Diabetes Volume 66, December 2017 2987 RNA Binding Protein Ybx2 Regulates RNA Stability During Cold-Induced Brown Fat Activation Dan Xu,1,2 Shaohai Xu,3 Aung Maung Maung Kyaw,2 Yen Ching Lim,1 Sook Yoong Chia,2 Diana Teh Chee Siang,2 Juan R. Alvarez-Dominguez,4 Peng Chen,3 Melvin Khee-Shing Leow,5,6,7 and Lei Sun2,8 Diabetes 2017;66:2987–3000 | https://doi.org/10.2337/db17-0655 Recent years have seen an upsurge of interest in brown and inducible/beige adipocytes. Classic BAT is located as a adipose tissue (BAT) to combat the epidemic of obesity discernible depot in the interscapular region in small mam- and diabetes. How its development and activation are regu- mals and human infants. Beige/inducible adipocytes exist in lated at the posttranscriptional level, however, has yet to be defined anatomical white adipose tissue (WAT) depots, par- fully understood. RNA binding proteins (RBPs) lie in the cen- ticularly in subcutaneous WAT, and express a gene program ter of posttranscriptional regulation. To systemically study more like WAT at thermoneutrality. In response to pro- fi > OBESITY STUDIES the role of RBPs in BAT, we pro led 400 RBPs in different longed cold exposure, chronic treatment of b-adrenergic adipose depots and identified Y-box binding protein receptor agonist, or intensive exercise, the number of beige 2 (Ybx2) as a novel regulator in BAT activation. Knock- adipocytes dramatically increases, accompanied by en- down of Ybx2 blocks brown adipogenesis, whereas its overexpression promotes BAT marker expression in brown hanced Ucp1 levels and mitochondria biogenesis, a process “ ” and white adipocytes. Ybx2-knockout mice could form BAT known as browning (2,5,6). but failed to express a full thermogenic program. Integrative Understanding the detailed mechanisms underlying BAT analysis of RNA sequencing and RNA-immunoprecipitation differentiation and function is an area of immense research study revealed a set of Ybx2’s mRNA targets, including interest. A vast array of factors has been identified that Pgc1a, that were destabilized by Ybx2 depletion during regulate BAT development and activity by acting at the cold-induced activation. Thus, Ybx2 is a novel regulator transcriptional level (6–16). How these processes are regu- that controls BAT activation by regulating mRNA stability. lated at the posttranscriptional level, however, has yet to be fully understood. RNA binding proteins (RBPs) comprise a largeanddiversegroup(17,18)thatlieatthecenterof Obesity has reached an epidemic scale in many countries, posttranscriptional regulation by governing the fate of resulting in a steep escalation in health care expenditures mRNA transcripts from biogenesis, stabilization, and trans- and a growing burden of chronic obesity-related morbidities lation to RNA decay. Several RBPs have been reported to (1). An attractive approach to improve metabolic health is modulate adipocyte development and lipid metabolism. to augment the mass and activity of brown adipose tissue SFRS10 (splicing factor arginine/serine-rich10) inhibits li- (BAT) (2–7). There are at least two types of thermogenic pogenesis by controlling the alternative splicing of LPIN1, a adipocytes in mammals, namely, classic brown adipocytes key regulator in lipid metabolism (19,20). Sam68 (the 1School of Laboratory Medicine and Life Science, Wenzhou Medical University, Corresponding author: Dan Xu, [email protected], or Lei Sun, sun.lei@duke- Wenzhou, Zhejiang, China nus.edu.sg. 2 Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, Received 9 June 2017 and accepted 12 September 2017. Singapore This article contains Supplementary Data online at http://diabetes 3School of Chemical & Biomedical Engineering, Nanyang Technological Univer- .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0655/-/DC1. sity, Singapore 4Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, © 2017 by the American Diabetes Association. Readers may use this article as Harvard University, Cambridge, MA long as the work is properly cited, the use is educational and not for profit, and the 5Clinical Nutrition Research Centre, Singapore Institute for Clinical Sciences, work is not altered. More information is available at http://www.diabetesjournals Agency for Science, Technology and Research (A*STAR), Singapore .org/content/license. 6Department of Endocrinology, Tan Tock Seng Hospital, Singapore 7Office of Clinical Sciences, Duke-NUS Medical School, Singapore 8Institute of Molecular and Cell Biology, Singapore 2988 RBP Affects RNA Stability During BAT Activation Diabetes Volume 66, December 2017 Src-associated substrate during mitosis of 68 kDa) is re- Retrovirus Transduction quired for WAT adipogenesis by regulating mTOR alter- Amurinestemcellvirus(MSCV)–based retroviral vector native splicing (21). Knockout of KSRP (KH-type splicing (MSCV-pgkGFP-U3-U6P-Bbs vector) (28) was used to gen- regulatory protein) promotes browning of WAT by reducing erate short hairpin (sh)RNAs to infect preadipocytes; miR-150 expression (22). IGF2 mRNA binding protein 2 XZ201 vector (29) was used to overexpress Ybx2 for gain- (IGF2BP2) is a widely expressed RBP, and a single nucleo- of-function studies. All of the retroviruses were packaged in tide polymorphism in its intron is associated with type 2 293T cells with the pCL-eco packaging vector and then used diabetes by genome-wide association studies (23). Knockout to transduce preadipocytes in the presence of 4 mg/mL of IGF2BP2 results in resistance to diet-induced obesity, Polybrene (Sigma-Aldrich), followed by induction of differ- largely resulting from an enhanced translational efficiency entiation. FuGENE 6 Transfection Reagent (Promega) was of Ucp1 and other mitochondria mRNAs in the knockout used for plasmid transfection according to the manufac- BAT (24). Paraspeckle component 1 (PSPC1) was recently turer’s instructions. identified as an essential RBP for adipose differentiation RNA Immunoprecipitation in vitro and in vivo by regulating the export of adipogenic Primary brown and white adipocytes were infected with RNA from the nucleus to the cytosol (25). Despite these retroviral Ybx2 and differentiated for 4 days. RNA immu- advances, our understanding of RBPs in adipocytes, partic- noprecipitation (RIP) was performed using the Magna RIP ularly in brown adipocytes, is still at its early stage, and the kit (Merck Millipore) according to the manufacturer’sin- functions of most RBPs remain unknown. structions. RNA samples retrieved from anti-Ybx2 (Abcam) In this study, we systemically profiled 413 RBPs in and IgG control with the Magna RIP kit were used for RNA different fat depots, during white fat browning and brown sequencing (RNA-seq). adipogenesis, and identified 5 BAT-enriched RBPs. We dem- onstrated the role of Y-box binding protein 2 (Ybx2) in the RNA Pull-Down development and activation of BAT in vitro and in vivo, which RNA pull-down was performed according to our published could be, at least partially, explained by stabilizing mRNA. protocol with a few modifications(30).Inthisstudy,we used tissue lysate from mouse BAT instead of primary cell RESEARCH DESIGN AND METHODS culturepreparedasdescribedabove.Thetissuelysatewas prepared as described in RNA IMMUNOPRECIPITATION.Therestof Animal Studies the experiment followed our published protocol (30). The animal experimental protocols in this study were ap- proved by the Singapore SingHealth Research Facili- Extracellular Flux Analysis ties Institutional Animal Care and Use Committee. Ybx2 Primary brown preadipocytes were seeded in an X-24 cell heterozygous mice (NSA [CF-1] background) were originally culture plate, infected by retroviral constructs, as indicated imported from Dr. Paula Stein (University of Pennsylvania). in the text, followed by induction of differentiation. C57BL6 mice were obtained from The Jackson Laboratory Differentiated cells were analyzed by Extracellular Flux and subsequently bred in house. All mice were maintained Analyzer (Seahorse Bioscience) according to the manufac- at the animal vivarium at Duke-NUS Medical School. For turer’s instructions. Oxygen consumption rates were nor- cold challenge experiments, animals were housed individu- malized by protein concentration. ally in a 4°C chamber for 6 h. The rectal body temperature Animals were kept at 4°C for 6 h before experiments. was recorded with a probe thermometer (Advance Technol- BAT and skeletal muscle (gastrocnemius) were harvested ogy) at a constant depth. and minced with a micromincer (Glen Mills Inc.). The Glucose tolerance tests and insulin tolerance tests were minced tissue was kept in ice-chilled mitochondrial respira- performed as previously described (26), and EchoMRI was tion media (MiR05) (EGTA, 0.5 mmol/L; MgCl2$6H2O, used to measure fat and lean mass. For the in vivo insulin- 3 mmol/L; lactobionic acid, 60 mmol/L; taurine, 20 mmol/L; signaling study, Ybx2 knockout (KO) and wild-type (WT) KH2PO4, 10 mmol/L; HEPES, 20 mmol/L; D-sucrose, mice were fasted for 6 h at room temperature or 4°C. Then 110 mmol/L; and BSA, 1 g/L). Tissue lysate, 2 mg and the mice were injected with insulin (1 unit/kg body wt). 10 mg, respectively, was immediately loaded into Oroboros Mice were sacrificed after 5 min, and BAT was collected. Respirometry together with substrates, including glutamate Lipolysis assay was performed as previously described (26). (10mmol/L),malate(2mmol/L),pyruvate(5mmol/L),and ADP (5 mmol/L). The oxygen consumption rate (OCR) was Cell Culture monitored at basal level and when the samples