KSRP Ablation Enhances Brown Fat Gene Program in White Adipose Tissue Through Reduced miR-150 Expression

Diabetes, Sep 2014

Brown adipose tissue oxidizes chemical energy for heat generation and energy expenditure. Promoting brown-like transformation in white adipose tissue (WAT) is a promising strategy for combating obesity. Here, we find that targeted deletion of KH-type splicing regulatory protein (KSRP), an RNA-binding protein that regulates gene expression at multiple levels, causes a reduction in body adiposity. The expression of brown fat–selective genes is increased in subcutaneous/inguinal WAT (iWAT) of Ksrp−/− mice because of the elevated expression of PR domain containing 16 and peroxisome proliferator–activated receptor gamma coactivator 1α, which are key regulators promoting the brown fat gene program. The expression of microRNA (miR)-150 in iWAT is decreased due to impaired primary miR-150 processing in the absence of KSRP. We show that miR-150 directly targets and represses Prdm16 and Ppargc1a, and that forced expression of miR-150 attenuates the elevated expression of brown fat genes caused by KSRP deletion. This study reveals the in vivo function of KSRP in controlling brown-like transformation of iWAT through post-transcriptional regulation of miR-150 expression.

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KSRP Ablation Enhances Brown Fat Gene Program in White Adipose Tissue Through Reduced miR-150 Expression

This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db13 KSRP Ablation Enhances Brown Fat Gene Program in White Adipose Tissue Through Reduced miR-150 Expression Chu-Fang Chou 2 Yi-Yu Lin 2 Hsu-Kun Wang 2 Xiaolin Zhu 1 Matteo Giovarelli 0 Paola Briata 0 Roberto Gherzi 0 W. Timothy Garvey 1 Ching-Yi Chen 2 0 Gene Expression Regulation Laboratory, Istituto Nazionale per la Ricerca sul Cancro , Genova , Italy 1 Department of Nutrition Sciences, University of Alabama at Birmingham , Birmingham, AL 2 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham , Birmingham, AL Brown adipose tissue oxidizes chemical energy for heat generation and energy expenditure. Promoting brownlike transformation in white adipose tissue (WAT) is a promising strategy for combating obesity. Here, we find that targeted deletion of KH-type splicing regulatory protein (KSRP), an RNA-binding protein that regulates gene expression at multiple levels, causes a reduction in body adiposity. The expression of brown fat-selective genes is increased in subcutaneous/inguinal WAT (iWAT) of Ksrp2/2 mice because of the elevated expression of PR domain containing 16 and peroxisome proliferatoractivated receptor gamma coactivator 1a, which are key regulators promoting the brown fat gene program. The expression of microRNA (miR)-150 in iWAT is decreased due to impaired primary miR-150 processing in the absence of KSRP. We show that miR-150 directly targets and represses Prdm16 and Ppargc1a, and that forced expression of miR-150 attenuates the elevated expression of brown fat genes caused by KSRP deletion. This study reveals the in vivo function of KSRP in controlling brown-like transformation of iWAT through posttranscriptional regulation of miR-150 expression. - WAT is the main storage site of excess energy, primarily in the form of triacylglycerol (TG). Conversely, BAT is specialized to dissipate chemical energy for heat generation primarily through uncoupling protein 1 (UCP1). UCP1 uncouples mitochondrial electron transport from ATP synthesis by permeabilizing the inner mitochondrial membrane to allow intermembrane protons to leak back into the mitochondrial matrix (1). The activity of BAT inversely correlates with BMI in adult humans (2,3). Promoting BAT function has a potential to defend against obesity and obesity-associated disorders (4). A distinct type of UCP1-positive adipocytes, designated as beige or brite (brown-in-white) cells, is found sporadically in WAT of adult animals upon exposure to long-term cold or b-adrenergic agonists (5–7). This brown-like transformation of WAT is most prominent in the inguinal subcutaneous depot, whereas epididymal/perigonadal adipose tissue is less susceptible to browning (8), the formation of multilocular UCP1-positive adipocytes. The emergence of these inducible brown-like adipocytes is associated with a protection against obesity and metabolic diseases in rodent models (8–11). Classic brown adipocytes develop from Myf5-positive precursors through the action of transcriptional regulators PR domain containing 16 (PRDM16) and CCAAT/enhancer binding protein-b (12,13). However, the inducible brownlike adipocytes arise from a non-Myf5 cell lineage. The browning of WAT in rodents can be induced by hormones such as irisin (14) and FGF12 (15), pharmacological activation of peroxisome proliferator–activated receptor (PPAR)-g (16), and modulation through various transcriptional regulators including PRDM16 (8), PPARGC1a/ PPARg coactivator 1a (17), forkhead box class C2 (9), receptor interacting protein 140 (10,18), transcription intermediary factor 2 (19), pRb, and p107 (20,21). Recently, several microRNAs (miRNAs) were shown to regulate brown fat differentiation and brown-like transformation of WAT. miRNA (miR)-133 inhibits brown adipocyte differentiation by directly repressing Prdm16 expression (22), and also regulates the choice between myogenic and brown adipose determination using multipotent adult skeletal muscle stem cells (satellite cells) that can give rise to both myogenic and brown adipogenic lineages (23). miR-196a is essential for brown adipogenesis of white fat progenitor cells by repressing the expression of Hoxc8 (24). miR-193b and miR-365 are essential for brown fat differentiation by repressing myogenesis as well as by promoting brown adipogenesis (25). miR-155 inhibits brown and beige adipocyte differentiation by repressing CCAAT/enhancer binding protein-b (26). KH-type splicing regulatory protein (KSRP) is a multifunctional RNA-binding protein involved in post-transcriptional regulation of gene expression. This includes splicing (27), mRNA decay (28), primary miRNA (pri-miRNA) processing (29), and translation (30). KSRP binds the AU-rich elements in the 39 untranslated regions (UTRs) of inherently unstable mRNAs and promotes their decay by recruiting mRNA decay machineries (28,31). KSRP also interacts with the terminal loops of a subset of miRNA precursors and promotes their maturation in cultured cells (29). However, the in vivo function of KSRP in controlling mRNA decay and pri-miRNA processing, and the associated phenotypes resulting from KSRP deficiency have not been completely established. To do this, we have generated Ksrp-null mice (32). In the current study, we report that Ksrp2/2 mice exhibit decreased fat mass owing to a reduction in TG content. The expression of brown fat–selective and fatty acid oxidation genes is increased in WAT in the absence of KSRP. We also find that high-fat feeding upregulates miR150 and KSRP in inguinal WAT (iWAT) and the deletion of KSRP significantly decreases adiposity in diet-induced obesity (DIO). Thus, these findings establish KSRP and miR-150 as important regulators for brown-like transformation of iWAT and whole-body adiposity. RESEARCH DESIGN AND METHODS Animal Studies The generation of Ksrp-null mice on a C57BL/6J congenic background has been described (32). Mice were maintained under a 12-h light/dark cycle at a room temperature of 22°C and were fed a normal chow diet (NCD) or a high-fat diet (HFD) containing 45% kcal from fat (Harlan Laboratories). All experiments were performed using 10- to 16-week-old, age-matched, wild-type and Ksrp2/2 male mice, which were littermate offspring of Ksrp+/2 3 Ksrp+/2. For metabolic analysis, including food intake, activity, and indirect calorimetry, mice were individually housed. Food intake and physical activity were measured using the Comprehensive Lab Animal Monitoring System. VO2 and VCO2 were measured by indirect calorimetry. Energy expenditure (EE) was calculated using the equation of EE = (3.941 3 VO2) + (1.106 3 VCO2) and normalized with respect to lean mass. Body fat content and lean mass were measured by dual-energy X-ray absorptiometry. All animal studies were conducted in accordance with guidelines for animal use and care established by the University of Alabama at Birmingham Animal Resource Program and the Institutional Animal Care and Use Committee. Measurement of Adipocyte Number, Adipocyte Size, and TG Content Adipocytes were isolated from epididymal and inguinal fat depots by collagenase digestion as described previously (33). The floating adipocytes were collected and washed with Krebs-Ringer HEPES buffer. Adipocyte number and size were determined as described previously (34). Briefly, aliquots of evenly suspended isolated adipocytes were removed for optical sizing of cell diameter with ImageJ (National Institutes of Health) using a micrometer ruler as a reference for the diameter and for determination of the volume occupied by packed adipocytes using microhematocrit capillary tubes. Adipocyte number was derived by dividing the volume occupied by packed adipocytes with the mean adipocyte volume obtained by optical sizing of the diameter. Lipids were extracted from adipocytes in 1% Triton X-100, and TG content was determined using Infinity reagent (Thermo Scientific). mRNA Analysis Total RNA was extracted by TRIzol (Invitrogen). For quantitative real-time RT-PCR analysis, total RNA (1 mg) was reverse transcribed using a mixture of oligo(dT) and random hexamers. Amplification was performed by using an LightCycler 480 and the SYBR Green system (Roche). mRNA levels were normalized to that of b-actin or cyclophilin B mRNAs. The primer sequences are listed in Supplementary Table 1. miRNA Microarrays and Analysis RNA was isolated from iWATs of six wild-type and six Ksrp2/2 mice by miRNeasy (Qiagen). Individual RNA of wild-type mice and individual RNA of Ksrp2/2 mice were pooled together, respectively, and subjected to genomewide miRNA microarray analysis in triplicate, which was performed by Phalanx Biotech. The miRNAs whose signals were fivefold above the background signal (these miRNAs are arbitrarily considered to be expressed in the iWAT) and were elevated by more than twofold in the Ksrp2/2 iWAT were selected for further analysis. For miRNA expression analysis, total RNA was converted into cDNA by miScript II RT Kit (Qiagen) and was subjected to real-time PCR with a specific primer for miR-150 using miScript Primer Assay (Qiagen). miRNA levels were normalized to that of U6 small nuclear RNA. pri-miRNA In Vitro Processing Assays pri-miRNA processing assays were performed as described previously (29,35). Briefly, a DNA template producing primiR-150 was generated by PCR. 32P-labeled pri-miR-150 was synthesized by in vitro transcription and incubated with total extracts of wild-type and Ksrp2/2 iWATs in a processing buffer containing 100 mmol/L KCH3COOH, 2 mmol/L Mg (CH3COOH)2, 10 mmol/L Tris-Cl (pH 7.6), 2 mmol/L dithiothreitol, 10 mmol/L creatine phosphate, 1 mg of creatine phosphokinase, 1 mmol/L ATP, 0.4 mmol/L guanosine triphosphate, 0.1 mmol/L spermine, and 2 units of RNasin at 37°C. RNA was isolated and subjected to 10% polyacrylamideurea gel electrophoresis and autoradiography. Ribonucleoprotein Immunoprecipitation Assays Ribonucleoprotein immunoprecipitation assays were performed as described previously (35,36). Briefly, cell lysates were immunoprecipitated with Dynabeads (Invitrogen) coated with protein A/protein G and coupled with antiKSRP serum at 4°C overnight. Pellets were washed four times, and RNA was isolated from the immunocomplexes using miRNeasy, reverse transcribed, and amplified by quantitative PCR (qPCR). Immunoblotting and Antibodies Cells or tissues were lysed in radioimmunoprecipitation assay buffer (0.5% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mmol/L NaCl, 50 mmol/L Tris-Cl, pH 7.5). Proteins were separated using 7–10% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed with the following antibodies: anti-PRDM16 (R&D Systems), anti-PPARGC1a (Abcam), anti-PPARa (Santa Cruz Biotechnology), anti-KSRP (37), anti-UCP1 (Abcam), anti-b-actin (Abcam), and anti-a-tubulin (Sigma-Aldrich). Immunohistochemistry of UCP1 Paraffin-embedded sections were incubated with antiUCP1 antibodies (1:500; Abcam), followed by detection using the Vectastain ABC Kit (Vector Laboratories). Isolation, Differentiation, and Transfection of StromalVascular Fraction Cells Stromal-vascular fraction (SVF) cells were prepared from iWAT as described previously (33), cultured in DMEM/ F12 containing 10% FBS, and induced for differentiation by incubating confluent cells with DMEM/F12 medium containing 10% FBS, 5 mg/mL insulin, 0.5 mmol/L isobutylmethylxanthine, 1 mmol/L dexamethasone, 1 nmol/L triiodothyronine (T3), 125 nmol/L indomethacin, and 1 mmol/L rosiglitazone for 48 h. Cells were maintained in maintenance medium containing 10% FBS, 1 nmol/L T3, and 1 mmol/L rosiglitazone. The medium was replaced every 2 days. For transfection of SVF cells, 80–90% of confluent cells were transfected with miRNA mimics (50 nmol/L), miRNA inhibitors (100 nmol/L), or small interfering RNAs (siRNAs) (60 nmol/L) using HiPerFect (Qiagen). The transfected cells were induced for differentiation the following day and collected for analysis 5 days postinduction. Measurement of Oxygen Consumption Rates SVF cells were seeded (15,000 cells/well) in XF24 culture microplates (Seahorse Bioscience) and induced for differentiation. At day 5 of differentiation, the oxygen consumption rate (OCR) was measured using an XF24 analyzer. Basal respiration was assessed in untreated cells, and uncoupled respiration was assessed after the addition of 10 mmol/L oligomycin. Nonmitochondrial respiration was measured after the addition of 4 mmol/L antimycin A and 1 mmol/L rotenone. Luciferase Reporters and Assays Dual luciferase reporters, expressing firefly luciferase containing the 39 UTRs of Prdm16 and Ppargc1a, and Renilla luciferase, were purchased from GeneCopoeia. A control reporter, pEZX-MT01, was also purchased from GeneCopoeia. The predicted miR-150 target sites were mutated by PCRmediated site-directed mutagenesis, and mutations were confirmed by DNA sequencing. NIH3T3 cells were transfected with 1 mg of a reporter together with an miRNA mimic (50 nmol/L) using Lipofectamine (Invitrogen). Cells were collected 36–48 h after transfection, and luciferase activities were measured using the Luc-Pair miR Lu ciferase Assay Kit (GeneCopoeia). Firefly luciferase activity was normalized to internal control Renilla luciferase activity. miRNA Mimics, miRNA Inhibitors, and siRNAs miR-150 mimic and inhibitor were purchased from Qiagen. siGENOME SMARTpool siRNAs against Prdm16 and Ppargc1a were purchased from Thermo Scientific. Control miRNA mimic and inhibitor and a control siRNA were purchased from Qiagen. Statistical Analysis All data are presented as the mean 6 SEM. Comparisons between two groups were performed using an unpaired two-tailed Student t test. One-way ANOVA, followed by Fisher least significant difference test, was used for multiple comparisons. Ksrp2/2 Mice Exhibit Reduced Adiposity Ksrp2/2 mice were lean with a 10% reduction in body weight in males eating a NCD compared with wild-type littermates (Fig. 1A) resulting from a reduction in the mass of distinct fat pads (Fig. 1B). A similar reduction in body weight and fat mass was also observed in female Ksrp2/2 mice (Supplementary Fig. 1A and B). By contrast, no significant difference in the weights of organs, such as liver, spleen, kidney, and heart, was detected between the two groups (Supplementary Fig. 1C). Consistent with these findings, body composition analysis showed a reduction in whole-body fat mass, but not in lean mass, in Ksrp2/2 mice (Supplementary Fig. 1D and E). Figure 1—KSRP ablation causes a reduction in body weight, fat pad weight, TG content, and adipocyte size, but not adipocyte number. A: Body weights of wild-type (n = 8) and Ksrp2/2 male mice (n = 8) from week 5 to week 12. B: Fat pad weights of 14-week-old wild-type (n = 8) and Ksrp2/2 (n = 8) male mice. mWAT, mesenteric WAT; prWAT, perirenal WAT. Number (C) and TG content (D) of adipocytes isolated from the eWAT and iWAT of 12-week-old wild-type (n = 4) and Ksrp2/2 (n = 4) mice. E: Hematoxylin-eosin staining of paraffin-embedded sections of the eWAT and iWAT of wild-type and Ksrp2/2 mice. F: Average diameters of adipocytes determined by optical sizing. Results were obtained from four wild-type and four Ksrp2/2 mice, and ;100 adipocytes/mouse. All data are represented as the mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Reduction in adipose tissue mass can result from less Ksrp2/2 mice exhibited a moderate increase in EE as meaTG storage, impaired adipocyte differentiation, or both. sured by indirect calorimetry during the dark period (SupWe isolated primary adipocytes from epididymal WAT plementary Fig. 1H). Collectively, these data suggest that (eWAT) and iWAT, and determined the adipocyte number the reduced adiposity in Ksrp2/2 mice results from a reducand total TG content. While no difference in adipocyte tion in TG content in WAT partly due to increased EE, but number was observed (Fig. 1C), TG content was signifi- not reduced food consumption or increased activity. cantly reduced in Ksrp2/2 eWAT and iWAT (Fig. 1D). Consistent with a decrease in TG content, adipocytes of Enhanced Expression of Brown Fat–Selective Genes in Ksrp2/2 eWAT and iWAT were smaller in size, as revealed Ksrp2/2 iWAT by histological analysis (Fig. 1E), and there was a 25% To determine the molecular mechanism leading to reduced decrease in the mean diameter of adipocytes in both the adiposity in Ksrp2/2 mice, we examined the expression of eWAT and iWAT of Ksrp2/2 mice (Fig. 1F). genes involved in adipocyte differentiation and lipid metab To further examine the cause of reduced fat mass, we olism in WAT. The expression of Pparg, but not of Cebpa, subjected wild-type and Ksrp2/2 mice to metabolic studies. was moderately elevated in Ksrp2/2 eWAT, and the expresNo significant difference in food consumption and locomo- sion of both Cebpa and Pparg was increased in Ksrp2/2 tor activity was observed between groups during the light iWAT (Supplementary Fig. 2A). There was no difference and dark periods (Supplementary Fig. 1F and G). However, in the expression of adipocyte makers such as fatty acid binding protein 4 (Fabp4) and perilipin 1 in either eWAT or and only selected miRNAs whose expression was reduced iWAT between the two groups (Supplementary Fig. 2A). We in the absence of KSRP. We identified only one miRNA, examined the expression of genes involved in the path- miR-150, whose expression was reduced by more than ways of TG synthesis (Gpam, Agpat2, Agpat6, and Dgat2), twofold in Ksrp2/2 iWAT (data not shown; see RESEARCH fatty acid synthesis (Fasn, Acaca/Acc1, and Scd1), and DESIGN AND METHODS). The expression of miR-150 was fatty acid uptake (Lpl, Cd36, and Fatp1). While no sig- higher in the iWAT than in the eWAT and BAT of wildnificant difference in their expression was observed in type mice, and there was a threefold reduction in Ksrp2/2 Ksrp2/2 eWAT, the expression of these genes, except for iWAT (Fig. 3A). Conversely, pri-miR-150 levels in Ksrp2/2 Dgat2, Lpl, Cd36, and Fatp1, was significantly increased iWAT were higher than that in wild-type iWAT (Fig. 3B), in Ksrp2/2 iWAT (Supplementary Fig. 2B–D). These suggesting that the reduction in miR-150 levels was likely results suggest that KSRP ablation increases the expres- due to impaired pri-mR-150 processing. To determine the sion of Cebpa, Pparg, and most of the genes involved in role of KSRP in pri-miR-150 processing, we performed TG synthesis and de novo fatty acid synthesis in iWAT. ribonucleoprotein immunoprecipitation assays and obHowever, the elevated expression of these genes is not served that KSRP physically associated with pri-miR-150 consistent with a reduction in TG content in the WAT of in wild-type iWAT extracts (Fig. 3C). In contrast, pri-miRKsrp2/2 mice. 34a was not associated with KSRP (Fig. 3C). The residual We also observed that genes selectively expressed in signals of pri-miR-34a in the anti-KSRP immunoprecipiBAT, including Ucp1, Cidea, Cox8b, and Dio2, were tates were likely due to a nonspecific interaction of antisignificantly upregulated in the iWAT of Ksrp2/2 mice KSRP serum with pri-miR-34a. We carried out in vitro (Fig. 2A). The fold increase in brown fat–selective genes pri-miRNA processing assays and found that production was much higher than that of TG synthesis and fatty of pre-miR-150 was significantly reduced using the cell lysate of acid synthesis genes (compare Fig. 2A with Supplemen- Ksrp2/2 iWAT (Fig. 3D). The addition of recombinant tary Fig. 2). The expression of some brown fat genes was KSRP to Ksrp2/2 lysate restored pre-miR-150 production also moderately increased in Ksrp2/2 eWAT, although (Fig. 3E). By contrast, processing of a control pri-miR-23b their expression was much less than that in iWAT (Fig. was equivalent between wild-type and Ksrp2/2 lysates 2A). In addition to brown fat markers, the expression of (Fig. 3F). These data indicate that decreased miR-150 genes encoding brown fat transcriptional regulators, in- expression in Ksrp2/2 iWAT is indeed due to a reduction cluding Ppargc1a and Ppara, but not Prdm16, was also in pri-miR-150 processing. elevated in the iWAT and eWAT of Ksrp2/2 mice (Fig. 2B). By contrast, no difference in the expression of these genes was observed between wild-type BAT and Ksrp2/2 BAT (Supplementary Fig. 3A and B). The lack of an alteration in brown fat gene expression in Ksrp2/2 BAT is likely due to a lower KSRP expression in BAT compared with iWAT (Supplementary Fig. 3C). Protein levels of PRDM16 (while Prdm16 mRNA was not elevated), PPARGC1a, and PPARa were significantly increased in Ksrp2/2 iWAT (Fig. 2C). Consistent with the elevated expression of PPARGC1a and PPARa, important regulators of fatty acid oxidation, the expression of Cpt1b, Acadm, and Acadl was also significantly increased in Ksrp2/2 iWAT and eWAT (Fig. 2D). We also detected elevated levels of UCP1 protein and increased numbers of UCP1-positive adipocytes in Ksrp2/2 iWAT, which contained clusters of multilocular cells (Supplementary Fig. 4A and B). Thus, the deletion of KSRP leads to brown-like transformation of iWAT that is characterized by the enhanced expression of brown fat–selective and fatty acid oxidation genes. Enhanced Brown Fat–Selective Gene Expression and Reduced miR-150 Expression in Differentiated SVF Cells of Ksrp2/2 iWAT To determine whether the increased brown-like transformation is cell autonomous, we isolated SVF cells of iWAT that are able to express brown fat–selective genes upon differentiation to adipocytes (8). After differentiation, the expression of Ucp1, Cidea, Cox8b, Ppargc1a, and Ppara, but not of Prdm16, was elevated in Ksrp2/2 SVF cells (Fig. 4A and B). By contrast, no difference in the expression of adipocyte markers common to white and brown adipocytes, such as Fabp4 and Adipoq, and a brown adipocyte–selective marker, Elovl3, was detected between wild-type and Ksrp2/2 SVF cells (Fig. 4C). The expression of PRDM16, PPARGC1a, and PPARa was elevated in differentiated Ksrp2/2 SVF cells (Fig. 4D). We examined miR-150 expression and observed a fivefold increase upon differentiation of wild-type SVF cells; we also observed that this increase was completely blunted in Ksrp2/2 SVF cells (Fig. 4E). Furthermore, pri-miR-150 levels were decreased in differentiated wild-type SVF cells, but those levels were not altered in differentiated Ksrp2/2 SVF cells compared with undifferentiated cells (Fig. 4E). These data indicate that the induction of miR150 upon adipocyte differentiation occurs primarily through pri-miR-150 processing due to an increase in KSRP levels (Fig. 4D), and that the elevated expression of brown fat–selective genes and decreased miR-150 Decreased miR-150 Expression in Ksrp2/2 iWAT Due to Impaired pri-mRNA Processing KSRP regulates the maturation of some miRNAs by facilitating pri-miRNA processing. To determine whether the impaired expression of miRNAs plays a role in enhancing brown fat gene expression in Ksrp2/2 iWAT, we subjected RNA samples to miRNA microarray analysis Figure 2—KSRP ablation elevates the expression of brown fat–selective genes, genes encoding brown fat transcriptional regulators, and genes involved in fatty acid oxidation in WAT. A: Expression of brown fat–selective genes, including Ucp1, Cidea, Cox8b, and Dio2, analyzed by qPCR in the eWAT and iWAT of wild-type (n = 8) and Ksrp2/2 (n = 8) mice. The expression level of each gene in wild-type eWAT was set at 1. B: Expression of genes encoding brown fat transcriptional regulators, including Prdm16, Ppargc1a, and Ppara, analyzed by qPCR in the eWAT and iWAT of wild-type (n = 8) and Ksrp2/2 (n = 8) mice. C: Immunoblot analysis of iWAT extracts of wild-type and Ksrp2/2 mice using antibodies against PRDM16, PPARGC1a, PPARa, KSRP, and a-tubulin. D: Expression of fatty acid oxidation genes, including Cpt1b, Acadm, and Acadl, analyzed by qPCR in the eWAT and iWAT of wild-type (n = 8) and Ksrp2/2 (n = 8) mice. All data are represented as the mean 6 SEM. *P < 0.05, **P < 0.01. expression in the absence of KSRP can be reproduced in adipocytes derived from SVF cells of iWAT. miR-150 Negatively Regulates Expression of Prdm16 and Ppargc1a and Mitochondrial Aspiration of Differentiated SVF Cells of Ksrp2/2 iWAT Target prediction analysis indicated that Prdm16 and Ppargc1a are targets of miR-150. To determine the molecular mechanism leading to enhanced expression of brown fat genes and whether it is due to a reduction in miR-150 expression, we manipulated miR-150 levels in SVF cells of iWAT. The inhibition of miR-150 expression in wild-type SVF cells increased mRNA levels of Ucp1, Cidea, Cox8b, Ppargc1a, and Ppara, but not of Prdm16 (Fig. 5A and B). Conversely, ectopic miR-150 expression in Ksrp2/2 SVF cells markedly repressed the expression of Ucp1, Cidea, Cox8b, Ppargc1a, and Ppara, but not of Prdm16 (Fig. 5A and B). By contrast, neither reduction nor overexpression of miR-150 changed the expression of Fabp4 and Adipoq (Fig. 5C). The inhibition of miR-150 expression in wildtype SVF cells moderately enhanced the expression of PRDM16 and PPARGC1a, and, conversely, the overexpression of miR-150 in Ksrp2/2 SVF cells caused a marked decrease in the protein levels (Fig. 5D). To correlate the brown fat gene program with mitochondrial functions, we examined mitochondrial aspiration in differentiated SVF cells. Ksrp2/2 adipocytes exhibited higher OCRs for basal and uncoupled (treated with oligomycin) aspirations, but not for nonmitochondrial aspiration (treated with antimycin A and rotenone), than wild-type adipocytes (Fig. 5E, left panel). Importantly, the treatment of Ksrp2/2 adipocytes with a miR-150 mimic decreased basal and uncoupled aspirations (Fig. 5E, right panel). These data indicate that KSRP deficiency increases mitochondrial respiration activity because of reduced miR-150 expression. To demonstrate that miR-150 can directly target Prdm16 and Ppargc1a mRNAs, we cloned their 39 UTRs into a dual luciferase reporter. We subcloned two overlapping fragments from each 39 UTR owing to their large size (.3.5 kb). Fragment (F) 2 of Prdm16 contains Figure 3—Decreased miR-150 expression in Ksrp2/2 iWAT resulting from impaired pri-miR-150 processing. A: miR-150 levels analyzed by qPCR in eWAT, iWAT, and BAT of wild-type (n = 8) and Ksrp2/2 (n = 8) mice. B: pri-miR-150 levels analyzed by qPCR in the iWAT of wildtype (n = 8) and Ksrp2/2 (n = 8) mice. All data are represented as the mean 6 SEM. *P < 0.05, **P < 0.01. C: Association of pri-miR-150 with KSRP. Extracts of wild-type and Ksrp2/2 iWATs were immunoprecipitated with a control serum or anti-KSRP serum and anti-KSRP serum, respectively. pri-miR-150 and pri-miR-34a transcripts in the precipitates were analyzed by qPCR. Data are represented as the mean 6 SEM of three independent experiments. **P < 0.01. IP, immunoprecipitation; P.I., preimmune serum. D: In vitro pri-miR-150 processing performed using extracts of wild-type or Ksrp2/2 iWAT. Extracts prepared from two different wild-type and Ksrp2/2 mice were used. E: In vitro pri-miR-150 processing performed using extracts of wild-type iWAT or Ksrp2/2 iWAT in the absence or presence of recombinant KSRP. F: In vitro pri-miR-23b processing performed using extracts of wild-type or Ksrp2/2 iWATs. a predicted miR-150 site, and both fragments (F1 and F2) derepression of Prdm16 and Ppargc1a through a decrease of Ppargc1a contain one predicted site (Fig. 6A). The ex- in miR-150 expression. pression of miR-150 decreased the expression of luciferase reporters harboring F2, but not F1, of Prdm16, and Downregulation of PRDM16 and PPARGC1a F2, but not F1, of Ppargc1a compared with a control Attenuates Expression of Brown Fat–Selective Genes miRNA (Fig. 6B). By contrast, miR-150 expression did To determine whether the increase in brown fat–selective not alter the expression of a control reporter (MT01) with- gene expression in the absence of KSRP is due to increased out an insertion (Fig. 6B). Mutations of the miR-150 target expression of PRDM16 and PPARGC1a, we downregulated sites in F2 of Prdm16 and F2 of Ppargc1a resisted inhibition their expression by siRNAs in SVF cells. The downregulaby miR-150 (Fig. 6B). These data indicate that miR-150 tion of PRDM16 in wild-type and Ksrp2/2 SVF cells dedirectly regulates the expression of PRDM16 and creased the expression of Ucp1, Cidea, Cox8b, Ppargc1a, and PPARGC1a, leading to changes in the expression of their Ppara. PPARGC1a knockdown decreased the expression downstream target genes, such as Ucp1, Cidea, Cox8b, and of Ucp1, Cidea, Cox8b, and Ppara, but not of Prdm16, in Ppara, and suggest that the absence of KSRP leads to wild-type and Ksrp2/2 SVF cells (Supplementary Fig. 5A Figure 4—Elevated expression of brown fat–selective genes in differentiated SVF cells of Ksrp2/2 iWAT. A: Wild-type and Ksrp2/2 iWAT SVF cells were induced for adipocyte differentiation. Expression of Ucp1, Cidea, and Cox8b was analyzed by qPCR before (d0) and 6 days (d6) after differentiation. B: Expression of Prdm16, Ppargc1a, and Ppara analyzed by qPCR in SVF cells at d0 and d6 after differentiation. C: Expression of Fabp4, Adipoq, and Elovl3 analyzed by qPCR in SVF cells at d0 and d6 after differentiation. D: Expression of PRDM16, PPARGC1a, PPARa, KSRP, and a-tubulin analyzed by immunoblotting in SVF cells at d0 and d6 after differentiation. E: Expression of miR150 and pri-miR-150 analyzed by qPCR in SVF cells at d0 and d6 after differentiation. All data are represented as the mean 6 SEM from three independent preparations of SVF cells. *P < 0.05, **P < 0.01. and B). By contrast, the downregulation of PRDM16 or PPARGC1a did not alter the expression of Fabp4, Adipoq, and Elovl3 (Supplementary Fig. 5C), indicating that adipocyte differentiation was not affected under these conditions. These data strongly indicate that the increase in the brown fat gene program in the absence of KSRP is indeed due to increased expression of PRDM16 and PPARGC1a. Elevated Expression of miR-150 and Ksrp in DietInduced Obesity We examined the expression of miR-150 and Ksrp in a model of DIO, and found elevated expression of miR150 and Ksrp in the iWAT of wild-type mice on an HFD and reduced miR-150 levels in HFD-fed Ksrp2/2 iWAT compared with HFD-fed wild-type iWAT (Fig. 7A). HFD feeding also increased mRNA levels of Prdm16, Ppargc1a, Fabp4, Ucp1, Cox8b, Cidea, Dio2, Cpt1b, and Acadl in wildtype iWAT (Fig. 7A–D), and levels of Ucp1, Cox8b, Cidea, Cpt1b, and Acadl were further upregulated in HFD-fed Ksrp2/2 mice (Fig. 7B–D). We also detected an increase in KSRP and PPARGC1a, but not of PRDM16, in wildtype mice fed an HFD compared with a NCD, and a further upregulation of PPARGC1a and PRDM16 in HFD-fed Ksrp2/2 mice (Fig. 7E). Since the mRNA levels of Prdm16 and Ppargc1a were not elevated in HFD-fed Ksrp2/2 mice compared with HFD-fed wild-type mice, we suggest that the increase in protein levels was likely due to increased translation through reduced miR-150 levels. More importantly, body weight and fad pad weights were reduced in HFD-fed Ksrp2/2 mice (Fig. 7F and G). These data suggest a model in which elevated Figure 5—miR-150 regulates brown fat–selective gene expression and mitochondrial aspiration. The expression of Ucp1, Cidea, and Cox8b (A); Prdm16, Ppargc1a, and Ppara (B); and Fabp4 and Adipoq (C) was analyzed by qPCR in wild-type SVF cells transfected with a control miRNA inhibitor or a miR-150 inhibitor, and Ksrp2/2 SVF cells were transfected with a control miRNA mimic or a miR-150 mimic after 5 days of differentiation. Data are represented as the mean 6 SEM from three independent preparations of SVF cells. **P < 0.01, ***P < 0.001. D: Expression of PRDM16, PPARGC1a, and a-tubulin analyzed by immunoblotting in wild-type SVF cells treated with a control miRNA inhibitor or an miR-150 inhibitor and in Ksrp2/2 SVF cells treated with a control miRNA mimic or an miR-150 mimic after 5 days of differentiation. Similar results were obtained using another preparation and transfection of wild-type and Ksrp2/2 SVF cells. E: OCRs in differentiated wild-type and Ksrp2/2 SVF cells (left panel) and differentiated Ksrp2/2 SVF cells transfected with a control miRNA mimic or an miR-150 mimic (right panel). OCRs from untreated cells and cells treated with oligomycin and with both antimycin A and rotenone (AA/Rot) were measured and normalized with protein concentrations. The data are represented as the mean 6 SEM from nine wells for each condition. **P < 0.01, ***P < 0.001. Figure 6—miR-150 directly represses Prdm16 and Ppargc1a. A: Schematic diagrams showing 39 UTRs of Prdm16 and Ppargc1a. Two overlapping fragments (F1 and F2) from each 39 UTR were subcloned into a dual luciferase reporter, pEZX-MT01. miR-150 target sites are indicated, and mutations in the seed motif are also denoted. B: Relative luciferase activities in NIH3T3 cells cotransfected with a control reporter (MT01) or reporter constructs containing F1 or F2 of Prdm16, mutated F2 of Prdm16 (F2M), F1 or F2 of Ppargc1a, or mutated F2 of Ppargc1a (F2M) and a control mimic or an miR-150 mimic. Data are represented as the mean 6 SEM from three independent experiments. **P < 0.01. expression of both KSRP and miR-150 is likely permissive for the development of obesity, and in which the absence of KSRP promotes a reduction in adiposity in DIO by enhancing brown-like transformation of iWAT through the increased expression of PPARGC1a and PRDM16. This study shows that targeted deletion of KSRP enhances brown fat–selective gene expression in iWAT through the elevated expression of PRDM16, PPARGC1a, and PPARa, which are important regulators for the thermogenic program in BAT and for brown-like remodeling in WAT. We demonstrated that KSRP is involved in the processing of pri-miR-150 in iWAT and its absence results in downregulation of miR-150. Mechanistically, miR-150 directly represses Prdm16 and Ppargc1a expression. Thus, reduction in miR-150 levels in Ksrp2/2 iWAT leads to elevated expression of PRDM16 and PPARGC1a, thereby increasing the brown fat gene program. In addition, we also found increased expression of fatty acid oxidation genes in Ksrp2/2 iWAT, likely due to the upregulation of PPARGC1a and PPARa, which are critical regulators for enhancing fatty acid use (38–40). The expression of brown fat–selective and fatty acid oxidation genes in the eWAT of Ksrp2/2 mice was also increased. By contrast, KSRP ablation did not alter the brown fat gene program in BAT, likely due to a lower expression of KSRP and miR-150 in BAT. These findings suggest that KSRP is a critical factor for balancing energy storage and EE in white adipocytes, and its absence favors EE, partly through post-transcriptional regulation of miR150 expression. Targeted deletion of miR-150 leads to B-cell expansion and an enhanced humoral immune response due to c-Myb upregulation (41). The body weight and fat mass of Mir1502/2 mice compared with wild-type mice were not reported. Consistent with the observation with HFD feeding (Fig. 7A), miR-150 was also reported to be upregulated in subcutaneous adipose tissue of obese human subjects (42). While Ppargc1a was predicted to be a target of miR150, its levels were not altered in obese adipose tissue (42). Using adipocytes derived from SVF cells of iWAT, we demonstrate that ectopic expression of miR-150 attenuates Prdm16, Ppargc1a, and Ppara expression, and, conversely, that the inhibition of miR-150 expression increases their expression. Although Prdm16 and Ppargc1a are directly targeted by miR-150, Ppara does not contain any predicted target site. The regulation of Ppara expression by miR-150 is likely indirect due to the altered expression of Prdm16 and Ppargc1a (see below). Our results point to miR-150 as a negative regulator of inducible brown-like adipocytes in iWAT by repressing Prdm16 and Ppargc1a. PRDM16 is a determinant of brown fat lineage and is able to induce browning of subcutaneous WAT (8,12,13). PPARGC1a is recognized as a critical regulator of thermogenesis and oxidative metabolism (43). PPARa is also critical for the expression of brown fat and fatty acid oxidation genes in BAT (44). We found that PRDM16 knockdown in SVF cells attenuated the expression of Ppargc1a, Ppara, and brown fat markers, and that PPARGC1a knockdown decreased the expression of Ppara, but not of Prdm16, and brown fat markers (Supplementary Fig. 5). These results strongly suggest that the enhanced brown-like transformation in Ksrp2/2 iWAT at least in part results from the elevated expression of Prdm16, Ppargc1a, and Ppara. These data are also consistent with previous studies showing that PRDM16 lines upstream of PPARGC1a and PPARa (8,25,45), and that PRDM16 and PPARGC1a are positive regulators for brown fat gene expression (8,17). While it was shown that PPARa activates Ppargc1a expression through coactivation by PRDM16 (46), our data suggest that PPARGC1a also regulates Ppara expression. Thus, there is a mutual regulation between PPARGC1a and PPARa. While Ksrp2/2 mice have reduced fat mass and increased EE, it will be of interest to see whether the enhanced brown fat gene program in iWAT is the sole contribution to these phenotypes. Using mice with both Figure 7—HFD feeding increases the expression of miR-150 and Ksrp, and KSRP ablation reduces adiposity in DIO. The expression of miR-150, pri-miR-150, Ksrp, Prdm16, Ppargc1a, and Fabp4 (A); Ucp1 and Cox8b (B); Cidea and Dio2 (C); and Cpt1b, Acadm, and Acadl (D) in wild-type mice (n = 8) fed a NCD and wild-type (n = 7) and Ksrp2/2 (n = 7) mice fed an HFD. E: Expression of KSRP, PPARGC1a, PRDM16, and b-actin in wild-type mice fed a NCD and wild-type and Ksrp2/2 mice fed an HFD. F: Body weights of wild-type (n = 10) and Ksrp2/2 male mice (n = 10) fed an HFD from week 5 to week 12. G: Fat pad weights of 14-week-old wild-type (n = 10) and Ksrp2/2 (n = 10) male mice fed an HFD. All data are represented as the mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. mWAT, mesenteric WAT; prWAT, perirenal WAT. KSRP ablation and adipose-specific overexpression of miR-150 should provide an answer. Furthermore, since our mouse model is a global KSRP knockout, other effects on lipid metabolism in metabolic tissues, such as muscle and liver, may also contribute to the observed leanness. Further studies using adipose-specific KSRP knock-out mice should reveal additional functions of KSRP in controlling whole-body adiposity and lipid metabolism. Nevertheless, our observations in Ksrp2/2 mice are consistent with a large body of previous studies (8–11,21,24,47–52) revealing that increasing the browning of WAT shows resistance to DIO and improved glucose metabolism. In summary, this work demonstrates the in vivo role of KSRP in post-transcriptional regulation of miRNA expression to control brown-like remodeling of iWAT, and suggests that modulation of KSRP-dependent pri-miR-150 processing could potentially lead to therapeutics for obesity and metabolic disorders. Acknowledgments. The authors thank Dr. Maria S. Johnson, University of Alabama at Birmingham, Animal Models Core, for conducting body composition analysis and indirect calorimetry; Dr. Martin Young, University of Alabama at Birmingham, for metabolic analysis using the Comprehensive Lab Animal Monitoring System; Dr. Chih-Hsuan Wang, Auburn University, for biostatistics analysis; and Dr. Douglas Moellering, University of Alabama at Birmingham-Comprehensive Diabetes Center, Core Diabetes Research Center Redox Biology Core (supported by National Institutes of Health grant P60 DK079626), for advice on and design of the measurement of oxygen consump2010-2306205) and the Association for International Cancer Research (#10-0527) to R.G., and by National Institutes of Health grant GM068758 (C.-Y.C.). Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. C.-F.C. helped to design and performed the experiments, analyzed the data, and wrote the manuscript. 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Chu-Fang Chou, Yi-Yu Lin, Hsu-Kun Wang, Xiaolin Zhu, Matteo Giovarelli, Paola Briata, Roberto Gherzi, W. Timothy Garvey, Ching-Yi Chen. KSRP Ablation Enhances Brown Fat Gene Program in White Adipose Tissue Through Reduced miR-150 Expression, Diabetes, 2014, 2949-2961, DOI: 10.2337/db13-1901