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RIP140 Represses the “Brown-In-White” Adipocyte Program Including a Futile Cycle of Triacyclglycerol Breakdown and Synthesis

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RIP140 Represses the “Brown-In-White” Adipocyte Program Including a Futile Cycle of Triacyclglycerol Breakdown and Synthesis Original citation: Kiskinis, Evangelos, Chatzeli, Lemonia, Curry, Edward, Kaforou, Myrsini, Frontini, Andrea, Cinti, Saverio, Montana, Giovanni, Parker, Malcolm G. and Christian, Mark. (2014) RIP140 represses the “Brown-in-White” adipocyte program including a futile cycle of triacyclglycerol breakdown and synthesis. Molecular Endocrinology, Volume 28 (Number 3). pp. 344-356. Permanent WRAP url: http://wrap.warwick.ac.uk/63874 Copyright and reuse: The Warwick Research Archive Portal (WRAP) makes this work of researchers of the University of Warwick available open access under the following conditions. This article is made available under the Creative Commons Attribution- 3.0 Unported (CC BY 3.0) license and may be reused according to the conditions of the license. For more details see http://creativecommons.org/licenses/by/3.0/ A note on versions: The version presented in WRAP is the published version, or, version of record, and may be cited as it appears here. For more information, please contact the WRAP Team at: [email protected] http://wrap.warwick.ac.uk/ ORIGINAL RESEARCH RIP140 Represses the “Brown-in-White” Adipocyte Program Including a Futile Cycle of Triacyclglycerol Breakdown and Synthesis Evangelos Kiskinis, Lemonia Chatzeli, Edward Curry, Myrsini Kaforou, Andrea Frontini, Saverio Cinti, Giovanni Montana, Malcolm G. Parker, and Mark Christian Department of Stem Cell and Regenerative Biology (E.K.), Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts 02138; Institute of Reproductive and Developmental Biology (L.C., E.C., M.G.P.), Faculty of Medicine, Imperial College London, W12 0NN, United Kingdom; Department of Mathematics (M.K., G.M.), Statistics Section, Imperial College London, London SW7 2AZ, United Kingdom; Department of Experimental and Clinical Medicine (A.F., S.C.), University of Ancona, (Politecnica delle Marche), 60126 Ancona, Italy; Division of Metabolic and Vascular Health (M.C.), Warwick Medical School, University of Warwick, Coventry, CV4 7AL, United Kingdom Receptor-interacting protein 140 (RIP140) is a corepressor of nuclear receptors that is highly expressed in adipose tissues. We investigated the role of RIP140 in conditionally immortal preadipocyte cell lines prepared from white or brown fat depots. In white adipocytes, a large set of brown fat-associated genes was up-regulated in the absence of RIP140. In contrast, a relatively minor role can be ascribed to RIP140 in the control of basal gene expression in differentiated brown adipocytes because significant changes were observed only in Ptgds and Fabp3. The minor role of RIP140 in brown adipocytes correlates with the similar histology and uncoupling protein 1 and CIDEA staining in knockout com- pared with wild-type brown adipose tissue (BAT). In contrast, RIP140 knockout sc white adipose tissue (WAT) shows increased numbers of multilocular adipocytes with elevated staining for uncoupling protein 1 and CIDEA. Furthermore in a white adipocyte cell line, the markers of BRITE adipocytes, Tbx1, CD137, Tmem26, Cited1, and Epsti1 were repressed in the presence of RIP140 as was Prdm16. Microar- ray analysis of wild-type and RIP140-knockout white fat revealed elevated expression of genes asso- ciated with cold-induced expression or high expression in BAT. A set of genes associated with a futile cycle of triacylglycerol breakdown and resynthesis and functional assays revealed that glycerol kinase and glycerol-3-phosphate dehydrogenase activity as well as [3H]glycerol incorporation were elevated in the absence of RIP140. Thus, RIP140 blocks the BRITE program in WAT, preventing the expression of brown fat genes and inhibiting a triacylglycerol futile cycle, with important implications for energy homeostasis. (Molecular Endocrinology 28: 344–356, 2014) here are two types of adipose tissue with opposing within the same depot, and their relative amount deter- Tfunctions: white adipose tissue (WAT) is associated mines the color of the depot (2). In mice, BAT is found with dynamic energy storage, whereas brown adipose tis- mainly in the interscapular depot whereas WAT is pre- sue (BAT) is associated with energy expenditure (1). dominant in all other depots. However, cellular compo- White and brown adipocytes are often interspersed sition can change depending on the genetic background, ISSN Print 0888-8809 ISSN Online 1944-9917 Abbreviations: BAT, brown adipose tissue; CoA, coenzyme A; ERR, estrogen related re- Printed in U.S.A. ceptor; GYK, glycerol kinase; NR, nuclear receptor; PGC, PPAR coactivator; PPAR, perox- This article has been published under the terms of the Creative Commons Attribution isome proliferator activated receptor; PPRE, PPAR response element; Q-RT-PCR, quanti- License (CC-BY), which permits unrestricted use, distribution, and reproduction in any tative real-time PCR; RIP140, receptor-interacting protein 140; TAG, triacylglycerol; WAT, medium, provided the original author and source are credited. Copyright for this article is white adipose tissue. retained by the author(s). Author(s) grant(s) the Endocrine Society the exclusive right to publish the article and identify itself as the original publisher. Received August 21, 2013. Accepted January 22, 2014. First Published Online January 30, 2014 344 mend.endojournals.org Mol Endocrinol, March 2014, 28(3):344–356 doi: 10.1210/me.2013-1254 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 November 2014. at 04:47 For personal use only. No other uses without permission. All rights reserved. doi: 10.1210/me.2013-1254 mend.endojournals.org 345 sex, age, environmental temperature, and nutritional Due to the critical role of RIP140 in the control of status (3). Chronic exposure to cold, treatment with energy homeostasis, elucidating its role in the regulation ␤3-adrenergic agonist, or overfeeding induces the devel- of WAT and BAT activity might provide new therapeutic opment of brown-like adipocytes (beige or BRITE adi- targets for obesity and diabetes. Here, we have found that pocytes) within WAT, mainly in subcutaneous (sc) depots RIP140 has only a minor role in gene regulation in brown (4). Although originally thought to be present only in adipocytes compared with its function in white adi- neonates, functional BAT has recently been identified in pocytes, where it repressed genes associated with energy adult humans (5). Because the thermogenic activity of utilization. Furthermore, a group of genes associated with BAT mass is associated with protection against overnu- a futile cycle of TAG breakdown and resynthesis is con- trition and glucose intolerance (6) it is important to char- trolled by RIP140, highlighting an additional mechanism acterize the regulators of the BAT-specific program. for heat generation in adipose tissues. Receptor-interacting protein 140 (RIP140) is a nuclear receptor (NR) coregulator highly expressed in metabolic and reproductive tissues (7). The physiological role of Materials and Methods RIP140 was indicated by the phenotype of RIP140-null mice, which are lean with a 70% reduction in total body fat Microarray analysis of gene expression mass and have higher oxygen consumption (8, 9). When Total RNA was isolated from 3 samples each of RIPKO-1 challenged with high-fat diet RIP140-knockout mice are re- and RIPKO-L cells after adipocyte differentiation (10 days). Two RIP140-lentivirus-transduced cell lines were used for pro- sistant to obesity and have increased insulin sensitivity and filing in order to avoid variation due to differences in the sites of glucose tolerance. They are also resistant to age- and diet- incorporation of the virus. For adipose tissue analysis, RNA was induced hepatic steatosis, indicating that they do not use extracted from sc WAT of wild-type and RIP140Ϫ/Ϫ 3-month- alternative fat depots. Importantly, RIP140-null mice have old male mice maintained on standard chow. Mice used in this elevated expression of Ucp1 and carnitine palmitoyltrans- study were backcrossed 8 generations to C57BL/6J background. ferase 1b (Cpt1b) in WAT, indicating that the lean pheno- All animal studies were carried out according to UK Home Office guidelines. type is due to altered energy balance (8). Affymetrix array hybridization and scanning were per- RIP140 was first identified as a cofactor that is re- formed by the CSC/IC Microarray Centre, Imperial College cruited to the estrogen receptor in presence of 17␤-estra- London, Hammersmith Campus, using Affymetrix MOE 430 diol (10). Subsequently, it was shown to interact with v2.0 GeneChips. For the array on RNA from the cell lines, data nearly all the NRs and despite its ligand-dependent re- were analyzed with d-CHIP software (16). The microarray data cruitment, it usually acts to suppress their activity (7). In are available at http://www.ebi.ac.uk/arrayexpress/ under ac- cession number E-MIMR-42. WAT and muscle, RIP140 interacts with key NRs such as For the array of wild-type and RIP140-knockout WAT ar- peroxisome proliferator-activated receptors (PPARs), ray, background correction, probe-level summarization and thyroid hormone receptors, and estrogen-related recep- normalization of raw data from Affymetrix CEL files were per- tor (ERR)␣ to suppress catabolic signaling pathways. formed across the full set of samples using Robust Multichip Expression analysis of differentiated adipocytes from Average (17) as implemented in the Affy package of Bioconduc- RIP140-null mouse embryo fibroblasts and from 3T3-L1 tor. Genes with a significant difference
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