International Journal of Obesity (2008) 32, 863–870 & 2008 Nature Publishing Group All rights reserved 0307-0565/08 $30.00 www.nature.com/ijo ORIGINAL ARTICLE Effects of rimonabant, a cannabinoid CB1 receptor ligand, on energy expenditure in lean rats

I Kunz1, MK Meier1, A Bourson1, M Fisseha2 and W Schilling1

1F Hoffmann-La Roche Pharmaceuticals Ltd, Department of Metabolic and Vascular Diseases, Basel, Switzerland and 2Healthcare Project Management, Geneva, Switzerland

Objective: To determine the effect of rimonabant on energy expenditure (O2 consumption) in rats at different metabolic states and in cannabinoid CB1 receptor-deficient (CB1RÀ/À) mice. Design: Animals were exposed to light–dark cycles and fed only during dark cycles. Rimonabant or vehicle was administered together with food (absorptive), following overnight feeding (postabsorptive) or following a whole day of no food (fasting). Indirect calorimetric measurements, physical activity and food intake were measured continuously. Results: Compared with vehicle-treated rats, rats administered 3 and 10 mg kgÀ1 rimonabant showed an 18 and 49% increase in O2 consumption, respectively after 3 h. A second dose of rimonabant administered 9–14.5 h after the first one failed to affect O2 consumption, suggesting the development of tolerance. Similarly, stereotypic behaviors and ambulatory activity increased following the first dose but these effects were not observed after the second dose. Respiratory quotients revealed no effect of rimonabant on rates of carbohydrate and fat oxidation. Analysis of the correlation between O2 consumption and physical activity indicated that factors other than increased physical activity may contribute to the increase in O2 consumption. Similar À/À studies in mice demonstrated that wild type but not CB1R mice showed a change in O2 consumption and physical activity following rimonabant administration, suggesting that these effects are mediated by the cannabinoid CB1 receptor. Conclusion: Previous studies suggested that reduced food intake alone may not explain the weight reduction observed with rimonabant. Our studies suggest that rimonabant stimulates significant acute energy expenditure in non-obese rodents, which could not be completely accounted for by an increase in physical activity. However, with the observation that there is rapid development of tolerance, these results suggest that there may be additional mechanism(s) that lead to weight loss in these rodents. International Journal of Obesity (2008) 32, 863–870; doi:10.1038/ijo.2008.3; published online 5 February 2008

Keywords: O2 consumption; stereotypic behavior; development of tolerance; rimonabant; cannabinoid CB1 receptor

Introduction antagonist that is highly specific and has been shown to be a potent appetite suppressant.3,4 Its weight-reducing effects The endocannabinoid system, which consists of cannabi- have been demonstrated in various rodent models,5–9 and noid receptors, endocannabinoids, and the enzymes recent large clinical trials in obese patients have shown that involved in the biosynthesis and degradation of rimonabant reduces body weight and cardiometabolic risk endocannabinoids, is involved in a wide range of physiolo- factors.10–14 gical functions.1 The demonstration that the endocannabi- Although reduction of food intake is considered to be the noid system plays an important role in the control of food main mode of action for the weight-reducing effect of intake offers opportunities for the treatment of obesity.2 rimonabant, several studies have suggested that other Antagonists of the cannabinoid receptor type 1 (CB1) display metabolic processes may also be involved.1,15 In studies in appetite-suppressant effects.2 Rimonabant is one such which diet-induced as well as genetically obese (ob/ob) mice were treated with rimonabant for 3 and 7 days, respectively, body weight was reduced in comparison to pair-fed control Correspondence: Dr I Kunz, DSM Nutritional Products Ltd, R&D Human animals.7,16 By contrast, rimonabant-treated obese (fa/fa) Nutrition and Health, PO 3255, Building 203/858, CH 4002, Basel, Zucker rats9 and diet-induced obese rats17 did not exhibit Switzerland. greater weight loss compared with pair-fed obese animals, E-mail: [email protected] Received 29 August 2007; revised 18 December 2007; accepted 21 December suggesting that in these animal models drug-induced weight 2007; published online 5 February 2008 loss could be explained by reduced food intake. Rimonabant effect on energy expenditure I Kunz et al 864

Data on the direct effect of rimonabant on energy consumption and CO2 production), physical activity and expenditure are scarce. In genetically obese mice (ob/ob) food intake were measured continuously (described below).

treated with rimonabant for 7 days, O2 consumption after 22 h following the last dose showed an increase.16 Recently, it has been shown that AVE1625, a new CB1 receptor Rimonabant administration antagonist increased energy expenditure independent of Rimonabant [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichloro- physical activity and also increased fat oxidation in non- phenyl)-4-methylpyrazole-3-carboxamide] was synthesized obese rats.18 Furthermore, although some studies show that in-house and administered orally as a microsuspension with rimonabant increases stereotypic behaviors, such as scrat- 0.3% Tween 80 as vehicle (2 ml kgÀ1 (rats), 4 ml kgÀ1 (mice)). ching and grooming in mice and rats,9,19–21 data on whether Treatment (rimonabant or vehicle) occurred at: 1730 h rimonabant increases locomotor (ambulatory) activity, an together with food (absorptive/fed state), at 0800 h following energy-consuming behavior, appear to be controversial.19,22–24 overnight feed (postabsorptive state), or at 1600 or 1700 h Thus, the objective of the current studies was to determine after a whole day without food (fasted state). whether rimonabant stimulates an increase in energy expenditure in lean rodents that is sufficiently sustained to contribute to the described weight-reducing properties of Determination of the effects of rimonabant the drug. Groups of rats or mice were administered different doses of rimonabant at different metabolic states. Calorimetric para- meters, physical activity and stereotypic behaviors were Materials and methods measured as described below. For the studies correlating O2 consumption and physical activity, four individual SD rats Animals were administered with 10 mg kgÀ1 rimonabant and vehicle Male Sprague–Dawley (SD) rats (10 to 12 weeks old and on separate occasions (following a night of unrestricted food weighing 340–380 g) were obtained from Charles River, intake) and moved into the respiratory chamber at 1000 h Lyon, France. Cannabinoid CB1 receptor-deficient mice after each administration. The effects of treatment on À/À (CB1R ) (6-month-old and weighing 26.5±1.0 g) and their O2 consumption and physical activity were measured wild-type littermates (CB1R þ / þ ) (weighing 28.0±1.5 g) were continuously every minute from 60 to 120 min after obtained from an in-house breeding colony. Breeding and treatment. Administration of either vehicle or rimonabant genotyping were performed as described previously.25 was repeated within 1–2 weeks. One-minute data pairs were We certify that institutional and governmental regulations analyzed by linear regression (see statistical analysis section). concerning the ethical use of animals were followed during Two groups each of seven CB1R þ / þ or CB1RÀ/À littermate this research. mice were administered with either rimonabant or vehicle. O2 consumption and physical activity were continuously measured, beginning at 1800 h on the day before adminis- Experimental designs tration until 1700 h the day after administration (that is, 9 h In general, animals were housed in groups of two in cages after the administration). that were maintained at 22 1C with fixed 12 h light–dark cycle (0600–1800 h lights-on). They were supplied with tap water and a standard powdered chow (KLIBA NAFAG, Indirect calorimetry and physical activity measurements

Kaiseraugst, Switzerland), with protein, fat and carbohydrate Consumption of O2 and CO2 production of individual providing 22.3, 12.3 and 65.4% of energy, respectively. For animals were measured in an open-circuit indirect calori- the determination of baseline food intake and indirect metric system consisting of one pair of air analyzers and calorimetric measurements, animals were caged individually. eight airtight respiratory chambers for both rats and mice Baseline food intake of individual animals was determined (Oxymax Equal Flow System, Columbus Instruments, OH, during the week preceding indirect calorimetric measure- USA). Each chamber was equipped with an infrared system ments by providing animals with powdered food and tap (OPTO-M3, Columbus Instruments, OH, USA), which con- water ad libitum during the lights-off period (1800–0600 h), sisted of two rows of twelve infrared beams at two levels and animals were accustomed to oral administration of tap above the bottom of the cage (2.5 cm between individual water twice daily for 3–4 days. Subsequently, weight- beams) to record physical activity of the animals. Condi- matched animals were grouped into eight and adapted to tioned fresh air with a temperature of 28±0.5 1C and relative the respiratory chambers for 24–36 h, during which food was humidity of 50±5% was pumped into the respiratory supplied only during the lights-off period. chambers at a flow rate of either 2 l minÀ1 (rats) or 0.6 l minÀ1 During indirect calorimetric measurements food supply in (mice). In each measuring cycle of 18 min, air was sequen- the lights-off period was restricted to 70% of baseline intake tially sampled at a flow rate of 0.4 l minÀ1 and analyzed by to ensure that all food provided would be eaten during the taking samples from inflow air for 2 min followed by samples

night before treatment. Indirect calorimetric parameters (O2 of exit air from each chamber. Samples were drawn through

International Journal of Obesity Rimonabant effect on energy expenditure I Kunz et al 865 drying tubes (magnesium perchlorate) before delivery to Po0.05 were considered significant. Comparisons of beha- the analyzers. Of the 2-min sampling time, 1 min was for vioral effects (experiment 2) were made by ANOVA followed analyzer conditioning (settle time) and 1 min for measuring. by Dunnett’s post hoc test.

O2 was analyzed electrochemically (measuring range: 19.3–21.5% volume, accuracy: 0.015% volume) and CO2 by infrared (measuring range: 0–0.9% volume accuracy: 0.025% Results volume). Analyzers (Carbagas, Basel, Switzerland) were calibrated before each indirect calorimetry experiment using Effect of rimonabant on O2 consumption, RQ, physical activity a standard gas mixture of O2:CO2:N2 (20.5:0.5:79% volume, and food intake (experiment 1) respectively). Single oral dose of 10 mg kgÀ1 but not 3 mg kgÀ1 of

Oxygen disappearance and CO2 appearance in the rimonabant significantly increased O2 consumption of rats chambers were calculated as the difference between gas in the postabsorptive state (ANOVA and Dunnett’s post hoc amounts of inflow and outflow per minute (flow rate for dry test, Po0.0005). Compared with baseline (0–2 h before drug air multiplied by O2 or CO2 concentrations). The outflow administration), mean O2 consumption, calculated for 3 h rate was corrected by Haldane equation as described after drug administration, was 18 and 49% higher after previously.26,27 Respiratory quotient (RQ) was calculated as administration of 3 and 10 mg kgÀ1, respectively (Figure 1a).

CO2 production/O2 consumption. All values for O2 By contrast, control rats showed a typical diurnal pattern of consumption and CO2 production were expressed in terms O2 consumption with increased values during the lights-off of standard temperature (0 1C), pressure (760 mm Hg) and period (Figures 1a and b). There was no significant increase dryness. Values for O2 consumption and CO2 production in O2 consumption (P ¼ 0.69) in response to the second dose were calculated as l hÀ1 per kg body weight and from mean of rimonabant administered after 9 h (1700 h) in the fasted values derived from sequential measuring cycles and were rats compared with vehicle-treated controls (Figure 1a). corrected for corresponding changes in the vehicle-treated Similarly, rats administered the first oral dose of rimona- control group. Values for physical activity of the animals bant (10 mg kgÀ1) during the fed state (before the onset of during the indirect calorimetric measurements were extra- the lights-off period, group IV) exhibited a significant polated for 1 h and expressed as numbers of infrared beam increase in O2 consumption (Po0.005). Mean O2 consump- breaks per animal per hour. tion calculated for 3 h after the first dose increased by 32% relative to baseline (0–2 h before drug administration) in these rats. However, response to the second and third doses Measurements of stereotypic behavior administered at 0800 and 1700 h the next day (14.5 and Stereotypic behaviors associated with elevation of physical 23.5 h, respectively after the first dose) were comparable to activity such as scratching (bursts of numerous rapid group II and III (Figure 1b). scratches), head shakes, wet dog shakes, yawning and penile All groups of animals had low physical activity during the erections) were categorized and scored 1 h after administra- lights-on period (before drug administration). During the tion by watching the animals and counting stereotypic lights-off period rats receiving vehicle (groups I, II and III) behaviors in the respiratory chambers for 15 min. Scoring increased activity by about three times and those receiving was performed by investigators (one for two animals) who rimonabant (group IV) by about five times (Student’s t-test, had no knowledge of the treatment. Po0.005 for the comparison between the control group and group IV). Compared with controls (group I), rats that were administered their first dose of rimonabant in the Statistical analysis postabsorptive state (groups II and III) showed a dose- Results are given as mean±standard error of the mean dependent increase in physical activity (Po0.005 for both (s.e.m.). Pairwise comparisons were made by Student’s t-test comparisons). However, a second dose of rimonabant and adjusted according to Bonferroni in case of multiple administered during the postabsorptive state (group IV) or testing. In cases of multiple group testing, analysis of in the fasted state (groups II and III) did not increase physical variance (ANOVA) was followed by a Dunnett’s post hoc test activity compared with control rats (data not shown). (StatView, Version 5.0.1, SAS Institute Inc., NC, USA). Control rats had a typical diurnal pattern of RQ, with

To calculate the relationship between O2 consumption and increased values during the lights-off period (Figure 2). physical activity, 1-min data pairs between 60 and 120 min Neither a single dose of rimonabant (3 and 10 mg kgÀ1) after drug administration was obtained from the individual administered in the postabsorptive condition (0800 h) nor a rats after treatment with either rimonabant or vehicle were second dose administered in the fasted state (1700 h) analyzed by linear regression. The intercept of the regression produced a significant effect on RQ (groups II and III, line with the y-axis provided an approximate value for O2 Figure 2a). Rats administered the first dose of rimonabant consumption at rest (physical activity ¼ 0). For experimental (10 mg kgÀ1) in the fed state (before the onset of the lights- repeats the values for y-axis intersections were averaged, and off period; group IV) exhibited a significant decrease in RQ intercepts were compared by Student’s t-test. Differences at (Po0.05) compared with vehicle-treated controls (Figure 2b).

International Journal of Obesity Rimonabant effect on energy expenditure I Kunz et al 866

Figure 2 Mean±s.e.m. respiratory quotient of Sprague–Dawley rats À1 À1 (n ¼ 5–8 per group) after two oral administrations of 3 and 10 mg kgÀ1 given Figure 1 Mean±s.e.m. O2 consumption (l h kg ) of Sprague–Dawley rats (n ¼ 5–8 per group) administered vehicle or different concentrations of at 0800 h (postabsorptive) and 1700 h (fasted) to groups II and III (a) and À1 rimonabant during the fed (1730 h), postabsorptive (800 h) and fasted three consecutive administrations of 10 mg kg given at 1730 h (absorptive), (1700 h) states. Group I received vehicle only at all metabolic states. 0800 h (postabsorptive) and 1700 h (fasted) to group IV (b) (experiment 1). Rimonabant was administered to groups II (3 mg kgÀ1) and III (10 mg kgÀ1) Arrows indicate the time of administrations. during the postabsorptive and fasted states, respectively (a), and to group IV (10 mg) during all three metabolic states (b) (experiment 1). Arrows indicate the times of administration of either vehicle or drug. Tolerance development in SD rats (experiment 2) This experiment was performed to further evaluate the development of tolerance following the first administration of rimonabant that was observed in experiment 1. Rats that The second and third doses of rimonabant administered at were administered two consecutive doses of rimonabant had

0800 and 1700 h did not produce additional significant a significant increase in both O2 consumption (ANOVA and decreases in RQ, although these values remained lower than Dunnett’s post hoc test, Po0.005) and physical activity those of control rats. (Po0.0001) only after the first administration (Figures 3a In this experiment, intake of rats in group IV (10.1±1.1 g) and b). The second dose, administered 8 h later, had no was 54% lower than baseline intake (21.8±2.0 g) during effect on either parameter. Administration of a single dose adaptation to the respiratory chambers in the preceding of rimonabant at fasted state (group II) also increased

lights-off period. Furthermore, their intake was significantly O2 consumption and physical activity. Relative to baseline lower than that of vehicle-treated rats (16.8±1.1 g). (0–2 h before drug administration), the increase in mean

International Journal of Obesity Rimonabant effect on energy expenditure I Kunz et al 867 increase in both parameters after the first administration but none after the fifth.

O2 consumption at rest (experiment 3) Mean O2 consumption at rest (zero physical activity) measured in four SD rats was significantly higher when they were administered rimonabant than when they were administered vehicle (0.937±0.083 vs 0.758±0.069 À1 À1 O2 consumption (l h kg ), respectively (Po0.05, paired student’s t-test). This suggests that rimonabant stimulates

O2 consumption beyond that explained by elevated physical activity.

Effect of rimonabant on CB1R þ / þ and CB1RÀ/À mice (experiment 4) A single administration of rimonabant significantly

increased O2 consumption (ANOVA and Dunnett’s post hoc test Po0.05) and physical activity (Po0.005) in postabsorp- tive CB1R þ / þ but not CB1RÀ/À mice (Figure 4a). The mean

O2 consumption calculated for 3 h after drug administration in CB1R þ / þ mice was increased by 50% relative to baseline (0–2 h before drug administration). The effect of rimonabant

on O2 consumption was more prolonged than its effect on physical activity (Figure 4b). Rimonabant did not have any effect on RQ in either CB1R þ / þ or CB1RÀ/À mice (data not shown).

Discussion

Although reduction in food intake is known to be the main mechanism by which rimonabant functions in decreasing

À1 À1 weight gain, other mechanisms might also be involved. Our Figure 3 Mean±s.e.m. O2 consumption (l h kg )(a) and physical activity (number of beam breaks per h) (b) of Sprague–Dawley rats (n ¼ 5–8 studies show that a single administration of rimonabant to per group) after vehicle administration (group I), a single oral administration non-obese rats and mice increases O2 consumption in a dose- of 10 mg kgÀ1 given at 1600 h (group II) and two consecutive administrations dependent manner, independent of the metabolic state. This À1 of 10 mg kg given at 0800 and 1600 h (group III) (experiment 2). Arrows was paralleled by a simultaneous increase in physical activity indicate the time of the two administrations of either vehicle or drug. and frequency of some stereotypic behaviors, consistent with previous observations.20,21 Regression analysis of the rela-

tionship between O2 consumption and physical activity O2 consumption 3 h following the first administration of suggests that although the increase in physical activity could rimonabant was similar in both groups of rats (30 and 33%). explain some of the increase in O2 consumption, our results One hour following the first administration of rimonabant suggest the possible involvement of other mechanisms. As (10 mg ÀkgÀ1), rats showed a significant increase in the our studies were performed with only four rats, future studies number of scratching and wet dog shakes compared with with a larger number of animals will need to be performed control animals (Table 1). By contrast, no appreciable to confirm these results. However, similar results were increase in the frequency of head shakes, yawnings and presented in a recent study by Herling et al.18 In this study, penile erections was observed. Furthermore, there was no which had a similar design as ours, lean Wistar rats were apparent increase in any of the stereotypic behaviors after administered another CB1 receptor antagonist, AVE162, the administration of the second dose of rimonabant once daily. These authors also reported an increase in energy (Table 1, group III). Rimonabant had similar effects on the expenditure independent of physical activity. However, frequency of O2 consumption and stereotypical behavior in unlike our observations in SD rats, they reported no Wistar rats that were administered 10 mg kgÀ1 rimonabant difference in physical activity between control rats and daily for 5 days (data not shown). There was a significant those given AVE1625. Furthermore, their results showed that

International Journal of Obesity Rimonabant effect on energy expenditure I Kunz et al 868 Table 1 Effects of rimonabant on stereotypic behaviors of male Sprague–Dawley rats (experiment 2)

Group I (control) vehicle Group II vehicle Group III rimonabant at 0800 and 1600 h at 0800 h, rimonabant at 1600 h at 0800 and 1600 h

First administration (0800 h) Scratching 1.6±1.1 (2) 2.6±1.7 (4) 44.6±13.9a (8) Head shakes 0.1±0.1 (1) 0 0.4±0.4 (1) Wet dog shakes 0 0 3.6±1.2a (7) Yawning 0.3±0.3 (1) 0.1±0.1 (1) 0 Penile erection 0 0 0.3±0.2 (2)

Second administration (1600 h) Scratching 1.5±0.8 (3) 71.9±11.2b (8) 4.3±1.6 (5) Head shakes 0 0.3±0.3 (1) 0 Wet dog shakes 0 5.1±2.2b (7) 0.5±0.5 (1) Yawning 1.0±0.8 (2) 0.3±0.3 (1) 0.9±0.5 (3) Penile erection 0 0.3±0.2 (2) 0.1±0.1 (1)

Data are mean number of counts per 15 min±s.e.m. of eight animals per group. Numbers in parentheses indicate the number of animals affected. aPo0.05. bPo0.01 ANOVA followed by Dunnett’s post hoc test as compared to control (group I).

a single administration of AVE162 at the beginning of the In our study, RQ increased in rats during the fed state light period results in an increase in fat oxidation whereas (lights-off period) (Figure 2a), reflecting the increase in we did not observe this change. These results suggest that the carbohydrate oxidation during intake of carbohydrate-rich two compounds may act via different mechanisms. food. During the postabsorptive state (lights-on period), Our studies on CB1R þ / þ mice also showed that the there was a similar pattern of decline in RQ in all groups,

rimonabant-induced increase in O2 consumption was not regardless of rimonabant administration, reflecting the always paralleled by a corresponding increase in physical progressive increase in fat oxidation after termination of activity, further suggesting the involvement of factors other feeding. In contrast, the significant decrease in RQ in rats than physical activity in the stimulation of energy expendi- that were administered their first dose of rimonabant during ture. As a similar effect on CB1RÀ/À mice was not observed, it the fed state (before lights off) compared with control

suggests that the rimonabant-stimulated O2 consumption in animals (Figure 2b), likely reflects the anorectic property of mice is mediated by the cannabinoid CB1 receptor. rimonabant leading to a delay in feeding and overall The exact mechanism underlying the induction of energy reduction of intake. expenditure by rimonabant remains to be elucidated, Development of tolerance has been reported previously, but there is evidence for the involvement of the endocanna- but published data on this topic is still contradictory. binoid system. Adipose tissue expresses various secretory Tolerance to the anorectic property of rimonabant has been proteins, including leptin, tumor necrosis factor-a demonstrated in obese rat models and diet-induced obese and adipocytokines such as adiponectin and visfatin, which mice.6,9,17,32 Development of tolerance to the behavioral and have been shown to increase fat oxidation in skeletal muscle locomotion stimulation effects of rimonabant have also been and hepatocytes28,29 and regulate glucose metabolism.30 reported previously.9,36 Contrary to these results, Liu et al.,16 Perwitz et al.31 reported that cannabinoid signaling directly reported the absence of tolerance to thermogenic stimula- influences expression of adiponectin and visfatin in brown tion in genetically obese (ob/ob) mice treated with rimona- and white adipose tissues, implicating its control of energy bant for 7 days with 10 mg kgÀ1 intraperitoneally. Our and glucose homeostasis. Furthermore, rimonabant has studies in SD (and Wistar) rats showed the rapid develop- been shown to increase adiponectin mRNA and protein ment of tolerance to the rimonabant effects on both 10 levels in overweight and obese humans and in diet- O2 consumption and physical activity. Our initial experi- induced obese mice.6 This increase has also been shown in ment (experiment 1) suggested that rats that were adminis- pre-adipocyte cultured cells treated with rimonabant.32,33 tered a second dose of rimonabant (9–14.5 h after their first

These observations suggest that rimonabant may affect dose) showed very little or no difference in O2 consumption energy expenditure via its effects on adiponectin level. and no difference in physical activity compared with control However, this is still not clear since a cross-sectional study animals. This was confirmed by the second experiment, in Pima Indians failed to show a correlation between which was designed specifically to address the issue of adiponectin levels and 24-h energy expenditure and RQ.34 tolerance. This experiment also showed that whereas rats Alternative mechanisms for the effect of rimonabant on had increased frequency of scratching and wet dog shakes energy expenditure have been suggested by studies of gene after the first administration of rimonabant, they developed expression in diet-induced obese mice, which indicate the tolerance to these behavioral effects of rimonabant by the involvement of enzymes in the b-oxidation pathway and second administration. It is possible that part of tricarboxylic acid cycle.35 the difference in tolerance development in our studies and

International Journal of Obesity Rimonabant effect on energy expenditure I Kunz et al 869 days. Tolerance development is probably different in humans. Rimonabant has been administered as 5 or 20 mg doses daily in large clinical trials that have lasted for 1–2 years.12,14 Thus far, development of tolerance to rimonabant in humans has not been reported. In conclusion, our studies suggest that in addition to reducing appetite, rimonabant also leads to an increase in energy expenditure that may not be completely explained by a corresponding increase in physical activity. The involve- ment of additional factors contributing to increased energy expenditure had been suggested from previous studies but had not been demonstrated in vivo with rimonabant. Elucidating the mechanism through which rimonabant acts

to increase O2 consumption in obese animal models could pave the way for the development of new drugs to treat obesity. It will also be important to understand the mechanism of tolerance development in these animals.

Acknowledgements

We thank Dr Laurence Ozmen for providing the CB1RÀ/À mice, Dr Stephan Roever for providing the in-house synthesized compound rimonabant, Dr Thomas Mindt and Dr Uwe Totzke for their valuable contributions, and Joseph Schoerlin, Nadine Petit and Anthony Vandjour for excellent technical help.

References

1 Pagotto U, Marsicano G, Cota D, Lutz B, Pasquali R. The emerging role of the endocannabinoid system in endocrine regulation and energy balance. Endocr Rev 2006; 27: 73–100. 2 Black SC. Cannabinoid receptor antagonists and obesity. Curr À1 À1 Figure 4 Mean±s.e.m. O2 consumption (l h kg )(a) and physical Opin Investig Drugs 2004; 5: 389–394. activity (number of beam breaks per mouse per h) (b) of cannabinoid CB1 3 Meschler JP, Kraichely DM, Wilken GH, Howlett AC. Inverse receptor-deficient mice (CB1RÀ/À) and wild-type littermates (CB1R þ / þ ) agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1- (n ¼ 7 per group) after a single oral administration of 10 mg kgÀ1 at 0800 h (2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (experiment 4). Arrows indicate the time of the administration of either (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)- vehicle or drug. 1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor. Biochem Pharmacol 2000; 60: 1315– 1323. 4 Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B, those of Liu et al. may be due to the difference in animal Congy C et al. SR141716A, a potent and selective antagonist of models, since tolerance development may take longer in the brain cannabinoid receptor. FEBS Lett 1994; 350: 240–244. obese rats9,17 due to their higher peripheral cannabinoid CB1 5 Colombo G, Agabio R, Diaz G, Lobina C, Reali R, Gessa GL. receptor levels compared to lean rats.37 Indeed, similar to our Appetite suppression and weight loss after the cannabinoid antagonist SR 141716. Life Sci 1998; 63: PL113–PL117. results, development of tolerance has also been reported for 6 Poirier B, Bidouard JP, Cadrouvele C, Marniquet X, Staels B, the anorectic activity of rimonabant in lean rats.5 The rapid O’Connor SE et al. The anti-obesity effect of rimonabant is development of tolerance that was observed in our studies associated with an improved serum lipid profile. Diabetes Obes Metab 2005; 7: 65–72. support the concept that differences in the ‘tone’ of 7 Ravinet Trillou C, Arnone M, Delgorge C, Gonalons N, Keane P, endoncannabinoid system exist between lean and obese Maffrand JP et al. Anti-obesity effect of SR141716, a CB1 receptor animals.1,38,39 Doyon et al.40 recently demonstrated that antagonist, in diet-induced obese mice. Am J Physiol Regul Integr development of tolerance in obese fa/fa rats was slower than Comp Physiol 2003; 284: R345–R353. 8 Verty AN, McGregor IS, Mallet PE. Consumption of high in lean Fa/? rats. Following oral treatment with 10 mg kgÀ1, carbohydrate, high fat, and normal chow is equally suppressed lean rats returned to their normal food intake after 4 days by a cannabinoid receptor antagonist in non-deprived rats. whereas in the obese rats food intake remained low until 14 Neurosci Lett 2004; 354: 217–220.

International Journal of Obesity Rimonabant effect on energy expenditure I Kunz et al 870 9 Vickers SP, Webster LJ, Wyatt A, Dourish CT, Kennett GA. 24 Navarro M, Hernandez E, Munoz RM, del Arco I, Villanua MA, Preferential effects of the cannabinoid CB1 receptor antagonist, Carrera MR et al. Acute administration of the CB1 cannabinoid SR 141716, on food intake and body weight gain of obese (fa/fa) receptor antagonist SR 141716A induces anxiety-like responses in compared to lean Zucker rats. Psychopharmacology (Berl) 2003; the rat. Neuroreport 1997; 8: 491–496. 167: 103–111. 25 Ibrahim MM, Deng H, Zvonok A, Cockayne DA, Kwan J, Mata HP 10 Despres JP, Golay A, Sjostrom L. Effects of rimonabant on et al. Activation of CB2 cannabinoid receptors by AM1241 metabolic risk factors in overweight patients with dyslipidemia. inhibits experimental neuropathic pain: pain inhibition by N Engl J Med 2005; 353: 2121–2134. receptors not present in the CNS. Proc Natl Acad Sci USA 2003; 11 Gelfand EV, Cannon CP. Rimonabant: a cannabinoid receptor 100: 10529–10533. type 1 blocker for management of multiple cardiometabolic risk 26 Even PC, Mokhtarian A, Pele A. Practical aspects of indirect factors. J Am Coll Cardiol 2006; 47: 1919–1926. calorimetry in laboratory animals. Neurosci Biobehav Rev 1994; 18: 12 Pi-Sunyer FX, Aronne LJ, Heshmati HM, Devin J, Rosenstock J. 435–447. Effect of rimonabant, a cannabinoid-1 receptor blocker, on 27 Simonson DC, DeFronzo RA. Indirect calorimetry: methodo- weight and cardiometabolic risk factors in overweight or obese logical and interpretative problems. Am J Physiol 1990; 258: patients: RIO-North America: a randomized controlled trial. E399–E412. JAMA 2006; 295: 761–775. 28 Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, 13 Scheen AJ, Finer N, Hollander P, Jensen MD, Van Gaal LF. Efficacy Yen FT et al. Proteolytic cleavage product of 30-kDa adipocyte and tolerability of rimonabant in overweight or obese patients complement-related protein increases fatty acid oxidation in with type 2 diabetes: a randomised controlled study. Lancet 2006; muscle and causes weight loss in mice. Proc Natl Acad Sci USA 368: 1660–1672. 2001; 98: 2005–2010. 14 Van Gaal LF, Rissanen AM, Scheen AJ, Ziegler O, Rossner S. Effects 29 Gil-Campos M, Canete RR, Gil A. Adiponectin, the missing link of the cannabinoid-1 receptor blocker rimonabant on weight in insulin resistance and obesity. Clin Nutr 2004; 23: 963–974. reduction and cardiovascular risk factors in overweight patients: 30 Sethi JK, Vidal-Puig A. Visfatin: the missing link between 1-year experience from the RIO-Europe study. Lancet 2005; 365: intra-abdominal obesity and diabetes? Trends Mol Med 2005; 11: 1389–1397. 344–347. 15 Smith RA, Fathi Z. Recent advances in the research and 31 Perwitz N, Fasshauer M, Klein J. Cannabinoid receptor signaling development of CB(1) antagonists. IDrugs 2005; 8: 53–66. directly inhibits thermogenesis and alters expression of 16 Liu YL, Connoley IP, Wilson CA, Stock MJ. Effects of the adiponectin and visfatin. Horm Metab Res 2006; 38: 356–358. cannabinoid CB1 receptor antagonist SR141716 on oxygen 32 Bensaid M, Gary-Bobo M, Esclangon A, Maffrand JP, Le Fur G, consumption and soleus muscle glucose uptake in Lep(ob)/ Oury-Donat F et al. The cannabinoid CB1 receptor antagonist Lep(ob) mice. Int J Obes Relat Metab Disord 2005; 29: 183–187. SR141716 increases Acrp30 mRNA expression in adipose tissue of 17 Thornton-Jones ZD, Kennett GA, Benwell KR, Revell DF, Misra A, obese fa/fa rats and in cultured adipocyte cells. Mol Pharmacol Sellwood DM et al. The cannabinoid CB1 receptor inverse 2003; 63: 908–914. agonist, rimonabant, modifies body weight and adiponectin 33 Gary-Bobo M, Elachouri G, Scatton B, Le Fur G, Oury-Donat F, function in diet-induced obese rats as a consequence of reduced Bensaid M. The cannabinoid CB1 receptor antagonist rimona- food intake. Pharmacol Biochem Behav 2006; 84: 353–359. bant (SR141716) inhibits cell proliferation and increases markers 18 Herling AW, Gossel M, Haschke G, Stengelin S, Kuhlmann J, of adipocyte maturation in cultured mouse 3T3 F442A preadipo- Muller G et al. CB1 receptor antagonist AVE1625 affects primarily cytes. Mol Pharmacol 2006; 69: 471–478. metabolic parameters independently of reduced food intake in 34 Stefan N, Vozarova B, Funahashi T, Matsuzawa Y, Ravussin E, Wistar rats. Am J Physiol 2007; 293: E826–E832. Weyer C et al. Plasma adiponectin levels are not associated with 19 Costa B, Colleoni M. SR141716A induces in rats a behavioral fat oxidation in humans. Obes Res 2002; 10: 1016–1020. pattern opposite to that of CB1 receptor agonists. Acta Pharmacol 35 Jbilo O, Ravinet-Trillou C, Arnone M, Buisson I, Bribes E, Sin 1999; 20: 1103–1106. Peleraux A et al. The CB1 receptor antagonist rimonabant reverses 20 Darmani NA, Pandya DK. Involvement of other neuro- the diet-induced obesity phenotype through the regulation of transmitters in behaviors induced by the cannabinoid CB1 lipolysis and energy balance. FASEB J 2005; 19: 1567–1569. receptor antagonist SR 141716A in naive mice. J Neural Transm 36 Rubino T, Vigano D, Zagato E, Sala M, Parolaro D. In vivo 2000; 107: 931–945. characterization of the specific cannabinoid receptor antagonist 21 Jarbe TU, DiPatrizio NV, Li C, Makriyannis A. The cannabinoid SR141716A: behavioral and cellular responses after acute and receptor antagonist SR-141716 does not readily antagonize open- chronic treatments. Synapse 2000; 35: 8–14. field effects induced by the cannabinoid receptor agonist 37 Engeli S, Jordan J. The endocannabinoid system: body weight and (R)-methanandamide in rats. Pharmacol Biochem Behav 2003; 75: metabolic regulation. Clin Cornerstone 2006; 8 (Suppl 4): S24–S35. 809–821. 38 Engeli S, Bohnke J, Feldpausch M, Gorzelniak K, Janke J, Batkai S 22 Compton DR, Aceto MD, Lowe J, Martin BR. In vivo characteri- et al. Activation of the peripheral endocannabinoid system in zation of a specific cannabinoid receptor antagonist human obesity. Diabetes 2005; 54: 2838–2843. (SR141716A): inhibition of delta 9-tetrahydrocannabinol-in- 39 Matias I, Di Marzo V. Endocannabinoids and the control of duced responses and apparent agonist activity. J Pharmacol energy balance. Trends Endocrinol Metab 2007; 18: 27–37. Exp Ther 1996; 277: 586–594. 40 Doyon C, Denis RG, Baraboi ED, Samson P, Lalonde J, 23 Masserano JM, Karoum F, Wyatt RJ. SR 141716A, a CB1 Deshaies Y et al. Effects of rimonabant (SR141716) on fasting- cannabinoid receptor antagonist, potentiates the locomotor induced hypothalamic-pituitary-adrenal axis and neuronal stimulant effects of amphetamine and apomorphine. Behav activation in lean and obese Zucker rats. Diabetes 2006; 55: Pharmacol 1999; 10: 429–432. 3403–3410.

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