The Cannabinoid CB 1 Receptor Antagonist Rimonabant ( SR 141716 ) Inhibits Cell Proliferation and Increases Markers of Adipocyte Maturation in Cultured Mouse 3 T 3 F 442 A Preadipocytes

Adipocyte cell proliferation is an important process in body fat mass development in obesity. Adiponectin or Acrp30 is an adipocytokine exclusively expressed and secreted by adipose tissue that regulates lipid and glucose metabolism and plays a key role in body weight regulation and homeostasis. Adiponectin mRNA expression in adipose tissue and plasma level of adiponectin are decreased in obesity and type 2 diabetes. In obese rodents, the selective CB1 receptor antagonist rimonabant reduces food intake and body weight and improves lipid and glucose parameters. We have reported previously that rimonabant stimulated adiponectin mRNA expression in adipose tissue of obese fa/fa rats, by a direct effect on adipocytes. We report here that rimonabant (10–400 nM) inhibits cell proliferation of cultured mouse 3T3 F442A preadipocytes in a concentration-dependent manner. In parallel to this inhibitory effect on preadipocyte cell proliferation, rimonabant (25–100 nM) stimulates mRNA expression and protein levels of two late markers of adipocyte differentiation (adiponectin and glyceraldehyde-3-phosphate dehydrogenase) with a maximal effect at 100 nM, without inducing the accumulation of lipid droplets. Furthermore, treatment of mouse 3T3 F442A preadipocytes with rimonabant (100 nM) inhibits basal and serum-induced p42/44 mitogen-activated protein (MAP) kinase activity. These results suggest that inhibition of MAP kinase activity by rimonabant may be one of mechanisms involved in the inhibition of 3T3 F442A preadipocyte cell proliferation and stimulation of adiponectin and GAPDH expression. The inhibition of preadipocyte cell proliferation and the induction of adipocyte late “maturation” may participate in rimonabant-induced antiobesity effects, particularly the reduction of body fat mass. Obesity is a complex metabolic disorder resulting from an imbalance between energy intake and expenditure. This dysregulation may have a genetic and/or behavioral origin, involving the quality and quantity of food intake as well as lifestyle (Carpino, 2000). Obesity is characterized by an increase in body weight and adipose tissue hyperplasia and hypertrophy with excessive fat storage (Hausman et al., 2001; Spiegelman and Flier, 2001). Adipose tissue is now considered an active endocrine organ that produces and secretes various hormones and biologically active proteins called adipocytokines, including leptin, adipsin, resistin, tumor necrosis factor , interleukin 6, transforming growth factor , angiotensinogen, plasminogen activator inhibitor-1, and adiponectin (Trayhurn and Beattie, 2001; Fain et al., 2004; Trayhurn and Wood, 2004). Through autocrine, paracrine, and endocrine mechanisms, these adipocytokines play multiple and crucial roles in several pathophysiological processe, such as obesity, and associated chronic diseases, including diabetes, dyslipidemia, cardiovascular disorders (atherosclerosis, hypertension, myocardial infarction, coronary diseases), and inflammation (Trayhurn and Beattie, 2001; Fortuno et al., 2003; Lyon et al., 2003; Rajala and Scherer, 2003; Reilly and Rader, 2003; Ruan and Lodish, 2003; Borst, 2004; Fain et al., 2004; Matsuzawa et al., 2004; Pischon et al., 2004; Trayhurn and Wood, 2004; Wisse, 2004). In obesity, excessive visceral and abdominal body fat mass development is generally accompanied by changes in the structural and cellular composition of adipose tissue associated with dramatic dysregulation of synthesis and release of adipocytokines and of enzyme activities involved in lipid and Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.015040. ABBREVIATIONS: Acrp30, adipocyte complement-related protein of 30 kDa (adiponectin); SR141716, rimonabant; DMEM, Dulbecco’s modified Eagle’s medium; PTX, Bordetella pertussis toxin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TBST, Tris-buffered saline/Tween 20; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase. 0026-895X/06/6902-471–478$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 2 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 15040/3080294 Mol Pharmacol 69:471–478, 2006 Printed in U.S.A. 471 at A PE T Jornals on Jne 4, 2017 m oharm .aspeurnals.org D ow nladed from glucose metabolism (Dugail et al., 1988, 1992; Rolland et al., 1995; Fried and Russell, 1998; Guerre-Millo, 2002). This dysregulation of adipocytokines and adipoenzymes with resulting metabolic and behavioral dysfunctions may be the principal cause of obesity and associated diseases (Lyon et al., 2003; Rajala and Scherer, 2003; Reilly and Rader, 2003; Borst, 2004; Grundy, 2004; Matsuzawa et al., 2004; Pischon et al., 2004; Wisse, 2004). Structural and functional integrity of adipose tissue plays a pivotal role in maintaining metabolic regulation, body weight, and physiological homeostasis. In fact, biological abnormality of adipose tissue may be a key step contributing to the emergence of pathologic features that characterize the metabolic syndrome and particularly obesity associated diseases. Adipocyte cell proliferation (hyperplasia) is an important process in body fat mass development in obesity. Studies in humans and in animal models indicate that increase in adipocyte cell size (adipocyte hypertrophy) often precedes increase in adipocyte cell number (Hausman et al., 2001), and the development of hyperplastic adipose tissue is currently associated with the most severe form of obesity and associated diseases (Hirsch et al., 1989). Based on these observations, drugs capable of restoring structure and functionality of adipose tissue may represent a novel approach to the therapeutic treatment of obesity and associated diseases. Adipocyte complement-related protein of 30 kDa (Acrp30), or adiponectin, is an adipocytokine exclusively expressed and secreted by adipose tissue that has been shown to regulate lipid and glucose metabolism and to play a key role in body weight regulation and homeostasis. Adiponectin has also been reported to be involved in obesity and with associated metabolic diseases. In fact, adiponectin mRNA expression in adipose tissue and its plasma level are decreased in obesity and associated pathologies (Hu et al., 1996; Arita et al., 1999; Fruebis et al., 2001; Weyer et al., 2001). Rimonabant (SR141716), a selective CB1 receptor antagonist (Rinaldi-Carmona et al., 1994), has been shown to possess potent antiobesity effects: it reduced food intake, body weight, and fat mass and improved lipid parameters and insulin sensitivity in obese rodents (Arnone et al., 1997; Chaperon et al., 1998; Colombo et al., 1998; Di Marzo et al., 2001; Ravinet-Trillou et al., 2002; Bensaid et al., 2003; Poirier et al., 2005). We have reported recently that rimonabant stimulates adiponectin mRNA expression in adipose tissue of obese fa/fa rats by a direct effect on adipocytes and reduces hyperinsulinemia associated with this animal model (Bensaid et al., 2003). These results demonstrated for the first time that rimonabant regulates hormones implicated in the control of lipid and glucose metabolism and that it could exert a metabolic “peripheral” action, which might account for its antiobesity effect (Bensaid et al., 2003). The aim of the present study was to evaluate the effect of rimonabant on the cell proliferation, endocrine profile, and enzyme expression in cultured mouse 3T3 F442A preadipocytes and to identify possible molecular mechanisms involved in these effects. Materials and Methods Cell Culture. Mouse 3T3 F442A preadipocytes were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum and cultured as described previously (Green and Kehinde, 1973; Kuri-Harcuch and Green, 1977). Cell Proliferation Assays. Mouse 3T3 F442A preadipocytes were seeded at 5 10 cells per 35-mm plastic dish containing DMEM supplemented with 10% calf serum and exposed or not to increasing concentrations of rimonabant (10–400 nM) added every day. Five days after seeding, cell cultures were harvested by trypsin incubation, and cell densities were determined with a Coulter counter (Beckman Coulter, Fullerton, CA). Triplicate dishes were used for each experimental point, and data are the mean S.E.M. of three different experiments. RNA Preparation and Northern Blot Analysis. -Actin and GAPDH cDNA probes were purchased from Clontech (Mountain View, CA). Adiponectin cDNA was produced as described previously (Bensaid et al., 2003). Total RNA was prepared from 3T3 F442A preadipocyte cultured in DMEM containing 10% calf serum and treated or not with rimonabant or Bordetella pertussis toxin (PTX) or with the combination of rimonabant and PTX, at indicated concentrations, using TRIzol reagent (Invitrogen, Carlsbad, CA). For Northern blot analysis, 20 g of total RNA was electrophoresed, and transferred to a nylon membrane (Hybond N ; GE Healthcare, Little Chalfont, Buckinghamshire, UK). The membranes were hybridized successively with Adiponectin, GAPDH, and -actin probes labeled with [ -P]dCTP using a random priming kit (GE Healthcare). Membranes were scanned on a Storm PhosphorImager (GE Healthcare). Relative quantification of RNA expression levels was performed with the Image-Quant program (GE Healthcare). Results were normalized against the -actin mRNA expression and were presented as a percentage of control values. Western Blot Analysis of Adiponectin and GAPDH Expression. 3T3 F442A preadipocyte cellular proteins were dissolved in the lysis buffer (50 mM Tris-HCl, pH 7.5, 1% SDS, 10 mM EDTA, 100 mM NaCl, and 1% -mercaptoethanol), containing protease inhibitors (Roche Diagnostics), and centrifuged at 12,000g for 15 min at 4°C. The supernatants were collected and protein concentrations were determined by BCA protein assay kit (Pierce). 3T3 F442A adipocyte cellular protein extracts (200 g) and conditioned medium (lyophilized 100 l) were analyzed on the Novex precast 4 to 20