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Research Report

Olanzapine-induced accumulation of adipose tissue isassociated with an inflammatory state

Montserrat Victorianoa,b,c, Renaud de Beaurepairec,⁎, Nadia Naourd,e,Michèle Guerre-Millod,e, Annie Quignard-Boulangéa,b, Jean-François Huneaua,b,Véronique Mathéa,b, Daniel Toméa,b, Dominique Hermiera,b

aUMR914, INRA, Physiologie de la Nutrition et du Comportement Alimentaire, 16 rue Claude Bernard, F-75005 Paris, FrancebUMR914, AgroParisTech, Physiologie de la Nutrition et du Comportement Alimentaire, 16 rue Claude Bernard, F-75005 Paris, FrancecLaboratoire de Psychopharmacologie, Centre Hospitalier Paul-Guiraud, 54 avenue de la République, 94806 Villejuif, FrancedUniversité Pierre et Marie Curie-Paris 6, Centre de Recherche des Cordeliers, UMRS 872, Paris, FranceeINSERM, U872, Paris, France

A R T I C L E I N F O

⁎ Corresponding author. Fax: +33 142117089.E-mail address: debeaurepaire@wanadoo

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.05.060

A B S T R A C T

Article history:Accepted 19 May 2010Available online 4 June 2010

Second-generation antipsychotics are widely used in the treatment of all forms ofpsychoses, but they often produce undesirable side effects, among which are weight gainand other elements of metabolic syndrome. The mechanisms of these adverse effects arenot known. The liver and adipose tissue are the principal candidate organs implicated in thedevelopment of antipsychotic-induced metabolic adverse effects. The present studyinvestigated in the rat the effects on liver and white adipose tissue of a chronic treatment(46 days) with olanzapine 2 mg/kg or haloperidol 1 mg/kg, as compared with a controlsolution. In the liver, the expression of key genes involved in glucose transport and lipidmetabolism and of regulatory transcription factors, as well as the TNFα gene, was notaltered in response to either antipsychotic. Similarly, key genes involved in glucosetransport and lipid metabolism were not changed in adipose tissue. However, the whiteadipose tissue was inflammatory in olanzapine-treated rats, with extensive macrophageinfiltration and a significant increase in TNFα expression. In the plasma, TNFα and IL-1βconcentrations were slightly elevated. Chronic olanzapine treatment therefore produces alow-grade inflammatory state, likely initiated in the adipose tissue. Such an inflammatorystate is known to be associated with an increased risk of insulin-resistance andcardiovascular diseases. This antipsychotic-induced inflammatory syndrome mayparticipate in the inflammatory syndrome often observed in patients with schizophrenia.The strong and rather selective effect of olanzapine on TNFα expression may open newtherapeutic opportunities for the prevention of olanzapine-induced metabolicabnormalities.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Adipose tissueAntipsychoticOlanzapineInflammationCytokineLipid metabolism

.fr (R. de Beaurepaire).

er B.V. All rights reserved.

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1. Introduction

Second-generation antipsychotics (SGAs) are widely pre-scribed for the treatment of schizophrenia. However, approx-imately 50% of antipsychotic-treated patients gain weight(Baptista, 1999); this effect being much greater with certainSGAs than with first-generation antipsychotics (FGA) (Allisonet al., 1999). In 2004, a consensus of the American DiabetesAssociation and the American Psychiatric Association claimedthat treatment with olanzapine or clozapine, two major SGAs,is associated with greater risk of weight gain and associatedmetabolic disorders (Consensus Development Conference,2004).

Because both liver tissue and white adipose tissue (WAT)play important roles in lipid metabolic pathways resulting inadiposity, it has been proposed that these peripheral tissuesare the principal targets of SGAs that produce adversemetabolic effects (Fernø et al., 2009; Minet-Ringuet et al.,2007; Raeder et al., 2006). However, the underlying mechan-isms of these adverse effects remain largely unknown. As faras adipose tissue is concerned, only a few studies haveinvestigated WAT morphology and found either hypertrophy(Minet-Ringuet et al., 2007) or hyperplasia (Baptista et al.,2004). Increased adiposity results from enhanced lipid storageand/or impaired lipid mobilization. The hypothesis of in-creased lipid storage is supported by studies showing thatcultured rat adipocytes treated with SGAs such as olanzapineand clozapine show enhanced lipogenesis (Vestri et al., 2007;Yang et al., 2007). Similarly, adipocytes isolated from chronicolanzapine-treated rats exhibit an increase in fatty acidsynthase (FAS) expression (Minet-Ringuet et al., 2007). Inparallel, the lipolytic capacity of the adipose tissue is impairedin response to SGAs, as demonstrated by reduced expressionof hormone-sensitive lipase (HSL) (Minet-Ringuet et al., 2007)and by decreased lipolytic activity (Fernø et al., 2009; Minet-Ringuet et al., 2007; Vestri et al., 2007).

In the liver, acute exposure to clozapine results in lipogen-esis stimulation through activation of transcription factorSREBP, as found in cultured humanhepatocytes and in rat liver(Fernø et al., 2009; Raeder et al., 2006). In addition, as in WAT,clozapine depresses lipolysis capacity by down-regulatinghepatic lipase and HSL expression (Fernø et al., 2009). Thesedata suggest that SGA-induced adverse metabolic effects maybe attributed, at least in part, to direct disturbances of lipidhomeostasis in the liver.

In parallel, altered inflammatory responses have beenfound in schizophrenia, leading to the hypothesis thatabnormalities in the inflammatory system are involved inthe physiopathology of the illness (Smith and Maes, 1995).

Table 1 –White adipose tissue characteristics after 46 days of a

Control Olanz

Body weight (g) 379±6 387±Epididymal WAT (g) 5.45±0.30a 6.39±Adipocyte diameter (μm) 26.5±1.1 27.2±Adipocyte number (n per slides) 38.3±2.5 36.9±

Values are means±SEM of six rats per group. Values in the same row sha

However, the mechanisms underlying the inflammatoryalterations in schizophrenia remain elusive. A few studieshave shown that patients treated with risperidone andclozapine exhibit a significant increase in plasma concentra-tions of proinflammatory cytokines, such as IL-6 and tumornecrosis factor-α (TNFα) (Maes et al., 1996, 1997; Pollmächeret al., 1996). Thus, the possibility that some of these alterationsare epiphenomena in schizophrenia, resulting from SGAtreatments rather than from the illness itself, is conceivable.Indeed, obesity, particularly that with splanchnic localization(visceral fat), is associated with a low-grade inflammatorystate (Weisberg et al., 2003). These low-grade inflammatoryalterations are associated with an increased abundance ofmacrophages in adipose tissue in humans and rodents(Weisberg et al., 2003; Cancello et al., 2005), which maycontribute to the production of inflammatory molecules andinduce the development of obesity-related comorbidities (VanGaal et al., 2006). To our knowledge, the possible relationsbetween obesogenic effects of antipsychotics, WAT morphol-ogy, and adipose tissue inflammation have never beeninvestigated.

In human patients, body weight gain during olanzapinetreatment has been shown to be due to WAT accumulation(Eder et al., 2001). Our previous studies in the male rat showedthat chronic olanzapine treatment results in increasedadiposity, which makes this model a very useful tool toinvestigate the specific effect of olanzapine on adipose tissue(Minet-Ringuet et al., 2007; Victoriano et al., 2009). Oneadvantage of this model is the use of antipsychotic dosesthat take into account the different half-lives of the moleculesin rats and humans; these values are consistent with thoseobserved in human patients. The aim of the present studywasto investigate, in this model, the direct effects of olanzapinetreatment on peripheral tissues (WAT and liver), with aspecific emphasis on inflammatory status and its possiblerelationship with WAT status.

2. Results

2.1. Effects of antipsychotics on adipose tissuecharacteristics

Body weight and body weight gain were not affected byantipsychotic treatments (Table 1). The absolute weight ofepididymal WAT was significantly higher in olanzapine-treated rats than in control and haloperidol-treated rats,which did not differ significantly (Table 1). Expressed aspercentage of body weight (%), the adipose tissue proportions

ntipsychotic treatment.

apine Haloperidol ANOVA F

4 380±6 0.3964 0.970.19b 5.48±0.16a 0.0292 4.541.6 26.0±1.1 0.1235 2.412.6 37.9±3.8 0.9363 0.07

ring the same letters or without letters did not differ at P<0.05.

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were 1.44±0.08, 1.63±0.05, and 1.44±0.04 for control-, olanza-pine-, and haloperidol-treated rats, respectively (P=0.0448).The mean diameter of adipose cells did not differ significantlybetween the groups (Table 1). Themean adipocyte number perslide was similar in all groups.

In biopsies from control rats, CD68+ cells were totallyabsent. In the olanzapine-treated rats, various reactive CD68+

cells were dispersed throughout the parenchyma of epididy-mal WAT (Fig. 1), whereas a single CD68+ cell was observed inepididymal WAT from haloperidol-treated rats. Thus, totalCD68+ cells' infiltration was 2.82% for olanzapine- and 0.07%for haloperidol-treated rats (Fig. 1).

2.2. RT–PCR analysis of gene expression

In the liver, mRNA levels of key genes involved in glucosetransport and lipid metabolism (GLUT2, ACC, FAS, ACOX, andCPT1) did not change notably in response to either antipsy-chotic treatment. No change was observed in mRNA levels ofregulatory transcription factors (PPARα and SREBP-1c) or TNFα(data not shown). Similarly, in adipose tissue, the expressionof proteins regulating glucose transport (GLUT4), as well aslipid uptake (LPL), synthesis (ACC and FAS), or release (HSL)was not significantly modified by olanzapine or haloperidoltreatment (data not shown). Among adipokines, TNFα expres-sion was 2-fold higher in the epididymal WAT from olanza-pine-treated rats as compared to that from control andhaloperidol-treated rats (Fig. 2). Other proinflammatory cyto-kines (IL-6, IL-1β, and PAI-1), as well as proteins involved inmacrophage recruitment (MCP-1, PLAUR, and CD11b) were notaffected by either antipsychotic.

Fig. 1 – Identification and quantification of CD68+ cells in epididyRepresentative section of epididymal adipose tissue taken at 20×group. Values in the same row sharing the same letters did not

2.3. Plasma analyses

After the 46-day treatment, no significant abnormalities inplasma inflammatory cytokines, leptin, PAI-1, or MCP-1concentrations were found in response to either antipsychotic(Table 2). Compared to controls, TNFα concentration wasincreased twice, and IL-1β was increased four times inolanzapine-treated rats, but these elevations did not reachstatistical significance.

3. Discussion

There is increasing evidence that certain metabolic sideeffects of SGAs are due, at least in part, to direct effects onperipheral tissues (Fernø et al., 2009). Liver and adipose tissueare the two main organs involved in the physiopathology ofmetabolic syndrome, in relation to obesity. This study wasdesigned to investigate the effects of olanzapine, as comparedto haloperidol, on these two organs, with special emphasis onthe relationship between adiposity, lipid metabolism, andinflammation. The results suggest that haloperidol andolanzapine do not alter liver metabolism and that haloperidoldoes not alter adipose tissue but that olanzapine has apronounced effect on adipose tissue cells and inflammatorystatus.

3.1. Antipsychotic effects on liver and epididymal WAT

The effects of antipsychotics on key enzymes in the lipogenicand lipolytic pathways were investigated in the liver and the

mal adipose tissue of control and antipsychotic-treated rats.: 1 pixel=0.1692 μm. Values are means±SEM of six rats perdiffer at P<0.05.

Fig. 2 – Gene expression in rat epididymal adipose tissue after 46 days of treatment. Dark bar, control; gray bar, olanzapine;white bar, haloperidol. Values are means±SEM of six rats per group. Results were expressed as arbitrary units correspondingfor each gene to the mRNA abundance normalized to 18S RNA. Values in the same row sharing the same letters or withoutletters did not differ at P<0.05. TNFα: tumor necrosis factor α; IL6: interleukin 6; IL1-β: interleukin 1-β; PAI-1: plasminogenactivator inhibitor-1; MCP-1: monocyte chemotactic protein-1; PLAUR: plasminogen activator, urokinase receptor.

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epididymalWAT. Although the liver plays amajor role in bodyhomeostasis by synthesizing lipids for storage in adiposetissue, there are very few studies dealing with the directeffects of antipsychotic drugs on this organ. In human liver-derived cultured hepatocytes, acute exposure to haloperidol,clozapine, and, to a lesser extent, olanzapine, resulted in anactivation of SREBP-1c, the main transcription factor promot-ing lipogenesis, without enhanced expression of the stearoyl-CoA desaturase gene (Raeder et al., 2006). In the livers offemale rats treated with a single high dose of clozapine(50 mg/kg), an initial up-regulation of lipogenic SREBP-1ctarget genes was followed by a marked and sustained down-regulation of the same genes as well as a down-regulation ofgenes involved in lipolysis and fatty acid oxidation (Fernøet al., 2009). In the present study, olanzapine and haloperidolwere chronically administrated tomale rats for 46 days,mixedin with the food, which allowed the drug to act throughout a24-h cycle. Calculated daily doses considered correspondinghalf-lives in humans and rats and were consistent with dosesused in humans (Victoriano et al., 2009). Under theseexperimental conditions, we observed no significant effectsof olanzapine or haloperidol on the expression of the twomajor transcription factors involved in hepatic lipid homeo-stasis (PPARα and SREBP-1c), nor did we observe any change inthe expression of target enzymes that control fatty acidsynthesis (ACC, FAS) or β-oxidation (ACOX, CPT1). This isconsistent with the absence of changes in the plasma lipidprofile, especially in triglyceride concentration that wereported in a previous study (Victoriano et al., 2009). Thismay suggest that in male rats chronically administered

Table 2 – Plasma leptin and cytokines of rats after 46 days of tr

Control Olanzapine

TNFα (pg/ml) 0.41±0.27 1.05±0.60IL-1β (pg/ml) 3.6±1.0 14.7±7.1IL-6 (ng/ml) 1.45±0.53 1.57±0.87MCP-1 (ng/ml) 0.23±0.05 0.27±0.12PAI-1 (ng/ml) 0.17±0.09 0.22±0.03Leptin (ng/ml) 1.69±0.49 2.38±0.83

Values are means±SEM of six rats per group.

olanzapine, the liver does not contribute significantly to themetabolic alterations resulting from such treatment.

Adipose tissue was significantly increased in response toolanzapine, but not to haloperidol, in agreementwith previousstudies (Table 1) (Minet-Ringuet et al., 2005, 2006, 2007).Because mean adipocyte diameter was not increased inolanzapine-treated rats, and mean adipocyte number wasthe same as in control rats (Table 1), it is likely that thisenhanced adiposity results from adipocyte hyperplasia (in-creased number of total cells in the adipose pad). Adipocytehyperplasia in response to SGAs was reported in a studyperformed in female rats (Baptista et al., 2002), but this differsfrom a previous study showing adipocyte hypertrophy in themale rat (Minet-Ringuet et al., 2007). This discrepancy may bedue to the important variability in the metabolic responsive-ness to SGAs in rodents, which resulted in amore pronouncedeffect of olanzapine on adiposity in our previous study (Minet-Ringuet et al., 2007) than in the present one. Nevertheless, theabsence of cell hypertrophy in our study is consistent with theabsence of enhanced expression of glucose transporter(GLUT4) and lipogenic enzymes (ACC and FAS) as well as thedownregulated expression of lipolytic enzymes (LPL and HSL).

3.2. Functional changes in adipose tissue

Olanzapine-treated animals showed only a modest, althoughsignificant, increase in the proportion of epididymal WAT(Table 1). Qualitative features of this adipose tissue weremarkedly altered in response to olanzapine. A significantinfiltration of CD68+ cells, indicative of the presence of

eatment.

Haloperidol ANOVA F

0.25±0.16 0.3373 1.1711.0±4.4 0.2880 1.350.88±0.45 0.7455 0.300.17 ±0.07 0.7048 0.360.35±0.09 0.1664 2.041.24±0.19 0.6082 0.52

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macrophages (Fig. 1), was accompanied by a 2-fold increase inthe expression of the TNFα gene (Fig. 2). Both modificationsare indicative of low-grade inflammation. There have beencontroversies concerning the status of the immune/inflam-matory system in patients with schizophrenia. Alterations inplasma cytokine levels and in immune responses have beenfound in schizophrenia, making it possible that abnormalitiesin the inflammatory system are involved in the pathophysi-ology of the illness (Smith and Maes, 1995). However, studiesdealing with plasma levels of cytokines in schizophrenia areinconsistent (Potvin et al., 2008). Inflammatory abnormalitiesmay be involved in the pathophysiology of schizophrenia, butsome inflammatory abnormalities may also be epiphenome-na. For example, patients with schizophrenia are sensitive tostress, and stress activates inflammatory responses in psy-chiatric patients (Pace et al., 2006). Another epiphenomenalfactor may be smoking, which has been shown to lowercirculating levels of cytokines (Zhang et al., 2008). Alterationsin circulating cytokines may also be secondary to adiposetissue accumulation. Obesity is associated with a low-gradeinflammatory state (Weisberg et al., 2003). Abnormalities incirculating cytokines have been found during obesity-relatedinflammation, along with increased levels of TNFα (Dandonaet al., 1998; Katsuki et al., 1998) and IL-1β (Di Renzo et al., 2007).Elevated TNFα levels in obese patients decline with weightloss (Dandona et al., 1998). In inflammatory adipose tissue,non-fat cells such as macrophages account for nearly allproinflammatory cytokine production (Weisberg et al., 2003).Therefore, significantly increased levels of TNFα mRNAexpression in adipose tissue in our experiments, as well asthe 2- to 3-fold increase of TNFα and IL-1β in the plasma, arelikely related to the infiltration of CD68+ cells (identified asmacrophages) in the adipose tissue secondary to chronicolanzapine treatment. The fact that the increase in plasmalevels of TNFα and IL-1β did not reach significance, despite a 2-to 3-fold increase,may not be surprising because of the limitedduration of the treatment, the modest increase in adiposity,the limited number of animals (with important interindividualdifferences), and also because increases in plasma cytokinesduring olanzapine treatment may not be linear, as shown inhumans by Kluge et al. (2009). Chronic treatment withantipsychotics that induce weight gain may therefore alterplasma levels of cytokines, producing a low-grade inflamma-tory state in patients with schizophrenia, independent of thepotentially primary inflammatory pathophysiology of theillness. In our model, chronic treatment with olanzapineappears to activate mostly TNFα, and at a lower level IL-1β,while levels of other cytokines such as IL-6, MCP-1, and PAI-1(which have been assayed in the present study) are increasedduring low-grade inflammation in obesity in the generalpopulation. Therefore, the adipose tissue-dependent inflam-matory syndrome following chronic olanzapine treatmentmay differ somehow from that in obesity in the generalpopulation. Given that the most significant effect of olanza-pine on adipose tissue in our study was its ability to increasethe expression of TNFα and that TNFα inhibitorsmay be usefulin the treatment of inflammatory disorders (Pappas et al.,2009), the effects of TNFα inhibitors in patients showingmetabolic abnormalities during olanzapine treatment shouldbe considered. Bupropion has shown efficacy in reducing

weight gain in olanzapine-treated patients, and its efficacyhas been attributed to its dopamine/norepinephrine reuptakeinhibition properties (Gadde et al., 2006). However, bupropionis also a TNFα and IL-1β inhibitor (Brustolim et al., 2006), andthe role of TNFα/IL-1β inhibition in the possible beneficialeffects of bupropion in olanzapine-treated patients maydeserve further investigation.

On the other hand, antipsychoticsmayhave peripheral anti-inflammatory properties (Maes et al., 2002). Chronic treatmentwith antipsychotics has been shown to alter plasma cytokinelevels, sometimes in the sense of an anti-inflammatory effect(Kim et al., 2009), but a recent meta-analysis showed that thestudies were inconsistent and the results, difficult to interpret(Potvin et al., 2008). Nevertheless, several studies have demon-strated that antipsychotics affect the release or plasma levels ofcytokines, in particular TNFα and IL-1β. It has been shown thatantipsychotics that produce weight gain increase circulatingTNFα (Pollmächer et al., 1996; Birkas Kovats et al., 2005) or othermarkers of inflammation (Meyer et al., 2009), whereas anti-psychotics which do not increase weight, such as haloperidol,do not increase circulating proinflammatory cytokines, includ-ing TNFα (Pollmächer et al., 1997). In a recent study, Kluge et al.(2009) showed that TNFα levels increasedwith bodymass indexin olanzapine-treated patients, while IL-6 levels were notaffected. According to our results, olanzapine may be specifi-cally involved in the accumulation of CD68+ cells (identified asmacrophages) in theadipose tissue and in theoverexpressionofTNFα by these immune cells. There have been controversies asto whether TNFα overexpression has a primary or a secondaryrole in the development of weight gain in antipsychotic-treatedpatients (Kluge et al., 2009). Our results would favor thehypothesis that macrophage in the adipose tissue precedestheoverexpressionof TNFα, since, in ourmodel, overexpressionof TNFα seems to be secondary to the accumulation ofmacrophages in the adipose tissue. However, antipsychoticstrigger very early events in cell physiology and metabolism, inthevery first hours following their administration (Vik-Mo et al.,2009), so that the real temporal sequence of events involved inCD68+ cells mobilization and cytokine release cannot beestablished from our experiments. The present study ispreliminary, and further work is needed to determine ifolanzapine has a role in the mobilization of macrophages andtheir migration into the adipose tissue. Also, macrophageinfiltration has to be investigated not only in epididymaladipose tissue but also in other adipose tissues, includingsubcutaneous tissue, to determine the possible regionalizationof the inflammatory process. Nevertheless, in an effort todelineate the sequence of events that determines the occur-rence of metabolic syndrome during antipsychotic treatments,wehavepreviously shown that an alteration in feeding patternsprecedes the occurrence of glucose metabolism abnormalitiesin olanzapine-treated rats (Victoriano et al., 2009).We raised thehypothesis that an early event in the effects of olanzapine couldbe the activation of a feeding-initiation system (i.e., themelanin-concentrating hormone system) located in the lateralhypothalamus and projecting to the nucleus accumbens(Georgescu et al., 2005). Activation of this system would lead toan enhanced incentive to eat. There may be potential relationsbetween such an enhanced incentive to eat and the increasedaccumulation of adipose tissue during chronic olanzapine

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treatment. If such a relation exists, itmay be the increase in sizeof the adipose tissue itself that triggers the mobilization ofmacrophages, independent of an effect of olanzapine. None-theless, the low-grade inflammation initiated in the adiposetissue and the subsequent increase in cytokines have beenrepeatedly associated with an increased risk of insulin resis-tance and cardiovascular diseases (Guerre-Millo, 2004).

In conclusion, our results indicate that the adipose tissuemay be a key target of antipsychotics not only through thepreviously known increase in adiposity but also through theoccurrence of low-grade inflammation. Both of these phe-nomena contribute to the etiology of metabolic syndrome thatis frequently observed in SGA-treated patients.

4. Experimental procedures

4.1. Animals

Male Sprague–Dawley rats (Harlan, Gannat, France), initiallyweighing 175–220 g, were individually housed in a roommaintained at 23±1 °C with an artificial 12:12 h light–darkcycle (lights on at 04:00 a.m.). The animals had free access totap water from bottles attached to the cage. After 1 week ofadaptation to the laboratory conditions, the rats were dividedin three groups with the same mean weight (284 g). Twogroups (n=6 per group) received antipsychotic treatments for46 days, while the third group was kept as a control (n=6) (seebelow).

The study was performed according to the Declaration ofHelsinki and the guidelines of the French Ministry ofAgriculture for the use and care of laboratory animals.

4.2. Study design

During the adaptation week, rats were allowed free access tothe powdered experimental diet, composed of 140 g/kg wholemilk protein, 538.1 g/kg cornstarch, 87.6 g/kg sucrose, and137 g/kg soybean oil. This diet was supplemented withminerals and vitamins and provided 17.5 kJ/g (UPAE, INRA,Jouy en Josas, France). Itwasmoderately high in lipid inorder toapproach the human western diet and provided (in percentageenergy) 14% protein, 31% lipid, and 55% carbohydrate. Duringthesecondweek, the twoexperimental groupsweregiveneitherolanzapine (2 mg/kg body weight, n=6) (Zyprexa-Velotab, Lilly,Suresnes, France) or haloperidol (1 mg/kg body weight, n=6)(Haldol, Janssen-Cilag, Issy-les-Moulineaux, France). Because ofthe short half-life of these compounds in the rat, drugs weregiven in the food to favor a more continuous deliverythroughout the day–night cycle than by injection or gavages.Food was prepared daily, as described previously (Victorianoet al., 2009). Briefly, olanzapine tablets, which have beendesigned for a rapid dissolving in water, were solubilized inwater (1 mg of active substance/1 ml) and blended in the plaindiet. Haloperidol was provided in aqueous solution and wasdirectly blended in the plain diet. The control group continuedreceiving theplaindiet. Food intakewasmeasured twiceaweek,so that the amounts of olanzapine and haloperidol to be addedwere adjusted every week according to the mean food intake ofthe previous week. As previously noticed, daily food intake or

repartition of meals throughout the 24-h cycle was not affectedby either treatment (Victoriano et al., 2009).

After 46 days of treatment, the rats were fasted overnight,then deeply anesthetized (intraperitoneal injection of 200mg/100 g of sodium pentobarbital; Sanofi Santé Animale, Paris,France). Blood was taken by intracardiac puncture (2 ml) andwas collected in tubes containing EDTA 10% (0.1 ml solution/mlof blood). Samples were centrifuged at 4 °C for 10min at1700×g, then plasma was collected, sampled, frozen, andstored at −20 °C for future use.

Anesthetized rats were then killed by exsanguination, andliver and epididymal WAT were dissected immediately andweighed. Samples of liver and epididymal WAT were imme-diately frozen in liquid nitrogen and stored at −80 °C until geneexpression and immunohistochemistry analysis.

4.3. Biochemical analyses

Plasma concentrations of cytokines (TNFα, IL-1β, IL-6,monocyte chemotactic protein-1 [MCP-1], and plasminogenactivator inhibitor-1 [PAI-1]) were determined simultaneous-ly by multiplex immunoassay (Linco Research, Missouri,USA) on a Luminex-200 analyzer (Bio-Rad Laboratories,Marnes-la-Coquette, France). Plasma leptin concentration wasdetermined by immunoassay (Linco-Millipore, Molsheim,France) on the same Luminex-200 analyzer.

4.4. Immunomorphologic analysis of adipose tissue

After thawing, biopsies of epididymal WAT were fixedovernight at 4 °C in 4% paraformaldehyde, then processedfor standard paraffin embedding. Sections of 5 μm wereroutinely stained (hematoxylin and eosin, Masson's tri-chrome, picrosirius red, and Perls' staining) and observedunder a Zeiss 20 Axiostar Plus microscope (Zeiss, Göttingen,Germany). Adipocyte diameters were measured, using thePerfect Image software (Claravision, Orsay, France). Immuno-histochemical detection of CD68+ cells by immunoreactivityfor the CD68 antigen (Morphosys AbD, Düsseldorf, Germany)was performed using the avidin–biotin peroxidase method(Hsu and Raine, 1981), as described previously (Cancello et al.,2006). Slides were counterstained with Mayer's hematoxylin.Method specificity tests were performed by omission ofprimary antibodies and use of preimmune serum. Digitalimages were captured by a triCCD camera (Sony, Paris,France). Adipocyte diameter and CD68+ cells were counted in10 randomly chosen areas in each processed slide under 20×magnification. Visual inspection based on expertise withadipose tissue immunohistochemistry allowed identifyingthe reddish CD68+ cells as macrophages. However, resultsrefer to CD68+ cells rather than to macrophages.

4.5. Quantitative real-time RT–PCR

Gene expression of proteins involved in macrophage recruit-ment and inflammation was assessed in the epididymalWAT.The expression of key enzymes involved in lipogenic andlipolytic pathways was measured in the liver and theepididymal WAT (Table 3). Total RNA was extracted fromliver and adipose tissue using Trizol reagent (Invitrogen). Four

Table 3 – Primer sequences used for quantitative RT–PCR.

Gene General function Primers Tissue tested

GLUT 2 Liver glucose carriers Forward 5′-GGCACAGACACCCCACTCAT-3′ LiverReverse 5′-GTCACACCAGCACATACG-3′

GLUT 4 Adipose tissue and muscle glucose carriers Forward 5′-CTTGGCTCCCTTCAGTTTGG-3′ Adipose tissueReverse 5′-GGTGCCTTGTGGGATGGA-3′

PPARα Transcription factors Forward 5′-CCGAATAGTTCGCCGAAAGAAG-3′ LiverReverse 5′-CCAGCCTACTCATCATTGGGATCA-3′

PPARγ Transcription factors Forward 5′-TGCTGGCCTCCCTGATGA-3′ Adipose tissueReverse 5′-GGCTTCCGCAGGTTTTTGA-3′

SREBP-1c Transcription factors Forward 5′-GGAGCCATGGATTGC-3′ LiverReverse 5′-GCTTCCAGAGAGGAG-3′ Adipose tissue

LPL Hydrolysis of circulating triacylglycerol Forward 5′-AGGACTGAGGATGGCAAGC-3′ Adipose tissueReverse 5′-GGCAGGGTGAAGGCAATGTT-3′

HSL Mobilization of fatty acids Forward 5′-CCTACATGGCTCAACTCC-3′ Adipose tissueReverse 5′-GGTTCTTGACTATGGGTGA-3′

ACC Biosynthesis of fatty acids Forward 5′-CAACGCCTTCACACCACCTT-3′ LiverReverse 5′-GAGCCCATTACTTCATCAAAGATCCT-3′ Adipose tissue

FAS Biosynthesis of fatty acids Forward 5′-TGCTCCCAGCTGCAGGC-3′ LiverReverse 5′-GCCCGGTAGCTCTGGGTGTA-3′ Adipose tissue

ACOX Fatty acid oxidation Forward 5′-GGAGCCATGGATTGC-3′ LiverReverse 5′-GCTCCAGAGAGGAG-3′

CPT1 Fatty acid oxidation Forward 5′-CAGCCATGCCACCAAGATC-3′ LiverReverse 5′-CTTGGGCAGTGATGTTTGGA-3′

TNF α Inflammatory cytokine Forward 5′-TGTCTTTGAGATCCATGCCATT-3′ LiverReverse 5′-TCGTAGCAAACCACCAAGCA-3′ Adipose tissue

Il6 Inflammatory cytokine Forward 5′AAAGTCGGAGGCTTAATTACATATGTTC-3′ Adipose tissueReverse 5′-TCATCGCTGTTCATACAATCAGAA-3′

Il1- β Inflammatory cytokine Forward 5′-AGTTGACGGACCCCAAAAGAT-3′ Adipose tissueReverse 5′-GGACAGCCCAGGTCAAAGG-3′

PAI-1 Inflammatory cytokine Forward 5′-GATCTTGACCTTTTGTAGTGCTTGTG-3′ Adipose tissueReverse 5′-TGGGCATGACTGACATCTTCA-3′

MCP-1 Recruited macrophages to inflammation areas Forward 5′-CGCTCAGCCAGATGCACTTAA-3′ Adipose tissueReverse 5′-CCAGCCTACTCATCATTGGGATCA-3′

PLAUR Macrophage attraction Forward 5′-CAGGACCTCTGCAGAACCA-3′ Adipose tissueReverse 5′-GCACAGGCCTCTGGTCAC-3′

CD11b Cell adhesion integrin Forward 5′-ACTCTGATGCCTCCCTTGG-3′ Adipose tissueReverse 5′-CCTGGACACGTTGTTCTCAC-3′

PPARα and γ, peroxisome proliferator-activated receptors α and γ; SREBP-1c, sterol-regulatory element-binding protein-1c; LPL, lipoproteinlipase; HSL, hormone-sensitive lipase; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; ACOX, acyl-CoA oxidase; CPT1, carnitine palmitoyltransferase 1; TNFα, tumor necrosis factor α; Il6, interleukin 6; Il1-β, interleukin 1-β; PAI-1, plasminogen activator inhibitor-1; MCP-1, monocytechemotactic protein- 1; PLAUR, plasminogen activator, urokinase receptor.

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hundred nanograms of total RNA was converted to cDNAusing the High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Villebon, France) (Nefti et al., 2009) on aPTC-200 thermocycler (MJ Research, Biorad, Marnes-la-Co-quette, France). Real time PCR amplifications were performedwith a Prism7300 sequence detection systemusing SYBRGreenMasterMix (Applied Biosystems). Ribosomal 18S RNAwas usedto normalize variability due to inequalities in the initialquantities of cDNA sampled. Gene expressionwas determinedusing the 2−ΔΔCt formula where ΔCt=(Ct target gene −Ct 18S).

4.6. Statistical analyses

All results were expressed as mean±SEM. Statistical analyseswere performed using the SAS statistical Package (SAS/STATversion 6.12 forWindows 95; SAS Institute, Cary, USA). ANOVAwas used for quantitative traits and Kruskal–Wallis tests fornonparametric values (adipocyte number). When the ANOVAtest was significant, a post-hoc Tukey's test was used to

compare between-group means. Differences were consideredsignificant at P<0.05.

Acknowledgments

Montserrat Victoriano gratefully acknowledges the financialsupport of the Servier Institute (France). We thank AngéliqueFoucault-Simonin for animal care and help with dissection,and Patricia Bonjour (Division d'Anatomopathologie, Hôtel-Dieu, Paris, France) for her skilful histological preparations.

Conflicts of interest: none.

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