) or MCH + MCH1R antagonist (1 μg) dissolved in artificial cerebrospinal fluid (aCSF), and food intake was monitored for 2 h. The injection was given 1 h before the onset of the dark cycle. To examine the effect on baseline food intake, rats were injected either with the MCH1R antagonist or aCSF under the same conditions.
Mouse chronic ICV infusion.
Experiment 1. ICV infusion of MCH1R antagonist in diet-induced obesity of C57BL/6J mice.
Mice were anesthetized with sodium pentobarbital (80 mg/kg, ip; Dinabot), and a sterile brain infusion cannula (28 gauge; Durect Corp., Cupertino, CA) was stereotaxically implanted into the right lateral ventricle. The stereotaxic coordinates used were 0.4 mm posterior to the bregma, 0.8 mm lateral to the midline, and 2.0 mm from the surface of the skull, using a flat skull position. Cannulae were attached to the skull with dental cement. The infusion cannula was connected via polyvinylchloride tubing to an osmotic minipump (model 2004; Durect Corp.) filled with 30% propylene glycol in water. The pump was implanted under the skin of the mouse’s back, and antibiotic (Cefamedin , 50 mg/kg; Fujisawa Pharmaceutical Co., Ltd., Tokyo, Japan) was injected sc. The placement of the cannula was confirmed at the end of the experiment by injection of 0.5% Evans blue dye. After a 2-wk recovery period, mice were fed a MHF diet. Five mice were kept on regular chow as a control. After 2 wk of exposure to the MHF diet, the mice were divided into two groups to match average values of basal food intake, body weight, and diet-induced body weight gain. The infusion pump was replaced with a new pump filled with the MCH1R antagonist (7.5 μg/d; n = 14) or its vehicle (30% propylene glycol; n = 14) under isoflurane anesthesia. The infusion pumps in the regular chow-fed mice were also replaced with new ones filled with vehicle (n = 5). Food intake and body weight were measured. After a 4-wk ICV infusion, the mice were fasted for 2 h, and blood samples were collected from the infraorbital vein under conscious condition, for measurement of plasma glucose, insulin, and leptin levels. Under isoflurane anesthesia, the mice were then killed via the collection of whole blood from the heart. Epididymal, retroperitoneal, and mesenteric adipose tissue and the liver were excised and weighed. All tissue was kept at –20 C for carcass analysis.
Experiment 2. ICV infusion of MCH1R antagonist in diet-induced obesity of wild-type and MCH1R-deficient mice.
Mice were anesthetized with ketamine (100 mg/kg, im) and domitor (0.75 mg/kg, im), and a sterile osmotic pump connector cannula (28 gauge; Plastics One, Inc.) was stereotaxically implanted into the dorsal third ventricle. The stereotaxic coordinates used were 0.22 mm posterior, 0.3 mm lateral, and 3.3 mm ventral to the bregma. Cannulae were attached to the skull with cyanoacrylate adhesive followed by dental cement. The infusion cannula was connected via polyvinylchloride tubing to an osmotic minipump (model 1007D; Durect Corp.) filled with aCSF (Harvard Apparatus, Inc., Holliston, MA). The pump was implanted under the skin of the mouse’s back, and antisedan (5 mg/kg) was injected im. After a 5-d recovery period, mice were anesthetized with ketamine (100 mg/kg) and domitor (0.75 mg/kg), and a dual-energy x-ray absorptiometry (DEXA) measurement was performed to evaluate body composition. After the DEXA measurements, the infusion pump was replaced with an osmotic minipump (model 2002; Durect Corp.) filled with the MCH1R antagonist (7.5 μg/d; n = 9–10) or its vehicle (aCSF; n = 10–11), and antisedan (5 mg/kg) was injected im. On the day of pump replacement, all mice were switched to a MHF diet, and food intake and body weight were measured. After a 2-wk ICV infusion, all mice were once again subjected to DEXA measurements under ketamine and domitor anesthesia.
Carcass analysis.
Carcasses were homogenized and dried at 135 C for 2 h. The carcass powder was extracted with diethyl ether to determine body fat (18). After using the Kjeldahl method to measure nitrogen content, protein content was then calculated by multiplying the nitrogen content by 6.25 (19).
Measurement of hormone and blood chemistry.
Plasma glucose, triglyceride, total cholesterol, high-density lipoprotein (HDL)-cholesterol, and low-density lipoprotein (LDL)-cholesterol levels were measured using commercial kits [Determiner GL-E, L TGII, L TCII, L HDL-C, and L LDL-C (Kyowa Medex, Tokyo, Japan)]. Plasma free fatty acids were determined using commercial kits [NEFA-HA Testwako (II) (Wako Pure Chemical Industries, Ltd., Osaka, Japan)]. Insulin and leptin levels were measured by ELISA (Morinaga, Yokohama, Japan).
Measurement of motor activity.
Another set of mice, which were ICV infused with the MCH1R antagonist, were prepared for measurement of spontaneous motor activity. The MCH1R antagonist was ICV infused for 4 wk under regular chow-fed conditions. Motor activity was measured in home cages during the last 3 d of the 4-wk treatment using an activity monitoring system (NS-AS01; Neuroscience, Tokyo, Japan). In brief, the activity monitor was composed of an infrared ray sensor placed over each home cage, a signal amplification circuit, and a control unit. The sensor detected the movement of the animal on the basis of the released infrared radiation associated with its body temperature (20, 21). The motor activity data were collected at 10-min intervals and analyzed with a computer-associated analyzing system (AB System-24A; Neuroscience).
Statistical analysis
Data are expressed as mean ± SEM. Body weight change was compared between groups using the repeated-measures ANOVA. For food intake, blood parameters, and tissue weights, a two-way ANOVA coupled to a post hoc Bonferroni test was performed. P values < 0.05 were considered significant.
Results
Affinity of the MCH1R antagonist
The binding affinities of MCH and the MCH1R antagonist were evaluated in CHO-K1 cells that stably expressed human MCH1R and MCH2R (Table 1). The MCH1R antagonist exhibited high affinity for MCH1R, with a Ki value of 9.9 nM and about 1000-fold selectivity over MCH2R, although the affinity for MCH1R was 140-fold less than that of MCH. The MCH1R antagonist showed antagonistic activity in a functional assay, with an IC50 value of 15 nM.
TABLE 1. In vitro profile of the MCH1R antagonist
Effect of MCH1R antagonist on ICV MCH-induced food intake in SD rats
ICV injection of MCH (5 μg) produced approximately a 2-fold increase of food intake in satiated SD rats (Fig. 1). Coadministration of the MCH1R antagonist at 1 μg completely suppressed the MCH-induced food intake, compared with the basal level of food intake in the aCSF-injected group. The MCH1R antagonist alone had no significant effect but tended to decrease short-term spontaneous food intake, as compared with the aCSF-injected group (Fig. 1), and did not produce any changes in gross behavior (data not shown).
FIG. 1. Effect of the MCH1R antagonist on MCH-induced food intake and on spontaneous food intake in SD rats. MCH (5 μg) and the MCH1R antagonist (1 μg) were injected simultaneously or the MCH1R antagonist (1 μg) was injected alone into the third ventricle at 1 h before the dark cycle. Food intake was measured 2 h after injection. Values are the mean ± SEM of eight to 16 rats per group. #, P < 0.05 vs. vehicle-treated (Veh.) group; *, P < 0.05 vs. MCH-treated group.
Effect of chronic ICV infusion of MCH1R antagonist in DIO mice
To investigate the antiobese effect of the MCH1R antagonist, the MCH1R antagonist was chronically ICV infused in DIO mice for 4 wk. Chronic treatment of the MCH1R antagonist significantly prevented diet-induced body weight gain and further decreased body weight in DIO mice (–1.1 ± 0.6 g) (Fig. 2A), whereas the vehicle-treated control group gained 5.5 ± 0.5 g (Fig. 2A). Treatment with the MCH1R antagonist significantly decreased food intake, and significant feeding suppression was still observed at the end of the treatment (3.58 ± 0.11 g vs. 4.02 ± 0.10 g at d 27; P < 0.01), although the feeding suppression tended to wane gradually. The MCH1R antagonist significantly decreased cumulative food intake (88.5 ± 2.8 g vs. 103.2 ± 2.1 g; P < 0.01).
FIG. 2. Effect of chronically ICV-infused MCH1R antagonist on body weight (B.W.) (A) and food intake (B) in C57BL/6J DIO mice. The MCH1R antagonist, at a dose of 7.5 μg/d, was chronically administered to the left lateral ventricle of the brain for 4 wk. Values are the mean ± SEM of 14 mice per group. #, P < 0.05; *, P < 0.01 vs. vehicle-treated group.
After the 4-wk treatment, DIO mice treated with vehicle or the MCH1R antagonist weighed 44.0 ± 0.8 g and 38.1 ± 0.8 g, respectively (P < 0.01), and regular chow-fed mice weighed 32.9 ± 0.8 g. DIO mice had a high percentage of body fat compared with the regular chow-fed mice (31.1 ± 1.0% vs. 13.6 ± 0.9%) (Fig. 3) and a relatively low protein content (15.6 ± 0.3% vs. 20.1 ± 0.6%) (Fig. 3). Treatment with the MCH1R antagonist significantly decreased the fat mass in DIO mice (22.5 ± 1.2%) (Fig. 3) and increased the relative protein content (17.5 ± 0.4%) (Fig. 3). Treatment with the MCH1R antagonist also significantly decreased the visceral adipose tissue weights, epidydimal, retroperitoneal, and mesenteric fat weight (Table 2), which confirms carcass analysis results. In addition, the MCH1R antagonist decreased liver weight to a level similar to that of the regular chow-fed mice.
FIG. 3. Effect of chronically ICV-infused MCH1R antagonist on body composition in DIO mice. The MCH1R antagonist, at a dose of 7.5 μg/d, was chronically administered to the left lateral ventricle of the brain for 4 wk. Body composition was quantified by chemical extraction methods. Values are the mean ± SEM of five to 14 mice per group. ##, P < 0.01 vs. vehicle-treated regular chow-fed group; **, P < 0.01 vs. vehicle-treated MHF diet-fed group.
TABLE 2. Effect of MCH1R antagonist on blood parameters and tissue weight in DIO mice
DIO mice exhibited obesity-related abnormalities of blood parameters, hyperinsulinemia, hyperglycemia, hyperleptinemia, and hypercholesterolemia, compared with the regular chow-fed mice (Table 2). The MCH1R antagonist significantly decreased all these parameters, plasma glucose, insulin, leptin, total cholesterol, and LDL-cholesterol.
Effect of chronic ICV infusion of the MCH1R antagonist in MCH1R-deficient and wild-type mice
To validate the selectivity of the MCH1R antagonist, it was chronically ICV infused at a dose of 7.5 μg/d in MCH1R-deficient and wild-type mice for 2 wk. After 2 wk of exposure to a MHF diet, wild-type mice gained 4.0 ± 0.6 g, and MCH1R deficient mice gained 1.8 g, respectively. The MCH1R antagonist significantly attenuated body weight gain only in wild-type mice (0.7 ± 0.4 g) but did not affect the body weight gain of MCH1R-deficient mice (Fig. 4A). Treatment with the MCH1R antagonist entirely prevented the gain in fat mass observed in wild-type mice treated with aCSF (Fig. 4C). In contrast, treatment with the MCH1R antagonist had no effect on fat mass gains in MCH1R-deficient mice. MCH1R antagonist treatment tended to decrease cumulative food intake only in wild-type mice, although this effect did not achieve statistical significance (P = 0.098) (Fig. 4B).
FIG. 4. Effect of chronically ICV-infused MCH1R antagonist on body weight (A), cumulative food intake (B), and fat mass change (C) in MCH1R-deficient and wild-type mice. The MCH1R antagonist, at a dose of 7.5 μg/d, was chronically administered to the dorsal third ventricle of the brain for 2 wk. Fat mass change was measured before and after treatment using DEXA. Values are the mean ± SEM of nine to 11 mice per group. ##, P < 0.01 vs. vehicle-treated group.
Effect of chronic ICV infusion of MCH1R antagonist on motor activity in mice
Because it has been reported that MCH1R-deficient mice showed increased motor activity (7, 22), we measured the motor activity to evaluate whether the antiobese effect of the MCH1R antagonist was due to the increased motor activity or not. In another set of regular chow-fed mice, the MCH1R antagonist was ICV infused for 4 wk at a dose of 7.5 μg/d, and the spontaneous motor activity was measured at the end of the treatment periods. Chronic treatment of the MCH1R antagonist slightly, but significantly, reduced body weight in the regular chow-fed mice. The MCH1R antagonist did not affect food intake throughout the treatment period (data not shown) and did not produce any change in motor activity during either the light or the dark cycle (Fig. 5).
FIG. 5. Effect of chronically ICV-infused MCH1R antagonist on body weight (A) and motor activity (B) in C57BL/6J lean mice. The MCH1R antagonist, at a dose of 7.5 μg/d, was chronically administered to the left lateral ventricle of the brain for 4 wk. Motor activity was measured at the end of the treatment period and expressed as cumulative activity during light and dark cycles. Values are the mean ± SEM of eight mice per group.
Discussion
To elucidate the physiological role of MCH1R in the development of obesity, we evaluated a selective, peptidic MCH1R antagonist in a diet-induced obesity model. The peptidic MCH1R antagonist was highly selective for the MCH1R, with more than a 1000-fold selectivity over human MCH2R, and did not show any significant cross-reactivity with 120 other binding assays and enzyme assays for targets that are considered to be involved in feeding regulation (data not shown). The MCH1R antagonist significantly decreased food intake and completely suppressed body weight gain in DIO mice. Similar results were previously reported with nonpeptidic MCH1R antagonists that produced antiobese effects in obese rodents (16). However, small molecules often cause nonmechanism-based feeding suppression and body weight reductions. Indeed, recently it was reported that a potent and selective Y5 antagonist reduced food intake in neuropeptide Y (NPY) Y5 receptor-deficient mice (23). Consequently, validation of an agent’s selectivity in an appropriate target receptor-deficient mouse was essential to prove the concept. In the present study, we further conducted validation of the compound’s selectivity in MCH1R-deficient mice. In the MCH1R-deficient mice, the antiobese effect of the peptidic MCH1R antagonist was completely abolished. As reported previously, the MCH1R-deficient mice are DIO resistant (7, 22); the diet-induced body weight gain was about half that of wild-type mice in present study. Thus, we cannot exclude the possibility that a relatively small increase in body weight of MCH1R-deficient mice may mask nonmechanism based body weight reductions caused by the MCH1R antagonist. However, the MCH1R antagonist had no effect on body weight gain, food intake, and fat mass gain in MCH1R-deficient mice, whereas it significantly inhibited body weight gain, even in lean mice (Fig. 5). Taken together, the antiobese effect of the MCH1R antagonist, which was observed in DIO mice, was not due to a nonselective toxic effect. Therefore, the current observations represent the first mechanism-based demonstration that MCH1R antagonism produces antiobese effects in DIO mice.
The MCH1R antagonist produced sustained reductions of food intake throughout the treatment periods, indicating that feeding suppression is one of the mechanisms of the MCH1R antagonist. Additionally, acute ICV administration of the MCH1R antagonist tended to decrease short-term spontaneous food intake in MHF diet-fed rats (Fig. 1). These data are in agreement with the observation that the exogenous administration of MCH stimulates food intake in rats (3, 6). Thus, MCH1R plays a key role in feeding regulation.
The MCH1R antagonist produced fat-selective reduction in DIO mice (Fig. 3). In general, body weight reduction that is derived merely through food restriction reduces both the fat and lean masses. Thus, the fat-selective reduction in the current experiment indicates that the MCH1R antagonist may have additional effects on the metabolic pathway beyond feeding suppression. We have demonstrated that chronic ICV infusion of MCH produced significant obesity; and even when hyperphagia was prevented, MCH still produced an increase in the fat mass (10). These data suggest that MCH regulates both energy intake and expenditure and makes it likely that the MCH1R antagonist produces an antiobesity effect by both reducing food intake and increasing energy expenditure. Further studies to elucidate the effect of the MCH1R antagonist on energy expenditure by measuring oxygen consumption or through the conduction of pair-feeding studies are needed to confirm this hypothesis.
Because the MCH1R-deficient mice showed increased motor activity (7, 22), the MCH1R antagonist might produce an antiobesity effect by increasing motor activity. However, after 4 wk of treatment with the MCH1R antagonist, no changes in spontaneous motor activity were recorded relative to the controls. Therefore, the antiobesity effect of the MCH1R antagonist in the present study is not due to hyperactivity.
We also observed that the efficacy of the MCH1R antagonist is clearly different between lean and DIO mice. The MCH1R antagonist remarkably reduced body weight in the DIO mice, whereas it reduced it only slightly in the lean mice. Because we administered the MCH1R antagonist via the ICV route, the exposure level of the MCH1R antagonist should be the same in both the DIO and lean mice. Therefore, these data indicate that the MCH pathway may be activated in obesity. In support of this idea, it has been reported that, in some obesity models, there are increased levels of hypothalamic MCH mRNA (3, 4, 5). Also, we have previously reported that the body weight gain produced by chronic ICV infusion of MCH is more pronounced under a high-fat diet-fed condition than a regular chow-fed condition (8). These results suggest that MCH may play a dominant role in energy homeostasis during obesity or high-fat intake.
Thus far, two types of MCH receptors have been identified. The rodent possesses only MCH1R, whereas humans possess both types of MCH receptors (11, 12, 13, 14, 15). The physiological role of MCH1R in species like humans, who have both receptors, is still unclear. In addition, the mechanism of MCH-mediated feeding regulation is unclear in rodents. Several studies have reported that there might be some interaction between MCH and other feeding-regulated neuropeptides. MCH neurons in the LH are innervated by NPY-, agouti-related protein (AgRP)-, and proopiomelanocortin (POMC)-containing fibers originating in the arcuate nucleus (24). ICV administration of MC4R antagonists, AgRP or SHU9119, increases MCH expression (25). However, MCH1R-deficient mice showed normal brain neuropeptides expression, such as NPY, AgRP, orexin, POMC, galanin, and cocaine- and amphetamine-regulated transcript (CART) (7, 22), and 2 wk treatment with the peptidic-MCH1R antagonist did not affect NPY and POMC mRNA levels in the arcuate nucleus (9). Further investigations using this MCH1R antagonist may clarify the pathway of MCH-mediated feeding regulation, and investigations in species with both MCH receptors will provide guidance in the elucidation of the role of MCH in human obesity.
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