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International Journal of Obesity (2005) 29, 864–871 & 2005 Nature Publishing Group All rights reserved 0307-0565/05 $30.00 www.nature.com/ijo

PAPER Fenofibrate prevents Rosiglitazone-induced body weight gain in ob/ob mice MC Carmona1, K Louche1, M Nibbelink1, B Prunet1, A Bross1, M Desbazeille1, C Dacquet2, P Renard3, L Casteilla1 and L Peńicaud1* 1 UMR 5018 CNRS-UPS, IFR 31, CHU Rangueil, Toulouse, France; 2Division of Metabolic Diseases, Institut de Recherches Servier (IdRS), Suresnes, France; and 3Division of Research Scientific and Technological Strategic Evaluation, Institut de Recherches Servier (IdRS), Suresnes, France

AIMS/HYPOTHESIS: Fibrates and thiazolidinediones are commonly used for the treatment of dyslipidemia and type 2 diabetes, respectively. The aim of this study was to investigate the effects on body weight as well as on glucose and lipid homeostasis of ligands for PPARa and PPARg, Fenofibrate and Rosiglitazone, alone or in association. METHODS: Ob/ob mice were divided into four groups: control, and mice daily injected (intraperitoneally), either with 10 mg/kg Rosiglitazone, 100 mg/kg Fenofibrate or both molecules. Body weight and food intake were monitored daily. After 13 days of treatment, mice were killed, and blood samples were collected for posterior metabolite quantification. The liver and adipose tissues were dissected and weighed. RESULTS: Body weight was significantly reduced or increased by Fenofibrate and Rosiglitazone, respectively. The effect of Rosiglitazone was prevented by coadministration of Fenofibrate. This was accompanied by a normalization of the daily food efficiency. Compared to those treated with Rosiglitazone, animals treated with Fenofibrate alone or in combination presented a decreased white adipose tissue mass. Fenofibrate or Rosiglitazone alone significantly reduced the levels of plasma lipid parameters. Surprisingly, Fenofibrate also decreased blood glucose levels in ob/ob mice, despite having no effect on insulin levels. By contrast, both glucose and insulin levels were decreased by Rosiglitazone treatment. Coadministration of both drugs improved all parameters as with Rosiglitazone. Fenofibrate restored almost normal hepatocyte morphology and significantly reduced the triglyceride content of the liver. This was accompanied by an increase in fatty acid oxidation in the liver in all groups receiving Fenofibrate. CONCLUSION/INTERPRETATION: These biological effects suggest that combined therapy with a PPARa and a PPARg ligand is more effective in ameliorating, specifically, lipid homeostasis than in activating any of this receptor separately. Furthermore, Fenofibrate prevents one of the most undesirable effects of Rosiglitazone, namely increased adiposity and body weight gain. International Journal of Obesity (2005) 29, 864–871. doi:10.1038/sj.ijo.0802943 Published online 10 May 2005 Keywords: fibrate; thiazolidinedione; food intake; liver; adipose tissues

Introduction Thiazolidinediones (TZD) are potent insulin-sensitizer drugs used in the treatment of type 2 diabetes.1 They are specific ligands for the peroxisome proliferator-activated receptor gamma (PPARg) transcription factor, a protein that belongs to the nuclear hormone receptor family.2 In adults, PPARg is preferentially expressed in both adipose tissues (white and brown), although it may also be present at lower rates in the skeletal muscle, heart and liver.3–5 PPARg directs adipocyte

ˆ pital Rangueil, *Correspondence: Dr L Peńicaud, UMR 5018 CNRS-UPS, Ho TSA 50032, 31059 Toulouse Cedex 9, France. E-mail: [email protected] Received 21 September 2004; revised 7 January 2005; accepted 16 January 2005; published online 10 May 2005

differentiation both in vitro and in vivo in collaboration with other transcription factors of the CCAAT/enhancer binding protein family.6 In accordance with this role, TZD drugs (including Rosiglitazone, Pioglitazone and Troglitazone, of which the last has been withdrawn because of liver toxicity), despite normalizing glycemia and insulinemia, promote body weight gain both in animals and humans7–12 and adipogenesis in vitro.13–15 This is one of the main side effects of TZD treatment, and current investigation focuses on correcting this undesirable consequence. PPARa was the first member of the PPAR family to be described.16 It is mainly expressed in tissues with high metabolic activity such as the liver, heart, kidney, muscle and brown adipose tissue,17,18 as well as in vascular endothelial cells, macrophages and monocytes.19,20 Fibrates

Fibrate prevents body weight gain in ob/ob mice MC Carmona et al

865 are synthetic ligands for PPARa and are used for the treatment of dyslipidemia, especially hypertriglyceridemia and hypercholesterolemia.21 The former has been proposed to contribute to the development of insulin resistance and type 2 diabetes in humans. Thus, fibrates lower plasma triglycerides and cholesterol in these patients21,22 as well as in murine models of insulin resistance and type 2 diabetes, probably by ameliorating liver function and lipid catabolism in this organ.23 In contrast to TZD, fibrates have been reported to induce no change in body weight or even to decrease it in some models of obesity in rodents24,25 and in patients with metabolic X syndrome.26 In the past few years, dual PPARa and PPARg agonists have been developed and their utility as antidiabetic drugs has been suggested.27–31 Nevertheless, only few reports have explored the effects of cotreatment with PPARa and PPARg agonists,7,30 particularly when focusing on body weight regulation. The aim of the present study was to investigate the effects on glucose and lipid homeostasis as well as on body weight of two well-known specific ligands for PPARa and PPARg, Fenofibrate and Rosiglitazone, alone or in association, the hypothesis being that they would exert additional beneficial effects for the treatment of type 2 diabetes compared to separately activating both receptors.

Methods Chemicals Rosiglitazone was synthesized at the Pharmaceutical Chemical Institute, EA 1043, Faculty of Pharmacy Lille (Professor D Lesieur). Fenofibrate, fatty acid-free bovine serum albumin, palmitic acid, ATP, NAD þ , cytochrome c, coenzyme A, L-carnitine and L-malate were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France).

Animals and treatments The care and the use of mice were in accordance with the European Community Council Directive 86/609/EEC and approved by the Comite´ d’Ethique et d’Expe´rimentation Animale of the University Paul Sabatier from Toulouse. All animals were purchased from Harlan (Gannat, France). Obese (ob/ob) 8–12-week-old male C57BL/6 mice were kept under standard conditions of housing (12 h light/dark cycles), feeding (diet: 3.81 kcal/g; reference 229H, UAR, Villemoissson sur Orge, France; food and water ad libitum) and environmental temperature (211C). Mice were randomly divided into four groups (vehicle control DMSO 10%/Solutol HS 15 (BASF) 15% and sterile water 75% (v/v/v), 10 mg/kg Rosiglitazone, 100 mg/kg Fenofibrate, and a combination of both molecules at the same doses used for each one alone; n ¼ 6–7 animals per group), and injected intraperitoneally (i.p.) once daily (5 ml/kg). Body weight and food intake were monitored daily. After 13 days of treatment, mice were killed

by decapitation after CO2 anesthesia, and blood samples were collected for immediate assessment of glucose levels. Serum was frozen for posterior metabolite quantification. The liver, interscapular brown adipose tissue, and inguinal and epidydimal white adipose tissues were dissected and weighed. Liver samples were collected for histological analysis, triglyceride content and fatty acid oxidation measurements.

Metabolite measurements Blood glucose levels were measured using Glucotrends and Accu-Checks active systems (Roche; Mannheim, Germany). Serum NEFA was determined using an acyl-CoA oxidasebased colorimetric kit (NEFA-C, Wako Pure Chemicals Industries; Neuss, Germany). Serum and liver triglycerides were measured using enzymatic colorimetric methods (Triglycerides Enzymatic PAP150, BioMe´rieux; Marcy-l’Etoile, France). Serum insulin levels were determined by an immunoassay enzymatic system (Mercodia Ultrasensitive Mouse Insulin ELISA, Mercodia; Uppsala, Sweden). Serum cholesterol levels were measured using a colorimetric kit from Sigma-Aldrich (Saint Quentin Fallavier, France).

Histological analysis Liver specimens were fixed on 100% ethanol for histological analysis and embedded in paraffin. Sections were cut at a thickness of 5 mm and stained with hematoxylin/eosin.

Liver triglyceride extraction and determination Liver triglyceride extraction was performed as previously described.32 Frozen liver samples (200–600 mg) were minced in chloroform and methanol (2:1 ratio) and incubated overnight at 41C. After filtration to remove tissue debris, 0.9% NaCl was added, and samples were centrifuged at 1500 r.p.m. for 10 min. Lipids in the organic phase were transferred to a new tube, air-dried and dissolved again with X-Triton, methanol and tert-butilic alcohol (1:1:3 ratio).

Fatty acid oxidation in liver Palmitate oxidation rates were measured in liver homogenates as previously described.33 Small fragments of liver were homogenized in Set buffer (250 mM sucrose, 2 mM EDTA, 10 mM Tris, pH 7.4) and 75 ml were incubated in 300 ml of oxidation buffer (27 mM KCl, 10 mM KH2PO4, 5 mM MgCl2, 1 mM EDTA, 25 mM sucrose, 75 mM Tris, 5 mM ATP, 1 mM NAD þ , 25 mM cytochrome c, 0.1 mM coenzyme A, 0.5 mM L-carnitine, 0.5 mM L-malate, pH7.4) in glass vials. After a 5-min preincubation at 371C in a shaking water bath, the reaction was started by adding 100 ml of 600 mM [1-14C]palmitic acid (Amersham Pharmacia Biotech, Orsay, France) bound to fatty acid-free bovine serum albumin in a 5:1 M ratio. Incubation was carried out for 30 min and stopped by International Journal of Obesity


866 adding 200 ml of 3 M perchloric acid. The 14CO2 produced was trapped in 300 ml ethanolamine/ethylene glycol (1:2 v/v) and measured using a liquid scintillation counter. After 90 min at 41C, the acid incubation mixture was centrifuged (5 min at 10 000 g) and 500 ml of supernatant containing the 14 C-perchloric acid acid-soluble products was assayed for radioactivity by liquid scintillation. Palmitate oxidation rates were calculated from the sum of 14CO2 and 14C-percholic acid-soluble products and expressed in nmol of palmitate per min per g tissue weight, or pmol of palmitate per min per mg of protein.

difference between groups. When appropriate, the unpaired t-test was performed.

Results Fenofibrate prevents Rosiglitazone-induced body weight gain in ob/ob mice Fenofibrate significantly reduced body weight in ob/ob mice compared to control mice whereas Rosiglitazone induced a significant increase (Figure 1). Thus, at the end of the treatment, the body weight change was 2.7470.42 g for control-vehicle mice, 0.8470.92 g for Fenofibrate-treated mice (Po0.001 vs control and Rosiglitazone-treated animals) and 6.9670.94 g for Rosiglitazone-treated mice (Po0.001 vs control). The increase in body weight associated with Rosiglitazone treatment was prevented by the coadministration of Fenofibrate. Thus, at day 13, these mice had a body weight not significantly different from that of controls or Fenofibrate-treated mice (0.6771.02 g, Po0.001 vs Rosiglitazone group).

Whole-body fat composition A PIXIMUS densitometer (LUNAR Corporation, Madison, WI, USA) was used for measurement of whole-body fat. Calibration was performed with a defined standard (phantom). After decapitation, mice were placed on a specimen tray and put in a PIXIMUS imaging area for analysis. The PIXIMUS software automatically calculated the whole-body fat and lean mass content.

Fenofibrate prevents hyperphagia in Rosiglitazonetreated mice Changes in body weight were associated with differences in food consumption. Rosiglitazone-treated mice were hyperphagic compared to controls (5.6070.15 and 5.0870.09 g/ day, respectively, Po0.01) (Figure 2a), whereas Fenofibratetreated animals, or mice treated with both molecules, had a

Statistical analysis Data are expressed as means7s.e.m. Prism Software (GraphPad software, San Diego, CA, USA) was used for all statistical analysis. A one-way analysis of variance followed by Tukey’s multiple comparison test was used to assess the statistical

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Days of treatment Figure 1 Fenofibrate prevents Rosiglitazone-induced body weight gain in ob/ob mice. Mice were treated daily with i.p. injection of vehicle (control mice, black circles), Rosiglitazone (10 mg/kg, black squares) and a combination of both drugs, Rosiglitazone and Fenofibrate (10 and 100 mg/kg, respectively, white squares), for 13 days. Body weight was measured daily. Body weight gain was calculated as the difference between each day and day 0. *Significantly different from control mice Po0.05; **Po0.01; ***Po0.001. *Significantly different from Rosiglitazone-treated mice Po0.05; þ þ Po0.01; þ þ þ Po0.001. *Significantly different from Fenofibrate-treated mice Po0.05; ***Po0.001.

International Journal of Obesity


867

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Daily food intake 6.0

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Figure 2 Fenofibrate prevents hyperphagia in Rosiglitazone-treated mice. (a) Daily food intake in animals treated with Rosiglitazone, Fenofibrate or a combination of both molecules. (b) Daily food efficiency calculated as gram of daily body weight gain per gram of daily food intake. C control; R, Rosiglitazone; F, Fenofibrate; RF, Rosiglitazone and Fenofibrate treated-mice. **Significantly different from control mice Po0.01; ***Po0.001. þþ Significantly different from Rosiglitazone-treated mice Po0.01; þþþ Po0.001.

reduced daily food intake (3.8470.25 g/day for Fenofibrate, Po0.001 vs control and vs Rosiglitazone; 4.1970.21 g/day, Po0.01 vs control, Po0.001 vs Rosiglitazone). Furthermore, Fenofibrate administration normalized the daily food efficiency of mice treated with Rosiglitazone (0.033170.0157, Po0.01 vs Rosiglitazone) (Figure 2b).

Blood chemistry in ob/ob treated mice As expected, Fenofibrate alone significantly reduced serum triglyceride (42% reduction, Po0.001. vs control), nonesterified fatty acid (5% reduction, Po0.001 vs control) and total cholesterol levels (50% reduction, Po0.001 vs control). Surprisingly, it also decreased blood glucose levels in ob/ob mice (34% reduction, Po0.01 vs control), despite having no effect on insulin levels (Table 1). Rosiglitazone alone had similar effects to Fenofibrate on lipid parameters, being even more efficient than Fenofibrate in reducing cholesterol levels (Po0.001). Furthermore, glucose and insulin levels were also decreased by Rosiglitazone treatment. The coadministration of both drugs improved all parameters as with Rosiglitazone administration.

Fenofibrate decreases liver triglyceride content, stimulates fatty acid oxidation and normalizes hepatocyte morphology Despite no significant changes in liver weight, Fenofibrate treatment significantly reduced the triglyceride content of the liver both when used alone (48.4%. reduction) or in combination with Rosiglitazone (62% reduction) (Table 2). Palmitic acid oxidation was increased by Fenofibrate treatment, either alone or in combination with Rosiglitazone. Notably, the association of both molecules further increased lipid oxidation compared to Rosiglitazone treatment alone (118% increase per g of the liver and 113% increase per mg of the protein vs the Rosiglitazone group, Po0.001 and Po0.01, respectively). Histological sections of the liver reflected these biochemical changes. Thus, Fenofibratealone , and Rosiglitazone plus Fenofibrate treatments restored hepatocyte morphology, with a great reduction in the size and number of lipid droplets compared to hepatocytes from control and Rosiglitazone-treated mice (Figure 3).

Fenofibrate effects on fat mass in ob/ob mice Rosiglitazone treatment significantly increased inguinal and epidydimal white adipose tissue (WAT) weights compared to control mice (15 and 24% increase, respectively), resulting in a total WAT weight increase of 19% (sum of inguinal and epidydimal depots) (Table 3). Compared to those treated with Rosiglitazone, animals treated with Fenofibrate alone or in combination had less white adipose tissue (20 and 21% reduction, respectively, for inguinal depot, and 19 and 10% for epidydimal pad) (Table 3). Total WAT of Fenofibratetreated mice (alone and in combination with Rosiglitazone) was also significantly reduced compared to Rosiglitazonetreated mice (Po0.001). X-ray densitometer analysis (PIXIMUS) showed that the percentage of body fat mass was diminished in animals treated with Fenofibrate alone or in combination with Rosiglitazone (6% reduction vs Rosiglitazone for both situations). It is interesting to note that absolute fat mass was increased in Rosiglitazone-treated International Journal of Obesity


868 Table 1

Rosiglitazone and Fenofibrate effects on glucose, triglyceride, nonesterified fatty acid and insulin serum levels in ob/ob mice

Ob/ob mice

GLC (mM)

INS (ng/ml)

TG (g/l)

NEFA (mM)

CHOL (g/l)

Control Rosiglitazone (10 mg/kg/day) Fenofibrate (100 mg/kg/day) Rosiglitazone Fenofibrate

12.371.4 6.571.3* 8.170.9* 5.570.3*

4974 1472w 3977z 1074w,y

1.0770.15 0.6670.07w 0.6270.03w 0.5070.07w

1.9770.30 0.5370.14w 0.8470.09w 0.3670.09w

3.0270.24 0.7170.08w 1.5370.15w,z 0.6370.08w,y

Values are mean7s.e. (n ¼ 6–7). One-way analysis of variance denoted significant differences between groups for glucose (Po0.0017), insulin (Po0.0000), triglyceride (Po0.001), NEFA (Po0.0000) and total cholesterol levels (Po0.0000). *Significantly different from control mice Po0.01; wPo0.001. zSignificantly different from Rosiglitazone-treated mice Po0.001. ySignificantly different from Fenofibrate-treated mice Po0.001. GLC, glucose; INS, insulin; TG, triglycerides; NEFA, nonesterified fatty acids; CHOL, total cholesterol.

Table 2

Rosiglitazone and fenofibrate effects on liver parameters in ob/ob mice

Ob/ob mice

Control

Rosiglitazone

Fenofibrate

Rosi+Feno

Liver (g) Liver TG Content (mg/g)

4.9670.18 12478

4.9970.17 129720

4.5970.25 64714*,z

4.5270.20 47710*,z

Palmitic acid oxidation nmol/min/g liver pmol/min/mg protein

3372 13478

6476w 263726

11577w,y 380759*

141716w,y 5607113*,z

Values are mean7s.e. (n ¼ 6–7). One-way analysis of variance denoted significant differences between groups for liver triglyceride content (Po0.0022), palmitic acid oxidation per g of liver (Po0.0000) and per mg of protein (Po0.0040). *Significantly different from control mice Po0.01; wPo0.001. zSignificantly different from Rosiglitazone-treated mice Po0.01; yPo0.001.

Figure 3 Liver sections of mice treated with vehicle (a), Rosiglitazone (b), Fenofibrate (c) or a combination of both molecules (d). As shown in (c and d), Fenofibrate restored hepatocyte morphology. Compared to control and Rosiglitazone-treated livers, Fenofibrate treatment produced a great reduction in the size and number of lipid droplets. (a–d) 20 magnification.

animals compared to controls (10.8% increase, Po0.001), and diminished in Fenofibrate groups (12% decrease in Fenofibrate-treated mice vs control mice and 20% vs International Journal of Obesity

Rosiglitazone-treated mice, Po0.001; 10% decrease in Rosiglitazone plus Fenofibrate-treated animals vs control and 19% vs Rosiglitazone group, Po0.001).


869 Table 3

Rosiglitazone and fenofibrate effects on adipose tissue weightand mass in ob/ob mice

Ob/ob mice iBAT (mg) iWAT (g) eWAT (g) tWAT (g) % FAT Fat mass (g) Lean mass (g)

Control 194718 3.8370.21 2.7270.14 6.5570.21 60.6770.76 32.2471.18 20.8370.34

Rosiglitazone a

387783 4.3970.18b 3.3970.08b 7.7870.18c 61.0270.65 35.7571.28c 22.7770.37b

Fenofibrate

Rosi+Feno

290724 3.5270.13e 2.7570.14e 6.2670.24f 57.5971.17b,e 28.5471.13c,f 20.9770.58d

383745a 3.4670.10e 3.0870.08b 6.5470.15f 57.3570.60b,e 28.9570.76c,f 21.7270.68

Values are mean7se (n ¼ 6–7). One-way analysis of variance denoted significant differences between groups for iBAT (Po0.0216), iWAT (Po0.0027), eWAT (Po0.0016), tWAT (Po0.0002), %FAT (Po0.0076) and Fat mass (Po0.0005). Unpired t-test for lean mass denoted significant differences between control and Rosiglitazone groups, and Rosiglitazone vs Fenofibrate-treated mice. Significantly different from control mice aPo0.05; bPo0.01;cPo0.01; Significantly different from Rosiglitazone-treated mice; dPo0.05; ePo0.01; fPo0.05. iBAT, interescapular brown adipose tissue; iWAT, inguinal white adipose tissue; eWAT, epididimal white adipose tissue; tWAT, total white adipose tissue; % Fat, percentage of fat versus the whole body calculated using the PIXIMUS densitometer; FAT MASS, absolute fat mass calculated using the PIXIMUS densitometer; LEAN MASS, absolute lean mass calculated using the PIXIMUS densitometer.

Discussion In this study, we describe the benefits of combining PPARg and PPARa ligands for the treatment of type 2 diabetes. As reported repeatedly, Rosiglitazone improves blood levels of glucose, insulin, triglycerides, NEFA and cholesterol.7,27,29,31,34,35 On the other hand, Fenofibrate alone was, indeed, able to reduce serum lipid levels (TG, NEFA and cholesterol).7,25,36 Surprisingly, it was also able to reduce significantly blood glucose concentration despite no effect on plasma insulin. Such an effect on blood glucose has already been reported in db/db mice,7 although the effect was found to be less pronounced in these later. Combined activation of the two receptors markedly prevents glucose and lipid dysregulations in the ob/ob mice as Rosiglitazone does. Recently, some investigations have reported the effects of dual PPARa/g agonist in animal models of dyslipidemia and type 2 diabetes.27–31 Our data are in good agreement with these reports, although in most of them, either no comparison was made with a specific PPARg agonist, or the dual agonist did not show a beneficial effect. The most striking result of the present study concerns the effect of Fenofibrate on body fat mass and body weight. Indeed, Fenofibrate alone decreased body weight in ob/ob mice. This effect is highly significant and persists over time. Such a decreased body weight and fat mass have already been reported in models of genetic or diet-induced obesity in rodents treated with Fenofibrate or PPARa agonist alone.24,25,31,36–39 One of the side effects of PPARg ligands is their tendency to increase body weight, which is a risk factor for type 2 diabetes. Rosiglitazone-treated animals had increased body and WAT weights. Furthermore, absolute lean mass was increased in this group in line with the fat mass, which explains the no-change observed in the percentage of body fat. Fenofibrate totally prevents the higher body weight gain induced by Rosiglitazone and reduces white fat mass. This clear-cut effect contrasts with those of the recent dual PPARa/g ligands developed, which are rather controversial. Some have been reported to increase body weight like Rosiglitazone does,27,28,30 whereas others have no effect.29,39

However, it should be noted that in the two last papers, Rosiglitazone was not reported to increase body weight as well. So this is the first report demonstrating that a treatment with a PPARa ligand is able to prevent body weight gain induced by a PPARg agonist. The changes in body weight might be related to the effects of the different treatment on food intake. Thus, Rosiglitazone and Fenofibrate alone induced hyperphagia and hypophagia, respectively. The effect of Fenofibrate appears to be predominant since cotreatment resulted in a decreased food intake compared to controls. This is probably not due to variations in leptin levels, since our model is the ob/ob mouse that lacks this hormone,40 but rather to changes in metabolic parameters, which in turn would regulate body weight and adipose tissue mass, as already suggested by Guerre-Millo et al.37 Long-term PPARa activation is usually associated with hepatomegaly.24,31,41 In the present work, no changes in liver weight were observed whatever the treatment used. However, Fenofibrate alone or in combination with Rosiglitazone, significantly reduced liver TG content, probably by ameliorating lipid metabolism in this organ (increased lipid oxidation), compared to Rosiglitazone alone or to control animals. This may contribute to the reduction in serum lipid metabolites observed in the Fenofibrate-treated animals (in combination with Rosiglitazone or not). These results suggest that cotreatment with both molecules zmay ameliorate fatty liver without increasing liver mass, and thus contributes to the therapeutic potential of PPARa/g ligands. To summarize, the present study demonstrates that combined therapy with a PPARa and a PPARg ligand is more effective in ameliorating lipid homeostasis than activating any of these receptors separately, contributing to the amelioration of the diabetic state. Furthermore, Fenofibrate prevents one of the most undesirable effects of Rosiglitazone, namely increased adiposity and body weight gain. This sustains the current development of the PPARa/g compound as a valuable approach for the treatment of type 2 diabetes. International Journal of Obesity


870 Acknowledgements We acknowledge the help of the staff of the Animal Quarter from the IFR 31 and more specially JM Lerme and Y Barreira in taking care of the animals. We thank B Girier for providing the PIXIMUS apparatus and his help in analyzing the data and B Morio for setting the palmitic acid oxidation technique. This study was supported by the Centre National de la Recherche Scientifique and by a grant in aids from the Institut de Recherches Internationales Servier (IRIS).

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