Insulin Receptor Substrate‐1 Inhibits High‐Fat Diet

Received: 30 December 2019 | Revised: 13 June 2020 | Accepted: 1 July 2020 DOI: 10.1096/fj.201903283RR

RESEARCH ARTICLE

Insulin receptor substrate-1 inhibits high-fat diet-induced obesity by browning of white adipose tissue through miR-503

1Hunan Provincial Key Laboratory For Metabolic Bone Diseases, Department of Abstract Endocrinology and Metabolism, National Genetic variation of insulin receptor substrate 1 (IRS-1) was found to modulate the Clinical Research Center for Metabolic insulin resistance of adipose tissues, but the underlying mechanism was not clear. Diseases, The Second Xiangya Hospital, Central South University, Changsha, China To investigate how the IRS-1 was involved in the browning of white adipose tissue −/− 2Department of Nephrology, The Affiliated through miRNA, we identified a mutated Irs-1 (Irs-1 ) mice model and found that Hospital of Qingdao University, Qingdao, this mice had a reduced subcutaneous WAT (sWAT) and increased brown adipose China tissue (BAT) in the interscapular region. So we isolated the bone marrow stromal

Correspondence cells and analyzed differentially expressed miRNAs and adipogenesis-related genes −/− Hou-De Zhou, Hunan Provincial Key with miRNA arrays and PCR arrays. Irs-1 mice showed decreased miR-503 ex- Laboratory For Metabolic Bone Diseases, Department of Endocrinology and pression, but increased expression of its target, bone morphogenetic protein recep- Metabolism, National Clinical Research tor type 1a (BMPR1a). Overexpression of miR-503 in preadipocytes downregulated Center for Metabolic Diseases, The Second BMPR1a and impaired adipogenic activity through the phosphotidylinositol 3-kinase Xiangya Hospital, Central South University, Irs-1−/− Changsha, Hunan, China. (PI3K/Akt) pathway, while the inhibitor had the opposite effect. In both and Email: [email protected] cold-induced models, sWAT exhibited BAT features, and showed tissue-specific increased BMPR1a expression, PI3K expression, and Akt phosphorylation. Thus, our Funding information NSFC | National Natural Science results showed that IRS-1 regulated brown preadipocyte differentiation and induced Foundation of China, Grant/Award Number: browning in sWAT through the miR-503-BMPR1a pathway, which played important 81770880, 81800788 and 81970762; roles in high-fat diet-induced obesity. Science and Technology Department of Hunan Province, Grant/Award Number: 2015JC3012 and 2018SK52511 KEYWORDS BMPR1a, brown adipogenesis, IRS-1, miR-503, PI3K/Akt pathway

Abbreviations: BAT, brown adipose tissue; BMPR1a, bone morphogenetic protein receptor type 1a; BMSCs, bone marrow stromal cells; C/EBPa, transcription factor CCAAT/enhancer-binding protein alpha; FABP4, fatty acid binding protein 4; H&E, hematoxylin and eosin; IRSs, insulin receptor substrates; miRNAs, micro-RNAs; PI3K, phosphatidylinositol 3-kinase; PPARγ, peroxisome proliferator-activated receptor-gamma; qPCR, quantitative PCR; rhBMP7, recombinant human bone morphogenetic protein-7; sWAT, subcutaneous white adipose tissue; UCP1, uncoupling protein 1; UTR, untranslated region; WT, wild type. Xiao-Fei Man and Nan Hu contributed equally to this study.

wileyonlinelibrary.com/journal/fsb2 | 1 The FASEB Journal. 2020;00:1–16. 2 | MAN et al. 1 | INTRODUCTION range of cell types including adipocytes, osteocytes, and so on. We have previously found that miR-503 inhibited white Obesity is characterized by increased adipose tissue mass, adipogenesis,15 and miR-342 regulated bone formation in a the expansion of adipose depots can be driven by adipocyte time-dependent fashion.17 Using a mice strain carried a trun- hypertrophy. Hypertrophic adipocytes experience hypoxia, cated mutation in Irs-1 gene, this study aimed to investigate inflammation, necrosis, and ultimately lead to adipose tissue how the IRS-1 was involved in the browning of adipose tis- dysfunction, with elevated lipolysis, increased inflammatory sue through miRNA. Mice homozygous for this mutation −/− cytokines secretion, and reduced secretion of anti-inflamma- (Irs-1 ) had brown-like subcutaneous white adipose tis- tory adipokines like leptin and adiponectin. These are inten- sue (sWAT) and increased BAT in the interscapular region sively related to various diseases such as insulin resistance, (iBAT), and were protected against high-fat diet-induced type 2 diabetes, cardiovascular disease, and cancer.1-5 In the obesity. Which might be resulted from the fact that IRS-1 development of obesity and related metabolic disorder, glu- regulated both brown preadipocyte differentiation and the cose tolerance is impaired, muscle, and adipose tissues be- browning of sWAT, via the miR-503-BMPR1a pathway. come resistant to insulin. Previous studies have indicated that a central feature of these defects was dysregulation in the expression and activation of insulin receptor substrates (IRSs). 2 | MATERIALS AND METHODS IRSs are key mediators in insulin signaling, and they play important roles in maintaining basic cellular functions, in- 2.1 | Animals cluding growth, differentiation, and metabolism.6,7 The first identified member in the IRS family, IRS-1, transduces the We purchased mice with a heterozygous spontaneous mu- insulin signal cascade by activating the phosphatidylinositol tation in Irs-1 gene (Jackson Laboratory, stock number: 3-kinase (PI3K) pathway and subsequent Akt/Foxo1 phos- 007240). These mice were mated to produce homozygous phorylation.8 Abnormal expression level of IRS-1 in human mice. The spontaneous mutation (C to A transition) in the Irs- SAT could be associated with increased risk of IR.9 1 gene resulted in an amino acid substitution, which produced At the cellular level, the development of insulin resis- a stop codon in place of serine at position 57 (S57X).18 Thus, tance arises from functional defects in IRSs expressed in the IRS-1 product was an N-terminally truncated peptide, this insulin-responsive cells, like adipocytes.10 Two functionally truncated 56-amino acid peptide could promote the brown and morphologically distinct types of adipocytes have been adipogenesis in vitro (PATENT NO: 201510004430.5). The described: the white adipocyte, which is the major compo- heterozygous cross produced progeny of three genotypes, −/− +/− nent of white adipose tissue (WAT); and the brown adipo- Irs-1 (homozygotes), Irs-1 (heterozygotes), and Irs- +/+ cyte, the main component of brown adipose tissue (BAT) and 1 (wild-type; WT). All protocols used in the mouse ex- beige adipose tissue. White adipocytes are the primary site periments were approved by the Animal Ethics Committee of of energy storage, which in excess leads to obesity and type the Central South University. Male littermates derived from 2 diabetes. Brown adipocytes oxidize substrates via the mito- the intercrosses were maintained in a temperature-controlled chondrial uncoupling protein 1 (UCP1), increase energy ex- room (22-24°C) with a 12/12-h light/dark cycle and had free penditure, and improve insulin sensitivity.11 Many factors are access to food and water until 4 weeks of age. Then, ho- involved in the network that regulates adipogenesis, includ- mozygotes (10.4 ± 1.2 g), heterozygotes (13.8 ± 1.3 g), and ing PPARγ and C/EBPs, which are central governing factors WT (14.6 ± 1.8 g), were, respectively, fed a standard chow in terminal-differentiation.12,13 IRS-1 is also regarded as an diet (n = 6) or a high-fat (HF) diet (n = 6) (composed of important role in adipogenesis. In cultured brown preadipo- 2.5% yolk, 20% sucrose, 10% lard, and 67.5% basic forage. cytes, it was expressed at relatively high level before and after GB1492413-2001, Guangzhou, China) for 3 months. We −/− differentiation into mature adipocytes.14 In our Irs-1 mice, then implemented glucose tolerance tests (GTTs) and insulin the white adipogenesis was inhibited.15 White preadipocytes tolerance tests (ITTs). Finally, we collected the retro-orbital from Irs-1 knockout mice exhibited severe defects in differ- blood sample to measure the free fatty acid in plasma by an entiation into mature fat cells and lipid accumulation, and enzymatic method (Ningbo Medicalsystem Biotechnology, showed significantly reduced expression of the adipogenic Zhejiang, China), and isolated sWAT from the inguinal re- markers, PPARγ, C/EBPa. The differentiation defect could gion and iBAT for further analyses. be nearly completely reversed by reintroducing expression of IRS-1, PPARγ, or C/EBPa. At the molecular level, these defects were caused by reduced insulin-stimulated PI3K activ- 2.2 | Glucose and insulin tolerance tests ity and subsequently reduced Akt/Foxo1 phosphorylation.16 Micro-RNAs (miRNAs) are small non-coding RNAs For GTT test, mice were remained unfed for 6-8 hours and that regulate the differentiation of stem cells into a broad then, administered with glucose (2 g/kg) via intraperitoneal MAN et al. | 3 injection. Small blood samples were taken by tail prick at 2.6 | Microarray analysis and 0, 15, 30, 60, 90, and 120 minutes relative to administration bioinformatics predictions for the measurement of blood glucose levels. For ITT test, mice were intraperitoneal injected with insulin (0.8 IU/kg) The 6th generation miRCURY LNA Array (v.16.0; Exiqon, following a 6 hours fast. At each time point after the adminis- Vedbaek, Denmark) was used to analyze BMSC RNA to −/− tration, Blood glucose levels were monitored by glucometer identify miRNAs that were expressed differentially in Irs-1 (Sinocare Inc, Changsha, China). and WT mice. We analyzed adipogenesis-related genes with a mouse adipogenesis PCR array, which displayed 84 key genes involved in adipocyte differentiation (SA Biosciences, 2.3 | Indirect calorimetry Frederick, MD, USA). RNA samples were hybridized to the arrays, and relative expression levels were calculated with the Mice (age = 90.3 ± 2.1 days) were placed in individual ΔΔCt (cycle threshold) method and reported as 2−ΔΔCt. After Nalgene cages equipped with wire lids. During a 72 h meas- normalization, differentially expressed miRNAs and genes urement period, mice were maintained in a SPF environ- were analyzed with Fold-Change filtering. Three prediction ment, with free access to standard chow diet, water, and algorithms (TargetScan, miRanda, and PicTar) were used to exercise wheels. Oxygen consumption (VO2), carbon dioxide identify the target genes of differentially expressed miRNAs. emission (VCO2), and food intake were measured with an Oxymax open-circuit indirect calorimetry system (Columbus Instruments, Columbus, USA). Energy expenditure was re- 2.7 | Histopathology and corded every 2 hours. immunohistochemistry

sWAT and iBAT were fixed, embedded in paraffin, and cut 2.4 | Enzyme-linked immunosorbent assay in 5 μm sections. Sections were placed on positively charged slides and stained with hematoxylin and eosin (H&E). For The retro-orbital blood sample were collected from mice of immunohistochemistry, sections were deparaffinized and three different genotypes at the age of 1 month, and then, treated with 0.25% trypsin for 30 minutes to retrieve anti- centrifuged at 3000 r/min for 10 minutes to get serum. gens. Endogenous peroxidase activity was blocked with 0.3% Serum insulin was detected using the commercial mouse H2O2. Then sections were incubated at 4°C overnight with insulin Enzyme-linked immunosorbent assay (ELISA) kit a rabbit polyclonal anti-UCP1 antibodies (ab10983; 1:150, (Cusabio, Wuhan, China) according to the instructions, Abcam, Cambridge, UK), and followed by incubation with a which utilized the quantitative sandwich enzyme immuno- secondary antibody. GTVision III Detection System/Mo&Rb assay technique. Adipose tissues from the inguinal region Kit (Gene Tech, Shanghai, China) was used to detect UCP1 of 1-month-old mice were isolated, flash freezed in liq- expression. Hematoxylin nuclear stain. Slides were analyzed uid nitrogen, and grounded to powder. Then tissues were under a 100 × oil objective with a Nikon E-400 fluorescence lysed in Tissue Protein Extraction Reagent (Thermo Fisher microscope. Scientific) in the presence of a protease inhibitor cocktail. Protein solutions were collected for PI3K activity detection by PI3-Kinase Activity ELISA kit (Echelon, Salt Lake City, 2.8 | Culture and differentiation of brown UT, USA). preadipocytes from three mouse genotypes

Interscapular BAT was removed from 2-day-old WT mice 2.5 | Bone marrow cell harvest and RNA and digested with 1 mg/mL collagenase. Digested tissues purification were filtered through a sterile 70 µm nylon mesh to remove red blood cells. Cells were cultured in Dulbecco's modified Bones were harvested from 1-month-old male mice. The essential medium-F12 nutrient mixture (DMEM/F12) con- ends of the bone were cut off, and the bone marrow was ex- taining 10% fetal bovine serum at 37℃ in a 5% CO2 atmos- pelled from the central channel with a jet of PBS from a sy- phere. To induce adipocyte differentiation, cells were plated ringe. The suspended bone marrow stromal cells (BMSCs) at 2.5 × 105/well in a 6-well plate, and were cultured in the were collected in polypropylene tubes and centrifuged for presence of 3.3 nM recombinant human bone morphogenetic 10 minutes. After discarding the supernatant, BMSCs were protein-7 (rhBMP7) for 3 days until they reached confluence washed twice with PBS. RNA was purified with TRIzol rea- (day 0). The cells were then induced to differentiate into gent (Invitrogen, Carlsbad, CA, USA). brown adipocytes with 20 nM insulin, 1 nM triiodothyronine 4 | MAN et al. (T3), 0.5 mM isobutylmethylxanthine (IBMX), 5 µM dexa- (28°C, controls), the other group (n = 5) was exposed to cold methasone, and 0.125 mM indomethacin for an additional temperature (4°C, cold-exposed), one mouse/cage. Mice 3 days (day 3). Then, cells were maintained in growth me- were maintained on a 12 hours/12 hours light/dark cycle with dium with 20 nM insulin and 1 nM T3, this medium was free access to water and food. After 7 days, iBAT and sWAT changed every 2nd day. were isolated and subjected to further analyses.

2.9 | Oil red O staining of brown adipocytes 2.13 | Real-time quantitative PCR analysis

Cells were washed twice in phosphate-buffered saline (PBS), RNA was extracted from sWAT, iBAT, and brown adipo- and fixed with 10% buffered formalin for 20 minutes, then cytes with TRIzol reagent according to the manufacturer's stained for 120 minutes at room temperature with filtered oil instructions (Thermo Fisher Scientific). For miRNA quan- red O (0.5% oil red O in isopropyl alcohol; Sigma, USA). tifications, 1.0 μg total RNA was reverse-transcribed with Cells were then washed and stored in PBS until examination a miRNA first-strand cDNA synthesis kit (Takara, Otsu, with an inverted microscope (Olympus). Japan). To measure target genes transcripts, total RNA was reverse transcribed with a reverse-transcription kit (Takara). Then, quantitative PCR (qPCR) was performed with SYBR 2.10 | Cell transfection in brown Green II Master Mix (Takara) and the LightCycler Real-time preadipocytes PCR system (Roche). U6 snRNA and β-actin were used as endogenous controls for miRNA and mRNA, respectively. Cell transfection was conducted as previously described.15 The sequences of all primers (Invitrogen, carlsbad, CA, USA; The transfection efficiency in brown preadipocytes was RiboBio, Guangzhou, China) were shown in Table S3. The about 75%. Mouse Bmpr1a-specific small, interfering RNAs cycling conditions were: 30 seconds polymerase activation at (mBmpr1a-siRNAs), or control scrambled siRNA (Qiagen, 95°C, followed by 40 cycles at 95°C for 5 seconds and 60°C West Sussex, UK), miR-503 mimic/negative control (NC), for 30-60 seconds. Relative miRNA and mRNA expression and miR-503 inhibitor/NC (Ribobio, Guangzhou, China) were was quantified with the comparative Ct method, and the fold- transfected into brown preadipocytes with Lipofectamine change was determined with the formula 2–ΔΔCt. 2000 (Invitrogen, Paisley, UK). After 6 hours of transfection, the medium was replaced with 3.3 nM rhBMP7 in complete medium for 72 hours, until the cells reached confluence (day 2.14 | Western blotting 0). Then these cells were induced to adipogenic differentiation with the methods mentioned above. Cells or tissues were lysed in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) in the presence of a protease inhibitor cocktail. The concentrations of 2.11 | Oxygen consumption rate analysis purified proteins were determined with the bicinchoninic acid protein assay (Thermo Fisher Scientific) using bovine Mitochondrial respiration assay was performed with the serum albumin (BSA) to standardize protein loading. The Seahorse Bioscience XF-24 analyzer (Seahorse Bioscience, lysates and protein markers (Thermo Fisher Scientific) USA). According to the manufacturer's protocol, cells were were separated on SDS-PAGE gels and transferred to incubated at a density of 2 × 104 cells/well in XF-24 micro- PVDF membranes (EMD–Millipore, Billerica, MA, USA). plates. Four wells/plate are left empty to correct for positional Membranes were blocked with 5% nonfat dry milk in Tris- temperature variations. Before the assay, cells were washed buffered saline-Tween (TBST) for 2 hours, and incubated twice with 1 mL un-buffered Seahorse assay DMEM and in- overnight at 4℃ with the following antibodies: anti-IRS1 cubated in a CO2-free incubator at 37°C for about 1 hour. The (ab52167; 1:1000), anti-BMPR1a (ab38560; 1:1000), anti- microplates were then loaded into the XF-24 Analyzer, and UCP1 (ab10983; 1:1000), anti-PGC1a (ab54481; 1:1000) the oxygen consumption rate (OCR) was measured. anti-LEPTIN (ab16227; 1:1000), and anti-FABP4 (ab92501; 1:1000) (Abcam, Cambridge, UK); anti-β-actin (48139; 1:1000, Signalway antibody, MD, USA); anti-AP2 (12802- 2.12 | Cold temperature exposure 1-AP; 1:5000, Proteintech, Wuhan, China); anti-PI3K experiments (#4292; 1:1000), anti-pAkt1 (#9271; 1:1000), anti-Akt1 (#9272; 1:1000), and anti-PPARγ (#2435; 1:1000) (Cell One-month-old C57BL/6 male mice were assigned into two Signaling Technology, Danvers, MA, USA). After wash- groups, one group (n = 5) was maintained at room temperature ing three times with TBST, the membrane was incubated MAN et al. | 5

−/− FIGURE 1 Phenotypic and metabolic changes in Irs-1 mice fed a chow-diet. Schematic overview of animal study (A). Body size (B) and −/− +/− +/+ average body weight changes over 16 weeks (C) of Irs-1 , Irs-1 , and Irs-1 littermates. GTT (D) and ITT (E) at 16 weeks. Serum insulin (F) at the age of 1 month. Respiratory quotient (G), oxygen consumption (H), activity test (I) and food intake (J) of 3-month-old mice with three different genotypes. sWAT (top row) and iBAT (bottom row) mass (K) as well as the percentage of fat to total body weight (L). H&E staining −/− +/− +/+ of sWAT and iBAT (M) in Irs-1 , Irs-1 and Irs-1 mice, Size bars indicate 200 μm. The percentage of brown-like cells (N) and their size distribution (O) in sWAT. n = 6 mice per genotype. *P ＜ .05 6 | MAN et al. (A) (B) (C)

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−/− +/− FIGURE 2 Irs-1 mice were protected against high-fat diet-induced obesity. High-fat diet-induced body weight increase in Irs-1 and +/+ −/− Irs-1 littermates, but not in Irs-1 mice (A,B). Body size (A), average body weight changes after 12 weeks of high-fat diets (B). Plasma free fatty acid (C). Expression levels of miR-503 and C/ebpa in sWAT (D, E). Protein expression of FABP4, LEPTIN, and PPARγ in sWAT of chow diet mice (F, G) and HFD mice (H, I). H&E staining of sWAT and iBAT (J), Size bars indicate 100 μm. *P ＜ .05 MAN et al. | 7 for 1 hour with peroxidase-conjugated secondary antibody remarkably reduced miR-503 and C/ebpa expression in sWAT (1:5000, Proteintech, Wuhan, China). Densitometric analy- compared to WT mice, the miR-503 and C/ebpa levels of ses were performed with ImageJ (NIH, USA). three different genotypes in HFD group were slightly higher than that in chow diet group (Figure 2D,E). The expression of FABP4, LEPTIN, and PPARγ were detectable in sWAT −/− 2.15 | Statistical analysis of Irs-1 mice in chow diet group, but their expression were reduced compared with that of wild type mice (Figure 2F,G). Quantitative data were expressed as the mean ± standard er- However, in HFD group, the expression of FABP4, LEPTIN, −/− rors of the means (SEM). Statistical differences between two and PPARγof Irs-1 mice were increased, and the PPARγ −/− means were assessed using Student's t test (unpaired, two- expression of Irs-1 mice was even higher than that of WT tailed). One-way analysis of variance (ANOVA) was used mice (Figure 2H,I). H&E staining showed that the HF diet to compare differences among multiple groups. The rate increased the sWAT area and the iBAT brown adipocyte cell −/− and constituent ratio were compared using chi-square test. size in WT mice, but not in Irs-1 mice, the morphology of +/− Statistical significance was defined as P < .05. adipose tissues in Irs-1 mice was between the two other genotypes (Figures 1M and 2J). Thus, we found a significant −/− reduction of sWAT and an increase of iBAT in Irs-1 mice, 3 | RESULTS which protected them from HF-induced obesity. 3.1 | Phenotypes and metabolic changes in Irs-1−/− mice fed a chow-diet 3.3 | Differentially expressed−/− adipogenesis genes and miRNAs in Irs-1 BMSCs −/− Less than one third of Irs-1 mice survived after birth. +/− −/− Compared to WT and Irs-1 mice, Irs-1 mice were slim- To elucidate the molecular mechanism underlying the signifi- −/− mer, their body weights were about two thirds that of WT cant differences of sWAT and iBAT between Irs-1 and WT mice (Figure 1B,C). GTT showed improved glucose toler- mice, we examined the expression of adipogenesis-related genes ance, ITT revealed insulin resistance, and serum insulin was and miRNAs in the three genotypes of mice. We found elevated −/− higher in Irs-1 mice compared with their heterozygotes and expression of 19 adipogenesis-related genes and reduced expres- −/− WT counterparts (Figure 1D-F). The respiratory quotient was sion of 13 genes in Irs-1 mice (Table S1). An analysis of −/− lower (Figure 1G), and oxygen consumption was increased miRNA expression in Irs-1 mice (Table S2) revealed 80 dif- −/− in Irs-1 mice (Figure 1H), whereas the activity (wheel ac- ferentially expressed miRNAs. We investigated the target genes cumulated) and food intake were not significantly different of all 80 differentially expressed miRNAs, and then, compared among three genotypes (Figure 1I,J). The sWAT weight, nor- the predicted target genes to the 32 differentially expressed genes −/− malized to body weight, was lower in Irs-1 mice, and the identified in the PCR array to explore the differentially expressed ratio of iBAT to body weight was higher, compared to WT miRNA/target gene pairs. We found three pairs of differentially +/− and Irs-1 mice (Figure 1K,L). Interestingly, H&E staining expressed miRNAs and their target genes: miR-143/Igf1r, miR- revealed significantly smaller adipocyte sizes in sWAT and 33/Irs2, and miR-503/Bmpr1a (Figure S1). Furthermore, we had −/− +/− iBAT of Irs-1 mice, and the cell phenotype in Irs-1 mice verified that miR-33 could regulate IRS-2 expression to com- −/− seemed to lie between the other two genotypes (Figure 1M). pensate for the insulin signal defects in Irs-1 mice,19 but there Apparently increased brown-like adipocytes were observed was no direct interaction between miR-143 and Igf1r by lucif- −/− in sWAT of Irs-1 mice (Figure 1N,O). erase activity analysis (Figure S2). However, we found miR-503 could specifically bind to the 3′-UTR of Bmpr1a and inhibits BMPR1a at the posttranscriptional level.15 Ultimately, we fo- −/− 3.2 Irs-1 mice are protected against HF cused on miR-503 and Bmpr1a. Microarray analysis showed | −/− diet-induced obesity that miR-503 was downregulated in BMSCs of Irs-1 mice, and its target gene Bmpr1a was upregulated. Based on the findings of increased iBAT and decreased sWAT −/− in Irs-1 mice, we tested the effect of a 3-month HF diet −/− −/− on the Irs-1 body weight and adipose histology. Both WT 3.4 Irs-1 mice show reduced miR- +/− | and Irs-1 mice exhibited higher total body weight than Irs- 503 and increased BMPR1a in sWAT and −/− 1 mice (Figure 2A,B). Plasma free fatty acid levels showed iBAT and increased phospho-Akt1 in iBAT no significant differences among the three genotypes of mice −/− when fed chow diet, but in HFD group, Irs-1 mice showed Next, we examined whether miR-503 and BMPR1a expres- a significantly low level of free fatty acid compared to WT sion were relevant to the increased iBAT and decreased sWAT −/− −/− mice (Figure 2C). Irs-1 mice undergoing HF diet showed observed in Irs-1 mice. We analyzed expression levels of 8 | MAN et al. (A) (B)

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−/− FIGURE 3 Downregulated miR-503 and upregulated BMPR1a in sWAT and iBAT from Irs-1 mice. sWAT and iBAT were dissected −/− +/− +/+ from 1-month-old Irs-1 , Irs-1 , and Irs-1 mice. Relative gene expression of miR-503, Bmpr1a, Ucp1, Pgc1a, and Cidea were measured with qPCR in sWAT (A) and iBAT (B). Western blot and the gray value analysis were used for detecting protein expression of BMPR1a, UCP1, PGC1a, p85α-PI3K, phospho-Akt1 (ser-473), and Akt1 in sWAT (C, D) and/or iBAT (E, F). PI3K activity measurement by ELISA (G). −/− +/− +/+ Immunohistochemistry (DAB) of tissue sections of sWAT (top) and iBAT (bottom) from Irs-1 , Irs1 , and Irs-1 mice showed UCP1 protein expression (brown stained) (H), Size bars indicate 100 μm. *P ＜ .05 miR-503, BMPR1a, and brown adipose markers in iBAT and confirmed that UCP1 expression was elevated both in iBAT sWAT from the three genotypes. Both iBAT and sWAT of and sWAT (Figure 3H). We also detected increased mRNA −/− Irs-1 mice showed downregulated miR503 (Figure 3A,B), levels of brown adipocyte markers such as Pgc1a and cell- −/− elevated mRNA (Figure 3A,B) and protein (Figure 3C-F) death activator (Cidea) in sWAT and iBAT of Irs-1 and Irs- +/− −/− levels of BMPR1a and UCP1. Immunohistochemistry results 1 mice (Figure 3A,B). Interestingly, Irs-1 mice showed MAN et al. | 9 a significant increase of PI3K level in iBAT and sWAT. adipogenesis (C/ebpa, Pparγ) were also clearly increased Besides, phospho-Akt up-regulations were detected in iBAT with maturation (Figure 4B,D,E). Ap2, a carrier protein for −/− +/− of Irs-1 and Irs-1 mice, despite the fact that both adipose fatty acids and an adipogenic differentiation marker, showed tissues showed increased BMPR1a expression (Figure 3C-G). a similar pattern (Figure 4C-E). All the fluctuations above, were consistent with what we have observed during white preadipocyte differentiation.15 Furthermore, brown adipo- 3.5 | Dynamic BMPR1a and miR-503 expression cyte markers (Ucp1, Pgc1a) were significantly increased during brown preadipocyte differentiation (Figure 4B,D,E).

Brown preadipocytes were induced for 7 days to differentiate into mature adipocytes with small lipid droplets (Figure 4A). 3.6 | Disruption of Bmpr1a expression Along with the lipid droplets development, BMPR1a ex- inhibits brown preadipocyte differentiation pression was significantly upregulated compared to day 0 (Figure 4B,D,E); conversely, miR-503 expression decreased We have previously validated that Bmpr1a interference in on days 3 and 7 (Figure 4B). Genes associated with general preadipocytes resulted in poor adipogenic activity.15 Here,

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FIGURE 4 Dynamic BMPR1a and miR-503 expression during brown preadipocyte differentiation. During the process of differentiation, oil red O staining (A) exhibited lipid droplets gradually accumulated, Size bars indicate 100 μm. QPCR analyses of genes and miRNA expression in adipogenesis process (B, C). Protein levels of BMPR1a and adipogenic-related markers during brown preadipocyte differentiation (D), quantification of protein expression (E). n = 3, *P ＜ .05 compared to Day 0 10 | MAN et al. we transfected brown preadipocytes with specific siRNA, to in Bmpr1a mRNA level was accompanied by down-expres- explore the effect of disrupted endogenous Bmpr1a expression of C/ebpa, Pparγ, Ucp1, Pgc1a, and Ap2 (Figure 5B). sion on brown preadipocyte differentiation. These cells had Accordingly, protein expression was decreased for UCP1 and significantly fewer lipid droplets compared to control group PGC1a in the Bmpr1a-siRNA group (Figure 5C,D). These and cells transfected with the NC (Figure 5A). The reduction results also indicated that Bmpr1a interference resulted in

(A) Control SiRNA NC Bmpr1a siRNA

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FIGURE 5 Bmpr1a disruption inhibited differentiation of brown preadipocytes. Brown preadipocytes were transfected with Bmpr1a-specific siRNA or negative control (NC). Oil red O staining of two groups (A), Size bars indicate 50 μm. mRNA expression of Bmpr1a and adipogenic- related markers at 72 hours after transfection (B). Western blots (C) and quantification of protein expression (D). n = 3, *P ＜ 0.05 compared to Control group MAN et al. | 11

(A) mimicNCmiR-503 mimic (B)

inhibitorNC miR-503 inhibitor

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FIGURE 6 MiR-503 negatively regulated brown preadipocyte differentiation by inhibiting BMPR1a expression. Brown preadipocytes were transfected with a miR-503 mimic, a miR-503 inhibitor, or corresponding negative controls (NC). Lipid droplets accumulation of the four groups (A), Size bars indicate 50 μm. (B,C) QPCR analyses of miR-503, Bmpr1a, Ucp1, Pgc1a, Ap2, C/ebpa, and Pparγ at 72 hours after transfection with a miR-503 mimic (B), a miR-503 inhibitor (C), or corresponding controls (NC). Western blots and gray values of whole cell lysates of brown adipocytes on day 3 after induction, β-actin was the internal control (D,E). n = 3, *P ＜ .05. Oxygen consumption rate (OCR) of brown preadipocytes transfected with miR-503 mimics, inhibitors, or corresponding NC for 32 hours, oxygen consumption was normalized to protein content (F). Schematic diagram summarizing the novel mechanism for IRS-1 deficiency leading to the browning of WAT and the differentiation of preadipocytes (G) 12 | MAN et al. lower adipogenic potential. In addition, PI3K and phospho- 4 | DISCUSSION Akt1 at ser-473 were downregulated by silencing Bmpr1a (Figure 5C,D). Insulin signaling pathway plays important roles in insulin resistance, diabetes, and obesity. It has been reported that mice with global knockout of the insulin receptor exhibit 3.7 | MiR-503 negatively regulates brown slight growth retardation.21 However, mice with a knock- preadipocyte differentiation by inhibiting out of the insulin receptor specifically in BAT are initially BMPR1a expression born with decreased BAT mass.22 Irs-1, as the down-stream of insulin receptor, a completely deficiency results in insulin It was previously reported that miR-503 could negatively resistance and reduced body weight.23 Our Irs-1 spontane- regulate insulin/PI3K-Akt signaling in a mouse model of ously mutated mice remaining 56 amino acids on N-terminus HF-diet-induced type 2 diabetes.20 We further investigated showed insulin resistance, even though the Adipoq, which the role of miR-503 in adipogenesis by targeting BMPR1a played a crucial role in protecting against insulin resistance, and examining the PI3K/Akt signaling pathway. We trans- was upregulated. Studies had shown that the cross-talk be- fected brown preadipocytes with a murine miR-503 mimic, a tween adiponectin and insulin relies on APPL1. As a signal- miR-503 inhibitor, or corresponding controls. We found that ing pathway mediator, it binds to the adiponectin receptors, the miR-503 mimic significantly reduced, and the miR-503 positively mediates adiponectin signaling, and then, interacts −/− inhibitor increased lipid droplet formation in induced adipo- directly with IRSs to activate insulin signaling. In Irs-1 cytes (Figure 6A). Furthermore, the miR-503 mimic caused mice, interaction between the two pathways was interrupted, significant reductions, and the miR-503 inhibitor caused sig- thus elevated adiponectin level could not improve insulin re- −/− nificant increases in mRNA expression of Bmpr1a, C/ebpa, sistance.24 The Irs-1 mice also had a lean phenotype and Pparγ, Ucp1, Pgc1a, and Ap2 (Figure 6B,C). The protein reduced WAT mass. Interestingly, these mice exhibited in- level of BMPR1a and adipogenic marker UCP1, showed the creased BAT mass, moreover, the sWAT exhibited profound same pattern (Figure 6D,E). Moreover, PI3K and phospho- morphological features of the BAT phenotype. Brown adi- Akt1 at ser-473 were inhibited by the miR-503 mimic and pocytes have a relatively high mitochondrial content, where activated by the miR-503 inhibitor (Figure 6D,E). In addi- the thermogenesis occurring is fueled by the energy stored tion, miR-503 inhibitor obviously increased OCR of brown in WAT.25 Thus, it could be an effective way to consume −/− preadipocytes. These results indicated that miR-503 inhib- excess fat. Accordingly, our Irs-1 mice had significantly ited brown adipogenesis by targeting BMPR1a and the PI3K/ lower respiration quotient and higher oxygen consumption, Akt signaling pathway, which suggested a new pathway that which might result from high level of lipid consumption IRS-1 deficiency leads to the browning of WAT and the dif- and active mitochondrial biogenesis. As brown adipocytes ferentiation of brown preadipocytes (Figure 6G). play a natural anti-obesity role, the brown adipogenesis in BAT and induced brown-like adipocytes in WAT might be expected to prevent or alleviate obesity. Based on the find- −/− 3.8 | Cold exposure promotes BMPR1a ings, we fed Irs-1 mice with HF diet for 3 months, as we expression and the browning of sWAT expected, Irs-1 knockout protected adipocyte from hypertrophy both in WAT and BAT, and protected the mice against To provide further evidence on the roles of miR-503 and HF diet-induced obesity. Numerous studies confirmed the BMPR1a in the browning of WAT, we examined their ex- vital role of IRS-1 in adipogenesis and lipid metabolism. To −/− pression in mice maintained in a 28°C or 4°C environment verify whether our Irs-1 mice were undergoing lipodys- for 7 days. H&E staining of sWAT showed that cold expo- trophy, we evaluated the adipogenic factors such as PPARγ, sure induced unilocular white adipocytes to switch to multi- as well as fatty acid binding protein 4 (FABP4, also known locular brown adipocytes, but no obvious histological change as AP2), an essential protein for mature adipocyte formation, was observed in brown adipocytes from iBAT (Figure 7A). along with adipokine like LEPTIN. Although the sWAT of −/− Cold exposure caused significant reduction in miR-503 ex- Irs-1 mice had the reduced proteins expression due to pression both in sWAT (Figure 7D) and iBAT (Figure 7E). IRS-1 truncation, they restored the functions in response to Additionally, cold exposure caused significant increases of HF diet. Besides, free fatty acid levels in plasma showed no Bmpr1a, Ucp1, Pgc1a, and Cidea mRNA expression in the significant differences among the three genotypes of mice −/− two adipose tissues (Figure 7D,E). Cold exposure signifi- fed with chow diet, and in HFD group, Irs-1 mice showed cantly increased BMPR1a, PI3K, and phospho-Akt1 protein a significantly low level of free fatty acid compared to WT −/− expression in sWAT, and tended to upregulate the protein lev- mice. All these results indicated that Irs1 mice were not els of BMPR1a, PI3K, and phospho-Akt1 in iBAT, although under lipodystrophic condition, and the anti-obesity role of the results were not statistically significant (Figure 7B,C). truncated IRS-1 was not contributed from lipodystrophy. MAN et al. | 13

(A) (B)

UCP1 sWAT BMPR1a

-actin

PI3K

p-Akt1

iBAT Akt1

-actin

(E)

FIGURE 7 Cold exposure promoted BMPR1a expression and the browning of sWAT in mice. C57BL/6 mice were maintained at room temperature (28°C) or exposed to cold (4°C) for 1 week. H&E staining of sWAT and iBAT after exposure to 4°C or 28°C (A), Size bars indicate 100 μm. Protein levels of UCP1, BMPR1a, p85α-PI3K, phospho-Akt1 (ser-473), and Akt1 in sWAT and iBAT were assessed by western blot (B) and quantified using ImageJ (C). (D,E) QPCR analyses of miR-503, Bmpr1a, and differentiation markers in sWAT (D) and iBAT (E). *P < .05

However, the exact molecular mechanism that gave rise to differentially expressed adipogenic genes and miRNAs in −/− −/− the Irs-1 phenotype was previously unknown. Irs-1 BMSCs, and identified 32 genes and 80 miRNAs Adipocytes are derived from MSCs, which first differ- (Tables S1 and S2). Upregulated genes such as Bmp4, Bmp7, entiate into preadipocytes, then into mature adipocytes. Wnt5a, Igf1R, and Ucp1 were reported to promote preadi- Preadipocyte differentiation requires many adipogenic cyto- pocyte differentiation,25-28 but Dlk-1 was a well-known ad- kines and regulatory miRNAs.25,26 Therefore, we analyzed ipogenic inhibitor.29 Downregulated genes, Klf15, Klf4, 14 | MAN et al. and Prdm16 were recognized as adipogenic promoters and active BMPR1a-induced adipocyte formation in 2T3 cells.35 Wnt10b was an inhibitory factor.25,26,30 miRNAs such as miR- Moreover, a BMPR1a antagonist suppressed adipogenesis −/− 193b, miR-365, and miR-711 were upregulated in Irs-1 and differentiation in BMSCs and reduced the expression of mice. Both miR-193b and miR-365 were reported to promote key adipogenic transcription factors.12 Conversely, another brown preadipocyte differentiation by inhibiting Runx1t1 study showed that BMPR1a increased osteoblast differen- expression.31 Among the 46 downregulated miRNAs, miR- tiation, but repressed preadipocyte differentiation.36 These 503 ranked the 13th, and among the differentially expressed discrepancies may have been due to the differences in cell genes, we surprisingly found that Bmpr1a was dramatically lines, induction conditions, and/or experimental designs. In −/− up-expressed in Irs-1 BMSCs, showing a trend contrary to the present study, we used primary cultured brown preadi- that of miR-503. So we first confirmed Bmpr1a was the tar- pocytes. Our results showed that BMPR1a was notably in- get gene of miR-503, and they played important roles in white creased during adipogenesis, together with an increase in adipogenesis.15 Liu et al had also confirmed the positive role brown adipocyte markers, interrupting Bmpr1a expression of BMPR1a in adipogenesis.32 The similar expression pattern resulted in reduced adipogenic activity and adipogenic mark- was also observed during preadipocyte differentiation in our ers, PPARγ and PGC1a, indicating that BMPR1a played a previously study.15 role in promoting the differentiation and maturation of brown To clarify whether the downregulation of miR-503 and preadipocytes. It was in accordance with the previous study upregulation of BMPR1a were associated with the phenotype by Schulz et al, that genetic ablation of Bmpr1a in brown −/− alteration in Irs-1 mice, BMPR1a and miR-503 expres- adipogenic progenitor cells led to a severe paucity of iBAT.37 sion in iBAT and sWAT of three different genotypes were In summary, the reduction of miR-503 enhanced BMPR1a detected, along with the brown adipose markers. BMPR1a expression and promoted the differentiation of brown pread- expression was increased, and miR-503 expression was de- ipocytes, which strongly supported our hypothesis that the −/− creased in Irs-1 sWAT. As white adipocytes transformed stimulation of brown adipogenesis might be one of the mech- −/− into small lipid morphology characteristic of brown adipo- anisms of the BAT mass gain in Irs-1 mice. −/− cytes, Irs-1 sWAT showed increased expression of UCP1, It is known that IRS-1-related adipogenesis is mediated which is responsible for the main function of BAT and is a by the PI3K/AKT pathway, which is upstream of transcrip- representative marker of brown adipocytes.33 These findings tional regulators, PPARγ and C/EBPa.16,38,39 However, in the suggested that miR-503-BMPR1a pathway may participate in present study, sWAT and iBAT from knockout mice exhibited −/− the browning of sWAT in Irs-1 mice. Except for increased up-expression of PI3K and Akt phosphorylation even with −/− mass, Irs-1 iBAT showed elevated activity, as evidenced by Irs-1 deficiency, which suggested that there exist a compensa- the markedly up-expressed brown adipocyte-specific mark- tion in canonical insulin pathway for Irs-1 deficiency, such as ers, UCP1 and PGC1a. Meanwhile, upregulated BMPR1a IRS-2/IRS-3 routes.6,19 The PI3K/AKT pathway was reported and downregulated miR-503 were also detected in iBAT of to possibly have a pivotal role in brown adipogenesis,39,40 and −/− Irs-1 mice. These results indicated that the reduced miR- was also an important signing pathway in browning of white 503 and elevated BMPR1a might promote the activity of adipocytes.40 Thus, activation of the pathway ultimately in- −/− BAT in Irs-1 mice. Induced WAT browning and activated duced adipose browning (including an increase of brown BAT increase energy consumption and non-shivering ther- adipocytes differentiation in BAT and transformation of mogenesis, thus inhibit weight gain.34 It explained why our brown-like adipocytes in WAT). Additionally, we found that −/− Irs-1 mice could be protected against high-fat diet-induced PI3K and Akt phosphorylation were blocked by a miR-503 obesity. mimic and Bmpr1a siRNA, and activated by miR-503 inhibi- We further elucidated the roles of miR-503 and BMPR1a tors and elevated BMPR1a in vitro, suggesting that BMPR1a in brown preadipocyte differentiation. During brown adipo- regulate adipose browning through PI3K/AKT pathway −/− genesis, miR-503 expression was inhibited. Furthermore, under the condition of Irs-1 knockout. Up to now, only a miR-503 overexpression reduced the differentiation potential handful of studies have focused on BMP/BMPR axis effect and lipid droplet formation. In contrast, miR-503 inhibition on PI3K/AKT pathway,41 crosstalk between insulin and BMP caused significantly more fat accumulation and upregula- signing in adipose browning remains to be investigated. tion of adipogenic markers, such as PGC1a and UCP1. In One of strong inducer of WAT browning is cold expo- addition, we evaluated the effect of miR-503 inhibitor/mimic sure.34,40 To verify the roles of miR-503, BMPR1a, and on OCR in brown preadipocytes, as we expected, miR-503 PI3K/AKT pathway in the browning of WAT, we treated inhibitor significantly increased cellular oxygen consump- WT mice with cold exposure. Cold exposure promoted the tion. These results showed that miR-503 negatively regu- browning of sWAT with UCP1 expressed at a higher level, lated brown adipogenesis. However, previous studies on the it also promoted BMPR1a expression but blocked miR-503 target gene of miR-503, Bmpr1a, reported conflicting roles expression. Moreover, PI3K/AKT pathway was active in in preadipocyte differentiation. Expression of constitutively sWAT under cold exposure. It has been reported that the MAN et al. | 15 PI3K/AKT signaling cascade exhibited a tissue-specific 2. Ortega FB, Lavie CJ, Blair SN. Obesity and cardiovascular disease. Circ Res pattern of regulation in WAT and BAT, which resulted in . 2016;118:1752-1770. 3. Malvi P, Chaube B, Singh SV, et al. Elevated circulatory levels the different responses to cold exposure.42 Similarly, Irs-1 of leptin and resistin impair therapeutic efficacy of dacarbazine in ablation could trigger different compensatory mechanisms melanoma under obese state. Cancer Metab. 2018;6:2. https://doi. affecting the development of WAT and BAT, inducing the org/10.1186/s40170-018-0176-5. contrary phenotype of the two adipose tissues. That is, in 4. Malvi P, Chaube B, Singh SV, et al. Weight control interventions -/- iBAT from Irs-1 mice, increased BMPR1a upregulated improve therapeutic efficacy of dacarbazine in melanoma by re- the Akt activity, whereas, in sWAT, the overexpression versing obesity-induced drug resistance. Cancer Metab. 2016;4:21. of BMPR1a could not completely compensate for the Akt https://doi.org/10.1186/s40170-018-0176-5. deficiency arising from the impaired standard canonical 5. Malvi P, Chaube B, Pandey V, et al. Obesity induced rapid mela- +/− noma progression is reversed by orlistat treatment and dietary in- pathway. In addition, for the half mutation in Irs-1 mice, tervention: role of adipokines. Mol Oncol. 2015;9:689-703. iBAT tended to have increased mRNAs of brown adipo- 6. Kubota T, Kubota N, Kadowaki T. Imbalanced insulin actions in cyte-specific markers compared to WT mice, which was obesity and type 2 diabetes: key mouse models of insulin signaling also consistent with the moderate cell size between the pathway. Cell Metab. 2017;25:797-810. other two genotypes. 7. Lavin DP, White MF, Brazil DP. IRS proteins and diabetic compli- In conclusion, our present study indicated that Irs-1 de- cations. Diabetologia. 2016;59:2280-2291. pletion led to downregulation of miR-503, which increased 8. Castorena CM, Arias EB, Sharma N, Cartee GD. Postexercise im- provement in insulin-stimulated glucose uptake occurs concomi- the expression of its target, Bmpr1a, in brown preadipocytes. tant with greater AS160 phosphorylation in muscle from normal BMPR1a contributed to brown preadipocyte differentiation, and insulin-resistant rats. Diabetes. 2014;63:2297-2308. which led to increased BAT mass and browning of sWAT −/− 9. Chen Z, Yu H, Shi X, et al. Functional screening of candidate in Irs-1 mice. The mechanism of BMPR1a action was to causal genes for insulin resistance in human preadipocytes and ad- increase PI3K expression and Akt phosphorylation. These re- ipocytes. Circ. Res. 2019;126:330-346. sults revealed a promising strategy for inducing the formation 10. Morley TS, Xia JY, Scherer PE. Selective enhancement of insu- of active BAT in vivo from stem cells and the browning of lin sensitivity in the mature adipocyte is sufficient for systemic Nat Commun WAT. metabolic improvements. . 2015;6:7906. https://doi. org/10.1038/ncomms8906. 11. Poher AL, Veyrat-Durebex C, Altirriba J, et al. Ectopic UCP1 ACKNOWLEDGMENTS overexpression in white adipose tissue improves insulin sen- This work was supported by the National Natural Scientific sitivity in Lou/C rats, a model of obesity resistance. Diabetes. Foundation of China [grant numbers: 81770880, 81800788, 2015;64:3700-3712. 81970762] and the Science & Technology Department 12. Mota de Sa P, Richard AJ, Hang H, Stephens JM. Transcriptional of Hunan Province [grant numbers: 2015JC3012, regulation of adipogenesis. Compr Physiol. 2017;7:635-674. 2018SK52511]. The authors gratefully thank the Metabolic 13. Montgomery MK, Bayliss J, Keenan S, Rhost S, Ting SB, Watt MJ. Syndrome Research Center at the Second Xiangya Hospital The role of Ap2a2 in PPARalpha-mediated regulation of lipolysis in adipose tissue. FASEB J. 2019;33:13267-13279. of Central South University for providing the Nalgene cages 14. Tseng YH, Kriauciunas KM, Kokkotou E, Kahn CR. Differential and Seahorse Bioscience XF-24 analyzer. And thanks for the roles of insulin receptor substrates in brown adipocyte differentia- work of Hui-Xuan Wu, Ying-Hui Zhou, and Hai-Qing Yue. tion. Mol Cell Biol. 2004;24:1918-1929. 15. Man XF, Tan SW, Tang HN, et al. MiR-503 inhibits adipogenesis DISCLOSURES by targeting bone morphogenetic protein receptor 1a. Am J Transl The authors declare no conﬂicts of interest. Res. 2016;8:2727-2737. 16. Fasshauer M, Klein J, Kriauciunas KM, Ueki K, Benito M, Kahn CR. Essential role of insulin receptor substrate 1 in differentiation AUTHOR CONTRIBUTIONS of brown adipocytes. Mol Cell Biol. 2001;21:319-329. 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Increased BMPR1A expression enhances How to cite this article: Man X-F, Hu N, Tan S-W, the adipogenic differentiation of mesenchymal stem cells in patients et al. Insulin receptor substrate-1 inhibits high-fat with ankylosing spondylitis. Stem Cells Int. 2019;2019:4143167. diet-induced obesity by browning of white adipose 33. Park A, Kim WK, Bae KH. Distinction of white, beige and brown tissue through miR-503. The FASEB Journal. World J Stem adipocytes derived from mesenchymal stem cells. 2020;00:1–16. https://doi.org/10.1096/fj.20190 Cells. 2014;6:33-42. 3283RR 34. Fischer C, Seki T, Lim S, et al. A miR-327-FGF10-FGFR2-mediated autocrine signaling mechanism controls white fat browning. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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