The Farnesoid X Receptor Promotes Adipocyte Differentiation and Regulates Adipose Cell Function in Vivo Giovanni Rizzo, Moises Disante, Andrea Mencarelli, Barbara Renga, Antimo Gioiello, Roberto Pellicciari, and Stefano Fiorucci Dipartimento di Medicina Clinica e Sperimentale, (G.R., M.D., A.M., B.R., S.F.) and Dipartimento di Tecnologia del Farmaco, (A.G., R.P.), University of Perugia, Perugia, Italy Received February 25, 2006; accepted June 15, 2006 ABSTRACT The differentiation of a preadipocyte into a mature adipocyte is a highly regulated process that requires a scripted program of transcriptional events leading to changes in gene expression. Several genes are associated with adipogenesis, including the CAAT/enhancer-binding protein (C/EBPs) and peroxisome pro- liferator-activated receptor (PPAR) families of transcription fac- tors. In this study, we have investigated the role of the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor, in regulating adipogenesis in a preadipocyte cell line (3T3-L1 cells). Our results show that FXR is expressed in the white adipose tissue of adult mice and in differentiated 3T3-L1 cells but not in undifferentiated preadipocytes. Exposure of 3T3-L1 cells to INT-747 (6-ethyl cheno-deoxycholic acid), a potent and selective FXR ligand, increases preadipocyte differentiation in- duced by a differentiating mixture containing insulin. Augmen- tation of differentiating mixture-induced differentiation of 3T3-L1 cells by INT-747 associated with induction of aP2, C/EBP, and PPAR2 mRNAs along with other adipocyte- related genes. This effect was reversed by guggulsterone, an FXR antagonist, and partially reverted by GW9662 (2-chloro-5- nitro-N-phenylbenzamide), a selective PPAR antagonist, indi- cating that FXR modulates adipocyte-related genes by PPAR- dependent and -independent pathways. Regulation of adipocyte-related genes by INT-747 was lost in FXR-/- mice, indicating that modulation of these genes by INT-747 requires an intact FXR. In addition, INT-747 enhances both insulin- induced serine phosphorylation of Akt and glucose uptake by 3T3-L1 cells. Taken together, these results suggest that acti- vation of FXR plays a critical role in regulating adipogenesis and insulin signaling. The farnesoid X receptor (FXR) is a nuclear receptor and bile acid sensor expressed in liver, intestine, kidney, and adrenal glands (Zhang et al., 2003; Bishop-Bailey et al., 2004). Upon activation, FXR regulates target gene expres- sion by binding to FXR response elements after heterodimer- ization with the retinoid X receptor (RXR). The optimal DNA binding sequence for the FXR/RXR heterodimer is an in- verted repeat of two AGGTCA half-sites spaced by one nu- cleotide (inverted repeat 1) (Forman et al., 1995). In target tissues, FXR ligands negatively regulate bile acid synthesis by decreasing the expression of cholesterol-7-hy- droxylase (Cyp7a1), the rate-limiting enzyme of the bile acid biosynthetic pathway. This process is partially mediated by induction of the small heterodimer partner (SHP), an atypi- cal nuclear receptor that lacks a DNA binding domain (Good- win et al., 2000; Lu et al., 2000). FXR controls cholesterol disposal and the enterohepatic circulation of bile acids by increasing the transcription of the intestinal ileal-bile acid binding protein (Grober et al., 1999), inhibiting the hepatic expression/function of the bile acid transporter Na + -tauro- cholate cotransporting polypeptide (Denson et al., 2001) and inducing the expression/function of the bile salt export pump (Ananthanarayanan et al., 2001) and multidrug resistance- associated protein 2 (Kast et al., 2002). A growing body of evidence also support the notion that FXR has an important role in regulating lipid (triglyceride and cholesterol) and glu- cose homeostasis (Sinal et al., 2000; Urizar et al., 2000; Lambert et al., 2003; Claudel et al., 2005). Consistent with this view, FXR gene ablation in mice is associated with increased blood cholesterol and triglyceride levels. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.023820. ABBREVIATIONS: FXR, farnesoid X receptor; RXR, retinoid X receptor; SHP, small heterodimer partner; INT-747, 6-ethyl chenodeoxycholic acid; CDCA, 6-ethyl chenodeoxycholic acid; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DIM, differentiation mixture; IBMX, 3-iso-butyl-1-methylxanthine; PCR, polymerase chain reaction; qRT-PCR, quantitative real-time PCR; C T , cycle threshold (the cycle number at which each PCR reaction reaches a predetermined fluorescence threshold); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; C/EBP, CAAT/enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; FABP, fatty acid binding protein; SREBP-1c, sterol-regulatory element binding protein-1c; TNF, tumor necrosis factor ; GW9662, 2-chloro-5-nitro-N-phenylbenzamide. 0026-895X/06/7004-1164 –1173$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 4 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 23820/3135537 Mol Pharmacol 70:1164–1173, 2006 Printed in U.S.A. 1164 at ASPET Journals on July 20, 2018 molpharm.aspetjournals.org Downloaded from