Original Article Effects of Rimonabant (SR141716) on Fasting-Induced Hypothalamic-Pituitary-Adrenal Axis and Neuronal Activation in Lean and Obese Zucker Rats Christian Doyon, Raphae¨l G. Denis, Elena-Dana Baraboi, Pierre Samson, Jose´e Lalonde, Yves Deshaies, and Denis Richard The effects of the cannabinoid-1 receptor (CB1) antagonist rimonabant on energy metabolism and fasting-induced hy- pothalamic-pituitary-adrenal (HPA) axis and neuronal ac- besity results from a prolonged energy imbal- tivation were investigated. Lean and obese Zucker rats ance during which intake exceeds expenditure. were treated orally with a daily dose of 10 mg/kg rimon- The difficulty to lose excess weight is tightly abant for 14 days. A comprehensive energy balance profile Olinked to the ability of the systems regulating based on whole-carcass analyses further demonstrated the energy balance to defend body weight. The complexity potential of CB1 antagonists for decreasing energy gain and redundancy within these systems, which involve an through reducing food intake and potentially increasing intricate network of peripheral signals and neuronal cir- brown adipose tissue thermogenesis. Rimonabant also re- cuits, constitute obstacles to finding potential targets for duced plasma glucose, insulin, and homeostasis model as- antiobesity treatments. Currently, one of the most prom- sessment of insulin resistance, which further confirms the ising targets for the pharmacological treatment of obesity ability of CB1 antagonists to improve insulin sensitivity. To test the hypothesis that rimonabant attenuates the effect is the cannabinoid-1 receptor (CB1). Rimonabant of fasting on HPA axis activation in the obese Zucker (SR141716), the first selective CB1 antagonist (1), acts as model, rats were either ad libitum–fed or food-deprived for a potent antiobesity agent when administered to diet- 8 h. Contrary to expectation, rimonabant increased basal induced obese mice (2). Rimonabant is presently in phase circulating corticosterone levels and enhanced the HPA III clinical trials for the treatment of obesity. The recently axis response to food deprivation in obese rats. Rimon- published results from clinical trials, known as Rimon- abant also exacerbated the neuronal activation seen in the abant in Obesity–Europe (3), Rimonabant in Obesity– arcuate nucleus (ARC) after short-term deprivation. In Lipids (4), and Rimonabant in Obesity–North America (5), conclusion, the present study demonstrates that CB1 indicate that rimonabant not only reduces body weight but blockade does not prevent the hypersensitivity to food also improves cardiovascular risk factors associated with deprivation occurring at the level of HPA axis and ARC obesity. activation in the obese Zucker rats. This, however, does not The precise mechanism responsible for the antiobesity prevent CB1 antagonism from exerting beneficial effects on effect of rimonabant remains unknown. It has been sug- energy and glucose metabolism. Diabetes 55:3403–3410, gested that the hypophagic effect of CB1 antagonists 2006 results from an attenuation of feeding-related reward processes (6,7) that could be under the modulation of hypothalamic centers regulating energy balance. Injection of the endocannabinoid anandamide in the ventromedial hypothalamic nucleus, an area rich in CB1 mRNA (8), increases food intake, and this effect is blocked by rimon- abant (9). Also, CB1 mRNA is co-expressed with hypotha- From the Merck Frosst/CIHR Research Chair in Obesity and Centre de lamic neuropeptides involved in the modulation of food recherche de l’Hoˆ pital Laval, Hoˆ pital Laval, Que´bec, Canada. intake, including the anorectic peptide corticotropin- C.D. is currently affiliated with the Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada. releasing factor (CRF) (10). The presence of CB1 in Address correspondence and reprint requests to Denis Richard, Direction CRF-positive neurons of the paraventricular hypothalamic de la recherche, Hoˆ pital Laval, 2725 chemin Sainte-Foy, Que´bec, Que´bec, nucleus (PVN) also suggests a possible connection be- Canada, G1V 4G5. E-mail: [email protected]. Received for publication 13 April 2006 and accepted in revised form 28 tween the cannabinoid system and the hypothalamic- August 2006. pituitary-adrenal (HPA) axis, the activity of which has a AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose major impact on energy balance regulation (11). However, tissue; CB1, cannabinoid-1 receptor; CRF, corticotropin-releasing factor; HOMA-IR, homeostasis model assessment of insulin resistance; HPA, hypo- the relationship between the cannabinoid system and the thalamic-pituitary-adrenal; MCH, melanin-concentrating hormone; MCR4, HPA axis remains unclear because both cannabinoid ago- melanocortin receptor-4; NEFA, nonesterified fatty acid; NPY, neuropeptide Y; nists and antagonists have been reported to activate the POMC, proopiomelanocortin; PVN, paraventricular hypothalamic nucleus; HPA axis (12–16). SON, supraoptic nucleus; UCP1, uncoupling protein-1. DOI: 10.2337/db06-0504 The potential interaction between the cannabinoid sys- © 2006 by the American Diabetes Association. tem and the HPA axis was examined by subjecting Zucker The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance rats to a mild physiological stress (8-h daytime food with 18 U.S.C. Section 1734 solely to indicate this fact. deprivation) after 2 weeks of treatment with rimonabant. DIABETES, VOL. 55, DECEMBER 2006 3403 RIMONABANT AND HPA/NEURONAL ACTIVATION The obese Zucker rat is not only hypercorticosteronemic They were then transferred to a paraformaldehyde-borax solution containing (17,18) and hypersensitive to stress (17,19), but it also 10% sucrose at least 12 h before cutting 30-␮m-thick coronal sections using a exhibits hyperactivity of the cannabinoid system. Defec- sliding microtome (HM 440E; Microm, Walldorf, Germany). Brain sections taken from the olfactory bulb to the brainstem were allocated to six sequential tive leptin signaling in obese Zucker rats is associated with sets in 24-well tissue culture plates containing a cold sterile cryoprotecting elevated hypothalamic levels of the endocannabinoid solution (50 mmol/l sodium phosphate buffer, 30% ethylene glycol, and 20% 2-arachidonoylglycerol (20). Based on these observations, glycerol) and stored at Ϫ30°C. we hypothesized that rimonabant attenuates HPA axis In situ hybridization histochemistry. In situ hybridization histochemistry hyperactivity, which is associated with the development of was used to determine c-fos, CRF, CRF1 receptor, agouti-related peptide obesity in Zucker fa/fa rats (21). The hypothesis was (AgRP), neuropeptide Y (NPY), proopiomelanocortin (POMC), and melanin- concentrating hormone (MCH) mRNA levels on tissue sections taken from the addressed in a study further aimed at investigating the hypothalamus. Our method was largely adapted from that of Simmons et al. effects of rimonabant on hypothalamic neuronal activation (26). Briefly, brain sections (one of every six sections) were rinsed in sterile induced by short-term food deprivation and on hypotha- 0.05 mol/l potassium PBS treated with diethyl pyrocarbonate, mounted onto lamic mRNA levels of neuropeptides known for their poly-L-lysine coated slides, and dehydrated in 100% ethanol. The sections were involvement in the regulation of energy balance and HPA successively fixed for 20 min in paraformaldehyde (4%), digested for 30 min at axis activity. 37°C with proteinase K (10 ␮g/ml in 100 mmol/l Tris-HCl containing 50 mmol/l EDTA, pH 8.0), acetylated with acetic anhydride (0.25% in 0.1 mol/l trietho- lamine, pH 8.0), and dehydrated through graded concentrations (50, 70, 95, and 100%) of ethanol. After drying for at least 2 h, 100 ␮l hybridization RESEARCH DESIGN AND METHODS mixture, which contained an antisense 35S-labeled cRNA probe (107 cpm/ml), Lean (Fa/?) and obese (fa/fa) male Zucker rats, aged 7–8 weeks, were was spotted on each slide. Slides were sealed under a coverslip and incubated purchased from Charles River Laboratories (St. Constant, Que´bec, Canada). overnight at 60°C. The next day, coverslips were removed, and slides were All rats were cared for and handled according to the Canadian Guide for the rinsed four times with 4ϫ sodium chloride–sodium citrate (0.6 mol/l NaCl and Care and Use of Laboratory Animals, and the protocol was approved by the 60 mmol/l trisodium citrate buffer, pH 7.0), digested for 30 min at 37°C with Universite´ Laval Animal Care Committee. The animals were housed individ- RNAse-A (20 ␮g/ml in 10 mmol/l Tris-500 mmol/l NaCl containing 1 mmol/l ually in wire-bottom cages, allowed unrestricted access to water, and, unless EDTA), washed in descending concentrations of sodium chloride–sodium specified, fed ad libitum with a ground stock diet (Charles River Rodent Diet citrate (2ϫ, 10 min; 1ϫ, 5 min; 0.5ϫ, 5 min; and 0.1ϫ, 30 min at 60°C), and 5075; Ralston Products, Woodstock, Ontario, Canada). They were subjected to dehydrated through graded concentrations of ethanol. After2hofdrying, a 12-h-dark/12-h-light cycle (lights on between 0700 and 1900) and kept under slides were exposed on an X-ray film (Eastman Kodak, Rochester, NY) for ambient temperature (23 Ϯ 1°C). Rats were separated in groups of equal initial 20 h. Slides were defatted in toluene, dipped in NTB2 nuclear emulsion average weights within each genotype the day preceding the treatment period. (Eastman Kodak), and exposed 3 (NPY and POMC), 4 (CRF), 5 (c-fos, AgRP Treated rats received a daily oral administration of 10 mg/kg rimonabant and MCH), or 22 (CRF1 receptor) days before being developed in D19 (SR141716; Sanofi-Aventis, Paris, France) for 14 days at 0830, except on the developer (Eastman Kodak) for 3.5 min at 14°C and fixed in rapid fixer last day where rimonabant was administered 6 h before death. This dose was (Eastman Kodak) for 5 min. Finally, tissues were rinsed in running water for previously shown to reduce body weight gain in the obese Zucker rat (22,23).