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Inhibition of Adipocyte Differentiation by Rorα

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Inhibition of Adipocyte Differentiation by Rorα FEBS Letters 583 (2009) 2031–2036 journal homepage: www.FEBSLetters.org Inhibition of adipocyte differentiation by RORa Hélène Duez a,b,c,1, Christian Duhem a,b,c,1, Saara Laitinen a,b,c, Prashant S. Patole a,b,c, Mouaadh Abdelkarim a,b,c, Brigitte Bois-Joyeux d, Jean-Louis Danan d, Bart Staels a,b,c,* a Institut Pasteur de Lille, Département d’Athérosclérose, Lille F-59019, France b INSERM UMR 545, Lille F-59019, France c Université Lille Nord de France, Faculté des Sciences Pharmaceutiques et Biologiques et Faculté de Médecine, Lille F-59006, France d Centre National de la Recherche Scientifique FRE3210, Faculté de Médecine René Descartes Paris 5, 75015 Paris, France article info abstract Article history: Here we show that gene expression of the nuclear receptor RORa is induced during adipogenesis, Received 8 April 2009 with RORa4 being the most abundantly expressed isoform in human and murine adipose tissue. Revised 27 April 2009 Over-expression of RORa4 in 3T3-L1 cells impairs adipogenesis as shown by the decreased expres- Accepted 8 May 2009 sion of adipogenic markers and lipid accumulation, accompanied by decreased free fatty acid and Available online 18 May 2009 glucose uptake. By contrast, mouse embryonic fibroblasts from staggerer mice, which carry a muta- Edited by Robert Barouki tion in the RORa gene, differentiate more efficiently into mature adipocytes compared to wild-type cells, a phenotype which is reversed by ectopic RORa4 restoration. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Adipogenesis RORa Nuclear receptors RAR-related orphan receptors (RORs) 1. Introduction [5,7] and ectopic Rev-erba expression in 3T3L1 pre-adipocytes promotes their differentiation into mature adipocytes and en- White adipose tissue is a central organ for whole-body energy hances lipid storage [5]. homeostasis. It serves as a buffering depot for excess energy and The RAR-related orphan receptors (RORs) are nuclear receptors releases fatty acids upon energy demand [1]. Adipose tissue is also closely related to the Rev-erbs. Rev-erba and RORa are co-ex- an endocrine organ which secretes numerous adipokines such as pressed in many tissues. In general, they compete for binding to leptin and adiponectin, and cytokines such as TNFa, which regulate specific monomeric response elements (RevRE/RORE) hence acting (patho)physiological processes such as insulin sensitivity, inflam- as a ying/yang loop to regulate common target genes in an oppo- mation or food intake. Several transcription factors act in concert site manner (RORa acts as an activator, whereas Rev-erba as a to orchestrate the differentiation of pre-adipocytes into adipocytes repressor) [8–10]. For instance, RORa regulates the expression of [2]. Peroxisome Proliferator-Activated Receptor c (PPARc) and several Rev-erba target genes, thereby playing a role in circadian CCAAT/enhancer-binding proteins (CEBP) a, b and d are master rhythm maintenance and plasma lipid control [11–17]. RORa also regulators, which trigger a cascade of transcriptional events hence plays a role in muscle lipid metabolism [18], and controls the initiating differentiation and instoring the mature phenotype. Re- expression of the gluconeogenic glucose-6-phosphatase gene [19]. cent evidence however indicates that this process can be modu- Here, we have investigated the role of RORa in adipogenesis and lated positively and negatively by other pathways [3]. For report that RORa is a potent inhibitor of adipocyte differentiation. example, we and others have shown that Rev-erba, a nuclear receptor regulating lipid and bile acid metabolism, the inflamma- tory process and a core clock machinery gene [4], plays also a mod- 2. Materials and methods ulatory role in the regulation of adipogenesis [5,6]. Indeed, Rev- erba gene expression is induced during the adipogenic process 2.1. Animals Inguinal and epididymal fat depots were from wild-type C57BL/ * Corresponding author. Address: UR545 INSERM, Institut Pasteur de Lille, BP 6 mice. See Supplementary data for details. 245, 1, Rue Calmette, 59019 Lille, France. Fax: +33 3 20 87 73 60. E-mail address: [email protected] (B. Staels). Animal care and experimental procedures were performed 1 These authors have equally contributed to this work. according to approved institutional guidelines. 0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.05.019 2032 H. Duez et al. / FEBS Letters 583 (2009) 2031–2036 2.2. Human biopsies insulin. At days 0 and 8, cells were lysed and homogenized for RNA isolation or fixed in 4% paraformaldehyde and stained with Human fat tissue samples were obtained from healthy adult do- Oil-red-O. nors, in agreement with French regulations. All subjects gave writ- ten informed consent prior sample collection. 2.6. FFA uptake assay 2.3. Viral production and infection Free fatty acid (FFA) uptake was measured using the commer- cially available QBT Fatty Acid Uptake Assay Kit (Molecular Devices, To generate cell lines that constitutively over-express RORa4, USA) as per the manufacturer’s instructions. See Supplementary the coding sequence of human RORa4 was inserted in the MFG ret- data for more details. rovirus plasmid [20] to generate the pMFG-RORa4 plasmid. A sim- ilar construct lacking the RORa4 sequence (pMFG-Neo) was used 2.7. Glucose uptake throughout the study as a negative control. Phoenix cells were transfected with these constructs and selected for resistance using Assays were performed as described previously [21]. See Sup- the geneticin analog G-418. 3T3-L1 cells were infected with the plementary data for details. supernatant from MFG-Neo or -RORa4 infected cells, and G-418- resistant infected cells were used within three passages after infec- 2.8. Gene expression tion. See Supplementary data for details. Total RNA was extracted from tissues or cells and retro-tran- 2.4. Cell culture and differentiation scribed as described earlier [21]. Gene expression was quantified by quantitative RT-PCR on a Mx4000 apparatus (Stratagene, La Jol- 3T3-L1 cells (ATCC) and MFG-Neo or -RORa4 infected 3T3-L1 la, CA) using specific oligonucleotides as described in the Supple- cells were cultured and differentiated as previously described mentary data. mRNA levels were corrected to an internal control [21]. See Supplementary data for details. (cyclophilin) and expressed as mean ± S.D. 2.5. MEF isolation and culture 2.9. Western blotting Mouse embryonic fibroblasts (MEFs) were derived from 13.5- See Supplementary data. day wild-type and staggerer mouse embryos. Adipocyte differenti- ation was initiated after 2 days confluence. MEFs were differenti- ated with AmnioMAX-C100 medium (Invitrogen), 7.5% 2.10. Statistics AmnioMAX-C100 supplement, 7.5% fetal calf serum, 0.5 mM IBMX, 1 lM dexamethasone, 5 lM insulin. From day 3 to day 8, cells were All values are presented as mean ± S.D. Comparison between incubated with the same AmnioMAXC100 medium with 5 lM two groups was performed by student’s t-test. Comparisons among 12 18 A hRORα1 hRORα4 B mRORα1 16 10 mRORα4 14 8 12 10 6 / cyclophilin / cyclophilin 8 α α 4 6 (fold induction) (fold induction) 4 hROR 2 mROR 2 0 0 Subcutaneous Visceral Inguinal Epididymal 10.0 C RORα1 RORα4 7.5 5.0 / cyclophilin α (fold induction) 2.5 mROR 0 0 2468 Day of differentiation Fig. 1. RORa1 and RORa4 are expressed in adipose tissue and differentiating adipocytes. RORa1 and RORa4 expression in human (A) and mouse (B) adipose tissues, and in differentiating 3T3-L1 cells. (C) mRNA levels were corrected to an internal control (cyclophilin) and expressed as mean ± S.D. of two independent experiments each in triplicate. (A) and (B) RORa1 expression in human visceral and mouse epididymal adipose tissue was set as 1; (C) expression levels of RORa1 and RORa4 were expressed as fold induction of their respective expression level at day 0 set as 1 (absolute Ct values in Table S1). H. Duez et al. / FEBS Letters 583 (2009) 2031–2036 2033 A B C 60 *** Neo α 4 α ROR 4 α MFG-ROR 4 40 ** 4 Lys α / cyclophilin α 20 MFG-Neo MFG-ROR ROR MFG-Neo (fold induction) # # # mROR 0 Day 0 Day 8 Day of differentiation DEFG PPARγ CEBPα aP2 SREBP1c/ADD1 # # # 14 3000 2.5 1.5 1.5 # # # 4000 Day 0 12 Day 0 2 Day 0 # # # # # # 2500 2.0 1.0 10 1.0 ** 3000 2000 8 1 1.5 0.5 0.5 2000 1500 6 1.0 0 0 # # # 4 1000 0 1000 *** (fold induction) *** 0.5 *** 2 *** 500 0 0 Normalized mRNA levels 0 0 Day 0 Day 8 Day 0 Day 8 Day 0 Day 8 Day 0 Day 8 Fig. 2. RORa4 over-expression impairs differentiation of 3T3-L1 pre-adipocytes. RORa4 mRNA (A) and protein (B) levels in 3T3-L1 over-expressing RORa4 (MFG-RORa4) vs control cells (MFG-Neo). Oil-red-O staining (C) and mRNA levels of PPARc, CEBPa, aP2 and SREBP1c/ADD1 (D–G) upon differentiation. ÃDifference between Neo and RORa4 over-expressing cells; #difference between day 0 and day 8. Gene expression levels were normalized to an internal control (cyclophilin) and expressed as mean ± S.D. They are from two independent experiments, each realized in triplicate, and expressed as fold induction of the MFG-Neo cells at day 0, set as 1. A FA uptake B glucose uptake 700 350 600 Neo 300 500 RORα4 250 400 200 *** 300 150 Relative unit 200 100 100 *** dpm/mg prot. 50 0 0 CDE CD36 FATP Glut-4 2500 1.4 60000 1.2 2000 50000 1.0 40000 1500 0.8 30000 1000 0.6 0.4 *** 20000 (fold induction) 500 *** 0.2 10000 *** Normalized mRNA levels mRNA levels Normalized 0 0 0 FGH FAS ACC PEPCK 4.5 300 1000 4.0 900 3.5 250 800 700 3.0 200 600 2.5 150 500 2.0 400 1.5 100 300 1.0 (fold induction) *** 50 200 0.5 ** 100 *** 0 0 Normalized mRNA levels Normalized mRNA levels 0 Fig.
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