Effects of Rimonabant (SR141716) on Fasting-Induced Hypothalamic-Pitui
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《糖尿病学杂志》
the Merck Frosst/CIHR Research Chair in Obesity and Centre de recherche de l’Hpital Laval, Hpital Laval, Quebec, Canada
AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; CB1, cannabinoid-1 receptor; CRF, corticotropin-releasing factor; HOMA-IR, homeostasis model assessment of insulin resistance; HPA, hypothalamic-pituitary-adrenal; MCH, melanin-concentrating hormone; MCR4, melanocortin receptor-4; NEFA, nonesterified fatty acid; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular hypothalamic nucleus; SON, supraoptic nucleus; UCP1, uncoupling protein-1
ABSTRACT
The effects of the cannabinoid-1 receptor (CB1) antagonist rimonabant on energy metabolism and fasting-induced hypothalamic-pituitary-adrenal (HPA) axis and neuronal activation were investigated. Lean and obese Zucker rats were treated orally with a daily dose of 10 mg/kg rimonabant for 14 days. A comprehensive energy balance profile based on whole-carcass analyses further demonstrated the potential of CB1 antagonists for decreasing energy gain through reducing food intake and potentially increasing brown adipose tissue thermogenesis. Rimonabant also reduced plasma glucose, insulin, and homeostasis model assessment of insulin resistance, which further confirms the ability of CB1 antagonists to improve insulin sensitivity. To test the hypothesis that rimonabant attenuates the effect of fasting on HPA axis activation in the obese Zucker model, rats were either ad libitum–fed or food-deprived for 8 h. Contrary to expectation, rimonabant increased basal circulating corticosterone levels and enhanced the HPA axis response to food deprivation in obese rats. Rimonabant also exacerbated the neuronal activation seen in the arcuate nucleus (ARC) after short-term deprivation. In conclusion, the present study demonstrates that CB1 blockade does not prevent the hypersensitivity to food deprivation occurring at the level of HPA axis and ARC activation in the obese Zucker rats. This, however, does not prevent CB1 antagonism from exerting beneficial effects on energy and glucose metabolism.
Obesity results from a prolonged energy imbalance during which intake exceeds expenditure. The difficulty to lose excess weight is tightly linked to the ability of the systems regulating energy balance to defend body weight. The complexity and redundancy within these systems, which involve an intricate network of peripheral signals and neuronal circuits, constitute obstacles to finding potential targets for antiobesity treatments. Currently, one of the most promising targets for the pharmacological treatment of obesity is the cannabinoid-1 receptor (CB1). Rimonabant (SR141716), the first selective CB1 antagonist (1), acts as a potent antiobesity agent when administered to diet-induced obese mice (2). Rimonabant is presently in phase III clinical trials for the treatment of obesity. The recently published results from clinical trials, known as Rimonabant in Obesity–Europe (3), Rimonabant in Obesity–Lipids (4), and Rimonabant in Obesity–North America (5), indicate that rimonabant not only reduces body weight but also improves cardiovascular risk factors associated with obesity.
The precise mechanism responsible for the antiobesity effect of rimonabant remains unknown. It has been suggested that the hypophagic effect of CB1 antagonists 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 rimonabant (9). Also, CB1 mRNA is co-expressed with hypothalamic neuropeptides involved in the modulation of food intake, including the anorectic peptide corticotropin-releasing factor (CRF) (10). The presence of CB1 in CRF-positive neurons of the paraventricular hypothalamic nucleus (PVN) also suggests a possible connection between the cannabinoid system and the hypothalamic-pituitary-adrenal (HPA) axis, the activity of which has a major impact on energy balance regulation (11). However, the relationship between the cannabinoid system and the HPA axis remains unclear because both cannabinoid agonists and antagonists have been reported to activate the HPA axis (12–16).
The potential interaction between the cannabinoid system and the HPA axis was examined by subjecting Zucker rats to a mild physiological stress (8-h daytime food deprivation) after 2 weeks of treatment with rimonabant. The obese Zucker rat is not only hypercorticosteronemic (17,18) and hypersensitive to stress (17,19), but it also exhibits hyperactivity of the cannabinoid system. Defective leptin signaling in obese Zucker rats is associated with elevated hypothalamic levels of the endocannabinoid 2-arachidonoylglycerol (20). Based on these observations, we hypothesized that rimonabant attenuates HPA axis hyperactivity, which is associated with the development of obesity in Zucker fa/fa rats (21). The hypothesis was addressed in a study further aimed at investigating the effects of rimonabant on hypothalamic neuronal activation induced by short-term food deprivation and on hypothalamic mRNA levels of neuropeptides known for their involvement in the regulation of energy balance and HPA axis activity.
RESEARCH DESIGN AND METHODS
Lean (Fa/) and obese (fa/fa) male Zucker rats, aged 7–8 weeks, were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). All rats were cared for and handled according to the Canadian Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Universite Laval Animal Care Committee. The animals were housed individually in wire-bottom cages, allowed unrestricted access to water, and, unless specified, fed ad libitum with a ground stock diet (Charles River Rodent Diet 5075; Ralston Products, Woodstock, Ontario, Canada). They were subjected to a 12-h-dark/12-h-light cycle (lights on between 0700 and 1900) and kept under ambient temperature (23 ± 1°C). Rats were separated in groups of equal initial average weights within each genotype the day preceding the treatment period. Treated rats received a daily oral administration of 10 mg/kg rimonabant (SR141716; Sanofi-Aventis, Paris, France) for 14 days at 0830, except on the last day where rimonabant was administered 6 h before death. This dose was previously shown to reduce body weight gain in the obese Zucker rat (22,23). Each day, a solution containing rimonabant (2 mg/ml) and Tween-80 (2 μl/ml) was administered at a dose of 5 ml/kg body wt. Rats were weighed and food intake was measured daily throughout the experiment. Rats were killed between 1400 and 1700 in either an ad libitum–fed state or after an 8-h food deprivation. Lean and obese rats were respectively anesthetized with 2 and 4 ml of a mixture containing ketamine (20 mg/ml) and xylazine (2.5 mg/ml). Blood was collected by intracardial puncture into syringes coated with 0.5 mol/l EDTA (Sigma-Aldrich, St. Louis, MO), and rats were perfused intracardially for 2 min with ice-cold isotonic saline. Brain and interscapular brown adipose tissue (BAT) were sampled immediately after the perfusion. BAT was flash frozen in liquid nitrogen and stored at –86°C.
Body gains in energy, fat, and protein.
Carcasses were autoclaved at 125 kPa for 15 min, homogenized in two volumes of water (w/v), and freeze-dried. Carcass energy content was determined by adiabatic bomb calorimetry, whereas carcass protein was determined using a FP-2000 Nitrogen Analyzer (Leco, St. Joseph, MI) with 250–300 mg dehydrated carcasses. Nonprotein matter energy was obtained by subtracting protein energy from total carcass energy. Values of 23.5 and 39.2 kJ/g were used for the calculation of the energy content of protein and fat, respectively (24). Initial energy, fat, and protein contents of the carcasses were estimated from the live body weight of lean and obese rats with reference to a baseline group of rats (six per phenotype) killed at the beginning of the experimental period. Such estimates allow gains in energy, fat, and protein to be determined for the treatment period. Rats in the initial group were identical in every respect (e.g., age and sex) to those of the experimental groups. Food efficiency was expressed as the ratio of energy gain to digestible energy intake multiplied by 100.
Plasma determinations.
Blood was harvested by cardiac puncture and centrifuged (1,500g, 15 min at 4°C), and plasma was stored at –20°C until later biochemical measurements. Plasma glucose concentrations were determined using an automated glucose analyzer YSI 2,300 Stat Plus (YSI, Yellow Springs, OH). Commercially available radioimmunoassay kits were used to determine plasma levels of corticosterone (MP Biomedicals, Toronto, ON), insulin, leptin, and adiponectin (Linco Research, St. Charles, MO), whereas enzymatic kits were used for triglycerides (Roche Diagnostics, Laval, Quebec, Canada) and nonesterified fatty acids (NEFAs; Wako Diagnostics, Richmond, VA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using plasma glucose and insulin levels of food-deprived rats as previously described (25).
Brain preparation.
Brains were essentially prepared as previously described (18). After their removal, brains were fixed into a 4% paraformaldehyde-3.8% borax solution for at least 7 days with frequent replacement of the solution. They were then transferred to a paraformaldehyde-borax solution containing 10% sucrose at least 12 h before cutting 30-μm-thick coronal sections using a sliding microtome (HM 440E; Microm, Walldorf, Germany). Brain sections taken from the olfactory bulb to the brainstem were allocated to six sequential sets in 24-well tissue culture plates containing a cold sterile cryoprotecting solution (50 mmol/l sodium phosphate buffer, 30% ethylene glycol, and 20% glycerol) and stored at –30°C.
In situ hybridization histochemistry.
In situ hybridization histochemistry was used to determine c-fos, CRF, CRF1 receptor, agouti-related peptide (AgRP), neuropeptide Y (NPY), proopiomelanocortin (POMC), and melanin-concentrating hormone (MCH) mRNA levels on tissue sections taken from the hypothalamus. Our method was largely adapted from that of Simmons et al. (26). Briefly, brain sections (one of every six sections) were rinsed in sterile 0.05 mol/l potassium PBS treated with diethyl pyrocarbonate, mounted onto poly-L-lysine coated slides, and dehydrated in 100% ethanol. The sections were successively fixed for 20 min in paraformaldehyde (4%), digested for 30 min at 37°C with proteinase K (10 μg/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 trietholamine, pH 8.0), and dehydrated through graded concentrations (50, 70, 95, and 100%) of ethanol. After drying for at least 2 h, 100 μl hybridization mixture, which contained an antisense 35S-labeled cRNA probe (107 cpm/ml), was spotted on each slide. Slides were sealed under a coverslip and incubated overnight at 60°C. The next day, coverslips were removed, and slides were rinsed four times with 4x sodium chloride–sodium citrate (0.6 mol/l NaCl and 60 mmol/l trisodium citrate buffer, pH 7.0), digested for 30 min at 37°C with RNAse-A (20 μg/ml in 10 mmol/l Tris-500 mmol/l NaCl containing 1 mmol/l EDTA), washed in descending concentrations of sodium chloride–sodium citrate (2x, 10 min; 1x, 5 min; 0.5x, 5 min; and 0.1x, 30 min at 60°C), and dehydrated through graded concentrations of ethanol. After 2 h of drying, slides were exposed on an X-ray film (Eastman Kodak, Rochester, NY) for 20 h. Slides were defatted in toluene, dipped in NTB2 nuclear emulsion (Eastman Kodak), and exposed 3 (NPY and POMC), 4 (CRF), 5 (c-fos, AgRP and MCH), or 22 (CRF1 receptor) days before being developed in D19 developer (Eastman Kodak) for 3.5 min at 14°C and fixed in rapid fixer (Eastman Kodak) for 5 min. Finally, tissues were rinsed in running water for 1–2 h, counterstained with thionin (0.25%), dehydrated through graded concentrations of ethanol, cleared in toluene, and coverslipped with DPX mounting medium (BDH; VWR, Mississauga, Ontario, Canada). Processed slides were examined by darkfield microscopy using an Olympus BX51 microscope (Olympus America, Melville, NY). Images were acquired with an Evolution QEi camera and analyzed with ImagePro plus v5.0.1.11 (MediaCybernetics, Silver Spring, MD). The system was calibrated for each set of analyses to prevent saturation of the integrated signal. Mean pixel densities were obtained by taking measurements from both hemispheres of one to four brain sections and subtracting background readings taken from areas immediately surrounding the region analyzed.
Antisense 35S-labeled riboprobes.
Complementary RNA probes were generated from rat cDNA fragments for c-fos (Dr. I. Verma, The Salk Institute, La Jolla, CA; GenBank accession no. V00727), CRF (Dr. K. Mayo, Northwestern University, Evanston, IL), CRF1 receptor (Dr. M.H. Perrin and Dr. W.W. Vale, The Clayton Foundation, La Jolla, CA; GenBank accession no. L24096), POMC (Dr. B.T. Bloomquist, Bayer, West Haven, CT), and MCH (Dr. R. Thompson and Dr. S.J. Watson, University of Michigan, Ann Arbor, MI), the XhoI-XbaI genomic fragment containing exon 2 of rat NPY (Dr. D.S. Larhammar, Uppsala University, Uppsala, Sweden), and a murine AgRP cDNA (Dr. M. Graham, Amgen, Thousand Oaks, CA). Radiolabeled antisense riboprobes were synthesized by incubating 250 ng linearized plasmid at 37°C for 60 min in the presence of 10 mmol/l NaCl, 10 mmol/l dithiothreitol, 6 mmol/l MgCl2, 40 mmol/l Tris (pH 7.9), 0.2 mmol/l ATP/GTP/CTP, [-35S]UTP, 40 units RNase inhibitor (Roche Diagnostics), and 20 units T7 (c-fos, CRF1 receptor, NPY, and AgRP), SP6 (CRF and MCH), or T3 (POMC) RNA polymerase (Promega, Madison, WI). The DNA templates were treated with 100 μl DNAse solution (1 μl DNAse, 5 μl 5 mg/ml tRNA, and 94 μl 10 mmol/l Tris/10 mmol/l MgCl2). Riboprobes were purified on RNeasy Mini Spin Columns (Qiagen, Mississauga, Ontario, Canada).
Real-time quantitative RT-PCR.
Total RNA was isolated from 60–90 mg BAT using the RNeasy Lipid Tissue mini kit (Qiagen). On-column DNA digestion was performed using the RNase-free DNase Set (Qiagen). First-strand cDNA was synthesized from 1 μg total RNA with Expand Reverse Transcriptase and oligo(dT) (Roche Diagnostics) and diluted 1:25 with diethyl pyrocarbonate–treated water. Rat uncoupling protein-1 (UCP1) amplicons were generated using the sense primer 5'-TGGTGAGTTCGACAACTTCC-3' and the antisense primer 5'-GTGGGCTGCCCAATGAATAC-3' (GenBank accession no. NM_012682). Rat L27 amplicons were generated using the sense primer 5'-CTGCTCGCTGTCGAAATG-3' and the antisense primer 5'-CCTTGCGTTTCAGTGCTG-3' (GenBank accession no. NM_022514). Amplification was carried out using Platinum Taq polymerase (Invitrogen), CYBR Green I (Cedarlane Laboratories, Hornby, Ontario, Canada), and a Rotor Gene 3000 (Corbett Research, Sydney, Australia) with the following program: 2 min denaturing at 94°C, then 40 cycles of denaturation at 94°C for 20 s, annealing at 64°C for 20 s, extension at 72°C for 20 s, and emission measurement at 85°C for 15 s. After the last cycle, the temperature was gradually increased from 72 to 99°C for the determination of a melting curve. The PCR reaction contained 5 μl diluted cDNA in a 20-μl PCR reaction. Results were analyzed using Rotor-gene v6.0 software (Corbett Research).
Statistics.
Results are presented as means ± 1 SE. Statistical differences in daily food intake and cumulative weight gain between control and rimonabant-treated rats were determined within each genotype using a crossed-nested design with repeated measurements. Cumulative weight gain data were log-transformed, and multivariate normality was verified with Mardia’s test. Statistical differences within each genotype were determined by Student’s t test or two-way ANOVA. Data for corticosterone, insulin, and c-fos mRNA were log-transformed, whereas a square root transformation was used for NEFA. Tukey’s multiple comparison tests followed two-way ANOVAs with significant interaction effect. Results were considered significant with P values <0.05. Statistical analyses were performed using SAS v9.1.3 software package (SAS Institute, Cary, NC) or SigmaStat v2.0 software (SPSS, Chicago, IL).
RESULTS
Body weight, food intake, and energy balance.
Treatment with the CB1 antagonist rimonabant led to a transient food intake reduction that remained significant for 10 days in obese rats compared with 3 days in lean rats (Fig. 1A and B). Initial weight did not differ within lean (control 290.2 ± 4.6 g; rimonabant 290.0 ± 5.1 g) or obese (control 463.0 ± 9.7 g; rimonabant 468.0 ± 5.3 g) rats. After an initial weight loss, cumulative body weight gain of rimonabant-treated rats remained below that of controls throughout the whole experiment (Fig. 1C and D). Treatment with rimonabant reduced total energy gain, as assessed by whole-carcass analyses, and this was mostly accompanied by a reduction in fat gain in obese rats, whereas lean rats showed a significant reduction in protein gain (Table 1). Rimonabant reduced food efficiency, and this reduction reached statistical significance in lean rats (Table 1). Rimonabant did not alter apparent energy expenditure (Table 1). BAT weight was increased in obese rats but reduced in lean rats by rimonabant (Table 1). BAT UCP1 mRNA levels were significantly increased in rimonabant-treated obese rats (Table 1).
Metabolic plasma variables.
Treatment with rimonabant reduced circulating triglyceride levels in both lean and obese rats (Table 2). NEFA levels were increased after food deprivation but not significantly modified by rimonabant. Circulating glucose levels of rimonabant-treated obese rats were reduced to values similar to those of lean and untreated food-deprived obese rats. Food deprivation reduced plasma insulin levels in both lean and obese rats, and these levels were further reduced by rimonabant. HOMA-IR was reduced in lean and obese rats treated with rimonabant. Rimonabant significantly reduced circulating leptin levels only in lean rats. Circulating adiponectin levels were increased in rimonabant-treated obese rats.
The HPA axis.
Food deprivation increased plasma corticosterone levels in obese rats (Fig. 2A). Treatment with rimonabant increased basal corticosterone levels and potentiated the fasting-induced elevation in plasma corticosterone in obese rats. In addition, treatment with rimonabant increased c-fos mRNA levels in the parvocellular division of the PVN (Fig. 2B). Rimonabant did not alter CRF or CRF1 receptor mRNA levels in the PVN (Fig. 2C and D).
Neuronal activation and neuropeptides in the arcuate nucleus, lateral hypothalamic area, and supraoptic nucleus.
Food deprivation increased NPY (Fig. 3A and B) and c-fos (Fig. 3E and F) mRNA levels in the arcuate nucleus (ARC) of obese rats and MCH mRNA in the lateral hypothalamic area of lean rats (Fig. 3H). Treatment with rimonabant enhanced ARC NPY (Fig. 3A and B), AgRP (Fig. 3C), and c-fos (Fig. 3E and F) mRNA levels in obese rats. Rimonabant also tended to increase POMC mRNA levels in the ARC of obese rats (P = 0.05; Fig. 3D). c-fos mRNA levels were elevated in the supraoptic nucleus (SON) of ad libitum–fed rats, and these levels were enhanced by rimonabant in obese rats (Fig. 3G).
DISCUSSION
The present results confirm the ability of CB1 antagonists to reduce weight gain in both lean and obese rats (22,23,27). Here, we extend previous findings with a complete energy balance profile based on carcass analyses. We show that the reduction in weight gain was largely accounted for by a reduction in fat gain, which is consonant with previous studies (2,28). The fact that rimonabant did not significantly decrease energy expenditure, which would be expected with a reduction in both total energy intake and energy gain, suggests that CB1 blockade exerted a stimulating effect on thermogenesis. This is further supported by the observation that, in obese rats, rimonabant increased BAT UCP1 mRNA levels, a widely used marker of thermogenic capacity. A recent study by Jbilo et al. (29) showed that rimonabant activates several BAT genes involved in the regulation of mitochondrial activity. Although increased BAT thermogenic activity likely contributed to rimonabant-induced reduction in energy gain, such reduction was obviously also attributable to the transient reduction in energy intake. The present study also further highlights the ability of CB1 antagonists to improve glucose metabolism. In obese Zucker rats, which are hyperinsulinemic (30) and glucose intolerant (31), rimonabant restored normal glycemia and reduced plasma levels of insulin and triglycerides. It is noteworthy that these marked changes were observed at a time when rimonabant no longer affected food intake. Our results are consistent with other studies that showed improved plasma profiles in Zucker rats (22), diet-induced obese mice (2,32), and obese humans (3,4).
In the present investigation, we tested the possibility that rimonabant may attenuate the food deprivation-induced stress response in obese Zucker rats. This hypothesis was based on the observation that the hyperactivity of the endocannabinoid system (20), which is thought to increase the incentive value of food (7,33), may in part explain the hypersensitivity of obese Zucker rats to food deprivation (19). Our results demonstrated that rimonabant did not block the effect of food deprivation on HPA axis activation in obese Zucker rats. In fact, rimonabant increased basal circulating corticosterone levels and enhanced the HPA axis response to a daytime food deprivation in obese Zucker rats. This enhanced activity of the HPA axis was associated with increased c-fos mRNA levels in the parvocellular division of the PVN. Thus, the response of obese Zucker rats was similar to that of lean ICR mice, in which rimonabant was recently shown to potentiate the corticosterone response to restraint stress (16). One possible mechanism for rimonabant-induced activation of the HPA axis is through increased CRF release. Because CB1 is primarily a presynaptic receptor that modulates neurotransmitter release (34), the presence of CB1 mRNA in >50% of CRF neurons in the PVN suggests that cannabinoids may directly influence CRF release (10). Finally, the observation of increased HPA axis activity is inconsistent with a reduced energy gain. Corticosteroids have been shown to promote energy deposition (35–37) and to inhibit BAT thermogenesis (37–39). This raises the possibility that rimonabant may impede the downstream effects of an activated HPA axis by obstructing the effects of chronically elevated plasma corticosterone on energy deposition and thermogenic activity. The enhancing effect of rimonabant on central insulin sensitivity would represent one mechanism through which rimonabant may counteract the detrimental metabolic effects of an increased HPA axis activity.
Obese Zucker rats exhibited a predictable increase in NPY mRNA levels in response to food deprivation, and this was accompanied by an increase in c-fos mRNA levels in the ARC. The NPY response was not observed in lean rats, which are less sensitive to food deprivation than obese rats (40). Nonetheless, lean rats showed a fasting-induced elevation in MCH mRNA levels in the lateral hypothalamus and reduction in c-fos mRNA levels in the SON. As previously observed (23,27,28), the hypophagic effect of rimonabant was transient despite continuous administration of the CB1 antagonist. In obese rats, rimonabant increased NPY and AgRP mRNA levels in both fed and fasted rats. These increases, which were paralleled by induction of the c-fos gene, likely occurred as a mechanism to compensate for the reducing effects of rimonabant on energy stores. However, the inductions, which were obtained at a time when food intake had normalized in both lean and obese rats, suggest that CB1 antagonists may block a possible NPY/AgRP-stimulated compensatory overfeeding. In agreement with this statement, previous studies showed that interrupting the administration of a CB1 antagonist leads to compensatory overfeeding (27,28). Also, rimonabant was shown to attenuate NPY-induced sucrose drinking in rats (41) and overeating in satiated mice (42). Together, these studies suggest that blockade of NPY action may have contributed to the rimonabant-induced hypophagia and/or inhibition of a compensatory overfeeding.
A recent study (43) showed that AM251, an analog of rimonabant, reduces NPY release and attenuates cannabinoid-induced NPY release from hypothalamic explants, suggesting that the effect of CB1 antagonists on food intake may result from a reduction in NPY synaptic tone. However, it is unlikely that cannabinoids act directly on NPY/AgRP neurons. A colocalization study failed to detect the presence CB1 mRNA in NPY-positive neurons of the ARC (10). Also, Di Marzo et al. (20) showed that the hypophagic effect of rimonabant remains present in NPY-deficient mice, suggesting that NPY and cannabinoids are independent orexigenic systems. Rimonabant-induced improvement in insulin sensitivity represents a potential mechanism whereby the drug could blunt the activity of the NPY/AgRP neurons. It is clear from our own HOMA-IR results and from those of other studies (3) that rimonabant has a strong positive action on insulin sensitivity. Whether this action of rimonabant is also apparent at the brain level is a likely possibility that certainly warrants further investigation.
Rimonabant tended to increase POMC mRNA, the precursor of the anorexigenic neuropeptide -melanocyte–stimulating hormone but did not affect the MCH system in obese rats. Involvement of these systems during the initial period of exposure, when food intake is reduced, cannot be excluded. A recent study demonstrated that rimonabant attenuated the feeding response induced by the melanocortin receptor-4 (MCR4) antagonist JKC-363 (44). This result indicates that rimonabant may act downstream from MCR4 and that it may block the orexigenic action of MCR4 antagonists, including AgRP. Of relevance to the present study is the fact that a stimulated melanocortin system, without concomitant activation of the MCH system, constitutes a condition promoting UCP1-induced thermogenesis (45).
Neuronal activation, as assessed by c-fos mRNA levels, in hypothalamic nuclei known for their involvement in the regulation of energy balance and the response to stress, argues in favor of an involvement of the hypothalamus in the effects of rimonabant. Increased c-fos mRNA in the PVN is in line with increased HPA axis activity, whereas neuronal activation in the ARC is consistent with increased NPY and AgRP mRNA levels. Rimonabant also increased c-fos mRNA levels in the SON of ad libitum–fed obese rats. The magnocellular neurosecretory cells of the SON secrete either arginine vasopressin or oxytocin (46), and these cells were shown to respond to feeding (47) and hypertonicity (48). Verty et al. (49) demonstrated an interaction between CB1 and oxytocin receptors; rimonabant attenuated the food and water intake induced by tocinoic acid, an oxytocin receptor antagonist. Therefore, rimonabant induced neuronal activation in brain structures that are consistent with its effect on neuropeptides involved in the regulation of energy balance.
In conclusion, our results further demonstrate the potential of CB1 antagonists to improve energy metabolism in obese rats, in particular by reducing energy gain and normalizing several plasma variables related to the metabolic syndrome and diabetes. The study also demonstrates that CB1 blockade does not prevent the hypersensitivity to food deprivation occurring at the level of HPA axis and ARC activation in the obese Zucker rats. This, however, does not prevent CB1 antagonism from exerting beneficial effects on energy and glucose metabolism.
ACKNOWLEDGMENTS
C.D. has received a postdoctoral fellowship from Fonds quebecois de la recherche sur la nature et les technologies. D.R. has received a grant from the Canadian Institutes of Health Research.
We thank Sanofi-Aventis for their supply of rimonabant. We also thank Serge Simard for statistical advice, and Julie Plamondon, Marie-Nolle Cyr, Sebastien Poulin, and Hakima Zekki for technical assistance.
FOOTNOTES
C.D. is currently affiliated with the Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
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 with 18 U.S.C. Section 1734 solely to indicate this fact.
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AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; CB1, cannabinoid-1 receptor; CRF, corticotropin-releasing factor; HOMA-IR, homeostasis model assessment of insulin resistance; HPA, hypothalamic-pituitary-adrenal; MCH, melanin-concentrating hormone; MCR4, melanocortin receptor-4; NEFA, nonesterified fatty acid; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular hypothalamic nucleus; SON, supraoptic nucleus; UCP1, uncoupling protein-1
ABSTRACT
The effects of the cannabinoid-1 receptor (CB1) antagonist rimonabant on energy metabolism and fasting-induced hypothalamic-pituitary-adrenal (HPA) axis and neuronal activation were investigated. Lean and obese Zucker rats were treated orally with a daily dose of 10 mg/kg rimonabant for 14 days. A comprehensive energy balance profile based on whole-carcass analyses further demonstrated the potential of CB1 antagonists for decreasing energy gain through reducing food intake and potentially increasing brown adipose tissue thermogenesis. Rimonabant also reduced plasma glucose, insulin, and homeostasis model assessment of insulin resistance, which further confirms the ability of CB1 antagonists to improve insulin sensitivity. To test the hypothesis that rimonabant attenuates the effect of fasting on HPA axis activation in the obese Zucker model, rats were either ad libitum–fed or food-deprived for 8 h. Contrary to expectation, rimonabant increased basal circulating corticosterone levels and enhanced the HPA axis response to food deprivation in obese rats. Rimonabant also exacerbated the neuronal activation seen in the arcuate nucleus (ARC) after short-term deprivation. In conclusion, the present study demonstrates that CB1 blockade does not prevent the hypersensitivity to food deprivation occurring at the level of HPA axis and ARC activation in the obese Zucker rats. This, however, does not prevent CB1 antagonism from exerting beneficial effects on energy and glucose metabolism.
Obesity results from a prolonged energy imbalance during which intake exceeds expenditure. The difficulty to lose excess weight is tightly linked to the ability of the systems regulating energy balance to defend body weight. The complexity and redundancy within these systems, which involve an intricate network of peripheral signals and neuronal circuits, constitute obstacles to finding potential targets for antiobesity treatments. Currently, one of the most promising targets for the pharmacological treatment of obesity is the cannabinoid-1 receptor (CB1). Rimonabant (SR141716), the first selective CB1 antagonist (1), acts as a potent antiobesity agent when administered to diet-induced obese mice (2). Rimonabant is presently in phase III clinical trials for the treatment of obesity. The recently published results from clinical trials, known as Rimonabant in Obesity–Europe (3), Rimonabant in Obesity–Lipids (4), and Rimonabant in Obesity–North America (5), indicate that rimonabant not only reduces body weight but also improves cardiovascular risk factors associated with obesity.
The precise mechanism responsible for the antiobesity effect of rimonabant remains unknown. It has been suggested that the hypophagic effect of CB1 antagonists 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 rimonabant (9). Also, CB1 mRNA is co-expressed with hypothalamic neuropeptides involved in the modulation of food intake, including the anorectic peptide corticotropin-releasing factor (CRF) (10). The presence of CB1 in CRF-positive neurons of the paraventricular hypothalamic nucleus (PVN) also suggests a possible connection between the cannabinoid system and the hypothalamic-pituitary-adrenal (HPA) axis, the activity of which has a major impact on energy balance regulation (11). However, the relationship between the cannabinoid system and the HPA axis remains unclear because both cannabinoid agonists and antagonists have been reported to activate the HPA axis (12–16).
The potential interaction between the cannabinoid system and the HPA axis was examined by subjecting Zucker rats to a mild physiological stress (8-h daytime food deprivation) after 2 weeks of treatment with rimonabant. The obese Zucker rat is not only hypercorticosteronemic (17,18) and hypersensitive to stress (17,19), but it also exhibits hyperactivity of the cannabinoid system. Defective leptin signaling in obese Zucker rats is associated with elevated hypothalamic levels of the endocannabinoid 2-arachidonoylglycerol (20). Based on these observations, we hypothesized that rimonabant attenuates HPA axis hyperactivity, which is associated with the development of obesity in Zucker fa/fa rats (21). The hypothesis was addressed in a study further aimed at investigating the effects of rimonabant on hypothalamic neuronal activation induced by short-term food deprivation and on hypothalamic mRNA levels of neuropeptides known for their involvement in the regulation of energy balance and HPA axis activity.
RESEARCH DESIGN AND METHODS
Lean (Fa/) and obese (fa/fa) male Zucker rats, aged 7–8 weeks, were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). All rats were cared for and handled according to the Canadian Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Universite Laval Animal Care Committee. The animals were housed individually in wire-bottom cages, allowed unrestricted access to water, and, unless specified, fed ad libitum with a ground stock diet (Charles River Rodent Diet 5075; Ralston Products, Woodstock, Ontario, Canada). They were subjected to a 12-h-dark/12-h-light cycle (lights on between 0700 and 1900) and kept under ambient temperature (23 ± 1°C). Rats were separated in groups of equal initial average weights within each genotype the day preceding the treatment period. Treated rats received a daily oral administration of 10 mg/kg rimonabant (SR141716; Sanofi-Aventis, Paris, France) for 14 days at 0830, except on the last day where rimonabant was administered 6 h before death. This dose was previously shown to reduce body weight gain in the obese Zucker rat (22,23). Each day, a solution containing rimonabant (2 mg/ml) and Tween-80 (2 μl/ml) was administered at a dose of 5 ml/kg body wt. Rats were weighed and food intake was measured daily throughout the experiment. Rats were killed between 1400 and 1700 in either an ad libitum–fed state or after an 8-h food deprivation. Lean and obese rats were respectively anesthetized with 2 and 4 ml of a mixture containing ketamine (20 mg/ml) and xylazine (2.5 mg/ml). Blood was collected by intracardial puncture into syringes coated with 0.5 mol/l EDTA (Sigma-Aldrich, St. Louis, MO), and rats were perfused intracardially for 2 min with ice-cold isotonic saline. Brain and interscapular brown adipose tissue (BAT) were sampled immediately after the perfusion. BAT was flash frozen in liquid nitrogen and stored at –86°C.
Body gains in energy, fat, and protein.
Carcasses were autoclaved at 125 kPa for 15 min, homogenized in two volumes of water (w/v), and freeze-dried. Carcass energy content was determined by adiabatic bomb calorimetry, whereas carcass protein was determined using a FP-2000 Nitrogen Analyzer (Leco, St. Joseph, MI) with 250–300 mg dehydrated carcasses. Nonprotein matter energy was obtained by subtracting protein energy from total carcass energy. Values of 23.5 and 39.2 kJ/g were used for the calculation of the energy content of protein and fat, respectively (24). Initial energy, fat, and protein contents of the carcasses were estimated from the live body weight of lean and obese rats with reference to a baseline group of rats (six per phenotype) killed at the beginning of the experimental period. Such estimates allow gains in energy, fat, and protein to be determined for the treatment period. Rats in the initial group were identical in every respect (e.g., age and sex) to those of the experimental groups. Food efficiency was expressed as the ratio of energy gain to digestible energy intake multiplied by 100.
Plasma determinations.
Blood was harvested by cardiac puncture and centrifuged (1,500g, 15 min at 4°C), and plasma was stored at –20°C until later biochemical measurements. Plasma glucose concentrations were determined using an automated glucose analyzer YSI 2,300 Stat Plus (YSI, Yellow Springs, OH). Commercially available radioimmunoassay kits were used to determine plasma levels of corticosterone (MP Biomedicals, Toronto, ON), insulin, leptin, and adiponectin (Linco Research, St. Charles, MO), whereas enzymatic kits were used for triglycerides (Roche Diagnostics, Laval, Quebec, Canada) and nonesterified fatty acids (NEFAs; Wako Diagnostics, Richmond, VA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using plasma glucose and insulin levels of food-deprived rats as previously described (25).
Brain preparation.
Brains were essentially prepared as previously described (18). After their removal, brains were fixed into a 4% paraformaldehyde-3.8% borax solution for at least 7 days with frequent replacement of the solution. They were then transferred to a paraformaldehyde-borax solution containing 10% sucrose at least 12 h before cutting 30-μm-thick coronal sections using a sliding microtome (HM 440E; Microm, Walldorf, Germany). Brain sections taken from the olfactory bulb to the brainstem were allocated to six sequential sets in 24-well tissue culture plates containing a cold sterile cryoprotecting solution (50 mmol/l sodium phosphate buffer, 30% ethylene glycol, and 20% glycerol) and stored at –30°C.
In situ hybridization histochemistry.
In situ hybridization histochemistry was used to determine c-fos, CRF, CRF1 receptor, agouti-related peptide (AgRP), neuropeptide Y (NPY), proopiomelanocortin (POMC), and melanin-concentrating hormone (MCH) mRNA levels on tissue sections taken from the hypothalamus. Our method was largely adapted from that of Simmons et al. (26). Briefly, brain sections (one of every six sections) were rinsed in sterile 0.05 mol/l potassium PBS treated with diethyl pyrocarbonate, mounted onto poly-L-lysine coated slides, and dehydrated in 100% ethanol. The sections were successively fixed for 20 min in paraformaldehyde (4%), digested for 30 min at 37°C with proteinase K (10 μg/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 trietholamine, pH 8.0), and dehydrated through graded concentrations (50, 70, 95, and 100%) of ethanol. After drying for at least 2 h, 100 μl hybridization mixture, which contained an antisense 35S-labeled cRNA probe (107 cpm/ml), was spotted on each slide. Slides were sealed under a coverslip and incubated overnight at 60°C. The next day, coverslips were removed, and slides were rinsed four times with 4x sodium chloride–sodium citrate (0.6 mol/l NaCl and 60 mmol/l trisodium citrate buffer, pH 7.0), digested for 30 min at 37°C with RNAse-A (20 μg/ml in 10 mmol/l Tris-500 mmol/l NaCl containing 1 mmol/l EDTA), washed in descending concentrations of sodium chloride–sodium citrate (2x, 10 min; 1x, 5 min; 0.5x, 5 min; and 0.1x, 30 min at 60°C), and dehydrated through graded concentrations of ethanol. After 2 h of drying, slides were exposed on an X-ray film (Eastman Kodak, Rochester, NY) for 20 h. Slides were defatted in toluene, dipped in NTB2 nuclear emulsion (Eastman Kodak), and exposed 3 (NPY and POMC), 4 (CRF), 5 (c-fos, AgRP and MCH), or 22 (CRF1 receptor) days before being developed in D19 developer (Eastman Kodak) for 3.5 min at 14°C and fixed in rapid fixer (Eastman Kodak) for 5 min. Finally, tissues were rinsed in running water for 1–2 h, counterstained with thionin (0.25%), dehydrated through graded concentrations of ethanol, cleared in toluene, and coverslipped with DPX mounting medium (BDH; VWR, Mississauga, Ontario, Canada). Processed slides were examined by darkfield microscopy using an Olympus BX51 microscope (Olympus America, Melville, NY). Images were acquired with an Evolution QEi camera and analyzed with ImagePro plus v5.0.1.11 (MediaCybernetics, Silver Spring, MD). The system was calibrated for each set of analyses to prevent saturation of the integrated signal. Mean pixel densities were obtained by taking measurements from both hemispheres of one to four brain sections and subtracting background readings taken from areas immediately surrounding the region analyzed.
Antisense 35S-labeled riboprobes.
Complementary RNA probes were generated from rat cDNA fragments for c-fos (Dr. I. Verma, The Salk Institute, La Jolla, CA; GenBank accession no. V00727), CRF (Dr. K. Mayo, Northwestern University, Evanston, IL), CRF1 receptor (Dr. M.H. Perrin and Dr. W.W. Vale, The Clayton Foundation, La Jolla, CA; GenBank accession no. L24096), POMC (Dr. B.T. Bloomquist, Bayer, West Haven, CT), and MCH (Dr. R. Thompson and Dr. S.J. Watson, University of Michigan, Ann Arbor, MI), the XhoI-XbaI genomic fragment containing exon 2 of rat NPY (Dr. D.S. Larhammar, Uppsala University, Uppsala, Sweden), and a murine AgRP cDNA (Dr. M. Graham, Amgen, Thousand Oaks, CA). Radiolabeled antisense riboprobes were synthesized by incubating 250 ng linearized plasmid at 37°C for 60 min in the presence of 10 mmol/l NaCl, 10 mmol/l dithiothreitol, 6 mmol/l MgCl2, 40 mmol/l Tris (pH 7.9), 0.2 mmol/l ATP/GTP/CTP, [-35S]UTP, 40 units RNase inhibitor (Roche Diagnostics), and 20 units T7 (c-fos, CRF1 receptor, NPY, and AgRP), SP6 (CRF and MCH), or T3 (POMC) RNA polymerase (Promega, Madison, WI). The DNA templates were treated with 100 μl DNAse solution (1 μl DNAse, 5 μl 5 mg/ml tRNA, and 94 μl 10 mmol/l Tris/10 mmol/l MgCl2). Riboprobes were purified on RNeasy Mini Spin Columns (Qiagen, Mississauga, Ontario, Canada).
Real-time quantitative RT-PCR.
Total RNA was isolated from 60–90 mg BAT using the RNeasy Lipid Tissue mini kit (Qiagen). On-column DNA digestion was performed using the RNase-free DNase Set (Qiagen). First-strand cDNA was synthesized from 1 μg total RNA with Expand Reverse Transcriptase and oligo(dT) (Roche Diagnostics) and diluted 1:25 with diethyl pyrocarbonate–treated water. Rat uncoupling protein-1 (UCP1) amplicons were generated using the sense primer 5'-TGGTGAGTTCGACAACTTCC-3' and the antisense primer 5'-GTGGGCTGCCCAATGAATAC-3' (GenBank accession no. NM_012682). Rat L27 amplicons were generated using the sense primer 5'-CTGCTCGCTGTCGAAATG-3' and the antisense primer 5'-CCTTGCGTTTCAGTGCTG-3' (GenBank accession no. NM_022514). Amplification was carried out using Platinum Taq polymerase (Invitrogen), CYBR Green I (Cedarlane Laboratories, Hornby, Ontario, Canada), and a Rotor Gene 3000 (Corbett Research, Sydney, Australia) with the following program: 2 min denaturing at 94°C, then 40 cycles of denaturation at 94°C for 20 s, annealing at 64°C for 20 s, extension at 72°C for 20 s, and emission measurement at 85°C for 15 s. After the last cycle, the temperature was gradually increased from 72 to 99°C for the determination of a melting curve. The PCR reaction contained 5 μl diluted cDNA in a 20-μl PCR reaction. Results were analyzed using Rotor-gene v6.0 software (Corbett Research).
Statistics.
Results are presented as means ± 1 SE. Statistical differences in daily food intake and cumulative weight gain between control and rimonabant-treated rats were determined within each genotype using a crossed-nested design with repeated measurements. Cumulative weight gain data were log-transformed, and multivariate normality was verified with Mardia’s test. Statistical differences within each genotype were determined by Student’s t test or two-way ANOVA. Data for corticosterone, insulin, and c-fos mRNA were log-transformed, whereas a square root transformation was used for NEFA. Tukey’s multiple comparison tests followed two-way ANOVAs with significant interaction effect. Results were considered significant with P values <0.05. Statistical analyses were performed using SAS v9.1.3 software package (SAS Institute, Cary, NC) or SigmaStat v2.0 software (SPSS, Chicago, IL).
RESULTS
Body weight, food intake, and energy balance.
Treatment with the CB1 antagonist rimonabant led to a transient food intake reduction that remained significant for 10 days in obese rats compared with 3 days in lean rats (Fig. 1A and B). Initial weight did not differ within lean (control 290.2 ± 4.6 g; rimonabant 290.0 ± 5.1 g) or obese (control 463.0 ± 9.7 g; rimonabant 468.0 ± 5.3 g) rats. After an initial weight loss, cumulative body weight gain of rimonabant-treated rats remained below that of controls throughout the whole experiment (Fig. 1C and D). Treatment with rimonabant reduced total energy gain, as assessed by whole-carcass analyses, and this was mostly accompanied by a reduction in fat gain in obese rats, whereas lean rats showed a significant reduction in protein gain (Table 1). Rimonabant reduced food efficiency, and this reduction reached statistical significance in lean rats (Table 1). Rimonabant did not alter apparent energy expenditure (Table 1). BAT weight was increased in obese rats but reduced in lean rats by rimonabant (Table 1). BAT UCP1 mRNA levels were significantly increased in rimonabant-treated obese rats (Table 1).
Metabolic plasma variables.
Treatment with rimonabant reduced circulating triglyceride levels in both lean and obese rats (Table 2). NEFA levels were increased after food deprivation but not significantly modified by rimonabant. Circulating glucose levels of rimonabant-treated obese rats were reduced to values similar to those of lean and untreated food-deprived obese rats. Food deprivation reduced plasma insulin levels in both lean and obese rats, and these levels were further reduced by rimonabant. HOMA-IR was reduced in lean and obese rats treated with rimonabant. Rimonabant significantly reduced circulating leptin levels only in lean rats. Circulating adiponectin levels were increased in rimonabant-treated obese rats.
The HPA axis.
Food deprivation increased plasma corticosterone levels in obese rats (Fig. 2A). Treatment with rimonabant increased basal corticosterone levels and potentiated the fasting-induced elevation in plasma corticosterone in obese rats. In addition, treatment with rimonabant increased c-fos mRNA levels in the parvocellular division of the PVN (Fig. 2B). Rimonabant did not alter CRF or CRF1 receptor mRNA levels in the PVN (Fig. 2C and D).
Neuronal activation and neuropeptides in the arcuate nucleus, lateral hypothalamic area, and supraoptic nucleus.
Food deprivation increased NPY (Fig. 3A and B) and c-fos (Fig. 3E and F) mRNA levels in the arcuate nucleus (ARC) of obese rats and MCH mRNA in the lateral hypothalamic area of lean rats (Fig. 3H). Treatment with rimonabant enhanced ARC NPY (Fig. 3A and B), AgRP (Fig. 3C), and c-fos (Fig. 3E and F) mRNA levels in obese rats. Rimonabant also tended to increase POMC mRNA levels in the ARC of obese rats (P = 0.05; Fig. 3D). c-fos mRNA levels were elevated in the supraoptic nucleus (SON) of ad libitum–fed rats, and these levels were enhanced by rimonabant in obese rats (Fig. 3G).
DISCUSSION
The present results confirm the ability of CB1 antagonists to reduce weight gain in both lean and obese rats (22,23,27). Here, we extend previous findings with a complete energy balance profile based on carcass analyses. We show that the reduction in weight gain was largely accounted for by a reduction in fat gain, which is consonant with previous studies (2,28). The fact that rimonabant did not significantly decrease energy expenditure, which would be expected with a reduction in both total energy intake and energy gain, suggests that CB1 blockade exerted a stimulating effect on thermogenesis. This is further supported by the observation that, in obese rats, rimonabant increased BAT UCP1 mRNA levels, a widely used marker of thermogenic capacity. A recent study by Jbilo et al. (29) showed that rimonabant activates several BAT genes involved in the regulation of mitochondrial activity. Although increased BAT thermogenic activity likely contributed to rimonabant-induced reduction in energy gain, such reduction was obviously also attributable to the transient reduction in energy intake. The present study also further highlights the ability of CB1 antagonists to improve glucose metabolism. In obese Zucker rats, which are hyperinsulinemic (30) and glucose intolerant (31), rimonabant restored normal glycemia and reduced plasma levels of insulin and triglycerides. It is noteworthy that these marked changes were observed at a time when rimonabant no longer affected food intake. Our results are consistent with other studies that showed improved plasma profiles in Zucker rats (22), diet-induced obese mice (2,32), and obese humans (3,4).
In the present investigation, we tested the possibility that rimonabant may attenuate the food deprivation-induced stress response in obese Zucker rats. This hypothesis was based on the observation that the hyperactivity of the endocannabinoid system (20), which is thought to increase the incentive value of food (7,33), may in part explain the hypersensitivity of obese Zucker rats to food deprivation (19). Our results demonstrated that rimonabant did not block the effect of food deprivation on HPA axis activation in obese Zucker rats. In fact, rimonabant increased basal circulating corticosterone levels and enhanced the HPA axis response to a daytime food deprivation in obese Zucker rats. This enhanced activity of the HPA axis was associated with increased c-fos mRNA levels in the parvocellular division of the PVN. Thus, the response of obese Zucker rats was similar to that of lean ICR mice, in which rimonabant was recently shown to potentiate the corticosterone response to restraint stress (16). One possible mechanism for rimonabant-induced activation of the HPA axis is through increased CRF release. Because CB1 is primarily a presynaptic receptor that modulates neurotransmitter release (34), the presence of CB1 mRNA in >50% of CRF neurons in the PVN suggests that cannabinoids may directly influence CRF release (10). Finally, the observation of increased HPA axis activity is inconsistent with a reduced energy gain. Corticosteroids have been shown to promote energy deposition (35–37) and to inhibit BAT thermogenesis (37–39). This raises the possibility that rimonabant may impede the downstream effects of an activated HPA axis by obstructing the effects of chronically elevated plasma corticosterone on energy deposition and thermogenic activity. The enhancing effect of rimonabant on central insulin sensitivity would represent one mechanism through which rimonabant may counteract the detrimental metabolic effects of an increased HPA axis activity.
Obese Zucker rats exhibited a predictable increase in NPY mRNA levels in response to food deprivation, and this was accompanied by an increase in c-fos mRNA levels in the ARC. The NPY response was not observed in lean rats, which are less sensitive to food deprivation than obese rats (40). Nonetheless, lean rats showed a fasting-induced elevation in MCH mRNA levels in the lateral hypothalamus and reduction in c-fos mRNA levels in the SON. As previously observed (23,27,28), the hypophagic effect of rimonabant was transient despite continuous administration of the CB1 antagonist. In obese rats, rimonabant increased NPY and AgRP mRNA levels in both fed and fasted rats. These increases, which were paralleled by induction of the c-fos gene, likely occurred as a mechanism to compensate for the reducing effects of rimonabant on energy stores. However, the inductions, which were obtained at a time when food intake had normalized in both lean and obese rats, suggest that CB1 antagonists may block a possible NPY/AgRP-stimulated compensatory overfeeding. In agreement with this statement, previous studies showed that interrupting the administration of a CB1 antagonist leads to compensatory overfeeding (27,28). Also, rimonabant was shown to attenuate NPY-induced sucrose drinking in rats (41) and overeating in satiated mice (42). Together, these studies suggest that blockade of NPY action may have contributed to the rimonabant-induced hypophagia and/or inhibition of a compensatory overfeeding.
A recent study (43) showed that AM251, an analog of rimonabant, reduces NPY release and attenuates cannabinoid-induced NPY release from hypothalamic explants, suggesting that the effect of CB1 antagonists on food intake may result from a reduction in NPY synaptic tone. However, it is unlikely that cannabinoids act directly on NPY/AgRP neurons. A colocalization study failed to detect the presence CB1 mRNA in NPY-positive neurons of the ARC (10). Also, Di Marzo et al. (20) showed that the hypophagic effect of rimonabant remains present in NPY-deficient mice, suggesting that NPY and cannabinoids are independent orexigenic systems. Rimonabant-induced improvement in insulin sensitivity represents a potential mechanism whereby the drug could blunt the activity of the NPY/AgRP neurons. It is clear from our own HOMA-IR results and from those of other studies (3) that rimonabant has a strong positive action on insulin sensitivity. Whether this action of rimonabant is also apparent at the brain level is a likely possibility that certainly warrants further investigation.
Rimonabant tended to increase POMC mRNA, the precursor of the anorexigenic neuropeptide -melanocyte–stimulating hormone but did not affect the MCH system in obese rats. Involvement of these systems during the initial period of exposure, when food intake is reduced, cannot be excluded. A recent study demonstrated that rimonabant attenuated the feeding response induced by the melanocortin receptor-4 (MCR4) antagonist JKC-363 (44). This result indicates that rimonabant may act downstream from MCR4 and that it may block the orexigenic action of MCR4 antagonists, including AgRP. Of relevance to the present study is the fact that a stimulated melanocortin system, without concomitant activation of the MCH system, constitutes a condition promoting UCP1-induced thermogenesis (45).
Neuronal activation, as assessed by c-fos mRNA levels, in hypothalamic nuclei known for their involvement in the regulation of energy balance and the response to stress, argues in favor of an involvement of the hypothalamus in the effects of rimonabant. Increased c-fos mRNA in the PVN is in line with increased HPA axis activity, whereas neuronal activation in the ARC is consistent with increased NPY and AgRP mRNA levels. Rimonabant also increased c-fos mRNA levels in the SON of ad libitum–fed obese rats. The magnocellular neurosecretory cells of the SON secrete either arginine vasopressin or oxytocin (46), and these cells were shown to respond to feeding (47) and hypertonicity (48). Verty et al. (49) demonstrated an interaction between CB1 and oxytocin receptors; rimonabant attenuated the food and water intake induced by tocinoic acid, an oxytocin receptor antagonist. Therefore, rimonabant induced neuronal activation in brain structures that are consistent with its effect on neuropeptides involved in the regulation of energy balance.
In conclusion, our results further demonstrate the potential of CB1 antagonists to improve energy metabolism in obese rats, in particular by reducing energy gain and normalizing several plasma variables related to the metabolic syndrome and diabetes. The study also demonstrates that CB1 blockade does not prevent the hypersensitivity to food deprivation occurring at the level of HPA axis and ARC activation in the obese Zucker rats. This, however, does not prevent CB1 antagonism from exerting beneficial effects on energy and glucose metabolism.
ACKNOWLEDGMENTS
C.D. has received a postdoctoral fellowship from Fonds quebecois de la recherche sur la nature et les technologies. D.R. has received a grant from the Canadian Institutes of Health Research.
We thank Sanofi-Aventis for their supply of rimonabant. We also thank Serge Simard for statistical advice, and Julie Plamondon, Marie-Nolle Cyr, Sebastien Poulin, and Hakima Zekki for technical assistance.
FOOTNOTES
C.D. is currently affiliated with the Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
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 with 18 U.S.C. Section 1734 solely to indicate this fact.
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