ARTICLE IN PRESS

Neuropharmacology xx (2007) 1e9www.elsevier.com/locate/neuropharm

+ MODEL

The cannabinoid CB1 receptor antagonist SR141716A (Rimonabant)enhances the metabolic benefits of long-term treatment with

oleoylethanolamide in Zucker rats

Antonia Serrano a, Ignacio del Arco a, Francisco Javier Pavon a, Manuel Macıas a,b,Vidal Perez-Valero a, Fernando Rodrıguez de Fonseca a,*

a Fundacion IMABIS, Hospital Carlos Haya de Malaga, Laboratorio de Medicina Regenerativa, Avenida Carlos Haya 82,7a planta, E-29010 Malaga, Spain

b CIBER Fisiopatologıa de la Obesidad y Nutricion, CB06/03, Instituto de Salud Carlos III, Madrid, Spain

Received 4 February 2007; received in revised form 9 March 2007; accepted 13 March 2007

Abstract

Anandamide and oleoylethanolamide (OEA) are lipid mediators that regulate feeding and lipid metabolism. While anandamide, a cannabinoidCB1 receptor agonist, promotes feeding and lipogenesis, oleoylethanolamide, an endogenous agonist of peroxisome proliferator activated recep-tor alpha (PPAR-a), decreases food intake and activates lipid mobilization and oxidation. The treatment with a cannabinoid CB1 receptorantagonist results in reduction of body weight gain and cholesterol in obese humans and rodents. In the present study, we show the benefitsof the treatment of obese Zucker rats with a combination of a cannabinoid CB1 receptor antagonist (Rimonabant) and oleoylethanolamide.This combinational therapy improved the separate effects of Rimonabant and OEA, and resulted in marked decreases on feeding, body weightgain, and plasma cholesterol levels. Additionally, the treatment with both drugs reduced the hepatic steatosis observed in Zucker rats, decreasingliver fat deposits and damage, as revealed by the levels of alanine aminotransferase activity in serum. The combined treatment inhibits theexpression of stearoyl coenzyme-A desaturase-1 (SCD-1), a pivotal enzyme in lipid biosynthesis and triglyceride mobilization that is linkedto obesity phenotypes. These results support the use of combined therapies with cannabinoid CB1 receptor antagonists and PPAR-a agonistsfor the treatment of obesity associated with dyslipemia.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Cannabinoid; Obesity; Zucker rat; Food intake; Rimonabant; Oleoylethanolamide

1. Introduction

Anandamide and related endocannabinoids control motiva-tion for appetitive stimuli, including food and drugs (Arnoneet al., 1997; Di Marzo et al., 1998; Gomez et al., 2002;Navarro et al., 2001), as well as peripheral metabolic pro-cesses, including lipid metabolism and glucose homeostasis(Bermudez-Silva et al., 2006; Cota et al., 2003; Fu et al.,

* Corresponding author. Tel.: þ34 951 291446; fax: þ34 951 291447.

E-mail address: fernando.rodriguez@fundacionimabis.org (F. Rodrıguez

de Fonseca).

0028-3908/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuropharm.2007.03.007

Please cite this article in press as: Serrano, A. et al., The cannabinoid CB1 rec

long-term treatment with oleoylethanolamide in Zucker rats, Neuropharmacolo

2003). The regulation of appetite and metabolism merits spe-cial attention because of the impact on the health systems ofentities such as feeding disorders and obesity. Preliminarystudies have described an overactivation of the endogenouscannabinoid system in human obesity and diabetes, suggestingthat this modulatory system might contribute to the physiopa-thology of both emerging epidemics (Di Marzo and Matias,2005; Engeli et al., 2005). Following this rationale, clinical tri-als on the effects of the cannabinoid receptor antagonistSR141716A (Rimonabant) on obesity have recently demon-strated the utility of cannabinoid receptor blockade as a therapyfor complicated obesity, i.e. obesity associated with

eptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of

gy (2007), doi:10.1016/j.neuropharm.2007.03.007

2 A. Serrano et al. / Neuropharmacology xx (2007) 1e9

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cardiovascular risk factors such as hypertriglyceridemia, hy-percholesterolemia and diabetes (Pi-Sunyer et al., 2006;Scheen et al., 2006; Van Gaal et al., 2005). Analysis of clinicaltrials with Rimonabant in patients with obesity has establishedthat the improvement in cardiovascular risk factors, includinglowering of plasma cholesterol and triglycerides, is not onlyattributable to the reduction in body weight originated bythe anorectic actions of the cannabinoid CB1 receptor blocker.It is also derived from the interference with cannabinoid recep-tor-mediated lipogenesis in the liver and in the adipose tissue(Cota et al., 2003; Osei-Hyiaman et al., 2005). However, themagnitude of the effects of Rimonabant on triglyceride andcholesterol levels is lower than those obtained after specifictherapies such as statins and fibrates, limiting the efficacy ofthese new compounds.

Oleoylethanolamide (OEA) is an endogenous fatty acidethanolamide that participates in the control of feeding be-havior. OEA is a structural analogue of the endocannabinoidanandamide but does not bind to or activates the cannabi-noid CB1 receptor (Rodriguez de Fonseca et al., 2001).This lipid is synthesized in cells of the small intestineand adipose tissue, where its levels are reduced by fastingand increased upon refeeding, in an opposite pattern tothat exhibited by anandamide (Fu et al., 2007; Gomezet al., 2002; Rodriguez de Fonseca et al., 2001). Different ex-perimental approaches have demonstrated that OEA inducessatiety, reduces the rate of body weight gain in both leanand obese animals, and stimulates lipolysis through activa-tion of the peroxisome proliferator-activated receptor alpha(PPAR-a) (Fu et al., 2003; Guzman et al., 2004; Rodriguezde Fonseca et al., 2001). OEA binds to PPAR-a receptorswith high affinity, and its effects are absent in mice thatlack PPAR-a, supporting a critical role for these receptorsin the metabolic effects of OEA (Fu et al., 2003; Guzmanet al., 2004).

PPAR-a is a member of the nuclear receptor superfamily ofligand-activated transcription factors that control multiple as-pects of energy balance. This receptor is abundantly expressedin the liver and intestine and is known to play a pivotal role incontrolling the transcription of genes involved in lipid metab-olism (Rosen, 2003). The fibrates, clinically tested low affinityPPAR-a ligands, are hypolipidaemic drugs that promote theinteraction of this nuclear receptor with transcriptionallyactive genes involved in lipid metabolism (Forman et al.,1997). Lower triglyceride levels have been found inOEA-treated rats, suggesting a possible participation of OEAin the control of energy expenditure and accumulation(Rodriguez de Fonseca et al., 2001).

Since OEA and anandamide seem to play opposite actionson feeding and lipid metabolism, we hypothesized that a com-binational therapy with OEA and a cannabinoid receptor an-tagonist might mutually enhance their respective beneficialactions as antiobesity agents. To this end, we used the Zuckerrats since they exhibit obesity and dyslipemia and were foundto be sensitive to the antiobesity actions of either cannabinoidreceptor blockers (Pavon et al., 2006; Vickers et al., 2003) oroleoylethanolamide (Fu et al., 2005).

Please cite this article in press as: Serrano, A. et al., The cannabinoid CB1 rec

long-term treatment with oleoylethanolamide in Zucker rats, Neuropharmacolo

2. Materials and methods

2.1. Reagents

Oleoylethanolamide (OEA) was synthesized in the laboratory as previously

described (Rodriguez de Fonseca et al., 2001); 5-(4-chlorophenyl)-1-(2,4-

dichlorophenyl)-3-hexyl-1H-1,2,4-triazole,N-piperidino-5-(4-chlorophenyl)-1-

(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR141716A, Rimonabant),

was a gift from Sanofi-Aventis (Montpellier, France).

The drugs were suspended with Tween 20 (5%) and diluted in saline as

vehicle and administered intraperitoneally (i.p.) in a volume of 1 mL kg�1 at

different doses according to acute, combined or subchronic treatments. For

acute treatments we used doses of 0.3, 1 and 3 mg kg�1 (SR141716A) or 1

and 5 mg kg�1 (OEA). When these drugs were combined, the doses used

were OEA (1 and 5 mg kg�1) and 0.3 mg kg�1 of SR141716A. Eventually

for subchronic treatments these compounds were administered at doses of

1 mg kg�1 (SR141716A) or 5 and 20 mg kg�1 (OEA). The drug solutions

were freshly prepared for each day.

2.2. Animals

Experiments were performed on male Wistar rats weighing 250e300 g

from the Animal Resource Centre, University of Malaga (Spain) and obese

male Zucker ( fa/fa) rats aged 8e9 weeks with their lean controls supplied

by Charles River Laboratories Espa~na, S.A. (Barcelona, Spain). Obese Zucker

( fa/fa) rats, which are homozygous for a mutated inactive form of the leptin

receptor, are an animal model of genetically obese rats used to explore the

anti-obesity properties of drugs (Phillips et al., 1996). Animals were housed

singly or in groups of two under a 12-h light/dark cycle in a temperature

and humidity controlled rooms. Water and standard chow pellets (Prolab,

RMH 2500) were available ad libitum.

All the procedures involving animals and their care were conducted in

adherence with the European Community Council Directive 86/609/EEC

regulating animal research.

2.3. Feeding experiments

2.3.1. Subchronic actions of OEA

For analyzing the subchronic effects (10 days of treatment) of OEA, free-

feeding Zucker rats were daily injected i.p. with OEA 20 mg kg�1 or vehicle

control (5% Tween 20 in saline). The amount of food eaten and the body

weight were registered daily. At the end of the subchronic treatment, animals

were killed by decapitation 24 h after the last injection in an independent

room, and the blood and liver tissue were collected.

2.3.2. Acute combinational therapyThe acute effects of drugs on feeding behavior were analyzed in Wistar

rats deprived of food for 24 h and habituated to handling (Gomez et al.,

2002). Thirty minutes before starting the test, drugs (SR141716A or OEA)

or vehicle control (5% Tween 20 in saline) were administered i.p. The bedding

material was removed from the cage and a small can containing weighed food

pellets was placed inside the cage. Food pellets were weighed at 30, 60, 120

and 240 min after starting the test, and the amount of food eaten was recorded.

The effects on feeding behavior observed after a single dose treatment with

a combination of SR141716A and OEA were analyzed in animals deprived

of food for 24 h and habituated to handling (Gomez et al., 2002) as well as

to acute saline injections. Thirty minutes before starting the test SR141716A

(one dose of 0.3 mg kg�1) was injected i.p. and then 30 min before OEA

(1 and 5 mg kg�1) was administered i.p. Food pellets and food spillage were

weighed at 120 min after starting the test.

2.3.3. Subchronic combined treatmentsFor analyzing the subchronic effects (10 days of treatment) of SR141716A,

OEA or its combination in an obesity model, free-feeding Zucker rats were

daily injected i.p. with SR141716A 1 mg kg�1, OEA 20 or 5 mg kg�1, the

combination of both drugs (SR141716A 1 mg kg�1 plus OEA 5 mg kg�1) or

eptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of

gy (2007), doi:10.1016/j.neuropharm.2007.03.007

3A. Serrano et al. / Neuropharmacology xx (2007) 1e9

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vehicle control (5% Tween 20 in saline). The amount of food eaten and the

body weight were registered daily. At the end of the subchronic treatment, an-

imals were killed by decapitation 24 h after the last injection in an independent

room and the blood and liver tissue were collected.

2.4. Biochemical analyses

Trunk blood was collected and serum was separated by centrifugation at

4 �C and stored at �20 �C. Serum cholesterol, triglycerides, glucose, aspartate

aminotransferase (AST) and alanine aminotransferase (ALT) were measured

using a Hitachi (Tokyo, Japan) 737 automatic analyzer as previously described

(Rodriguez de Fonseca et al., 2000).

2.5. Isolation and analysis of RNA

Total RNA was isolated from livers with Trizol reagent (Gibco BRL Life

Technologies, Baltimore, Maryland) following the supplier’s protocol. Hun-

dred micrograms of total RNA were treated with DNAase I amplification grade

(Qiagen) and then 2 mg of these RNA were used to generate first strand cDNA,

by using MmuLV reverse transcriptase (Roche Applied Science, Indianapolis,

USA) according to the manufacturer’s instruction. Negative controls included

reverse transcription reactions omitting reverse transcriptase. The cDNA ob-

tained was used as the template for real-time quantitative PCR. Primers for

the PCR reaction were designed based on NCBI database sequences of rat

cyclophilin and stearoyl-CoA desaturase-1 (SCD-1), mRNA and tested to

ensure amplification of single discrete bands with no primeredimers. Primer

sequences, obtained from Proligo (Proligo France SAS, Paris, France), were

Please cite this article in press as: Serrano, A. et al., The cannabinoid CB1 rece

long-term treatment with oleoylethanolamide in Zucker rats, Neuropharmacolog

as follows: cyclophilin (forward), 50-agaaggcatgagcattgtgg-30, cyclophilin

(reverse), 50-ttacagggtattgcgagcag-30 (189 bp product); SCD-1 (forward),

50-tgggttggctgcttgtg-30, SCD-1 (reverse), 50-gcgtgggcaggatgaag-30 (150 bp

product). PCR was carried out using SYBR Green (Qiagen) on an iCycler

PCR machine (Opticon 2, MJ Research). Once the absolute values for SCD-

1 and cyclophilin were obtained for each sample, we calculated the ratio

between SCD-1 and the housekeeping gene (cyclophilin). The values were

normalized in the treated groups with the control group ratio. Details on the

methods have been published elsewhere (Serrano et al., 2006).

2.6. Statistical analysis

Results are expressed as the mean� SEM for the experiments, which in-

cluded from eight to ten animals in each group. The significance of differences

between the groups was evaluated by one-way analysis of variance (ANOVA)

followed by post hoc analysis (Tukey’s test) for multiple comparisons or Stu-

dent’s t test. A p-value< 0.05 was considered significant.

3. Results

3.1. High dose of OEA reduces body weight gain andserum lipids in Zucker rats

Fig. 1 shows that subchronic treatment with a high dose ofOEA (20 mg kg�1) induced changes in feeding behavior and

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Fig. 1. Subchronic treatment with OEA reduces body weight gain and serum cholesterol and triglycerides in Zucker rats. Obese ( fa/fa) and lean Zucker rats were

administered (i.p.) once daily, with vehicle or OEA (20 mg kg�1). Rat body weight was controlled every day from the 1st day to 10 days of treatment. Data are

presented as relative weight gain (g/kg of body weight at day 1) (A). The amount of food eaten was controlled every day from the 1st day to 10 days of treatment.

Data are presented as cumulative food intake (g/kg of body weight) (B). Serum cholesterol (C) and triglycerides (D) were measured and expressed in mg dl�1. Data

are mean� SEM (n¼ 8). *p< 0.05; **p< 0.01; ***p< 0.001 versus vehicle-treated animals by Student’s t test.

ptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of

y (2007), doi:10.1016/j.neuropharm.2007.03.007

4 A. Serrano et al. / Neuropharmacology xx (2007) 1e9

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serum lipids in this animal model of obesity. The daily admin-istration of OEA for 10 days inhibited body weight gain(Fig. 1A) and reduced food intake (Fig. 1B) in obese andlean Zucker rats. The levels of cholesterol (Fig. 1C) and tri-glycerides (Fig. 1D) were greater in Zucker fatty rats com-pared with their lean littermates. Rats treated with OEA hada marked decrease in cholesterol levels (35% and 25% inobese and lean rats, respectively) compared with the controlanimals, whereas serum triglycerides were decreased by about37% in obese rats treated with OEA but there were no differ-ences in lean rats compared with the control group.

3.2. SR141716A enhances the feeding suppressioninduced by OEA in normal rats

Previous studies from our laboratory (Rodriguez de Fon-seca et al., 2001; Gomez et al., 2002) have shown that eitherthe administration of a cannabinoid CB1 receptor blocker(SR141716A) or the non-cannabinoid fatty-acid ethanolamideOEA caused a dose-dependent suppression of food consump-tion. As reported previously (Gomez et al., 2002), the i.p. ad-ministration of the cannabinoid CB1 receptor inverse agonistSR141716A (0.3, 1 and 3 mg kg�1) to food-deprived animals

Please cite this article in press as: Serrano, A. et al., The cannabinoid CB1 rece

long-term treatment with oleoylethanolamide in Zucker rats, Neuropharmacolog

resulted in a dose-dependent reduction in food intake(Fig. 2A). This effect was significant for doses of 0.3 mg kg�1

or greater, but only was significant at all the time points testedfor the 1 and 3 mg kg�1 doses.

A similar pattern of effects can be observed when oleoyle-thanolamide was administered to food-deprived Wistarrats (Fig. 2B). When combined, a sub-effective dose ofSR141716A (0.3 mg kg�1) potentiated feeding suppression in-duced by OEA (1 and 5 mg kg�1) in food-deprived animals(Fig. 2C).

3.3. Effects of subchronic administration of SR141716Aand OEA on Zucker rats

To further explore the effect of the combined administrationof SR141716A and OEA, we used genetically obese Zucker andtheir lean littermates. As described above, doses of 1 mg kg�1 ofSR141716A and 5 mg kg�1 of OEA have a moderate effect onfood intake, so we used a combination of these doses for thisstudy.

The daily administration of SR141716A (1 mg kg�1) orOEA (5 mg kg�1) for 10 days reduced cumulative food intake(14% and 19%, respectively) in obese Zucker rats (Fig. 3A),

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Fig. 2. Systemic administration of SR141716A reduces food intake in food-deprived rats and potentiates the feeding suppression induced by OEA. (A)

Dose-dependent effect of i.p. injection of 0.3, 1 or 3 mg kg�1 of SR141716A to 24-h food-deprived rats. Food intake was measured 30, 60 and 120 min after in-

jection of drug or vehicle. (B) Dose-dependent effect of i.p. injection of 1 or 5 mg kg�1 of OEA to 24-h food-deprived rats. Food intake was measured 60, 120 and

240 min after injection of drug or vehicle. (C) The combination of SR141716A (0.3 mg kg�1, injected 30 min before) and OEA (1 and 5 mg kg�1) to 24-h food-

deprived rats is more effective in feeding suppression than OEA alone. Food intake was measured 120 min after the injection of OEA. Data are mean� SEM

(n¼ 10). *p< 0.01 versus vehicle-treated animals; #p< 0.01 versus 0 dose.

ptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of

y (2007), doi:10.1016/j.neuropharm.2007.03.007

5A. Serrano et al. / Neuropharmacology xx (2007) 1e9

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whereas the combined administration of both drugs reducedfood intake by 22% compared with control group. In leanrats, there was not a significant effect of SR141716A orOEA (Fig. 3B), but the combination of both compounds de-creased cumulative food intake is a significant way ( p< 0.05).

3.4. SR141716A and OEA inhibit body weight gain

The daily administration of SR141716A (1 mg kg�1) for10 days reduced relative body weight gain in obese rats(Fig. 4A). Body weight gain was reduced up to 61% on day4 compared with the vehicle control. Furthermore, after the6th day of treatment, body weight gain of SR141716A-treatedrats continued to be significantly lower than that of vehicle-treated rats. In lean Zucker rats (Fig. 4B), SR141716A(1 mg kg�1) had no significant effect on body weight gain, ex-cept the 4th day of treatment that was reduced up to 45% incomparison with body weight gain of control animals that re-ceived vehicle. These results agree with those reported previ-ously (Vickers et al., 2003). Under the same conditions, OEA

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Fig. 3. Subchronic treatment with OEA reduces food intake in Zucker rats and

SR141716A potentiates this effect. Obese ( fa/fa) and lean Zucker rats were

administered (i.p.) once daily, with vehicle or SR141716A (1 mg kg�1) and/

or OEA (5 mg kg�1). The amount of food eaten was controlled every day

from the 1st day to 10 days of treatment. Data are presented as cumulative

food intake (g/kg of body weight) in obese (A) and lean (B) Zucker rats.

Data are mean� SEM (n¼ 8). *p< 0.05; ***p< 0.001 versus vehicle-treated

animals, one-way ANOVA followed by Tukey’s test.

Please cite this article in press as: Serrano, A. et al., The cannabinoid CB1 rec

long-term treatment with oleoylethanolamide in Zucker rats, Neuropharmacolo

(5 mg kg�1) had no effect, while combination of SR141716Aand OEA was significantly more potent than only SR141716A(Fig. 4A and B).

Fig. 4C shows the net body weight change in these animalsat the end of the experiment. In obese rats, the administrationof either SR141716A or the combination of SR141716A plusOEA had a significant effect on body weight change( p< 0.001), while in lean Zucker rats only the administrationof the combination of SR141716A plus OEA decreased( p< 0.05) the body weight gain.

3.5. SR141716A and OEA reduce lipid levelsin serum and liver

Because OEA reduces lipid levels in obese Zucker rats (Fuet al., 2005), we studied whether a combined treatment withSR141716A plus OEA improved this effect.

Table 1 depicts the effects of OEA and SR141716A onserum lipids (cholesterol and triglycerides), glucose andserum markers of liver damage (alanine transaminase (ALT)and aspartate aminotransferase (AST)) in obese Zucker rats.After 10 days of OEA (5 mg kg�1) administration, obese ratserum cholesterol and ALT levels were significantly( p< 0.01) lower in this group than in the control group, butno significant differences were found in serum triglycerides,glucose and AST levels. In the treatment with SR141716A(1 mg kg�1), glucose levels were significantly higher thanthose in control group ( p< 0.05 and p< 0.01, respectively),whereas the level of ALT was significantly ( p< 0.05)decreased. After the treatment with SR141716A plus OEA,serum levels of cholesterol were decreased by 33% comparedwith the control group and the serum level of ALT wassignificantly ( p< 0.01) reduced. In lean rats, there wereno significant differences between the different treatments(Table 2).

Leptin deficit results in hepatic steatosis. The treatmentwith OEA was previously reported to decrease fat depots inthe liver (Fu et al., 2005). Here we show that the combinedtreatment with OEA and SR141716A was more effectivethan the treatment with OEA. Total lipid content was extractedfrom frozen liver according to the method of Bligh and Dyer(1959). Subchronic administration of OEA or SR141716Adid not significantly modify the total percentage of liver fat(Fig. 5). In contrast, the combination of both drugs reducedthe total content of liver fat in obese (about 21%) and lean(about 35%) Zucker rats.

3.6. SR141716A and OEA regulate the expression ofSCD-1 gene

Stearoyl coenzyme-A desaturase 1 is a key enzyme in-volved in lipid biosynthesis and triglyceride export from theliver (Miyazaki et al., 2000; Ntambi, 1999) and its deletioncounteracts the hepatic steatosis derived of leptin deficit(Cohen et al., 2002). Previous data from our laboratory haveshown that OEA reduced the hepatic level of monounsaturatedfatty acids, a reduction that is related with SCD-1 inhibition

eptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of

gy (2007), doi:10.1016/j.neuropharm.2007.03.007

6 A. Serrano et al. / Neuropharmacology xx (2007) 1e9

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VehicleOEA (5mg/kg)SR141716A (1mg/kg)SR141716A (1mg/kg) +OEA (5mg/kg)

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Fig. 4. Subchronic treatment with SR141716A and OEA reduces body weight gain in Zucker rats. Obese ( fa/fa) and lean Zucker rats were administered (i.p.) once

daily, with vehicle, SR141716A (1 mg kg�1) and/or OEA (5 mg kg�1). Rat body weight was controlled every day from the 1st day to 10 days of treatment. Data are

presented as relative weight gain (g/kg of body weight at day 1) in obese (A) and lean (B) Zucker rats. Body weight change (body weight at day 10 minus body

weight at day 1 in obese and lean Zucker rats are represented in (C). Each experimental point represents the mean� SEM (n¼ 8). *p< 0.05; **p< 0.01;

***p< 0.001 versus vehicle-treated animals, one-way ANOVA followed by Tukey’s test.

(Serrano et al., 2006). In the current study, we tested whetherthe combined treatment with SR141716A plus OEA resultedin changes in the expression of SCD-1 gene. We measuredmRNA level of SCD-1 by real-time quantitative PCR. Thesubchronic administration of SR141716A plus OEA signifi-cantly decreased the expression of SCD-1 (Fig. 6) by 56%in obese rats. By contrast, in lean rats the combined adminis-tration of both drugs increased the mRNA expression of SCD-1 compared with the control group, potentially indicatinga compensatory effect derived of the decrease in fat storagein the liver of the lean animals.

Please cite this article in press as: Serrano, A. et al., The cannabinoid CB1 re

long-term treatment with oleoylethanolamide in Zucker rats, Neuropharmacol

4. Discussion

This study demonstrates, first, that subchronic daily treat-ment with either the selective CB1 receptor antagonistSR141716A or OEA, an endogenous agonist of peroxisomeproliferator-activated receptor alpha, reduces the food intakeand body weight gain in Zucker rats. Second, the combinedtreatment with both drugs improves feeding suppression ac-tions and ameliorates the metabolic syndrome associatedwith leptin deficiency. The results also suggest that a combinedtreatment with SR141716A and OEA reduces liver steatosis,

Table 1

Effects of OEA and SR141716A or its combination on serum lipids and glucose, and on serum activity of ALT and AST in obese Zucker rats

Obese Zucker rats

Vehicle OEA SR141716A SR141716AþOEA

Cholesterol (mg dl�1) 172.25� 8.55 142.38� 5.77** 166.00� 6.83 115.25� 8.99**#

Triglycerides (mg dl�1) 336.75� 20.36 344.38� 48.79 366.75� 24.90 373.25� 77.29

Glucose (mg dl�1) 127.75� 4.00 158.63� 16.51 145.00� 9.50* 141.50� 15.19

ALT (U/L) 102.38� 6.04 79.13� 2.69** 79.75� 3.93* 71.33� 3.89**

AST (U/L) 223.50� 25.59 178.63� 7.12 189.25� 8.84 188.75� 49.89

Vehicle (5% Tween 20 in saline, i.p.), OEA (5 mg kg�1, i.p.) and/or SR141716A (1 mg kg�1, i.p.) were administered once daily for 10 days. Serum cholesterol,

triglycerides, glucose, AST and ALT were measured as described under Section 2. Data are mean� SEM (n¼ 8). *p< 0.05; **p< 0.01 versus vehicle-treated

animals; #p< 0.05 versus OEA-treated animals, one-way ANOVA followed by post hoc analysis. ALT: alanine transaminase; AST: aspartate aminotransferase.

ceptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of

ogy (2007), doi:10.1016/j.neuropharm.2007.03.007

7A. Serrano et al. / Neuropharmacology xx (2007) 1e9

+ MODEL

ARTICLE IN PRESS

Table 2

Effects of OEA and SR141716A or its combination on serum lipids and glucose, and on serum activity of ALT and AST in lean Zucker rats

Lean Zucker rats

Vehicle OEA SR141716A SR141716AþOEA

Cholesterol (mg dl�1) 85.75� 7.93 84.57� 5.83 84.50� 2.73 82.00� 5.06

Triglycerides (mg dl�1) 92.63� 10.19 85.29� 3.85 64.50� 7.22 95.75� 6.49

Glucose (mg dl�1) 161.38� 18.32 151.33� 8.69 147.00� 13.62 153.50� 11.48

ALT (U/L) 55.00� 3.03 47.14� 3.83 53.00� 1.41 55.25� 1.44

AST (U/L) 210.14� 45.65 150.86� 32.45 167.50� 14.30 199.25� 34.90

Vehicle (5% Tween 20 in saline, i.p.), OEA (5 mg kg�1, i.p.) and/or SR141716A (1 mg kg�1, i.p.) were administered once daily for 10 days. Serum cholesterol,

triglycerides, glucose, AST and ALT were measured as described under Section 2. Data are mean� SEM (n¼ 8). ALT: alanine transaminase; AST: aspartate

aminotransferase.

decreasing lipid content in liver and depressing the SCD-1mRNA expression, a key enzyme for lipogenesis. These re-sults are in agreement with the physiology of both the endog-enous cannabinoid CB1 receptor ligand anandamide and thePPAR-a receptor agonist oleoylethanolamide, which haveopposite actions on feeding and metabolism. The metabolic ef-fects are not only dependent on the reduction of food intake,since we have observed (Serrano et al., unpublished observa-tions) that pair-fed animals (animals receiving the sameamount of food than those treated with these drugs) do notshow the same changes on metabolic parameters than those re-ceiving this type of compound. These feeding-independentmetabolic effects have also been observed in humans treatedwith Rimonabant (Schwimmer et al., 2006; Van Gaal et al.,2005).

During the 10-day study, significant reductions in bodyweight gain, food intake and lipid levels in blood were observedin Zucker rats treated with a high dose of OEA (20 mg kg�1).These results are in agreement with previous studies that de-scribe that OEA exerts anti-obesity effects in Zucker rats (Fuet al., 2005). A similar pattern was observed after chronic treat-ment with cannabinoid receptor blockers, as it has been reportedthat both, SR141716A and LH-21, a neutral cannabinoid CB1receptor antagonist, decrease food intake and body weightgain in Zucker rats (Pavon et al., 2006; Vickers et al., 2003).

0123456789

VehicleOEA (5mg/kg)SR141716A (1mg/kg)SR141716A (1mg/kg)+OEA(5mg/kg)

∗∗

∗∗

Liver fat (%

)

OBESE ZUCKER LEAN ZUCKER

Fig. 5. Subchronic administration of SR141716A plus OEA reduces the

amount of lipid content in liver of Zucker rats. Obese ( fa/fa) and lean Zucker

rats were treated once daily i.p. for 10 days, with vehicle, SR141716A

(1 mg kg�1) and/or OEA (5 mg kg�1). Total lipids were extracted from liver

and presented as total lipid in percent liver weight (%). Data are presented

as mean� SEM (n¼ 8). **p< 0.01 versus vehicle-treated animals, one-way

ANOVA followed by Tukey’s test.

Please cite this article in press as: Serrano, A. et al., The cannabinoid CB1 rec

long-term treatment with oleoylethanolamide in Zucker rats, Neuropharmacolo

Our findings are consistent with those previous reports and es-tablish a frame for the study of the combination of both typesof drugs as a new strategy for developing anti-obesitytreatments.

In genetically obese ( fa/fa) rats, a subchronic treatmentwith OEA (5 mg kg�1) or SR141716A (1 mg kg�1) produceda significant reduction of total food intake over the 10-daystudy period. In contrast to findings in obese animal, the ad-ministration of these compounds had no effect in the leanZucker rats, something reported previously for both types ofdrugs (Fu et al., 2005; Vickers et al., 2003).

We also used animals treated with both drugs to show that thereduction of total food intake was greater in this group comparedwith the rats treated only with SR141716A or OEA. Bodyweight gain in obese rats was significantly attenuated fromday 3 onwards in SR141716A-treated animals and from day10 onwards in OEA-treated rats. When we used both com-pounds, the effect on body weight gain was earlier and greaterthan in the other groups. Over the course of this study, the vehi-cle-treated controls gained an average of 43.60� 2.70 g, whilethe other groups gained an average of 36.84� 2.26 g (OEA),20.48� 3.47 g (SR141716A) and 15.40� 3.30 g (SR141716A plus OEA). In lean rats, only the combination of both drugshad a significant effect on body weight gain. These results showthat combined administration of SR141716A and OEA

VehicleOEA (5mg/kg)SR141716A (1mg/kg)SR141716A (1mg/kg)+OEA(5mg/kg)

mRNA SCD-1

Fo

ld

ch

an

ge

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

∗∗

∗∗

OBESE ZUCKER LEAN ZUCKER

Fig. 6. Subchronic administration of SR141716A plus OEA modifies liver de-

saturase mRNA expression in Zucker rats. Obese ( fa/fa) and lean Zucker rats

were treated once daily i.p. for 10 days, with vehicle, SR141716A (1 mg kg�1)

and/or OEA (5 mg kg�1). SCD-1 mRNA expression from livers was assessed

by real-time quantitative PCR, using cyclophilin gene as the internal control in

each sample. Data are presented as fold change (calculated after correction for

the levels of cyclophilin) as mean� SEM (n¼ 8). **p< 0.01 versus vehicle-

treated animals, one-way ANOVA followed by Tukey’s test.

eptor antagonist SR141716A (Rimonabant) enhances the metabolic benefits of

gy (2007), doi:10.1016/j.neuropharm.2007.03.007

8 A. Serrano et al. / Neuropharmacology xx (2007) 1e9

+ MODEL

ARTICLE IN PRESS

enhances the effects of each compound when given alone. Suchdata are in agreement with a preliminary study that suggests thatthe combined treatment with SR141716A and OEA decreasedeating in food-deprived Wistar rats, being the combinationmore effective than the different drugs given alone (Gomezet al., 2002). These results extend this notion to the metabolic ac-tions of both types of drugs. Obese Zucker rats have a mutationin the intracellular domain of the leptin receptor and are com-pletely insensitive to the action of leptin (Phillips et al., 1996).Therefore, these animals have significant increases in choles-terol and triglyceride levels. We measured serum lipid levelsand observed that obese rats had higher cholesterol and triglyc-eride levels in serum than their lean littermates (Tables 1 and 2).The cholesterol content decreased in the obese rats treated withOEA. However, the treatment with SR141716A plus OEA ame-liorated serum cholesterol level to a greater extent than the sep-arate treatments. In lean rats there was no effect. Considering thefact that either cannabinoid receptor blockers or fibrates are con-sidered independent therapies for obesity and dyslipemia, re-spectively, these results support the search of a combined formof treatment in complicated obesity on which a cannabinoid re-ceptor antagonist-based therapy may be improved by addinga PPAR-a ligand. The potential existence of additive/synergisticeffects of the combination of Rimonabant and OEA demandsmore thorough analysis, because its existence may help to im-prove the pharmacological spectrum of Rimonabant on feedingbehavior and metabolic processes reported in humans (Pi-Sunyer et al., 2006; Scheen et al., 2006; Van Gaal et al., 2005).

Additionally, the combinational therapy assayed in this ex-periment may also be beneficial in hepatic steatosis, a growingpathological condition that parallels the obesity epidemics(Schwimmer et al., 2006). We have used the Zucker rats toprovide experimental support for this pharmacological effectbecause they develop liver steatosis. The combination ofOEA and SR141716A decreases fat depots in the liver. Thiseffect is accompanied by a general improvement in liver func-tion as reflected in the lowering of cholesterol and transami-nases. The estimation in the serum of the level of alaninetransaminase (ALT) and aspartate aminotransferase (AST)(enzymes normally located in the cytosol and they are releasedinto the blood stream when the liver cell plasma membrane isdamaged) is a marker of the hepatocellular damage. In thisstudy, obese Zucker rats treated with vehicle had increasedALT release compared with lean rats treated with vehicle.However, we found a decrease in serum level of ALT in theobese rats treated with SR141716A, OEA or both compounds.In all cases, the biochemical estimation of ALT in these groupswas similar to lean rats, suggesting a normalizing effect ofthese drugs on the liver damage in Zucker rats with lack offunctional leptin receptors.

Although the identification of the molecular mechanisms ofthe improved actions derived from combining a cannabinoidreceptor blocker and a PPAR-a ligand is beyond the aims ofthis study, we provide the identification of a potential target,the stearoyl-CoA desaturase-1 (SCD-1). This is a key micro-somal enzyme involved in the biosynthesis of monounsatu-rated fatty acids from saturated fatty acids that are either

Please cite this article in press as: Serrano, A. et al., The cannabinoid CB1 rec

long-term treatment with oleoylethanolamide in Zucker rats, Neuropharmacolo

synthesized de novo or derived from the diet. Stearoyl-CoAdesaturase-1 introduces the cis double bond in the D9 position(between carbons 9 and 10) of saturated fatty acyl-CoA sub-strates. The preferred substrates are palmitoyl- and stearoyl-CoA to generate palmitoleoyl-CoA (16:1) and oleoyl-CoA(18:1), respectively (Enoch et al., 1976; Miyazaki et al.,2001). These products have diverse but crucial roles in thecells of living organisms. Changes in SCD-1 activity in tissuesare reflected in the cell composition of phospholipids, choles-terol esters and triglycerides. High SCD-1 activity and alter-ation in the balance between saturated and monounsaturatedfatty acids are implicated in a wide range of disorders, includ-ing cancer, diabetes, atherosclerosis and obesity (Ntambi,1999; Dobrzyn and Ntambi, 2004). Previous reports haveshown that anorectic actions of both OEA and stearoylethano-lamide are linked to SCD-1 inhibition (Serrano et al., 2006;Terrazzino et al., 2004). Our results indicate that combinedtreatment with a cannabinoid receptor blocker and OEA mayhave additive effects on the inhibition of SCD-1. Under thelight of the present studies, we recommend this hypothesisto be tested in future clinical trials exploring the efficacy ofcannabinoid receptor antagonists on obesity and metabolicdisorders.

Acknowledgements

This work was supported by MCYT SAF 2004-07762; FIS03/001; FIS 04/0834; FIS CP 04/0039; CB 06/03/0018 andPrograma de Redes Tematicas: Red de Obesidad y Red deTrastornos Adictivos (RD06/001); Plan Nacional SobreDrogas; Consejerias de Innovacion y de Salud de la Junta deAndalucıa and Fundacion Eugenio Rodriguez Pascual.

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