Effects of a Specific MCHR1 Antagonist (GW803430) on Energy

Original Article Obesity OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Effects of a Specific MCHR1 Antagonist (GW803430) on Energy Budget and Glucose Metabolism in Diet-induced Obese Mice Li-Na Zhang1, Rachel Sinclair1, Colin Selman1, Sharon Mitchell1, David Morgan2, John C. Clapham2 and John R. Speakman1

Objective: The melanin-concentrating hormone (MCH) is a centrally acting peptide implicated in the regulation of energy homeostasis and body weight, although its role in glucose homeostasis is uncertain. Our objective was to determine effects of MCHR1 antagonism on energy budgets and glucose homeostasis in mice. Methods: Effects of chronic oral administration of a specific MCHR1 antagonist (GW803430) on energy budgets and glucose homeostasis in diet-induced obese (DIO) C57BL/6J mice were examined. Results: Oral administration of GW803430 for 30 days reduced food intake, body weight, and body fat. Circulating leptin and triglycerides were reduced but insulin and nonesterified fatty acids were unaffected. Despite weight loss there was no improvement in glucose homeostasis (insulin levels and intraperitoneal glucose tolerance tests). On day 4-6, mice receiving MCHR1 antagonist exhibited decreased metabolisable energy intake and increased daily energy expenditure. However these effects had disappeared by day 22-24. Physical activity during the dark phase was increased by MCHR1 antagonist treatment throughout the 30-day treatment. Conclusions: GW803430 produced a persistent anti-obesity effect due to both a decrease in energy intake and an increase in energy expenditure via physical activity but did not improve glucose homeostasis.

Obesity (2014) 22, 681–690. doi:10.1002/oby.20418

Introduction obese (DIO) mice (11) induced hyperphagia and body weight gain, especially on a high-fat diet. In contrast, MCH/ mice were hypo- Melanin-concentrating hormone (MCH) is a 19-animo acid cyclic pep- phagic and lean compared to wild-type littermates (12), and genetic tide predominantly expressed in the lateral hypothalamus and zona ablation of the MCHR1 gene resulted in a lean phenotype accompa- incerta, with projections to the dorsal and ventral striatum, prefrontal nied by hypophagia and increased energy expenditure (13). Further- cortex, nucleus of the solitary tract, and the parabrachial nucleus (1). more, acute or chronic ICV injections of MCHR1 agonists in MCH acts via two G protein-coupled receptors, MCHR1 and MCHR2 rodents resulted in the same phenotype observed with MCH treat- (2) expressed in humans, rhesus monkeys (Macaca mulatta), dogs (Canis familiaris), and ferrets (Mustela putorius furo) with similar dis- ment (14) whereas central and peripheral administration of a tribution patterns (2-4). However, MCHR2 is not expressed in rodents. MCHR1 antagonist led to reduced food intake and body weight MCHR1 is widely distributed in the mouse and rat brain (5,6) includ- (15). In addition to effects on energy balance, MCH has also been ing areas associated with energy homeostasis such as the arcuate, ven- implicated in glucose homeostasis, but the relevant data are con- tro-medial and dorso-medial nuclei in the hypothalamus (5,7). fused. MCHR1 knockout mice had lower insulin levels compared with wild-type mice, indicative of increased insulin sensitivity (13). The MCH/MCHR1 pathway plays a key role in the regulation of Similarly, MCHR1 deficiency in ob/ob mice caused a lower blood feeding behavior and energy balance. Acute intra-cerebro-ventricu- glucose response and markedly lower insulin levels despite no dif- lar (ICV) injection of MCH stimulated food and water intake in rats ferences in body weight (16). In contrast, central MCH injection to (8,9). Chronic ICV infusion of MCH in rats (10) and diet-induced rats induced insulin resistance (17).

1 Integrative Physiology, Institute of Biological Sciences, University of Aberdeen, Aberdeen, Scotland, UK. Correspondence: John R. Speakman ([email protected]) 2 CVGI, Research and Development, AstraZeneca, Alderly Park, Cheshire, UK

Funding agencies: This work was funded by a studentship from the University of Aberdeen and AstraZeneca. Disclosure: The authors declare no conflicts of interests. Additional Supporting Information may be found in the online version of this article. Received: 27 November 2012 Accepted: 1 February 2013 Published online 20 March 2013. doi:10.1002/oby.20418

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Brain penetration is required for MCHR1 antagonists to inhibit food et al. (25). RMR was measured at baseline (week 15 of HFD) and intake and to reduce body weight (18). Recently a number of small- on day 19 of treatment using an open-flow respiratory system. molecule antagonists for MCHR1 have been developed for the treatment of obesity. However, relatively few studies have addressed the All data were expressed as means 6 SD. General linear modeling mechanisms underlying the antiobesity effects of these MCHR1 (GLM) with repeated measures was used to compare body mass, food antagonists. Huang et al. showed that rats treated with a MCHR1 an- intake, PA, Tb between MCHR1 antagonist and vehicle groups tagonist (1-(4-Amino-phenyl)-pyrrolidin-3-yl-amine and 6-(3-amino- throughout the experiment. Independent t test with Bonferroni correc- pyrrolidin-1-yl)-pyridin-3-yl-amine) lost significantly more weight tion was performed to compare differences between groups at a given than pair-fed matches (19). Acute administration of a highly selec- time point. Analysis of covariance (ANCOVA) was performed to tive and potent MCHR1 antagonist (20) that has high brain penetra- examine differences in MEI, DEE, and RMR using body mass as a bility and oral bioavailability increased metabolic rate in DIO mice covariate (26). MEI, DEE, and RMR were corrected for body mass (20). Moreover, mice chronically treated with this antagonist exhib- using the residual method in linear regression and corrected data were ited a significantly higher body temperature compared to pair-fed then compared by independent t tests. Body fat mass and corrected controls (20). However, neither body weight nor fat mass differed RMR data were analyzed using repeated measures to examine differ- between DIO mice treated with the MCHR1 antagonist SCH-A ences between baseline and treatment. Paired t test was performed to ((6)-N-[trans-5-(4-cyanophenyl)bicyclo[3.1.0]hex-2-yl]-N’-[4-fluoro- compare body fat mass before and after dosing. Group differences in 3-(trifluoromethyl)phenyl]-N-[3-(4-methyl-1-piperazinyl)propy- plasma leptin, insulin, TG, NEFA levels, and fat depots were exam- l]urea), and the pair-fed group during a 5-day treatment (21), which, ined with independent t test. P values < 0.05 were considered statisti- together with unchanged energy expenditure (21), suggests that the cally significant. All data were analyzed using SPSS 17.0 statistical effect of SCH-A on body weight was only due to suppression of package for Windows (SPSS, Chicago, IL). feeding. GW803430 is a potent and selective non-peptide MCHR1 antagonist (22) that has been suggested to have weight-reducing effects (23). To examine exactly how GW803430 affected energy Results balance and glucose homeostasis, we evaluated energy budgets at Body mass and fatness early (day 4-6) and late (day 22-24) stages of a 30-day oral treat- Prior to the treatment, there was no difference in body mass between ment, compared to a control group receiving only vehicle. Physical vehicle-treated control and MCHR1 antagonist-treated group (Vehi- activity (PA) and body temperature (Tb) were monitored throughout cle: 42.94 6 6.40 g; MCHR1 antagonist: 42.71 6 5.52 g. Mice the experiment, and resting metabolic rate (RMR) was measured on receiving MCHR1 antagonist showed a significant loss of body day 19 of treatment. We measured glucose homeostasis using i.p. weight throughout the experiment (GLM repeated measures: F(1,19) ¼ glucose tolerance tests and levels of circulating insulin in fasted ani- 43.55, P < 0.001, Figure 1A), and the relative weight change was sig- mals. This protocol permitted investigation of the effects of chronic nificantly greater in MCHR1 antagonist-treated mice than vehicle- MCHR1 antagonist administration on the different components of treated control (GLM repeated measures: F(1,19) ¼ 38.90, P < 0.001, energy expenditure and glucose regulation. Figure 1B). At the end of the study, the mean body mass of mice treated with MCHR1 antagonist was 15.7% lower than for the control Methods animals (t test on final body weight: t ¼ 2.19, df ¼ 19, P ¼ 0.041). Male C57BL/6 mice were fed on a high-fat diet (HFD) for 16 week to In line with body weight changes, there was a significant effect of establish diet-induced obesity and then were randomly divided into time (baseline and day 26 of treatment) on fat mass (F(1,19) ¼ 8.54, MCHR1 antagonist-treated group and vehicle-treated group. P ¼ 0.009) and a strong interaction between treatment group and GW803430, a specific MCHR1 antagonist, and its vehicle were orally time (F(1,19) ¼ 32.51, P < 0.001) though the between-group effects administered to two groups of mice on a daily basis for 30 days (for on fat mass did not reach a statistically significant level (F(1,19) ¼ more details see Supporting Information uploaded separately). 0.99, P ¼ 0.331) again showing that fat mass had declined in the treatment group over time but not in the controls (Figure 1C). After Body weight and food intake was measured every day. Body fatness 26 days of MCHR1 antagonist treatment there was a significant was evaluated using dual energy X-ray absorptiometry (DXA) at decrease in fat mass (Paired t test: t ¼ 5.43, df ¼ 9, P < 0.001) baseline and on day 26 of dosing. PA and Tb was constantly recorded whereas vehicle-treated mice showed no significant change in fat via Mini-Mitter and VitalViewTM system throughout the experiment. mass (Paired t test: t ¼ -2.23, df ¼ 10, P ¼ 0.05). At baseline, no All mice received an i.p. glucose tolerance test (GTT) on day 27 (see difference was detected in body fat mass between groups. Following Supporting Information uploaded separately). At the end of 30-day 26 days of MCHR1 antagonist treatment DIO mice had 35.5% less treatment, mice were sacrificed and blood samples were collected for fat mass than vehicle-treated controls (Independent t test: t ¼ 2.33, the determination of metabolite levels (leptin, insulin, triglyceride, df ¼ 19, P ¼ 0.031). Moreover, on day 30 when all animals were and nonesterfied fatty acid). See Zhang et al. (24) for details. dissected, MCHR1 antagonist-treated mice exhibited lower total fat mass (t ¼ 2.26, df ¼ 19, P ¼ 0.036) accompanied by less perirenal On days 4-6 and day 22-24, energy budget profile was obtained by fat (t ¼ 3.21, df ¼ 19, P ¼ 0.005) and less mesenteric fat (t ¼ 2.47, measuring energy intake, assimilation efficiency and energy expend- df ¼ 19, P ¼ 0.023) whereas the weights of gonadal fat and subcu- iture. We measured daily energy expenditure (DEE) using the dou- taneous fat did not differ significantly, although both effects were bly labeled water (DLW) technique. Feces accumulated during DEE on the borderline of significance (gonadal fat: t ¼ 2.03, df ¼ 19, P evaluation were collected and dried to a constant mass. The gross ¼ 0.057; subcutaneous fat: t ¼ 1.98, df ¼ 19, P ¼ 0.062; Figure energy (GE) content of the samples was measured using bomb calo- 1D). No differences were detected in lean mass either at baseline or rimetry. The apparent energy absorption efficiency (AEAE) and me- on day 26 of dosing (baseline: t ¼ 0.23, df ¼ 19, P ¼ 0.819; day tabolizable energy intake (MEI) was calculated as detailed by Krol 26 of dosing; t ¼ 0.30, df ¼ 19, P ¼ 0.770; Figure 1E).

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FIGURE 1 Chronic administration of a MCHR1 antagonist decreased body mass and fatness in DIO mice. The data are presented as mean 6 SD. A. Absolute body mass change of vehicle-treated group and MCHR1 antagonist-treated group during 30 days of dosing. B. Body mass changes presented as % of initial body weight. C. Body fat mass measured by DXA at baseline and on day 26 of treatment. Dashed line represents vehicle-treated group; solid line represents MCHR1 antagonist-treated group. Different letters over bars indicate significant differences between groups; *, P < 0.05; **, P < 0.01. D. Masses of different body fat depots after 30-day treatment, **, P < 0.01. E. Lean mass measured by DXA at baseline and on day 26 of treatment.

Glucose tolerance and plasma metabolites exhibited a better tolerance to glucose, suggesting that HFD On day 27 of dosing, mice from both groups exhibited similar fasted impaired glucose metabolism (Figure 2A). blood glucose levels (Vehicle: 9.44 6 0.55 (lgmL1); MCHR1 antagonist: 8.60 6 0.59 (lgmL1)). There were no significant differ- Plasma leptin levels were highly associated with body fat mass (R2 ences between vehicle-treated and MCHR1 antagonist-treated groups ¼ 0.94, F(1,19) ¼ 290.09, P < 0.001, Figure 2B). Despite an abso- in glucose tolerance (F(1,19) ¼ 0.36, P ¼ 0.558, Figure 2A). How- lute difference in leptin (Independent t test: t ¼ 2.48, df ¼ 19, P ¼ ever, age-matched non-DIO mice that were fed with a regular diet 0.022; Figure 2C), chronic MCHR1 antagonist treatment had no

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FIGURE 2 Effects of chronic administration of a MCHR1 antagonist on plasma metabolites and hormones. A. Glucose tolerance test (GTT) in DIO mice on day 27 of MCHR1 antagonist treatment. Open squares represent vehicle-treated group; Filled squares represent MCHR1 antagonist-treated group; Open triangles represent age-matched controls under a regular diet. B. Effects of chronic MCHR1 antagonist dosing on plasma leptin level; different letters over bars indicate significant differences between groups. C. Linear regression plot of plasma leptin level against body fat mass. D. Effects of chronic MCHR1 antagonist dosing on plasma triglyceride (TG) levels. E. Effects of chronic MCHR1 antagonist dosing on plasma insulin levels. F. Effects of chronic MCHR1 antagonist dosing on plasma nonesterified fatty acid (NEFA) levels. Empty bar represents vehicle-treated group; filled bar represents MCHR1 antagonist-treated group.

significant effect on circulating levels of leptin with the effect of fat MCHR1 antagonist dosing ((t ¼ 2.49, df ¼ 19, P ¼ 0.022, Figure mass removed (GLM: using fat mass as a covariate: F(1,18) ¼ 0.96, 2D). In contrast, no differences were observed in circulating insulin P ¼ 0.341; using square of fat mass as a covariate: F(1,18) ¼ 0.88, P (t ¼ 1.38, df ¼ 19, P ¼ 0.187; Figure 2E) and NEFA (t ¼ 0.95, df ¼ 0.36). Circulating TG levels were significantly reduced by chronic ¼ 19, P ¼ 0.353; Figure 2F).

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FIGURE 3 Effects of chronic administration of a MCHR1 antagonist on food intake in DIO mice. The data represent mean 6 SD A. Average daily food intake during 30-day treatment of a MCHR1 antagonist, *, P < 0.05. B. Cumulative food intake during 30-day treatment of a MCHR1 antagonist *, P < 0.05. Group differences were significant from day 1 till the end of experiment. Open squares represent vehicle-treated group; Filled squares represent MCHR1 antagonist-treated group.

Food intake and energy budget account for fat loss suggests an increase in energy expenditure was Chronic MCHR1 antagonist dosing markedly inhibited daily average also marked. food intake in DIO mice throughout the treatment (GLM repeated measures: group, F(1,19) ¼ 5.31, P ¼ 0.033; days of treatment, Indeed, chronic MCHR1 antagonist dosing altered energy budget on F(1,19) ¼ 12.72, P < 0.001; days of treatment group, F(1,19) ¼ day 4-6 of treatment, when mice treated with MCHR1 antagonist 2.49, P ¼ 0.008; Figure 3A). Cumulative food intake was signifi- showed an increase in DEE (ANCOVA, F(1,18) ¼ 7.52, P ¼ 0.013, Fig- cantly reduced by chronic MCHR1 antagonist administration (GLM ure 4A) and a significant reduction in MEI (ANCOVA, F(1,18) ¼ 4.90, repeated measures: group: F ¼ 7.121, P ¼ 0.015, days of treatment, P ¼ 0.040, Figure 4B). As the treatment progressed, these effects of F(1,19) ¼ 1638.26, P < 0.001, days of treatment group, F(1,19) ¼ MCHR1 antagonist on energy budget disappeared on day 22-24 (DEE, 4.559, P < 0.001; Figure 3B). Over the 30-day treatment, accumu- F(1,18) ¼ 0.02, P ¼ 0.899; MEI, F(1,18) ¼ 0.14, P ¼ 0.709; Figure lated food intake of DIO mice receiving the MCHR1 antagonist was 4C,D). When both MEI and DEE were corrected for body mass, con- 9.1% less than that of vehicle-treated counterparts (t test: t ¼ 2.14, trast tests revealed a 16.4% decrease in MEI (t ¼ 2.13, df ¼ 19, P ¼ df ¼ 19, P ¼ 0.046). On day 26 when body fatness was evaluated, 0.027) and a 10.8% increase in DEE (t ¼ -2.77, df ¼ 19, P ¼ 0.012) the difference in accumulated gross food intake between groups was during day 4-6 of treatment whereas no changes were observed on day 6.62 g, equivalent to 138.34 kJ (dry food intake energy content of 22-24 (MEI: t ¼ 0.61, df ¼ 19, P ¼ 0.548; DEE: t ¼ 0.41, df ¼ 19, P dry food). Body fat has an energy content of about 39 kJ g1, so the ¼ 0.688) (Figure 4E,F). MCHR1 antagonist treatment had no effects difference in mean fat mass on day 26 (5.25 g) was equivalent to on assimilation efficiency at both time points (day 4-6: t ¼ 0.24, df ¼ 204.75 kJ. The fact that reduced gross food intake did not fully 19, P ¼ 0.813; day 22-24: t ¼ -0.31, df ¼ 19, P ¼ 0.759; Figure 4G).

www.obesityjournal.org Obesity | VOLUME 22 | NUMBER 3 | MARCH 2014 685 FIGURE 4 Effects of chronic administration of a MCHR1 antagonist on energy budgets in DIO mice. The data represent mean 6 SD. A. Regression plot of daily energy expenditure (DEE) against body mass on day 4-6 of MCHR1 antagonist treatment. B. Regression plot of metabolizable energy intake (MEI) against body mass on day 4-6 of MCHR1 antagonist treatment. C. Regression plot of daily energy expenditure (DEE) against body mass on day 22-24 of MCHR1 antagonist treatment. D. Regression plot of metabolizable energy intake (MEI) against body mass on day 22-24 of MCHR1 antagonist treatment. Open squares represent vehicle-treated group; Filled squares represent MCHR1 antagonist-treated group. E. Corrected metabolizable energy intake (MEI) on day 4-6 and day 22-14 of MCHR1 antagonist treatment. F. Corrected daily energy expenditure (DEE) on day 4-6 and day 22-24 of MCHR1 antagonist treatment. G. The apparent energy absorption efficiency (AEAE) on day 4-6 and day 22-24 of MCHR1 antagonist treatment. H. Resting metabolic rate (RMR) on day 19 of MCHR1 antagonist treatment. Open bar represents vehicle-treated group; filled bar represents MCHR1 antagonist-treated group. Dif- ferent letters over bars indicate significant differences between groups. Original Article Obesity OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

No changes in RMR were observed following 19 days of MCHR1 an- reduced after chronic MCHR1 antagonist dosing. This observation is tagonist dosing (GLM repeated measures on corrected RMR: group: in agreement with the fact that MCHR1 antagonism is associated F(1,19) ¼ 0.02, P ¼ 0.901; time (baseline and day 19 of treatment), with the reduction of hepatic TG accumulation in ovariectomized F(1,19) ¼ 0.02, P ¼ 0.902, time group, F(1,19) ¼ 0.01, P ¼ 0.923; mice (27). Figure 4H), indicating the enhancement on DEE was attributed to changes in physical activity. Notably, normalizing the DEE data with Paradoxically, no improvement in glucose metabolism was observed lean mass instead of total body mass at day 22-24 did not alter the despite a significant reduction in body weight and fat mass. MCHR1 results. antagonist treatment recovered the weight gain induced by HFD but had no effect on impaired glucose tolerance. Moreover, chronic MCHR1 antagonist administration did not affect circulating insulin Body temperature or NEFA. Consistent with this finding, Kowalski et al. also demon- At baseline, mice treated with MCHR1 antagonist and with vehicle strated that 28 days of MCHR1 antagonist treatment had no effect showed a circadian rhythm in body temperature with higher temper- on circulating insulin levels (21). Notably, previous studies showed ature during the dark phase and lower temperature during the light that the effects of either MCHR1 antagonism or MCH administra- phase, and no differences between groups. Chronic MCHR1 antago- tion on glucose homeostasis were independent of body weight nist treatment did not alter body temperature throughout the experi- change (17,29). The absence of an effect on glucose homeostasis ment (see Supporting Information S8 for detailed data). and lipid profiles however may indicate that while MCHR1 antagonism may improve obesity its impact on the metabolic sequalae of high body fatness may be less profound. Physical activity Prior to the treatment, animals showed a circadian rhythm in daily In agreement with the changes in body mass and fatness, the physical activity, with most activity confined to the dark phase and MCHR1 antagonist exerted a persistent anorexic effect on food there were no differences in daily activity patterns between groups intake in DIO mice on a high-fat diet. Mashiko et al. reported that a (GLM repeated measures: group, F ¼ 0.399, P ¼ 0.535; time, F ¼ peptidic MCHR1 antagonist suppressed MCH-induced food intake in 22.860, P < 0.001 time group F ¼ 0.585, P ¼ 0.938; Figure satiated Sprague-Dawley rat (28). Furthermore, chronic 4-week infu- 5A). During the first 4 days of dosing, no changes in activity pattern sion of the antagonist reduced food intake, prevented body weight were observed (GLM repeated measures: group, F ¼ 0.062, P ¼ gain; and also further reduced body weight in these obese mice (21). 0.807; time, F ¼ 23.791, P < 0.001; time group, F ¼ 1.119, P ¼ Another study showed that the peptide antagonist blocked the 0.320; Figure 5B). In contrast, DIO mice receiving the MCHR1 an- increase in palatable food intake after icv administration of MCH tagonist showed significantly elevated physical activity levels on (30). The antagonist was more effective when rats were fed a highly day 18-22 of dosing (group, F ¼ 9.531, P ¼ 0.006; time, F ¼ palatable diet, suggesting that the reward system is involved in the 26.623, P < 0.001; time group, F ¼ 2.167, P ¼ 0.002; Figure feeding suppression effect of MCHR1 antagonism. In support of this 5C). Furthermore, chronic MCHR1 antagonist administration signifi- notion, studies on MCH/ mice showed that expression of dopa- cantly increased physical activity levels during the dark phase (F ¼ mine transporter was significantly elevated in the nucleus accum- 8.586, P ¼ 0.009; Figure 5D) despite no effects on activity during bens and that evoked dopamine release was significantly increased the light phase (F ¼ 0.119, P ¼ 0.734; Figure 5E) or on averaged in the nucleus accumbens shell (31). Hypothalamic MCH also mod- daily activity (F ¼ 3.134, P ¼ 0.095; Figure 5F). ulates nucleus accumbens activity (32).

To further understand the mechanisms underlying the weight-reducing effect of MCHR1 antagonist, we evaluated energy budgets of Discussion DIO mice at different stages of the treatment. Hu et al. demon- We have demonstrated here that chronic oral administration of strated that a brain permeable MCH1R antagonist resulted in a sig- GW803430, reduced body mass and adiposity in DIO mice fed a nificant reduction in body weight and fat mass in DIO mice and high fat diet, but with minimal effects on their glucose homeostasis. that this effect appeared to be as partially driven by the decrease Previous work has shown GW803040 inhibited MCHR1 binding in in food intake, which indicated an impact of blockade of MCHR1 different areas of the brain and exhibits substantial occupancy of the signaling on energy expenditure (18). Indeed, in our study we MCHR1 at doses of 1 mg kg1 or greater (23). These authors (23) found that on day 4-6 of dosing, MCHR1 antagonism significantly further evaluated the effects of the same compound at a dose of 10 inhibited metabolizable energy intake and enhanced daily energy mg kg1 in MCHR1þ/þ and MCHR1/ mice, respectively. The expenditure. Consistent with these observations, Shearman et al. fact that MCHR1/ mice did not show a decrease in body weight, observed that antagonism of MCHR1 in rats for 14 days resulted but MCHR1þ/þ mice did, suggested that GW803040 produced sig- in a decrease in caloric efficiency (expressed as mg body weight/ nificant reductions in body mass, fatness and food intake via kcal food intake) indicating an increase in energy expenditure. MCHR1 antagonism (23). Our findings are consistent with previous However, these effects on components from both sides of energy studies reporting reductions in body fat mass following administra- balance disappeared when the treatment progressed to day 22-24. tion of other MCHR1 antagonists (15,21,27,28). The loss of body At this stage, the modest surplus of energy expenditure over mass and body fat mainly stemmed from a negative energy balance energy intake might contribute to the maintenance of weight loss. induced by the MCHR1 antagonist. The MCHR1 antagonist also The animals could have adopted compensatory adaptations in the caused a reduction of circulating leptin levels, but this was second- energy budget in response to a sustained weight loss. With regard ary to the loss of body fat since the effect of the MCHR1 antagonist to energy expenditure, there has been inconsistent evidence show- on leptin disappeared after statistically accounting for body fat ing that acute (4 days) MCHR1 antagonism failed to increase mass. In addition, plasma triglyceride levels were significantly energy expenditure in DIO mice (21). It should be noted that in

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FIGURE 5 Effects of chronic administration of a MCHR1 antagonist on physical activity in DIO mice. The data represent mean 6 SD. A. Baseline 24-h physical activity (PA) pattern. B. 24-h physical activity (PA) during day 1-4 of MCHR1 antagonist treatment. C. 24-h physical activity (PA) during day 18-22 of MCHR1 antagonist treatment. D. Daily average physical activity (PA) over 30-day treatment of MCHR1 antagonist (F ¼ 3.134, P ¼ 0.095). E. Average physical activity (PA) during the light phase over 30-day treatment of MCHR1 antagonist (F ¼ 0.119, P ¼ 0.734). F. Average physical activity (PA) during the dark phase over 30-day treatment of MCHR1 antagonist (F ¼ 8.586, P ¼ 0.009). Open squares represent vehicle-treated group; Filled squares represent MCHR1 antagonist- treated group. MCHR1 antagonist-treated mice showed higher levels of physical activity during the dark phase, but not activity during the dark phase. the aforementioned study energy expenditure was measured for 23 Because energy expenditure consists of resting metabolic rate h using indirect calorimetry (Oxymax-CLAMS system), whereas (RMR), diet-induced thermogenesis, thermoregulation and physical daily energy expenditure in the current study was evaluated using activity, we specifically measured the effects of MCHR1 antagonism the doubly labeled water method. DLW measures are made in the on each component of energy expenditure. RMR was not affected animals’ home cage and therefore tend to involve fewer artifacts by MCHR1 antagonism after 19 days of treatment and nor was the than 24-h indirect calorimetry measures. The discrepancy in results respiratory quotient, which resembled the findings from DIO mice might be attributed to the different methods. treated with MCHR1 antagonist for 4 consecutive days (21). This

688 Obesity | VOLUME 22 | NUMBER 3 | MARCH 2014 www.obesityjournal.org Original Article Obesity OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY implies that MCHR1 antagonism has no effects on RMR regardless Acknowledgments of the duration of treatment. In our study, chronic MCHR1 antagonist administration had no effects on body temperature, which is in The authors would like to thank Peter Thomson and Paula Redman agreement with findings from Kowalski et al. who failed to detect for their technical help with isotope analysis for DLW. Thanks to any change in body temperature during the 6 h following acute Lambertus Benthem and Caroline Wingoff for the formulation and MCHR1 antagonist administration (21). In contrast, Ito et al. delivery of the MCHR1 antagonist. We thank Yuko Gamo for assis- recently demonstrated that MCHR1 antagonist treated mice showed tance with surgeries, Lobke Vaan Holt for her advice on statistics a significantly higher rectal temperature during the month of drug and Cathy Wyse for constructive comments on the manuscript. Spe- ¨ administration (20), but these previous data, based on rectal prob- cial thanks to Marianne Alholm Larsen Grønning, former senior ing to measure body temperature, are more prone to handling arti- director of Diabetes Research China, Novo Nordisk, for her gener- facts than the present study which employed completely noninva- ous support on the completion of this manuscript. sive measurements from implanted transmitters. On the other hand, VC 2013 The Obesity Society there has been evidence in support of a positive effect of MCH antagonism on body temperature. For example, mice centrally infused with MCH for 14 days significantly decreased body temperature (33). 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