laboratori de neurofarmacologia, departament de ciencies...

,1 ,1

*Laboratori de Neurofarmacologia, Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra, PRBB,

Barcelona, Spain

�Medimod pharmacology services GmbH, Reutlingen, Germany

�Institut d’Alta Tecnologia PRBB Fundacio Privada (IAT), PRBB, Barcelona, Spain

Obesity continues to grow as a worldwide health problemand represents a major concern in the health care system indeveloped and developing countries (Hagmann 2008).Multiple physiological systems are involved in the controlof food intake and metabolism and participate in thepathophysiological mechanisms leading to obesity. In thissense, several studies have recently identified the crucial roleplayed by the endocannabinoid system in the control ofenergy balance. The endocannabinoid system constitutes a‘silent’ mechanism that is activated in a transitory way tomaintain the homeostatic equilibrium (Di Marzo and Matias2005; Di Marzo 2008). This system includes the cannabi-noid receptors (CB1, CB2 and G-protein-coupled receptor55), the endogenous lipid ligands (endocannabinoids), andthe enzymatic machinery for their synthesis and inactivation.Obesity seems to be associated with a pathological over-activation of the endocannabinoid system revealed by anup-regulation of CB1 receptor and/or an enhancementof endocannabinoid levels (Di Marzo and Matias 2005;

Di Marzo 2009). Animal and clinical studies have revealedthat the overactivity of the endocannabinoid system is a keycomponent in the pathophysiological mechanisms leading toobesity and metabolic unbalance (Cota et al. 2003; Ravinetet al. 2004; Despres et al. 2005; Van Gaal et al. 2005;Vickers and Kennett 2005; Ward and Dykstra 2005; Pagottoet al. 2006; Pi-Sunyer et al. 2006; Scheen et al. 2006;Schafer et al. 2008). Therefore, the blockade of CB1

receptor was considered a potential pharmacological tool

Received September 11, 2009; revised manuscript received December 9,2009; accepted December 12, 2009.Address correspondence and reprint requests to Rafael Maldonado,

Departament de Ciencies Experimentals i de la Salut, Universitat Pom-peu Fabra, PRBB, C/Dr. Aiguader 88, 08003, Barcelona, Spain. E-mail:[email protected] authors contributed equally to this work.Abbreviations used: CB1, cannabinoid receptor 1; CB2, cannabinoid

receptor 2; CD, cafeteria diet; CT, computed tomography; GTPcS,guanosine 5¢-[c-thio] triphosphate; HU, Hounsfield; SC, standard chow.

Abstract

The endocannabinoid system plays a crucial role in the

pathophysiology of obesity. However, the clinical use of can-

nabinoid antagonists has been recently stopped because of its

central side-effects. The aim of this study was to compare the

effects of a chronic treatment with the CB1 cannabinoid

antagonist rimonabant or the CB1 inverse agonist taranabant

in diet-induced obese female rats to clarify the biological

consequences of CB1 blockade at central and peripheral

levels. As expected, chronic treatment with rimonabant and

taranabant reduced body weight and fat content. Interestingly,

a decrease in the number of CB1 receptors and its functional

activity was observed in all the brain areas investigated after

chronic taranabant treatment in both lean and obese rats. In

contrast, chronic treatment with rimonabant did not modify the

density of CB1 cannabinoid receptor binding, and decreased

its functional activity to a lower degree than taranabant. Six

weeks after rimonabant and taranabant withdrawal, CB1

receptor density and activity recovered to basal levels. These

results reveal differential adaptive changes in CB1 canna-

binoid receptors after chronic treatment with rimonabant and

taranabant that could be related to the central side-effects

reported with the use of these cannabinoid antagonists.

Keywords: autoradiography, CB1 cannabinoid receptor,

obesity, rimonabant, taranabant, withdrawal.

J. Neurochem. (2010) 112, 1338–1351.

JOURNAL OF NEUROCHEMISTRY | 2010 | 112 | 1338–1351 doi: 10.1111/j.1471-4159.2009.06549.x

1338 Journal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351� 2010 The Authors

to restore the normal endocannabinoid tone under thesepathological conditions.

In view of the promising results reported in the differentclinical trials, the CB1 receptor antagonist rimonabantinitially emerged as an effective treatment of obesity andmetabolic disorders (Despres et al. 2005; Pi-Sunyer et al.2006; Scheen et al. 2006; Van Gaal et al. 2008). In 2006, theEuropean regulatory authorities approved the use of rimo-nabant in obese patients (BMI ‡ 30 kg/m or > 27 kg/m withcomplications) and was in the market as Acomplia� from2006 to 2008 in more than 40 countries around the world.Taranabant, a CB1 receptor inverse agonist, was alsodeveloped and reached Phase-III clinical trials for thetreatment of obesity (Hagmann 2008). However, the Euro-pean Medicines Agency recommended the suspension of themarketing authorization for rimonabant in 2008 because ofthe presence of undesired central side-effects and the clinicaltrials with taranabant were stopped.

Animal models are required to investigate the biologicalmechanisms underlying obesity. A new model of compulsivefood seeking/taking has been recently validated in rats tostudy obesity (Hansen et al. 2005; Heyne et al. 2009; Shafatet al. 2009). It is based on the exposure to a feeding regimein which rats are offered a free choice of cafeteria diet (CD)(palatable chocolate-containing food) in addition to theirstandard chow (SC) diet. Under these conditions, animalshighly prefer CD over SC and most of them become obese.Furthermore, animals rapidly develop compulsive feedingreflected by a lack of flexibility in food taking with rejectionof SC when CD is transiently not available. This model is anexcellent tool to investigate the neurobiological basis under-lying obesity, and to evaluate new therapeutical approaches.

The aim of this study was to improve the knowledge of thecentral and peripheral consequences of chronic CB1 block-ade. For this purpose, we have compared the central andperipheral effects of a chronic treatment with the CB1

receptor antagonist rimonabant (10 mg/kg, once daily, sub-lingual) or with the CB1 receptor inverse agonist taranabant(3 mg/kg, once daily, sublingual) in this new model ofcompulsive food seeking/taking. Body weight and foodintake were periodically recorded, and both molecular andimaging analyses were performed. At the molecular level,autoradiography was used to evaluate the functional activityand density of CB1 receptors in these animals. The possiblelongitudinal variation in the content and distribution of bodyfat was also evaluated by using a customized computedtomography (CT) imaging approach.

Material and methods

Animals, housing and food regimenThe experiments were performed with 48 outbred female Wistar rats

that were 6 months old at the beginning of the study (Harlan,

Winkelmann, Germany). The rats were kept in groups of four

animals in Makrolon type IV cages (60 · 38 · 20 cm) with

heightened lids (5 cm) during acclimatization and the first 6 weeks

of obesity development. Subsequently, they were housed singly in

Makrolon type III cages (43 · 26 · 15 cm) with heightened lids

(5 cm) for the remaining period of the experimental sequence.

Animals were housed in rooms with controlled temperature

(20 ± 2�C) and humidity (40–60%) under a 12 h/12 h light dark

cycle. Twenty-four animals were assigned to the lean group and

were maintained on standard pellet chow (Altromin diet 1324 from

Altromin GmbH, Germany, 65% energy from carbohydrates, 24%

from protein and 9% from fat with in total 2.850 kcal/g). Another 24

animals (obese group) were exposed to a free choice between SC and

highly caloric palatable pellet diet (CD). This CD was manufactured

as previously described (Heyne et al. 2009) by a mix of equal

amounts of Bounty�, Snickers�, Mars� and Milka� chocolate

prepared as homogenous food pellets (65% energy from carbo-

hydrates, 6.5% from protein and 23% from fat with in total

4.846 kcal/g). Both types of food were presented separately on the

top of the grid of the cage (food container of the cage) with half of the

space (right/left) containing 150 g of each kind of food (lean group:

SC + SC or obese group: CD + SC). Water was always available

ad libitum. After 14 weeks exposed to their corresponding diets, both

lean and obese rats were randomly subdivided into three different

groups (n = 8 each) according to the pharmacological treatment

assigned (vehicle, rimonabant or taranabant). All protocols were

conducted following the standard ethical guidelines of the European

Communities Directive 86/609/EEC regulating animal research, and

according to the internal standard operating procedures of the three

laboratories where the experiments were carried out [medimod

pharmacology services, Reutlingen, Germany; Departament de Cien-

cies Experimentals i de la Salut, Universitat Pompeu Fabra (UPF),

Barcelona, Spain; Institut d’Alta Tecnologia (IAT), Barcelona, Spain].

Pharmacological treatmentRimonabant, taranabant and vehicle were administered sublingually

(application volume 0.5 mL per rat) once per day 1 h before the

beginning of the dark phase. Fresh drug solutions were prepared

daily. The selective CB1 receptor antagonist rimonabant [(Npiperi-din-1-yl)-5-(4-chlorophenyl)-1(2,4-dichlorophenyl)-4-methyl-1H-

pyrazole-3-carboxy amide] was administered at a dose of 10 mg/kg/

day. The CB1 receptor inverse agonist taranabant [MK-0364, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-

methyl-2-{[5-(trifluoromethyl) pyridine-2-yl]oxy}propanamide] was

administered at a dose of 3 mg/kg/day. Both drugs were dissolved in

a vehicle solution containing 0.5% natrosol (Sigma-Aldrich, Madrid,

Spain), 0.1% tween-80 (Sigma-Aldrich) and 99.4% distilled water.

Rimonabant was purchased from Chemos GmbH (Regenstauf,

Germany) and taranabant was a gift from Solvay Pharmaceuticals

(Hannover, Germany).

Study design

Experimental part in the German laboratory (MedimodPharmacology Services)Obesity development phase (14 weeks). After 1 week of acclimati-

zation, one group was exposed to a free choice between SC and CD

(obese group), whereas the other group received SC only (lean group).

� 2010 The AuthorsJournal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351

Central and peripheral effects of rimonabant and taranabant | 1339

Treatment phase I (6 weeks). The same food regimen was

maintained and the corresponding anti-obesity treatment (vehicle,

rimonabant or taranabant) was given once daily to each group of

animals. Transfer from the medimod pharmacology service to the

Spanish laboratory (UPF and IAT) was carried out at the end of this

period. The international shipment was handled by World Courier

following internal standard operating procedures for reducing stress

to the animals. Upon arrival, an acclimatization period of four days

was allowed before starting the next experimental sequence.

Experimental part in the Spanish laboratories (UPF and IAT)Treatment phase II (7 weeks). The same food regimen and anti-

obesity treatments were maintained as in treatment phase I.

Animals were scanned by CT for body fat content determination

on the first, third and seventhweek upon arrival, which corresponded

to days 44, 58 and 85 of treatment. One day after the last CT scan

session, 24 of the 48 animalswere killed to evaluate the density and the

functional activity of CB1 cannabinoid receptors by autoradiography.

Treatment withdrawal phase (6 weeks). For the next 6 weeks, the 24

remaining animals were maintained under the same food regimen,

but no pharmacological treatment was administered. Their body fat

content was evaluated by CT scans performed on weeks 3 and 6 of

this phase, which corresponded to days 21 and 39 after treatment

withdrawal. After the last CT scan, animals were killed to evaluate

the density and functional activity of CB1 cannabinoid receptor by

autoradiography.

Body weight and food intake controlBody weight was registered twice a week. Data are expressed as the

percentage of body weight gain taking as reference the weight of

each animal before the beginning of treatment. Food intake in the

Spanish laboratory was determined weekly by placing an estab-

lished quantity of 150 g of food (CD and/or SC) at the top of the

cage every Monday at 12:00 h. One week later, the remaining food

was removed, weighed and replaced by a same initial quantity

(150 g). Food intake was normalized for each animal based on its

body weight using the following equation: [(weekly food intake

(g) · (kcal/g))/body weight (g)] · 100.

Computed tomography imaging and data processingAnimals were anesthetized by 2.5% isoflurane inhalation using

oxygen as vehicle and data from two consecutive CT scans at

different voltage settings of the X-ray tube (140 and 80 kV, both at

100 mA) were acquired using a General Electric Discovery ST

device. Images were acquired with a matrix size of 256 · 256

yielding a 0.39 · 0.39 mm pixel size. Slice thickness was 1.25 mm

and the transversal field of view covered about 25 cm, which was

enough to image the whole animal, except for the tail. Immediately

after the second CT scan, animals were placed back into their cages,

allowed to wake up and housed as usual.

Two composite images were created from the 140 and 80 kV

images: one average image and one subtraction image. The average

image was used to segment the background, lungs, soft tissues and

bone by applying appropriate thresholds to the Hounsfield (HU)

units: background, below )600 HU; lungs, between )600 and )200HU; soft tissue, between )200 and 200 HU; bone, over 200 HU. A

mask was created to assign the scanner platform and the anesthesia

nozzle to the background. Fat was determined from the subtracted

image as those voxels presenting differences between 15 and 45 in

HU, and falling within the pre-calculated soft-tissue mask. To

estimate the total body weight and its distribution, the following

density values were assigned to the segmented tissues: lungs, 0.43 g/

cm3; soft tissue, 0.73 g/cm3; bone, 1.53 g/cm3; fat, 0.55 g/cm3.

Brain slicing for autoradiography studiesAnimals were killed by decapitation. Brains were removed and

quickly frozen by immersion in chilled 2-methyl-butane. Coronal

sections 20-lm-thick were cut in a cryostat, thaw-mounted on

gelatin/chrome-coated slides, dried briefly at 30�C and stored at

)80�C until the cannabinoid receptor and guanosine 5¢-[c-thio]triphosphate (GTPcS) binding experiments were performed.

Autoradiography of cannabinoid receptor bindingThe protocol used was based on the method described before

(Herkenham et al. 1991). Briefly, slide-mounted brain sections

were incubated for 2.5 h at 37�C in a buffer containing 50 mM

Tris with 5% bovine serum albumin (fatty acid-free), pH 7.4, and

10 nM [3H] CP-55 940 (NEN, Boston, MA, USA) prepared in the

same buffer, in the absence or presence of 10 lM nonlabeled

CP-55,940 (Tocris Bioscience, Ellisville, MO, USA) to determine

total and nonspecific binding, respectively. Following this incuba-

tion, slides were washed in 50 mM Tris buffer with 1% bovine

serum albumin (fatty acid-free), pH 7.4, for 4 h (2 · 2 h) at 0�C,dipped in ice-cold distilled water, then dried under a stream of cool

dry air.

Autoradiograms were generated by apposing the labeled tissues,

together with autoradiographic standards ([3H] microscales; Amer-

sham, UK), to tritium-sensitive film (Amersham Hyperfilm-3H;

Barcelona, Spain) for a period of 10 days and developed for 4 min

at 20�C. Densitometry determinations were carried out with a GS-

800 Calibrated Densitometer (Bio-Rad Laboratories, Hercules, CA,

USA) and analyzed with Quantity One 1D software (Bio-Rad),

using the standard curve generated from [3H]-standards. Specific

binding measured in each structure was determined by subtracting

the non-specific binding image from that of total binding.

Analysis of WIN-55,212–2-stimulated [35S]-GTPcS bindingThe protocol used was based on the method described before (Sim

et al. 1995). Briefly, slide-mounted brain sections were rinsed in

assay buffer (50 mM Tris, 3 mM MgCl2, 0.2 mM EGTA,

100 mM NaCl and 0.5% fatty acid-free bovine serum albumin,

pH 7.4) at 25�C for 10 min, then pre-treated for 15 min with an

excess concentration (2 mM) of GDP (Sigma Chemical Co.,

Madrid, Spain) in assay buffer. Afterwards, sections were

incubated at 25�C for 2 h in assay buffer containing 0.04 nM

[35S]-GTPcS (PerkinElmer Espana SL, Madrid, Spain), 2 mM

GDP, and 10 lM WIN-55,212–2 (Sigma Chemical Co.). Basal

activity was assessed in the absence of agonist, whereas non-

specific binding was measured in the presence of 10 lM unlabeled

GTPcS. In pilot experiments, additional brain sections were

incubated in the presence of rimonabant (3 lM) (Rinaldi-Carmona

et al. 1994) in addition to 0.04 nM [35S]-GTPcS, 2 mM GDP and

10 lM WIN-55,212–2. Slices were rinsed twice in 50 mM Tris

buffer, pH 7.4, at 4�C and deionized once in water, then dried

under a stream of cool dry air. Autoradiograms were generated by

Journal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351� 2010 The Authors

1340 | E. Martın-Garcıa et al.

apposing the labeled tissues to film (Biomax MS, Amersham) for

a period of 3 days and developed for 4 min at 20�C. Developedfilms were analyzed and quantified in a computerized image

analysis system (MCID, St Catharines, Ontario, Canada). Densi-

tometry determinations were carried out with a GS-800 Calibrated

Densitometer (Bio-Rad) and analyzed with Quantity One 1D

software (Bio-Rad).

Statistical analysisData from body weight gain, food intake and CT imaging were

analyzed using three-way ANOVA with diet (lean and obese) and

treatment (vehicle, rimonabant and taranabant) as between-subjects

factors and day as within-subject factor. Data analysis of CB1

receptor binding and of WIN-55,212–2-stimulated [35S]-GTPcSbinding was performed for each brain structure by two-way ANOVA

with diet and treatment as between-subjects factors. All these

analyses were carried out separately for the different periods

corresponding to the presence or absence of treatment. Percentage of

body weight gain was analyzed the last day of treatment and the last

day of the experiment using one-way ANOVA with treatment as

between-subjects factor. The percentage of body weight gain was

calculated taking as reference the value obtained before the

beginning of the pharmacological treatment. Differences were

considered significant at p < 0.05. Results are expressed as

mean ± SEM, unless otherwise stated. The statistical analysis was

performed using the Statistical Package for Social Science program

SPSS� 15.0 (SPSS Inc, Chicago, IL, USA).

Results

Body weightBody weight gain changes across days during treatment andtreatment withdrawal are shown in Fig. 1. The statisticalanalysis of body weight gain was performed consideringthree different periods, early treatment period (from day 1 to40), late treatment period (from day 42 to 89) andwithdrawal of treatment (from day 8 to 46 after withdrawal).Three-way ANOVA during the early period of treatmentrevealed a significant main effect of ‘diet’ [F(1,42) = 10.60;p < 0.01] confirming the higher body weight gain in obesethan lean animals. A significant main effect of ‘treatment’was also obtained [F(2,42) = 36.46; p < 0.001], which wasdependent on the ‘diet’ [F(2,42) = 5.23; p < 0.01]. Post hocanalyses revealed that pharmacological treatments haddifferential effects in reducing body weight in obese andlean animals. Thus, rimonabant produced higher reduction ofbody weight gain in obese than lean rats (p < 0.01). Incontrast, taranabant-induced reduction of body weight gainwas similar in both groups of animals. In both lean andobese animals, body weight gain similarly decreased afterrimonabant (p < 0.001) and taranabant (p < 0.001) treatmentcompared with vehicle. A significant main effect of ‘day’was revealed during the early treatment period

(a)

(c)

(b)

(d)

Fig. 1 Mean values of body weight gain evolution in lean (a) and

obese rats (c) during the treatment and withdrawal periods of the

experiment. Mean ± SEM values of last day of treatment or treat-

ment withdrawal in lean (b) and obese animals (d). The reference

value used to calculate the percentage of body weight variation was

the value obtained before the beginning of treatment. X-axis (a and

c) indicates day of the treatment period (from 1 to 89) and the

withdrawal phase (from 8 to 46). From beginning of treatment to day

89 of treatment, the number of rats per group was 8. During the

withdrawal phase (from 8 to 46), the number of animals per group

was 4. wp < 0.05; wwwp < 0.001 significant differences when

comparing vehicle to taranabant group. p < 0.01 significant

differences when comparing vehicle to rimonabant group (Newman-

Keuls).



[F(13,546) = 10.08; p < 0.001] with a significant interactionbetween ‘day’ and ‘diet’ [F(13,546) = 3.25; p < 0.001], aswell as an interaction between ‘day’ and ‘treatment’[F(26,546) = 3.93; p < 0.001], and a significant interactionbetween ‘day’, ‘diet’ and ‘treatment’ [F(26,546) = 1.51;p < 0.05].

Three-way ANOVA during the late period of treatment alsorevealed a significant main effect of ‘diet’ [F(1,42) = 15.04;p < 0.001] and ‘treatment’ [F(2,42) = 23.22; p < 0.001],which was dependent on the ‘diet’ [F(2,42) = 2.96;p < 0.05]. Accordingly to the previous period, post hocanalyses revealed that rimonabant produced higher reductionof body weight gain in obese than lean rats (p < 0.01). Incontrast, taranabant reduction of body weight gain wassimilar in both groups of animals. In obese rats, body weightgain similarly decreased after rimonabant (p < 0.001) andtaranabant (p < 0.01) treatment compared with vehiclegroup. In lean animals, both treatments also decreased bodyweight gain compared with vehicle (p < 0.001), althoughtaranabant was more efficacious than rimonabant (p < 0.05).A significant interaction between ‘day’ and ‘diet’[F(13,546) = 2.51; p < 0.01] was also revealed. No significantmain effect of ‘day’ [F(13,546) = 1.11; n.s.] or interactionbetween ‘day’ and ‘treatment’ [F(26,546) = 1.49; n.s.] orbetween ‘day’, ‘diet’ and ‘treatment’ [F(26,546) = 0.63; n.s.]were revealed during this late treatment period.

In the treatment withdrawal period, three-way ANOVA didnot reveal significant main effects of ‘diet’ [F(1,18) = 0.93;n.s.], ‘treatment’ [F(2,18) = 0.71; n.s.] or interaction between‘diet’ and ‘treatment’ [F(2,18) = 0.08; n.s.], ‘day’ and ‘treat-ment’ [F(22,198) = 1.52; n.s.] or between ‘day’, ‘diet’ and‘treatment’ [F(22,198) = 1.17; n.s.]. Significant main effects of‘day’ [F(11,198) = 11.67; p < 0.001], and significant inter-action between ‘day’ and ‘diet’ [F(11,198) = 8.52; p < 0.001]were found in this period.

The values obtained on the last day of treatment and thelast day of the experiment were analyzed separately for thetwo diet groups (lean and obese). In lean animals, one-wayANOVA showed a significant main effect of ‘treatment’[F(2,21) = 19.17; p < 0.001]. Post hoc Newman-Keuls anal-ysis showed decreased body weight gain in rimonabant(p < 0.001) and taranabant (p < 0.001) treated groups com-pared with vehicle. In the obese group, one-way ANOVA alsoshowed a main effect of ‘treatment’ [F(2,21) = 6.21; p < 0.01]and Newman-Keuls analysis showed decreased body weightgain in rimonabant (p < 0.001) and taranabant (p < 0.05)groups compared with vehicle. The last day of treatmentwithdrawal one-way ANOVA showed no significant maineffects of ‘treatment’ in lean [F(2,9) = 0.21; n.s.] or in obeseanimals [F(2,9) = 0.25; n.s.]. On this last day of treatment,rimonabant was more effective in reducing body weight gainin obese (13% of reduction) than lean (2% of reduction) rats,whereas taranabant was similarly efficacious in both groupsof rats (9% vs. 6% of reduction respectively).

Food intakeCaloric food intake for each group of rats is shown in Fig. 2(treatment from day 1 to 40) and table 1 (treatment from day42 to 89 and treatment withdrawal from day 8 to 46). Thestatistical analysis of caloric food intake was also performedconsidering three different periods, early treatment period(from day 1 to 40), late treatment period (from day 42 to 89)and withdrawal of treatment (from day 8 to 46 afterwithdrawal). Three-way ANOVA during the early period oftreatment showed a significant main effect of ‘diet’[F(1,42) = 33.34; p < 0.001], confirming the enhanced caloricintake in obese compared with lean animals. Significant maineffects of ‘treatment’ [F(2,42) = 14.03; p < 0.001] and inter-action between these two factors [F(2,42) = 3.37; p < 0.05]were also found. Significant main effects of ‘day’[F(7,294) = 47.38; p < 0.001], interaction between ‘day’ and‘diet’ [F(7,294) = 5.84; p < 0.001], ‘day’ and ‘treatment’[F(14,294) = 12.99; p < 0.001] and between ‘day’, ‘diet’ and‘treatment’ [F(14,294) = 3.71; p < 0.001] were also revealed.Considering the significant effect of treatment during thisperiod, a more detailed post hoc analysis was performed todetermine the time point when CB1 antagonists decreasedfood intake. Thus, post hoc Newman-Keuls showed inlean animals significant lower caloric consumption aftertaranabant administration than after vehicle on days 1

(a)

(b)

Fig. 2 Mean ± SEM values of energy intake in lean (a) and obese

rats (b) during first 40 days of treatment. wp < 0.05; wwp < 0.01;wwwp < 0.001 significant differences when comparing vehicle to tar-

anabant group. p < 0.001; p < 0.01 significant differences

when comparing vehicle to rimonabant group. #p < 0.05 significant

differences when comparing rimonabant to taranabant group

(Newman-Keuls) (n = 8 per group).



(p < 0.001), 2 (p < 0.001), 7 (p < 0.01) and 28 (p < 0.05) oftreatment. Lean animals under rimonabant treatment alsopresented lower caloric consumption than lean vehicle groupon days 1 (p < 0.001) and 2 (p < 0.001) of treatment. In obeseanimals, post hoc Newman-Keuls showed significant lowercaloric consumption after taranabant administration than aftervehicle on days 1 (p < 0.001) and 2 (p < 0.01) of treatment.Obese animals under rimonabant treatment also presentedlower caloric consumption than vehicle group on days 1(p < 0.001), 2 (p < 0.001), 7 (p < 0.01) and 14 (p < 0.05) oftreatment. In addition, rimonabant treated animals presentedlower caloric intake than obese taranabant treated rats ondays 1 (p < 0.05), 2 (p < 0.05) and 7 (p < 0.05) of treatment.

Three-way ANOVA for caloric food intake during the lateperiod of treatment showed a significant main effect of ‘diet’[F(1,42) = 6.71; p < 0.05], which confirmed the expecteddifferences in food intake between lean and obese animals.No significant main effects of ‘treatment’ [F(2,42) = 1.77;n.s.] or interaction between these two factors [F(2,42) = 0.84;n.s.] were found. This three-way ANOVA also showedsignificant main effects of ‘day’ [F(5,210) = 2.90; p < 0.05],indicating that all animals increased caloric intake acrossdays but this effect was independent of ‘diet’ or ‘treatment’as shown by the absence of a significant interaction between‘day’ and ‘diet’ [F(5,210) = 1.16; n.s.], ‘day’ and ‘treatment’[F(10,210) = 1.78; n.s.] or between ‘day’, ‘diet’ and ‘treat-ment’ [F(10,210) = 0.70; n.s.].

Three-way ANOVA was also performed for the period oftreatment withdrawal. The significant main effect of ‘diet’was maintained [F(1,18) = 13.89; p < 0.01] revealing that theobese animals maintained a higher caloric consumption thanthe lean group. A significant main effect of ‘treatment’ wasalso obtained during withdrawal [F(2,18) = 9.81; p < 0.01].Post hoc Newman-Keuls analyses showed significant highercaloric consumption after taranabant withdrawal than afterrimonabant (p < 0.05) or vehicle withdrawal (p < 0.001).Animals under rimonabant withdrawal also presented highercaloric consumption than those of the vehicle group(p < 0.05). No significant interaction between ‘diet’ and‘treatment’ was obtained [F(2,18) = 1.71; n.s.]. In contrast, asignificant main effect of ‘day’ [F(5,90) = 12.78; p < 0.001]and interaction between ‘day’ and ‘diet’[F(5,90) = 4.40;

p < 0.01] was obtained indicating that caloric intake evolveddifferently in obese and lean animals. No significantinteractions between ‘day’ and ‘treatment’ [F(10,90) = 1.27;n.s.] or between ‘day’, ‘diet’ and ‘treatment’ [F(10,90) = 0.35;n.s.] were revealed.

Computed tomography image derived body fat contentThree-way ANOVA analysis of the body fat content revealed asignificant main effect of ‘diet’ [F(1,41) = 33.26; p < 0.001]confirming the differences between lean and obese animals(see Fig. 3 for coronal CT images). A significant main effectof ‘treatment’ was also found [F(2,41) = 5.21; p < 0.05],which was dependent on the ‘diet’ [F(2,41) = 3.62; p < 0.05](Fig. 4). Newman-Keuls post hoc analyses revealed signif-icantly lower body fat content in obese animals chronicallytreated with both rimonabant (p < 0.05) and taranabant(p < 0.01) when compared with the obese vehicle group onthe CT scans performed on days 44, 58 and 85 of treatment(Fig. 4b). These results showed that both pharmacologicaltreatments were effective in reducing body fat content ofobese animals when compared with the vehicle treatment. Nosignificant main effect of ‘day of CT scan’ was revealedduring the treatment period [F(2,82) = 0.17; n.s.], but asignificant interaction between ‘day of scan’ and ‘diet’ wasobserved [F(2,82) = 4.42; p < 0.05]. These results confirmedthat the body fat content evolved in a different way in lean(Fig. 4a) and obese (Fig. 4b) animals during the time thatanimals were treated. No interactions between ‘day’ and‘treatment’ [F(4,82) = 1.27; n.s.] or between ‘day’, ‘diet’ and‘treatment’ [F(4,82) = 0.55; n.s.] were revealed.

Three-way ANOVA of the CT-estimated body fat contentduring the withdrawal period showed a significant maineffect of ‘diet’ [F(1,18) = 23.24; p < 0.001], thus confirmingpersistent differences in fat content between lean and obeseanimals. No significant effects of ‘treatment’ [F(2,18) = 0.09;n.s.] or interaction between ‘treatment’ and ‘diet’[F(2,18) = 0.001; n.s.] were found in the withdrawal period.Similarly to what was observed during the ‘treatment’ period,significant effects were obtained for ‘day of scan’[F(2,36) = 23.33; p < 0.001] and its interaction with ‘diet’[F(2,36) = 4.65; p < 0.05] and ‘treatment’ [F(2,36) = 3.51;p < 0.05], but no significant interaction between the three

Table 1 Caloric food intakeMean ± SEM

Treatment Treatment withdrawal

Lean Obese Lean Obese

Vehicle 104.46 ± 4.90 107.62 ± 9.86 101.70 ± 7.46 110.22 ± 8.84

Rimonabant 105.46 ± 4.75 115.45 ± 8.18 110.83 ± 8.84 119.25 ± 9.01

Taranabant 107.09 ± 5.17 121.32 ± 6.74 113.85 ± 7.60 136.07 ± 10.54

Food intake was normalized for each animal based on body weight using the following equation:

[(weekly food intake (g) · (kcal/g))/body weight (g)] · 100.



factors was observed [F(2,36) = 1.27; n.s.]. These resultsindicated that body fat content evolved differently duringtreatment withdrawal in lean and obese animals dependingon the previous pharmacological treatment.

Autoradiography of cannabinoid receptor bindingThe quantitative values of CB1 receptor binding obtainedafter treatment and withdrawal were analyzed for eachdifferent brain structure by two-way ANOVA with ‘diet’ and‘treatment’ as between-subjects factors (Fig. 5). Represen-

tative autoradiograms for cannabinoid receptor binding areshown in Fig. 6. In the nucleus accumbens, two-way ANOVA

of CB1 receptor binding after treatment revealed a signi-ficant main effect of ‘treatment’ [F(2,17) = 55.24; p < 0.001],but no significant effect of ‘diet’ [F(1,17) = 1.70; n.s.] orinteraction between these two factors [F(2,17) = 2.90; n.s.]was observed. Subsequent post hoc Newman-Keuls showedlower levels of CB1 receptors in animals treated withtaranabant than in those treated with rimonabant (p < 0.001)or vehicle (p < 0.001). Two-way ANOVA of CB1 receptor

Fig. 3 Mosaic image showing coronal CT

images of one representative individual for

each group under study over the five scans.

Amber signal corresponds to the estimated

fat distribution.

(a) (b)

Fig. 4 Evolution of the mean values of

body fat percentage in (a) lean and (b) ob-

ese rats during treatment and withdrawal

phase. wp < 0.05; wwp < 0.01 significant

differences when comparing vehicle to tar-

anabant group. p < 0.05 significant dif-

ferences when comparing vehicle to

rimonabant group (Newman-Keuls).



binding after withdrawal of treatment did not showsignificant main effects of ‘diet’ [F(1,18) = 0.02; n.s.],‘treatment’ [F(2,18) = 1.51; n.s.] or interaction between ‘diet’and ‘treatment’ [F(2,18) = 1.42; n.s.]. This result indicatesthat the changes induced on CB1 receptors binding recoverfollowing withdrawal.

In the striatum, two-way ANOVA of CB1 receptor bindingafter treatment showed a significant main effect of ‘treat-ment’ [F(2,17) = 43.29; p < 0.001], but no significant effectof ‘diet’ [F(1,17) = 3.42; n.s.] or interaction between ‘diet’and ‘treatment’ [F(2,17) = 0.58; n.s.]. Post hoc Newman-Keuls analysis showed lower density of CB1 receptors in rats

treated with taranabant than in those treated with rimonabant(p < 0.001) or vehicle (p < 0.001). In this brain structure,significant differences between vehicle and rimonabant werealso observed (p < 0.05). Two-way ANOVA of CB1 receptorbinding after treatment withdrawal did not show significantmain effects of ‘diet’ [F(1,18) = 0.23; n.s.], ‘treatment’[F(2,18) = 1.16; n.s.], or interaction between ‘diet’ and‘treatment’ [F(2,18) = 1.48; n.s.], indicating that changesinduced on CB1 receptors binding recover following with-drawal.

In the hippocampus, two-way ANOVA of CB1 receptorbinding after treatment revealed a significant main effect of

Fig. 5 Cannabinoid receptor binding in the

nucleus accumbens, striatum, hippocampus,

hypothalamus, prefrontal cortex and

cerebellum after chronic treatment with

taranabant and rimonabant (a) and after

treatment withdrawal (b). Data are

expressed as mean ± SEM. wp < 0.05;wwp < 0.01; wwwp < 0.001 significant dif-

ferences when compared with vehicle

group. p < 0.001 significant differences

when comparing taranabant to rimonabant

group. #p < 0.05 significant differences

when comparing vehicle lean to vehicle

obese group (Newman-Keuls) (n = 4 per

group).



‘treatment’ [F(2,17) = 10.70; p < 0.01], whereas no significanteffect of ‘diet’ [F(1,17) = 0.49; n.s.] or interaction betweenthese two factors [F(2,17) = 0.25; n.s.] was obtained. Again,subsequent post hoc Newman-Keuls revealed lower levels ofCB1 receptors in rats treated with taranabant than in thosetreated with rimonabant (p < 0.001) or vehicle (p < 0.01).Two-way ANOVA of CB1 receptor binding after treatmentwithdrawal did not show significant main effects of ‘diet’[F(1,18) = 0.44; n.s.], ‘treatment’ [F(2,18) = 3.32; n.s.] or inter-action between ‘diet’ and ‘treatment’ [F(2,18) = 1.97; n.s.].Similarly, a recovery of the changes induced on CB1 receptorsbinding by the pharmacological treatment was observed.

In the hypothalamus, two-way ANOVA of CB1 receptorbinding revealed a significant main effect of ‘treatment’[F(2,17) = 76.02; p < 0.001], but no significant effect of ‘diet’[F(1,17) = 0.97; n.s.] or interaction between ‘diet’ and ‘treat-ment’ [F(2,17) = 1.74; n.s.]. Subsequent post hoc Newman-Keuls analysis revealed again lower levels of CB1 receptorsin animals treated with taranabant than in those treated withrimonabant (p < 0.001) or vehicle (p < 0.001). During thetreatment withdrawal period, two-way ANOVA of CB1 recep-tor binding showed significant main effects of ‘diet’[F(1,18) = 6.64; p < 0.05] and obese animals showed higherdensity of CB1 receptors than lean rats. No significant maineffects of ‘treatment’ [F(2,18) = 1.49; n.s.] were obtained,which indicates a recovery of the changes induced on CB1

receptors binding after treatment withdrawal. Furthermore,a significant interaction between ‘diet’ and ‘treatment’[F(2,18) = 4.60; p < 0.05] was obtained indicating that thedifferences between lean and obese animals depended on theprevious treatment. Subsequent post hoc Newman-Keuls test

confirmed that the differences in CB1 receptor bindingbetween lean and obese animals were only observed invehicle animals (p < 0.05).

In the prefrontal cortex, two-way ANOVA of CB1 receptorbinding after treatment showed a significant main effect of‘treatment’ [F(2,18) = 9.98; p < 0.01], but no significanteffect of ‘diet’ [F(1,18) = 0.003; n.s.] or interaction between‘diet’ and ‘treatment’ [F(2,18) = 0.74; n.s.]. Post hocNewman-Keuls showed lower levels of CB1 receptor in animals treatedwith taranabant than in those treated with rimonabant(p < 0.001) or vehicle (p < 0.01). Two-way ANOVA of CB1

receptor binding after treatment withdrawal did not showsignificant main effects of ‘diet’ [F(1,18) = 0.22; n.s.], ‘treat-ment’ [F(2,18) = 0.27; n.s.] or interaction between ‘diet’ and‘treatment’ [F(2,18) = 1.20; n.s.], revealing the recovery of thechanges induced on CB1 receptors binding after treatmentwithdrawal.

Finally, in the cerebellum two-way ANOVA of CB1 receptorbinding after treatment showed a significant main effect of‘treatment’ [F(2,18) = 14.38; p < 0.001], but not significanteffect of ‘diet’ [F(1,18) = 0.19; n.s.] or interaction between‘diet’ and ‘treatment’ [F(2,18) = 2.01; n.s.]. Post hoc New-man-Keuls showed lower levels of CB1 receptors in animalstreated with taranabant than in those treated with rimonabant(p < 0.001) or vehicle (p < 0.01). Two-way ANOVA of CB1

receptor binding after withdrawal of the treatment did notshow significant main effects of ‘diet’ [F(1,18) = 0.23; n.s.],‘treatment’ [F(2,18) = 0.03; n.s.] or interaction between ‘diet’and ‘treatment’ [F(2,18) = 0.08; n.s.]. This result indicates arecovery of the changes induced on CB1 receptors bindingafter treatment withdrawal.

Analysis of WIN-55,212–2-stimulated [35S]-GTPcSbindingThe quantitative values of WIN-55,212–2-stimulated [35S]-GTPcS binding obtained after treatment and withdrawalwere analyzed for each different brain structure by two-wayANOVAwith ‘diet’ and ‘treatment’ as between-subjects factors(Fig. 7). Representative autoradiograms for cannabinoidreceptor functional activity are shown in Fig. 8. Aftertreatment, two-way ANOVA of WIN-55,212–2-stimulated[35S]-GTPcS binding in the nucleus accumbens showed asignificant main effect of ‘treatment’ [F(2,18) = 25.09;p < 0.001] but no significant effect of ‘diet’ [F(1,18) = 0.02;n.s.] or interaction between ‘diet’ and ‘treatment’[F(2,18) = 0.39; n.s.]. Subsequent post hoc Newman-Keulsshowed lower WIN-55,212–2-stimulated binding of [35S]-GTPcS in animals treated with taranabant than in thosetreated with rimonabant (p < 0.01) or vehicle (p < 0.001). Alower binding of [35S]-GTPcS was also observed in animalstreated with rimonabant than in animals treated with vehicle(p < 0.01). Two-way ANOVA after withdrawal of the treatmentdid not show significant main effects of ‘diet’ [F(1,16) = 0.01;n.s.], ‘treatment’ [F(2,16) = 1.32; n.s.] or interaction between

(a)

(b)

Fig. 6 Representative autoradiograms for cannabinoid receptor

binding of coronal brain sections from obese animals treated with

vehicle, rimonabant or taranabant during treatment period and treat-

ment withdrawal phase. The sections shown are from (a) the striatum

and nucleus accumbens, and (b) hippocampus and hypothalamus.



‘diet’ and ‘treatment’ [F(2,16) = 1.18; n.s.] indicating arecovery of the changes induced on CB1 receptors functionalactivity by the pharmacological treatment.

In the striatum, two-way ANOVA of [35S]-GTPcS bindingafter treatment showed a significant main effect of ‘treat-ment’ [F(2,18) = 43.29; p < 0.001], but no significant effectof ‘diet’ [F(1,18) = 3.42; n.s.] or interaction between thesetwo factors [F(2,18) = 0.58; n.s.]. In the same way, post hocNewman-Keuls showed lower WIN-55,212–2-stimulated[35S]-GTPcS binding in animals treated with taranabant thanin those treated with rimonabant (p < 0.05) or vehicle(p < 0.001). Lower WIN-55,212–2-stimulated [35S]-GTPcSbinding was also observed after treatment in animalsreceiving rimonabant than in those treated with vehicle(p < 0.05). Two-way ANOVA after withdrawal of the treatmentdid not show significant effects of ‘diet’ [F(1,16) = 0.03; n.s.],‘treatment’ [F(2,16) = 0.17; n.s.], or interaction between ‘diet’and ‘treatment’ [F(2,16) = 0.19; n.s.], indicating a recovery ofthe changes induced on CB1 receptors functional activity bythe pharmacological treatment.

In the hippocampus, two-way ANOVA of [35S]-GTPcSbinding after treatment showed a significant main effect of‘treatment’ [F(2,18) = 8.84; p < 0.01] without significanteffect of ‘diet’ [F(1,18) = 0.30; n.s.] or interaction between‘diet’ and ‘treatment’ [F(2,18) = 0.25; n.s.]. Again, post hoc

Fig. 7 WIN-55,212–2-stimulated [35S]-GTPcS

binding in the nucleus accumbens, striatum,

hippocampus, hypothalamus, prefrontal

cortex and cerebellum after chronic treat-

ment with taranabant and rimonabant (a)

and after treatment withdrawal (b). Data are

expressed as mean ± SEM, wp < 0.05;wwp < 0.01; wwwp < 0.001 significant dif-

ferences compared with vehicle group.

p < 0.05; p < 0.01 p < 0.001 sig-

nificant differences when comparing

taranabant to rimonabant group (Newman-

keuls).

(a)

(b)

Fig. 8 Representative autoradiograms for cannabinoid receptor

functional activity in coronal brain sections from obese animals

treated with vehicle, rimonabant or taranabant during treatment

period and treatment withdrawal. The sections shown are from (a)

the striatum and nucleus accumbens, and (b) hippocampus and

hypothalamus.



Newman-Keuls revealed lower WIN-55,212–2-stimulated[35S]-GTPcS binding in animals treated with taranabant thanin those treated with rimonabant (p < 0.05) or vehicle(p < 0.01). Similarly to previous results two-way ANOVA

after withdrawal of the treatment did not show significanteffects of ‘diet’ [F(1,16) = 0.08; n.s.], ‘treatment’ [F(2,16) =0.31; n.s.] or interaction between these two factors[F(2,16) = 0.32; n.s.], indicating a recovery of the changesinduced on CB1 receptors functional activity by thepharmacological treatment.

In the hypothalamus, two-way ANOVA of [35S]-GTPcSbinding after treatment showed a significant main effect of‘treatment’ [F(2,17) = 18.81; p < 0.001], but no significanteffect of ‘diet’ [F(1,17) = 0.51; n.s.] or interaction betweenthose factors [F(2,17) = 0.09; n.s.]. Post hoc Newman-Keulsshowed lower WIN-55,212–2-stimulated [35S]-GTPcS bind-ing in animals treated with taranabant than in those treatedwith rimonabant (p < 0.001) or vehicle (p < 0.001). Two-way ANOVA after withdrawal of the treatment showedsignificant main effects of ‘diet’ [F(1,16) = 6.03; p < 0.05]being higher WIN-55,212–2-stimulated [35S]-GTPcS bind-ing in lean animals. Furthermore, significant main effects of‘treatment’ [F(2,16) = 6.27; p < 0.05] without significantinteraction between ‘diet’ and ‘treatment’ [F(2,16) = 3.02;n.s.] were observed. Post hoc Newman-Keuls revealed lowerWIN-55,212–2-stimulated [35S]-GTPcS binding in animalstreated with vehicle than in those treated with rimonabant(p < 0.01) or taranabant (p < 0.01) indicating a recovery ofthe changes induced on CB1 receptors functional activity bythe pharmacological treatment.

Discussion

In this study, a decrease in the density and functional activityof CB1 cannabinoid receptor binding was observed in thenucleus accumbens, striatum, hippocampus and hypotha-lamus after chronic treatment with the inverse agonisttaranabant (3 mg/kg) in both lean and obese rats. In contrast,chronic treatment with rimonabant (10 mg/kg) only de-creased the functional activity of CB1 receptors in thenucleus accumbens and striatum without decreasing thedensity of cannabinoid receptor binding. This down-regula-tion and/or decreased functional activity disappeared aftertreatment withdrawal, indicating a recovery of the changes inall the brain areas studied. These biochemical results parallelthe effects of both drugs on the reduction of body weightgain and total amount of fat content observed during chronictreatment, both of which recovered after treatment with-drawal.

Chronic blockade of CB1 receptors after antagonist orinverse agonist treatment decreased binding and/or functionalactivity of central CB1 cannabinoid receptors. Differenteffects on CB1 receptor density and functional activity wererevealed after chronic rimonabant and taranabant treatment,

which could be explained by the preferential inverse agonistprofile of taranabant. Indeed, taranabant acts as a highlypotent and selective CB1 receptor inverse agonist that exertsthe opposite pharmacological effects to CB1 receptor agon-ists (Fong et al. 2007). CB1 receptors are coupled to Giproteins and their activation produces a decrease of intracel-lular cAMP concentrations (Devane et al. 1988; Howlettet al. 1990). Therefore, CB1 receptor inverse agonism resultsin an increase of cAMP levels, whereas neutral antagonistsdo not induce any signal transduction effects of their own(Lange and Kruse 2008). In this context, rimonabant wasinitially defined as a selective CB1 receptor antagonist(Rinaldi-Carmona et al. 1994, 1995; Pagotto et al. 2006).However, several studies have reported that rimonabantmight function as an inverse agonist mainly when adminis-tered at high doses (Xie et al. 2007). In this study, thedecreased functional activity after rimonabant treatment waslower than after taranabant, and the density of central CB1

receptors was only decreased after taranabant administration.One possible explanation for this result could be thatrimonabant preferentially acts as a neutral antagonist at thedose tested, while taranabant mainly acts as an inverseagonist in these experimental conditions. The chronicblockade of the activity of the endocannabinoid systemproduced by rimonabant would decrease CB1 receptorfunctional activity whereas the chronic inverse agonisteffects of taranabant would also decrease CB1 receptordensity. On the other hand, it has been demonstrated thatrimonabant occupies 50% of central CB1 receptors 8 h afteroral administration, whereas taranabant (Rinaldi-Carmonaet al. 1995) ocupies 25–30% of central CB1 receptors 24 hafter oral administration (Fong et al. 2007) when usingsimilar doses employed in this study. Therefore, thepredominant changes on CB1 receptor density observed aftertaranabant administration were not related to a higheroccupancy of central CB1 receptors by this antagonist sincerimonabant also occupies a high percentage of central CB1

receptors after oral administration. The undesired side-effectsof rimonabant and taranabant could be related with theadaptive changes on CB1 receptors revealed in this study indifferent brain structures involved in reward and motivation,such as the nucleus accumbens, hippocampus and prefrontalcortex. Thus, CB1 receptors are present in brain areasinvolved in the regulation of energy balance such as thehypothalamus, but also play an important role in theemotional control by acting on the prefrontal cortex,hippocampus and limbic system (Breivogel and Sim-Selley2009). The changes induced by these CB1 antagonists onCB1 cannabinoid receptors in the hypothalamus could berelated to adaptive changes on food intake after chronictaranabant treatment and after withdrawal of rimonabant andtaranabant administration. The changes produced by theseantagonists on CB1 receptors in the prefrontal cortex,hippocampus and nucleus accumbens could explain the



presence of psychiatric side-effects described in the literatureafter chronic rimonabant and taranabant administration thatinclude anxiety and depression (Akbas et al. 2009). Thesechanges induced by CB1 receptor antagonist/inverse agonistseem to be transitory since a complete recovery of CB1

receptor density and functional activity appeared aftertreatment withdrawal.

The successful reduction in body weight observed in thepresent work during chronic CB1 antagonist/inverse agonisttreatment is in agreement with the results previouslypublished in animal studies and clinical trials (Despres et al.2005; Di Marzo and Matias 2005; Pi-Sunyer et al. 2006;Kirkham 2008). In animal studies, mice chronically treatedwith rimonabant (Ward and Dykstra 2005) or lacking theCB1 cannabinoid receptor (Cota et al. 2003; Ravinet et al.2004) are leaner, have lower food-motivation, and present atransitory lower caloric intake than their correspondingcontrols. In humans, rimonabant administration producesweight reduction and improves several cardiometabolic riskfactors (Bellocchio et al. 2006). In all these studies, theeffects of rimonabant on weight gain were reported to bemore pronounced and prolonged than the reduction in foodintake (Vickers et al. 2003). In contrast to the differentialeffects of rimonabant and taranabant on central CB1

receptors, both CB1 ligands reduced body weight gain andfat content in obese rats suggesting a similar efficacy on thecentral and peripheral mechanisms leading to this metabolicunbalance. However, rimonabant was more effective in obese(13% of reduction) than lean (2% of reduction) rats, whereastaranabant was similarly efficacious in both groups ofanimals (9% vs. 6% of reduction respectively). The activityof the endogenous cannabinoid system is enhanced in thehypothalamus (Di Marzo et al. 2001) and in several periph-eral tissues such as the adipose tissue, liver, skeletal muscleand pancreas during obesity, which plays a major role in thephysiopathology of the metabolic unbalance (Matias et al.2006; Pagotto et al. 2006). Therefore, the present resultssuggest that taranabant might act as an inverse agonist in vivoand it does not need an underlying overactivity of theendocannabinoid system to reduce body weight in leananimals, whereas rimonabant could preferentially act as aneutral antagonist under these experimental conditions. Foodintake was decreased only at the beginning of chronicrimonabant and taranabant treatment under the presentexperimental conditions, in agreement with the transitoryeffects previously reported on food intake during chronicCB1 receptor blockade (Colombo et al. 1998; Carai et al.2005). In addition, adult CB1 knockout mice feed ad libitumpresented similar levels of food intake to wild-type litter-mates (Di Marzo et al. 2001). The transitory reduction offood intake observed in this study can be due to thedecreased CB1 receptor function found in hypothalamus and/or nucleus accumbens during chronic rimonabant andtaranabant treatment. After withdrawal of rimonabant and

taranabant treatment, an increase in food intake was observedwhich was associated with a recovery of body weight tovalues reached by obese rats receiving chronic vehicle. Thisincrease in food intake after treatment withdrawal wasparallel to the increased functional activity of CB1 cannab-inoid receptors in the hypothalamus, the main brain structureinvolved in the control of food intake. The enhancement ofCB1 receptor activity in the hypothalamus after treatmentwithdrawal could explain the presence of this rebound effecton food intake that contributes to the rapid recovery of bodyweight. These findings are consistent with previous reportshighlighting the relevance of endocannabinoid levels inlimbic forebrain and hypothalamus in the regulation ofappetite (Di Marzo et al. 2001; Kirkham et al. 2002).

CB1 receptors have been identified in multiple central andperipheral tissues involved in the regulation of metabolism,including adipocites, liver, muscle and pancreas (Cota 2007).The psychiatric side-effects produced by CB1 antagonists arebecause of brain CB1 receptor occupancy, whereas theireffects on weight-loss and metabolic unbalance are alsobecause of the occupancy of CB1 receptors located inperipheral tissues (Despres 2007; Addy et al. 2008; Belloc-chio et al. 2008; Di Marzo 2008, 2009). The important roleplayed by peripheral CB1 receptors on the metabolic effectsof CB1 antagonists opens new interesting strategies. Indeed,the design of CB1 antagonists with predominant effects onCB1 peripheral receptors remains an interesting experimentalapproach to develop new compounds devoid of the psychi-atric side-effects that were associated to the first generationof CB1 antagonists.

In conclusion, this study reveals differential effects afterchronic rimonabant and taranabant treatment in the adaptivechanges occurring in CB1 cannabinoid receptors in thecentral nervous system. Chronic treatment with taranabantdecreased the density and functional activity of CB1

cannabinoid receptor in the brain, whereas chronic rimona-bant only decreased functional activity of central CB1

receptors without modifying the density. These results areimportant for a better understanding of the central side-effects that have been associated to the anti-obesity drugsacting on the endocannabinoid system.

Acknowledgements

This work was supported by the USA National Institutes of Health –

National Institute of Drug Abuse (NIH-NIDA) (no. 1R01-DA01

6768-0111), the DG Research of the European Commission

(PHECOMP, no. LHSM-CT-2007-037669 and GENADDICT, no.

LSHM-CT-2004-05166), the Spanish ‘Instituto de Salud Carlos III’

(no. RD06/001/001), the Spanish ‘Ministerio de Educacion y

Ciencia’ (no. SAF2007-64062), the Catalan Government

(SGR2009-00131), the ICREA Foundation (ICREA Academia-

2008) and the Polish Ministry of Science and Higher Education

subsidiary grant 478/6. PR UE/2007/7. Elena Martın-Garcia was



supported by a post-doctoral fellowship from the Spanish ‘Instituto

de Salud Carlos III’. We thank Mª Dolors de la Fuente, and Begona

Penalba for invaluable technical assistance and Dr Patricia Robledo

for critical reading and revision of the manuscript.

Conflict of interest statement

Rafael Maldonado has received research grants from Sanofi-Aventis,

Esteve, and Ferrer. Olga Millan has received research grants from

Sanofi-Aventis, Esteve, GSK and Advancell. Neither of the other

authors have relevant financial interests to disclose, nor a conflict of

interest of any kind.

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