Glucocorticoids Suppress the Browning of Adipose Tissue via miR-19b in Male Mice

Glucocorticoids Suppress the Browning of Adipose Tissue via miR-19b in Male Mice Abstract Physiological levels of glucocorticoids (GCs) are required for proper metabolic control, and excessive GC action has been linked to a variety of pandemic metabolic diseases. MicroRNA (miRNA)-19b plays a critical role in the pathogenesis of GC-induced metabolic diseases. This study explored the potential of miRNA-based therapeutics targeting adipose tissue. Our results showed that overexpressed miR-19b in stromal vascular fraction (SVF) cells derived from subcutaneous adipose tissue had the same effects as dexamethasone (DEX) treatment on the inhibition of adipose browning and oxygen consumption rate. The inhibition of miR-19b blocked DEX-mediated suppression of the expression of browning marker genes as well as the oxygen consumption rate in differentiated SVF cells derived from subcutaneous and brown adipose tissue. Overexpressed miR-19b in SVF cells derived from brown adipose tissue had the same effects as DEX treatment on the inhibition of brown adipose differentiation and energy expenditure. Glucocorticoids transcriptionally regulate the expression of miR-19b via a GC receptor–mediated direct DNA binding mechanism. This study confirmed that miR-19b is an essential target for GC-mediated control of adipose tissue browning. It is hoped that the plasticity of the adipose organ can be exploited in the next generation of therapeutic strategies to combat the increasing incidence of metabolic diseases, including obesity and diabetes. Since the discovery of the beneficial effects of adrenocortical extract in treating adrenal insufficiency more than 80 years ago, glucocorticoids (GCs) and their cognate intracellular receptor, the GC receptor (GR), have been shown to be critical components of the delicate hormonal control system that regulates energy homeostasis in mammals. Although physiological levels of GCs are required for normal metabolic control, excessive GC activity has been linked to a variety of pervasive metabolic diseases, such as type 2 diabetes and obesity (1). Given its importance in human health, the molecular mechanisms of GC action have been extensively studied in biomedical research. In particular, elucidation of the tissue-specific functions of the GC pathway has identified therapeutic strategies for the treatment of severe metabolic disorders (2). Adipose tissue is made up of multiple depots located in two compartments of the body: some are below the skin (subcutaneous depots), and some are in the trunk (visceral depots). Adipose tissues thus make up a multi-depot organ that is involved in many critical survival functions, facilitating thermogenesis, lactation, and immune responses and serving as a fuel for metabolism. Traditionally, adipocytes have been divided into two types. White adipocytes compose the bulk of adipose tissue in most animals; they can be found in the marbling in steaks and in abdominal fat after weight gain (3). Brown adipocytes, in contrast, are highly specialized cells that dissipate stored chemical energy in the form of heat. This is mediated through the actions of uncoupling protein-1 (Ucp1), a brown adipose tissue (BAT)-specific protein located within the mitochondria, which are densely packed in these cells (4, 5). Numerous studies have demonstrated that Ucp1-expressing thermogenic adipocytes, so-called “brite” or “beige” adipocytes, can also be activated in white adipose tissue (WAT), thus resulting in WAT “browning,” which contributes to increased energy expenditure (6, 7). Recently, large depots of genuine BAT in adult humans have been identified on the basis of radiological observations of symmetrical [18F]-2-fluoro-D-2-deoxy-d-glucose positron emission tomography–positive loci in the supraclavicular and spinal regions (8, 9). The plasticity of the adipose organ might be exploited in the next generation of therapeutic strategies to combat the increasing incidence of metabolic diseases, including obesity and type 2 diabetes mellitus (10). MicroRNAs (miRNAs) are small (∼22 nucleotides) noncoding RNAs that are important regulators of mRNA expression. Recent findings have indicated that miRNAs are involved in the networks regulating many biological processes, including cell differentiation, animal development, metabolism, tumorigenesis, and other diseases, by regulating transcription factors and/or other genes (11, 12). Several miRNAs expressed in the adipocytes of mammals have been shown to play a role in adipogenesis and may affect adipogenesis dysfunction (13). In addition, many miRNAs are dysregulated in the metabolic tissues of obese animals and humans, thus potentially contributing to the pathogenesis of obesity-associated complications (14). In a previous study, the expression of more than 20 miRNAs regulated by GCs in human adipocytes has been examined (15). miR-19b is the most upregulated miRNA in response to GC treatment (15). Here, we found that the repression of BAT function, as well as browning in subcutaneous WAT by GCs, can be mediated by miR-19b. These results provide a basis for future research on miRNA-based therapeutics to target adipose tissue, which may lead to new strategies in the treatment of obesity and its associated complications. Materials and Methods Adipocyte culture and differentiation Inguinal, epididymal, and interscapular adipose tissues from 3-week-old male C57BL/6J mice were removed and minced with a scalpel. After digestion with collagenase and centrifugation, adipocyte precursors derived from the stromal vascular fraction (SVF) were cultured in growth medium [Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS)]. After 48 hours, cells were cultured in differentiation medium [DMEM containing 10% FBS, 1 µM dexamethasone (DEX), 0.5 mmol/L 3-isobutyl-1-methylxanthine, and 20 nM insulin] for 4 days. Cells were grown in maintenance medium containing 10% FBS and 20 nM insulin for the remainder of the culture period. For BAT in vitro differentiation, the cells were treated with growth medium containing 20 nM insulin and 1 nM tri-iodothyronine for 48 hours. After this induction period, the cells were washed and incubated in differentiation medium with an additional 1 µM DEX. The culture medium was changed every 2 days. Full differentiation was achieved after 6 days. The methods were carried out in accordance with the approved guidelines of the Animal Care and Use Committee of Nanjing Medical University. The experimental protocols were approved by the Animal Care and Use Committee of Nanjing Medical University. Murine adipocyte transfection SVF cells derived from subcutaneous adipose tissue (SAT)/visceral adipose tissue (VAT)/BAT were cultured in growth medium as described earlier. After the cells had grown to 80% confluence, they were transfected with either a mimic–miR-19b or mimic–miR–control transfection complex consisting of 50 nM miRNA and 1 μL of siPORT NeoFX transfection agent (Ambion, Austin, TX) in 50 μL of Opti-MEM. To inhibit miR-19b expression, the cells were transfected with 2 nM locked nucleic acid–modified anti–miR-19b oligonucleotides (methylene bridge between the 2′-O and the 4′-C atoms) (Exiqon, Copenhagen, Denmark) or locked nucleic acid–scrambled miR-19b control (anti–miR-control). RNA preparation and quantitative real-time polymerase chain reaction of mRNA Total RNA was extracted from adipose tissues or adipocytes by using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA (2 µg) was reverse-transcribed to cDNA using 200 U M-MLV reverse transcription (Promega, Madison, WI) in the presence of 0.5 mM deoxynucleotide triphosphate, 25 U RNase inhibitor, and 0.5 μg N15 random primers in a total volume of 25 μL. The sequences of the primers are shown in Supplemental Table 1. Each quantitative real-time polymerase chain reaction (PCR) reaction was carried out in triplicate in a 25 μL volume using SYBR Green Real-time PCR Master Mix (Roche, Basel, Switzerland). β-actin was used as an internal reference as described previously. The expression of each gene was arbitrarily set at 1 to facilitate comparison between several treatment groups. For miRNA quantitative real-time PCR, a miRNA-specific stem-loop reverse transcription primer was hybridized to the miRNA and then reverse-transcribed. Then, the reverse transcription product was amplified and monitored in real time using a miRNA-specific upstream primer and a universal downstream primer (Supplemental Table 1; universal downstream primer: 5′-GTGCAGGGTCCGAGGT-3′). The expression levels were normalized to that of small noncoding RNA U6. Western blotting The cell extracts were lysed in radio-immunoprecipitation assay buffer [0.5% NP-40, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, and 50 mM Tris-Cl, pH 7.5]. The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA), and probed with anti-Ucp1 (1:1000, Abcam, Cambridge, MA), anti-Adrb1 (1:1000, Sigma-Aldrich, Munich, Germany), and anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Abcam, Cambridge, MA) antibodies overnight at 4°C. The blots were treated with horseradish peroxidase–conjugated anti-rabbit IgG (1:10,000) (Vector Laboratories, Burlingame, CA) in Tris-buffered saline with Tween 20 containing 1% (w/v) bicinchoninic acid for 60 minutes, and immune complexes were detected using an enhanced chemiluminescence plus detection kit (Cell Signaling Technology, Danvers, MA). The bands were quantified using densitometric image analysis software (Quantity One; Bio-Rad, Hercules, CA). The relative expression of Ucp1 and Adrb1 was normalized to that of GAPDH. Chromatin immunoprecipitation assay Chromatin in the control and treated cells was cross-linked with 1% formaldehyde. The cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris, pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablets and phenylmethylsulfonyl fluoride. DNA was fragmented (∼500 bp) using a Branson 250 sonicator (Branson Ultrasonics Corp., Danbury, MA). Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-GR antibody (Cell Signaling Technology). The EpiQuik chromatin immunoprecipitation (ChIP) kit (Epigentek Group Inc., Farmingdale, NY) was used to measure DNA precipitated by particular antibodies. Precipitated genomic DNA was amplified by real-time PCR. Plasmid construction The plasmids comprising the full-length GR, the miR-19b promoter (sequence shown in Supplemental Material), or the miR-19b promoter with mutations in putative glucocorticoid response element (GRE) motifs (see Supplemental Material) were synthesized by Integrated DNA Technologies, annealed, and cloned into the pGL3 vector (Promega) for use in luciferase reporter assays. Site-directed mutagenesis The oligonucleotides comprising the wild-type or mutated miR-19b target sequence were synthesized by Integrated DNA Technologies, annealed, and cloned into the pMiRGLO vector (Promega) for luciferase reporter assays. The miR-19b target sequences within the 3′-untranslated regions (3′-UTRs) of Adrb1 were predicted by TargetScan. The sequence of Adrb1 wild-type was 5′-UGGUUGGAAGGCCAUUUUGCACA-3′, and the mutated sequence was 5′-UGGUUGGAAGGCCAUAAACGUGU-3′. Luciferase assay 3T3-L1 cells (RRID:CVCL_0123) were differentiated for 5 days by culturing of the cells in differentiation medium (DMEM containing 10% FBS, 1 µM DEX, 0.5 mmol/L 3-isobutyl-1-methylxanthine, and 20 nM insulin) for 3 days and in maintenance medium containing 10% FBS and 20 nM insulin for 2 days. Luciferase assays for the GR segment and miR-19b promoter were performed using 3T3-L1 cells after the completion of differentiation. Cells were transfected with luciferase reporter constructs containing the miR-19b promoter or the miR-19b promoter with mutations in GRE sites (mut-miR-19b) and cotransfected with or without GR constructs. Twenty-four hours after transfection, cells were incubated with or without DEX (1 µM), and 24 hours later, the cells were lysed, and extracts were prepared and analyzed using a luciferase reporter assay system (Promega). Luciferase assays for miR-19b and Adrb1 3′-UTR were performed using HEK293T cells (RRID:CVCL_0063). Cells were seeded in 96-well plates, and a plasmid containing the 3′UTR of the murine Adrb1 gene or a mutated version (mut 3′-UTR) was transfected together with mimic–miR-19b or mimic–miR–control using Lipofectamine 2000 (Invitrogen). Luciferase activities were assayed after transfection for 24 hours using a luciferase reporter assay system (Promega). Measurement of O2 consumption SVF cells were cultured in 24-well plates and differentiated as indicated (Seahorse Bioscience, North Billerica, MA). The medium was replaced with prewarmed unbuffered DMEM (DMEM basal medium supplemented with 25 mM glucose, 2 mM sodium pyruvate, 31 mM NaCl, 2 mM GlutaMax, 15 mg/L phenol red, and 2% essentially fatty acid-free bovine serum albumin, pH 7.4) and incubated at 37°C in a non-CO2 incubator for 1.5 hours. The oxygen consumption rate (OCR) was measured at basal glucose levels with oligomycin (ATP synthase inhibitor, 1 µM) (Sigma-Aldrich), which disrupts the respiratory chain. Ucp1-mediated uncoupling respiration was determined after isoproterenol (0.5 μM) stimulation (Sigma-Aldrich). The maximum respiratory capacity was assessed after carbonylcyanide-p-trifluorophenylhydrazone (FCCP) stimulation (1 μM) (Sigma-Aldrich). Finally, the mitochondrial respiration was blocked by 1 µM rotenone (Sigma-Aldrich). The residual OCR was considered nonmitochondrial respiration. Immunofluorescence staining Cells were fixed in 4% paraformaldehyde solution and incubated with primary antibody against Ucp1 (1:100, Abcam, Cambridge, MA) at 4°C overnight, followed by the FITC-conjugated secondary antibody (1:100, Jackson ImmunoResearch Inc., West Grove, PA) for 60 minutes at room temperature. To counterstain the nuclei, 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, Danvers, MA) was used. Cells were visualized under ×20 magnification by fluorescence microscopy (Olympus, Tokyo, Japan). Statistical analysis The results are presented as the mean ± standard error of the mean (SEM). Statistically significant differences were calculated using Student t test. A value of P < 0.05 was considered significant. Results GCs transcriptionally upregulate miR-19b in murine adipocytes We have previously demonstrated that GCs inhibit the function of brown fat and the browning activity of white fat (15, 16). A previous miRNA microarray analysis and other studies have shown that GCs transcriptionally regulate miR-27b expression, thereby promoting body fat accumulation by suppressing the browning of SAT and VAT. In this study, we focused on miR-19b because we have previously found it to be strongly upregulated by 1 µM DEX treatment in differentiated SVF cells derived from human SAT or VAT (15). SVF cells were derived from the SAT or VAT of 3-week-old male C57BL/6J mice in the presence of DEX at a concentration of 0 to 1 µM. Without DEX treatment, the constitutive expression level of miR-19b was significantly higher (1.3-fold) in 4-day differentiated SVF cells derived from VAT than cells derived from SAT [Fig. 1(a)]. DEX treatment elevated miR-19b expression in SAT- and VAT-derived differentiated cells in a dose-dependent manner, and peak levels were observed at 1 µM [Fig. 1(a)]. In contrast, the constitutive expression level of Ucp1 was higher in 4-day differentiated SVF cells derived from SAT (2.4-fold) than in cells derived from VAT. However, compared with that of SAT, the browning ability of VAT was weaker. Ucp1 mRNA expression was still significantly inhibited by DEX in a dose-dependent manner [Fig. 1(b)]. Reverse regulation of Ucp1 and miR-19b by DEX in murine primary adipocyte cultures suggested that DEX suppression of Ucp1 expression and the browning of SAT and VAT may be mediated by miR-19b. Figure 1. View largeDownload slide GCs transcriptionally upregulate miR-19b in murine adipocytes. (a, b) Relative miR-19b and Ucp1 mRNA levels in SVF cells derived from murine SAT or VAT and differentiated for 4 days in the presence of DEX at doses of 0, 0.1, and 1 µM. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05, **P < 0.01, DEX-treated SAT vs control; #P < 0.05, DEX-treated VAT vs control; $P < 0.05, $$P < 0.01, SAT vs VAT. (c) The 3T3-L1 cells were transfected with plasmids containing the miR-19b promoter (miR-19b) or the miR-19b promoter with mutations GRE motifs (mut-miR-19b) and cotransfected with or without GR constructs. Twenty-four hours after transfection, cells were incubated with or without DEX (1 µM) and, 24 hours later, cells were lysed and extracted for analysis of luciferase activity. The experiments were performed in triplicate wells and repeated three times. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (d) Differentiated SVF cells derived from SAT (s) or VAT (v) were treated with or without 1 µM DEX for 24 hours. ChIP assays were performed by using antibodies against GR (GR), RNA-pol II (pol II), and IgG (IgG). Quantitative PCR using primers of miR-19b promoter region (miR-19b) were performed on the chromatin precipitated with anti-GR, anti–RNA-pol II, and anti-IgG antibodies. Quantitative PCR using primers of Gilz and GAPDH were performed on the chromatin precipitated with anti-GR antibodies. Data are expressed as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01, con vs DEX. Figure 1. View largeDownload slide GCs transcriptionally upregulate miR-19b in murine adipocytes. (a, b) Relative miR-19b and Ucp1 mRNA levels in SVF cells derived from murine SAT or VAT and differentiated for 4 days in the presence of DEX at doses of 0, 0.1, and 1 µM. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05, **P < 0.01, DEX-treated SAT vs control; #P < 0.05, DEX-treated VAT vs control; $P < 0.05, $$P < 0.01, SAT vs VAT. (c) The 3T3-L1 cells were transfected with plasmids containing the miR-19b promoter (miR-19b) or the miR-19b promoter with mutations GRE motifs (mut-miR-19b) and cotransfected with or without GR constructs. Twenty-four hours after transfection, cells were incubated with or without DEX (1 µM) and, 24 hours later, cells were lysed and extracted for analysis of luciferase activity. The experiments were performed in triplicate wells and repeated three times. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (d) Differentiated SVF cells derived from SAT (s) or VAT (v) were treated with or without 1 µM DEX for 24 hours. ChIP assays were performed by using antibodies against GR (GR), RNA-pol II (pol II), and IgG (IgG). Quantitative PCR using primers of miR-19b promoter region (miR-19b) were performed on the chromatin precipitated with anti-GR, anti–RNA-pol II, and anti-IgG antibodies. Quantitative PCR using primers of Gilz and GAPDH were performed on the chromatin precipitated with anti-GR antibodies. Data are expressed as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01, con vs DEX. The effects of GCs are mediated through the GR, which activates the transcription of genes by binding to a consensus GRE. To determine whether GCs transcriptionally regulate miR-19b expression by binding to GRE sites in vitro, we performed luciferase reporter assays. We generated luciferase reporter constructs containing the miR-19b promoter region (miR-19b) or mutant miR-19b promoters with mutations of putative GRE motifs (mut–miR-19b). Then, 1 µM DEX was added to induce miR-19b activity in 3T3-L1 cells cotransfected with GR construct to more than 1.5-fold higher than that of cells in the absence of DEX, whereas there was about 20% activity induction by DEX observed in the cells without GR cotransfection compared with the untreated cells, thus indicating endogenous GR activity in 3T3-L1 cells. Additionally, mutations in the GREs resulted in the complete abrogation of miR-19b promoter activation by DEX, indicating that liganded GR bound to GRE motifs on the promoter regions of miR-19b [Fig. 1(c)]. To further verify that liganded GR transcriptionally regulated the expression of miR-19b, ChIP assay was performed. Our observation revealed that 1 µM DEX treatment led to more then 3.5-fold enhancements in GR binding to the miR-19b promoter in the 4-day differentiated SVF cells derived from SAT and VAT compared with the cells without DEX treatment, which were consistent with our luciferase assay. We detected a >2.5-fold elevation in RNA–pol II binding to the miR-19b promoter region with 1 µM DEX treatment, indicating the elevation of miR-19b’s transcription. Also, we examined a known GR target locus (Gilz) as positive control by PCR on the chromatin precipitated with anti-GR antibody. Treatment with 1 µM DEX led to more than twofold enhancement in GR binding to the Gilz promoter region. In addition, we detected GAPDH on the chromatin precipitated with anti-GR antibody. As a negative control of ChIP assay, GR barely bound to GAPDH promoter region, which obviously did not bring any significant change in GAPDH promoter region compared with the cells without DEX treatment [Fig. 1(d)]. These results indicated that GCs transcriptionally upregulate miR-19b expression via a GR-mediated DNA binding mechanism. miR-19b negatively regulates brown marker genes in differentiated SVF cells derived from SAT We next examined the expression of miR-19b in the primary adipocyte cultures derived from SAT or VAT over a 6-day time course of differentiation. During differentiation, the expression of miR-19b decreased and reached a minimum on day 4 (35% lower than on day 0) and then increased to the baseline level on day 6. In contrast, the Ucp1 mRNA expression increased during differentiation, peaking on day 4 (5.7-fold higher than on day 0) [Fig. 2(a) and 2(b)]. This inverse relationship between the mRNA expression of Ucp1 and miR-19b suggested that miR-19b might negatively regulate Ucp1 expression. Figure 2. View largeDownload slide Overexpression of miR-19b suppresses the browning effect on adipocytes. (a, b) Relative expression of miR-19b and Ucp1 mRNA during SVF cells differentiation under adipogenic conditions, which were isolated from SAT or VAT (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, SAT; #P < 0.05, VAT, compared with day 0. (c, d) miR-19b mRNA expression and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells from SAT by standard differentiation (Vehicle) or transfecting with either mimic-control (Vehicle + mimic-con) or mimic–miR-19b (Vehicle + miR-19b) after 4 days differentiation. A concentration of 1 mmol/L DEX (DEX + mimic-con) presents as positive control (n = 4). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con. (e) Representative images of Western blots of Ucp1 protein of whole cell lysates from differentiated SVF cells isolated from SAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). (f) Bands were quantified using densitometric image analysis software. The relative expression of Ucp1 was normalized to that of GAPDH (mean ± SEM; n = 3). **P < 0.01, SAT mimic-con vs mimic–miR-19b. (g, h) Immunofluorescence staining of Ucp1 (green) in 4 days differentiated SVF cells isolated from SAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity were quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, SAT mimic-con vs mimic–miR-19b. (i) OCRs of SVF cells derived from SAT were quantified in the cell cultures under the same conditions as in (c) and (d) under basal conditions (Basal) or with the following drugs disrupting the respiratory chain: OL, FCCP (a mitochondrial uncoupler), and Rot (mean ± SEM; n = 5). **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05, Vehicle + mimic-con vs DEX + mimic–miR-con. Figure 2. View largeDownload slide Overexpression of miR-19b suppresses the browning effect on adipocytes. (a, b) Relative expression of miR-19b and Ucp1 mRNA during SVF cells differentiation under adipogenic conditions, which were isolated from SAT or VAT (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, SAT; #P < 0.05, VAT, compared with day 0. (c, d) miR-19b mRNA expression and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells from SAT by standard differentiation (Vehicle) or transfecting with either mimic-control (Vehicle + mimic-con) or mimic–miR-19b (Vehicle + miR-19b) after 4 days differentiation. A concentration of 1 mmol/L DEX (DEX + mimic-con) presents as positive control (n = 4). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con. (e) Representative images of Western blots of Ucp1 protein of whole cell lysates from differentiated SVF cells isolated from SAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). (f) Bands were quantified using densitometric image analysis software. The relative expression of Ucp1 was normalized to that of GAPDH (mean ± SEM; n = 3). **P < 0.01, SAT mimic-con vs mimic–miR-19b. (g, h) Immunofluorescence staining of Ucp1 (green) in 4 days differentiated SVF cells isolated from SAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity were quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, SAT mimic-con vs mimic–miR-19b. (i) OCRs of SVF cells derived from SAT were quantified in the cell cultures under the same conditions as in (c) and (d) under basal conditions (Basal) or with the following drugs disrupting the respiratory chain: OL, FCCP (a mitochondrial uncoupler), and Rot (mean ± SEM; n = 5). **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05, Vehicle + mimic-con vs DEX + mimic–miR-con. The constitutive Ucp1 expression level was extremely low in VAT, which was considered to be classic white adipose and highly resistant to browning. It is widely accepted that subcutaneous fat (but not visceral fat) is highly susceptible to browning in rodents (17, 18). We therefore use SAT to clarify the function of miR-19b in regulating the browning activity of adipocytes. Mimic–miR-19b was transfected into SVF cells derived from SAT in the presence or absence of 1 µM DEX. Compared with expression in the vector control cells (mimic-con), DEX treatment led to a threefold increase in miR-19b expression, and expression increased by 18-fold in cells treated with mimic–miR-19b [Fig. 2(c)]. Accordingly, DEX-treated cells showed a substantial reduction in the expression of browning marker genes, including Ucp1, Cidea, Cox7a1, and Cox8b. Overexpression of miR-19b also led to a decrease in these genes [Fig. 2(d)]. Notably, the mRNA expression of Fabp4, a marker of mature white adipocytes, was not altered by mimic–miR-19b transfection. Western blot analyses also indicated that the Ucp1 protein level was decreased in SVF cells transfected with mimic–miR-19b- derived from SAT after 4 days of differentiation [Fig. 2(e) and 2(f)]. Additionally, immunofluorescence staining of Ucp1 showed a significant decrease in the overexpression of miR-19b (mimic–miR-19b) compared with the vector control cells (mimic-con) [Fig. 2(g) and 2(h)]. Consistent with the inhibited expression of genes for browning, the oxygen consumption rate (OCR) in basal condition (Basal) and the maximal respiration stimulated by the mitochondrial uncoupler FCCP of cells transfected with mimic–miR-19b and cultured under the same conditions as described for Fig. 2(c) were markedly inhibited to levels similar to those observed after DEX treatment. However, there were no significant differences in coupled respiration [oligomycin (OL)] and nonmitochondrial respiration [rotenone (Rot)] OCR [Fig. 2(i)]. We then transfected anti–miR-19b to induce cells’ browning abilities. Scrambled miR-19b oligonucleotides (scr-miR) were used as a control. After 4 days of differentiation of SVF cells derived from SAT in the presence or absence of 1 µM DEX, anti–miR-19b transfection successfully inhibited miR-19b expression in both DEX-treated (86%) and untreated cells (90%) [Fig. 3(a)]. Silencing of miR-19b led to a significant increase in the mRNA expression of Ucp1, Cidea, Cox7a1, and Cox8b and blocked the effect of DEX on the mRNA expression of these browning marker genes [Fig. 3(b)]. Fabp4 mRNA was not altered by either DEX or anti–miR-19b treatment. Western blot analyses indicated that the Ucp1 protein level was also substantially elevated in SVF cells transfected with anti–miR-19b derived from SAT after 4 days of differentiation [Fig. 3(c) and 3(d)]. Immunofluorescence staining of Ucp1 also markedly raised in the silence of miR-19b (anti–miR-19b) compared with the vector control cells (scr-miR) [Fig. 3(e) and 3(f)]. DEX treatment led to a significant decrease of OCR in basal condition (Basal), coupled respiration (OL), and cells stimulated by the FCCP. Furthermore, silencing of miR-19b blocked the inhibitory effects of DEX on OCR in basal conditions, coupled respiration (OL), and FCCP stimulation. There were no differences in nonmitochondrial respiration (Rot) OCR [Fig. 3(g)]. Figure 3. View largeDownload slide Inhibition of miR-19b promotes the browning effect in adipocytes. (a, b) Relative expression of miR-19b and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in differentiated SVF cells from SAT after transfection with scrambled (scr) control (Vehicle + scr-miR), anti–miR-19b (Vehicle + anti-miR-19b), or 1 µM DEX treatment (DEX + scr-miR, DEX + anti-miR-19b) after 4 days of differentiation (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05; ##P < 0.01; ###P < 0.001, Vehicle + scr-miR vs DEX + scr-miR. $P < 0.05; $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b). Experiments were repeated three times. (c) Representative images of Western blots of Ucp1 protein of whole cell lysates from differentiated SVF cells isolated from SAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti-miR-19b). (d) Bands were quantified using densitometric image analysis software. The relative expression of Ucp1 was normalized to that of GAPDH (mean ± SEM; n = 3). *P < 0.05, SAT scr-miR vs anti–miR-19b). (e, f) Immunofluorescence staining of Ucp1 in 4-day differentiated SVF cells isolated from SAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, SAT scr-miR vs anti–miR-19b. (g) OCRs were quantified in the cell cultures under the same conditions as in (a) and (b) under basal conditions (Basal) or with the following drugs disrupting the respiratory chain: OL, FCCP, and Rot (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05, Vehicle + scr-miR vs DEX + scr-miR. $P < 0.05, DEX + scr-miR vs DEX + anti–miR-19b. Figure 3. View largeDownload slide Inhibition of miR-19b promotes the browning effect in adipocytes. (a, b) Relative expression of miR-19b and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in differentiated SVF cells from SAT after transfection with scrambled (scr) control (Vehicle + scr-miR), anti–miR-19b (Vehicle + anti-miR-19b), or 1 µM DEX treatment (DEX + scr-miR, DEX + anti-miR-19b) after 4 days of differentiation (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05; ##P < 0.01; ###P < 0.001, Vehicle + scr-miR vs DEX + scr-miR. $P < 0.05; $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b). Experiments were repeated three times. (c) Representative images of Western blots of Ucp1 protein of whole cell lysates from differentiated SVF cells isolated from SAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti-miR-19b). (d) Bands were quantified using densitometric image analysis software. The relative expression of Ucp1 was normalized to that of GAPDH (mean ± SEM; n = 3). *P < 0.05, SAT scr-miR vs anti–miR-19b). (e, f) Immunofluorescence staining of Ucp1 in 4-day differentiated SVF cells isolated from SAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, SAT scr-miR vs anti–miR-19b. (g) OCRs were quantified in the cell cultures under the same conditions as in (a) and (b) under basal conditions (Basal) or with the following drugs disrupting the respiratory chain: OL, FCCP, and Rot (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05, Vehicle + scr-miR vs DEX + scr-miR. $P < 0.05, DEX + scr-miR vs DEX + anti–miR-19b. miR-19b negatively regulates the function of BAT A recent investigation has confirmed that GCs inhibit the function of BAT (16). The current study investigated the underlying mechanism by validating the expression of miR-19b in SVF cells derived from BAT. During brown adipocyte differentiation, the expression of miR-19b decreased and reached a minimum on day 6 (2.7-fold lower than day 0) [Fig. 4(a)]. In contrast, the expression of Ucp1 mRNA increased and reached a maximum on day 4 (2.1-fold higher than day 0) and remained at that level on day 6 (1.81-fold higher than day 0) during differentiation [Fig. 4(b)]. We then overexpressed miR-19b (mimic–miR-19b) in SVF cells derived from BAT and differentiated the cells for 4 days in the presence or absence of 1 µM DEX. Compared with expression in the vector control cells (mimic-con), DEX treatment led to a threefold increase in miR-19b expression, and expression increased by eightfold in cells treated with mimic–miR-19b [Fig. 4(c)]. Accordingly, DEX-treated cells showed a substantial reduction in the expression of brown adipocyte marker genes, including Ucp1, Cidea, Cox7a1, and Cox8b. Overexpression of miR-19b led to a decrease in these genes [Fig. 4(d)]. In addition, immunofluorescence staining showed that overexpression of miR-19b (mimic–miR-19b) dramatically repressed Ucp1 expression compared with the vector control cells (mimic-con) [Fig. 4(e) and 4(f)]. Importantly, the OCR of cells transfected with mimic–miR-19b in basal conditions and after stimulation with OL and FCCP was markedly inhibited to levels similar to those observed after DEX treatment [Fig. 4(g)]. We then silenced miR-19b in SVF cells derived from BAT. Cells transfected with either anti–miR-19b or scrambled miR-19b oligonucleotide control (scr-miR) were differentiated for 4 days in the presence or absence of 1 µM DEX. Anti–miR-19b transfection successfully inhibited miR-19b expression in both DEX-treated (61%) and untreated cells (66%) [Fig. 4(h)]. Compared with the expression in anti-control cells (scr-miR), anti–miR-19b led to a notable increase in the mRNA expression of Ucp1, Cidea, Cox7a1, and Cox8b and blunted the effect of DEX on the mRNA expression of these genes [Fig. 4(i)]. Accordingly, miR-19b deficiency group (anti–miR-19b) displayed a remarkable increase in the immunofluorescence staining of Ucp1 compared with the vector control group (scr-miR) [Fig. 4(j) and 4(k)]. Silencing of miR-19b significantly blocked the inhibitory effects of DEX on the OCR in basal conditions and after stimulation by OL and FCCP [Fig. 4(l)]. Together, these data indicated that GCs inhibit the function of BAT by activating the expression of miR-19b in vitro. Figure 4. View largeDownload slide miR-19b is a potent negative regulator of the functions of brown adipocytes. (a, b) Relative expression of miR-19b and Ucp1 mRNA during differentiation of SVF cells, which were isolated from BAT and differentiated under adipogenic conditions. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, compared with day 0. (c, d) miR-19b mRNA expression and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells from BAT by standard differentiation (Vehicle) or transfecting with either mimic-control (Vehicle + mimic-con) or mimic–miR-19b (Vehicle + miR-19b) after 4 days of differentiation. A concentration of 1 mmol/L DEX (DEX + mimic-con) presents as positive control (mean ± SEM; n = 4). **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con). (e, f) Immunofluorescence staining of Ucp1 in 4-day differentiated SVF cells isolated from BAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, BAT mimic-con vs mimic–miR-19b. (g) OCRs of SVF cells derived from BAT were quantified under basal conditions and after treatment with drugs disrupting the respiratory chain: OL, FCCP, and Rot (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con. (h, i) Relative expression of miR-19b and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells derived from BAT after transfection with scrambled (scr) control (Vehicle + scr-miR), anti–miR-19b (Vehicle + antimiR-19b), or 1 µM DEX treatment (DEX + scr-miR and DEX + anti–miR-19b) after 4 days of differentiation. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + scr-miR vs DEX + scr-miR. $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b. (j, k) Immunofluorescence staining of Ucp1 in 4 days differentiated SVF cells isolated from BAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti–miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity were quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). *P < 0.05, BAT scr-miR vs anti–miR-19b). (l) OCRs were quantified in the cells described in (h) (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. ##P < 0.01, Vehicle + scr-miR vs DEX + scr-miR. $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b. Figure 4. View largeDownload slide miR-19b is a potent negative regulator of the functions of brown adipocytes. (a, b) Relative expression of miR-19b and Ucp1 mRNA during differentiation of SVF cells, which were isolated from BAT and differentiated under adipogenic conditions. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, compared with day 0. (c, d) miR-19b mRNA expression and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells from BAT by standard differentiation (Vehicle) or transfecting with either mimic-control (Vehicle + mimic-con) or mimic–miR-19b (Vehicle + miR-19b) after 4 days of differentiation. A concentration of 1 mmol/L DEX (DEX + mimic-con) presents as positive control (mean ± SEM; n = 4). **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con). (e, f) Immunofluorescence staining of Ucp1 in 4-day differentiated SVF cells isolated from BAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, BAT mimic-con vs mimic–miR-19b. (g) OCRs of SVF cells derived from BAT were quantified under basal conditions and after treatment with drugs disrupting the respiratory chain: OL, FCCP, and Rot (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con. (h, i) Relative expression of miR-19b and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells derived from BAT after transfection with scrambled (scr) control (Vehicle + scr-miR), anti–miR-19b (Vehicle + antimiR-19b), or 1 µM DEX treatment (DEX + scr-miR and DEX + anti–miR-19b) after 4 days of differentiation. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + scr-miR vs DEX + scr-miR. $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b. (j, k) Immunofluorescence staining of Ucp1 in 4 days differentiated SVF cells isolated from BAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti–miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity were quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). *P < 0.05, BAT scr-miR vs anti–miR-19b). (l) OCRs were quantified in the cells described in (h) (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. ##P < 0.01, Vehicle + scr-miR vs DEX + scr-miR. $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b. miR-19b directly targets Adrb1 β-Adrenergic receptors are the major mediators of the facultative thermogenesis activated by the sympathetic nervous system and have a fundamental role in regulating energy expenditure (19). β-Adrenergic receptor-1 (Adrb1) knockout mice are cold intolerant and obese. Additionally, they are more prone to developing obesity when they are fed a high-fat diet (20). In humans, Adrb1 plays an important role in regulating nonshivering thermogenesis. The activation of Adrb1 increases Ucp1 mRNA and protein levels (21). According to miRNA target prediction analyses [TargetScan, miRanda, and Diana MicroT-CDS (22)], Adrb1 was a predicated target gene of miR-19b, and a putative miR-19b target site is present at a highly conserved octamer seed motif within the 3′-untranslated region (3′-UTR) of Adrb1 [Fig. 5(a)]. We therefore performed luciferase assays to investigate the direct targeting of the Adrb1 3′-UTR by miR-19b. HEK293T cells transfected with reporter plasmids containing the Adrb1 3′-UTR showed significantly decreased luciferase activity in the presence of miR-19b. Mutation of the conserved seed sequence (Mut 3′-UTR) abrogated the miRNA-induced repression of the Adrb1 3′UTR [Fig. 5(b)]. To verify the interaction between miR-19b and Adrb1 in adipose tissue, we examined the Adrb1 mRNA expression levels in response to the miR-19b mimics and inhibitors in the presence or absence of DEX in the 4-day differentiated SVF cells isolated from SAT. Overexpression of miR-19b significantly suppressed Adrb1 mRNA expression levels [Fig. 5(c)]. Silencing of miR-19b stimulated the expression of Adrb1 mRNA and substantially blunted the effect of DEX on the expression of Adrb1 mRNA [Fig. 5(d)], thus indicating that DEX suppressed Adrb1 mRNA expression via miR-19b. Additionally, Western blot analysis revealed an elevation of Adrb1 protein levels in SVF cells transfected with anti–miR-19b derived from SAT on day 4 after induction of differentiation [Fig. 5(e) and 5(f)]. Figure 5. View largeDownload slide miR-19b directly targets Adrb1. (a) The miRNA target prediction analyses from TargetScan and Diana microT-CDS. (b) The 3′-UTR of the Adrb1 gene contains the predicted binding site for miR-19b, as shown by the colored sequences, and was cloned into the pMiRGLO vector (Adrb1 3′UTR). In the mutant construct (mut 3′UTR of Adrb1), the corresponding binding sequences that were mutated are indicated in black. Relative luciferase activity in HEK293T cells transfected with plasmid reporter constructs containing the 3′-UTR (3′UTR) or mutated 3′-UTR (Mut 3′UTR) of Adrb1 is shown. The experiments were performed in triplicate wells and repeated three times (mean ± SEM). *P < 0.05, con vs miR-19b. (c) Relative Adrb1 mRNA expression in 4-day differentiated SVF cells derived from SAT after no treatment (Vehicle) or transfection with mimic-control (Vehicle + mimic-con), mimic-control with 1 µM DEX treatment (DEX + mimic-con), or mimic–miR-19b (Vehicle + mimic–miR-19b) (mean ± SEM; n = 4). **P < 0.01, Vehicle + mimic-con vs Vehicle + miR-19b. #P < 0.05, Vehicle + mimic-con vs DEX + mimic-con. (d) Relative Adrb1 mRNA expression in 4-day differentiated SVF cells derived from SAT or VAT after transfection with scrambled control (Vehicle + scr-miR), anti–miR-19b (Vehicle + anti–miR-19b), or 1 µM DEX treatment (DEX + scr-miR and DEX + anti-miR-19b). The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05, Vehicle + scr-miR vs DEX + scr-miR. $$$P < 0.001, DEX + scr-miR vs DEX + anti–miR-19b. (e) Representative images of Western blots of Adrb1 protein of whole cell lysates from 4-day differentiated SVF cells isolated from SAT after transfection with anti-control (scr-miR) or anti–miR-19b (antimiR-19b). (f) Bands were quantified using densitometric image analysis software. The relative expression of Adrb1 was normalized to that of GAPDH (mean ± SEM; n = 3). **P < 0.01, scr-miR vs anti–miR-19b. Figure 5. View largeDownload slide miR-19b directly targets Adrb1. (a) The miRNA target prediction analyses from TargetScan and Diana microT-CDS. (b) The 3′-UTR of the Adrb1 gene contains the predicted binding site for miR-19b, as shown by the colored sequences, and was cloned into the pMiRGLO vector (Adrb1 3′UTR). In the mutant construct (mut 3′UTR of Adrb1), the corresponding binding sequences that were mutated are indicated in black. Relative luciferase activity in HEK293T cells transfected with plasmid reporter constructs containing the 3′-UTR (3′UTR) or mutated 3′-UTR (Mut 3′UTR) of Adrb1 is shown. The experiments were performed in triplicate wells and repeated three times (mean ± SEM). *P < 0.05, con vs miR-19b. (c) Relative Adrb1 mRNA expression in 4-day differentiated SVF cells derived from SAT after no treatment (Vehicle) or transfection with mimic-control (Vehicle + mimic-con), mimic-control with 1 µM DEX treatment (DEX + mimic-con), or mimic–miR-19b (Vehicle + mimic–miR-19b) (mean ± SEM; n = 4). **P < 0.01, Vehicle + mimic-con vs Vehicle + miR-19b. #P < 0.05, Vehicle + mimic-con vs DEX + mimic-con. (d) Relative Adrb1 mRNA expression in 4-day differentiated SVF cells derived from SAT or VAT after transfection with scrambled control (Vehicle + scr-miR), anti–miR-19b (Vehicle + anti–miR-19b), or 1 µM DEX treatment (DEX + scr-miR and DEX + anti-miR-19b). The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05, Vehicle + scr-miR vs DEX + scr-miR. $$$P < 0.001, DEX + scr-miR vs DEX + anti–miR-19b. (e) Representative images of Western blots of Adrb1 protein of whole cell lysates from 4-day differentiated SVF cells isolated from SAT after transfection with anti-control (scr-miR) or anti–miR-19b (antimiR-19b). (f) Bands were quantified using densitometric image analysis software. The relative expression of Adrb1 was normalized to that of GAPDH (mean ± SEM; n = 3). **P < 0.01, scr-miR vs anti–miR-19b. Discussion Although physiological levels of GCs are required for normal metabolic control, aberrant GC activity has been linked to a variety of common metabolic diseases. Given the importance of GC signaling pathways in human health, studies of the molecular mechanisms underlying these pathways have become a major focus in biomedical research. Therefore, the current study investigated the effects of GC modulation on metabolic homeostasis, focusing on the tissue-specific contributions of the glucocorticoid axis to the control of energy metabolism. Our current study demonstrated that miR-19b plays a crucial role in the pathogenesis of the detrimental effects of high-dose GCs in adipose tissues and energy metabolism and consequently may be a potential target to prevent GC-induced obesity and metabolic syndromes (Fig. 6). Figure 6. View largeDownload slide Schematic model of the effects of GCs on adipose tissue function in mice. GCs transcriptionally upregulate miR-19b expression. The GC-induced miR-19b then suppresses the expression of its target gene Adrb1 and further inhibits the browning signature genes like Ucp1, Cidea, Cox7a1, and Cox8b. The inhibition of the browning of SAT and the function of BAT results in low energy expenditure. The low energy expenditure consequently causes or at least partly contributes to GC-induced fat accumulation and insulin resistance. Figure 6. View largeDownload slide Schematic model of the effects of GCs on adipose tissue function in mice. GCs transcriptionally upregulate miR-19b expression. The GC-induced miR-19b then suppresses the expression of its target gene Adrb1 and further inhibits the browning signature genes like Ucp1, Cidea, Cox7a1, and Cox8b. The inhibition of the browning of SAT and the function of BAT results in low energy expenditure. The low energy expenditure consequently causes or at least partly contributes to GC-induced fat accumulation and insulin resistance. We have recently reported that GCs inhibit the browning of SAT and VAT. A previous miRNA microarray analysis has found that miR-19b is the miRNA most strongly upregulated by 1 µM DEX treatment in differentiated SVF cells derived from human SAT and VAT (15). Therefore, we focused on the regulation of miR-19b in murine adipocytes. In the current study, we discovered that miR-19b is a GC-target miRNA. DEX treatment elevated miR-19b expression in 4-day differentiated SVF cells derived from SAT or VAT in a dose-dependent manner. Using luciferase assay, we proved that liganded GR regulated miR-19b through binding GRE motifs. We then performed ChIP assay to verify that GRs transcriptionally regulate the expression of miR-19b. By ChIP assay, we detected increased GR binding on miR-19b promoter region as well as an enhancement of RNA–pol II recruitment. Using luciferase assays and ChIP analyses, we eventually confirmed that miR-19b is transcriptionally upregulated by GCs via a GC receptor–mediated DNA binding mechanism. miR-19b is constitutively expressed in murine adipose tissue but was lower in SAT than VAT. In contrast, the gene expression of Ucp1 was higher in SAT than in VAT. Because VAT, the typical white adipose, was considered to be resistant to browning, mimic–miR-19b or anti–miR-19b was only transfected into SVF cells isolated from SAT. According to our studies, DEX treatment led to a decrease in BAT-specific gene expression and OCR, whereas overexpression of miR-19b further suppressed the expression of these genes. Silencing of miR-19b significantly blocked the inhibitory effect of DEX on the expression of Ucp1, Cidea, Cox7a1, and Cox8b and the OCR at day 4 of differentiation, thus indicating that miR-19b mediates the adverse effects of DEX through its inhibitory action on browning and oxygen consumption. The presence of BAT has been reported in adult humans, thus making this tissue a potential target for the treatment of obesity and metabolic syndromes (23). We have recently shown that the expression of BAT-specific genes, including Ucp1, Cidea, Cox7a1, and Cox8b, is significantly decreased in the BAT of DEX-treated mice (16). We therefore hypothesized that miR-19b may mediate GC regulation of BAT functions. Consistent with our observations in SAT, DEX treatment led to a decrease in BAT-specific gene expression and OCR, whereas overexpression of miR-19b further suppressed the expression of these genes. In contrast, anti–miR-19b significantly blocked the effect of DEX on the mRNA expression of BAT-specific genes and OCR. Thus, we confirmed that GCs inhibit the function of BAT by activating the expression of miR-19b in vitro. There is mounting evidence indicating miRNAs play essential roles in regulation of target genes. Using miRNA target prediction analyses, we identified a putative miR-19b target site in the 3′−UTR of Adrb1, a regulator of both the BAT function and the browning of white fat. Using luciferase reporter assays, we confirmed a role for miR-19b in the regulation of Adrb1. Mutation of the conserved target site abrogated the miRNA-induced repression of the Adrb1 3′-UTR. DEX treatment led to a decrease in Adrb1 mRNA expression, and overexpression of miR-19b further suppressed the expression of Adrb1 mRNA, whereas anti–miR-19b substantially blocked the effect of DEX on the mRNA expression of Adrb1 in differentiated SVF cells derived from SAT. As expected, a significant increase in the Adrb1 protein level was observed in SVF cells transfected with anti–miR-19b derived from SAT after 4 days of differentiation. These results indicate that DEX suppressed Adrb1 mRNA expression via miR-19b. The increase in Adrb1 by inhibition of GC-induced miR-19b may be essential for the observed effects, which requires further investigation. Overall, our data showing that miR-19b is upregulated by GCs provide a mechanism underlying browning in subcutaneous adipose tissue as well as BAT function. Owing to the increased importance of adipose functions, it would be interesting to investigate whether miR-19b is a potent therapeutic target for clinical purposes. Appendix. Antibody Table Peptide/Protein Target  Name of Antibody  Manufacturer, Catalog No.  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  Ucp1  AntiUcp1  Abcam, ab10983  Rabbit; polyclonal  1:1000  AB_2241462  Adrb1  Anti-Adrb1  Sigma-Aldrich, SAB4500573  Rabbit; polyclonal  1:1000  AB_10745010  GAPDH  Anti-GAPDH  Abcam, ab9485  Rabbit; polyclonal  1:1000  AB_307275  GR  Anti-GR  Cell Signaling Technology, 12041  Human; monoclonal  1:50  AB_2631286  Peptide/Protein Target  Name of Antibody  Manufacturer, Catalog No.  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  Ucp1  AntiUcp1  Abcam, ab10983  Rabbit; polyclonal  1:1000  AB_2241462  Adrb1  Anti-Adrb1  Sigma-Aldrich, SAB4500573  Rabbit; polyclonal  1:1000  AB_10745010  GAPDH  Anti-GAPDH  Abcam, ab9485  Rabbit; polyclonal  1:1000  AB_307275  GR  Anti-GR  Cell Signaling Technology, 12041  Human; monoclonal  1:50  AB_2631286  Abbreviation: RRID, Research Resource Identifier. View Large Abbreviations: 3′-UTR 3′-untranslated region BAT brown adipose tissue ChIP chromatin immunoprecipitation DAPI 4′,6-diamidino-2-phenylindole DEX dexamethasone DMEM Dulbecco's modified Eagle medium FBS fetal bovine serum GAPDH glyceraldehyde 3-phosphate dehydrogenase GC glucocorticoid GR glucocorticoid receptor GRE glucocorticoid response element miRNA microRNA OCR oxygen consumption rate OL oligomycin PCR polymerase chain reaction Rot rotenone SAT subcutaneous adipose tissue scr-miR scrambled miR-19b oligonucleotides SDS sodium dodecyl sulfate SEM standard error of the mean SVF stromal vascular fraction Ucp1 uncoupling protein-1 VAT visceral adipose tissue WAT white adipose tissue. Acknowledgments Financial Support: This work was supported by National Natural Science Foundation of China Grants 81370950 and 81170796 (to G.-X.D.) and by the Natural Science Foundation of Jiangsu Province Youth Project, Grant BK20151036 (to J.Y.). Author Contributions: Y.-F.L. and J.Y. wrote the main manuscript and performed the majority of the experiments. Y.-L.S. and M.H. collected the samples. X.-C.K., W.-J.D., and J.L provided oversight for the project and participated in editing of the manuscript. H.Z. participated in analysis and interpretation of data and editing the manuscript. G.-X.D. and H. L. conceived and designed the study. All authors reviewed the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Anagnostis P, Athyros VG, Tziomalos K, Karagiannis A, Mikhailidis DP. Clinical review. The pathogenetic role of cortisol in the metabolic syndrome: a hypothesis. J Clin Endocrinol Metab . 2009; 94( 8): 2692– 2701. Google Scholar CrossRef Search ADS PubMed  2. Buttgereit F, Burmester GR, Lipworth BJ. Optimised glucocorticoid therapy: the sharpening of an old spear. Lancet . 2005; 365( 9461): 801– 803. Google Scholar CrossRef Search ADS PubMed  3. Cinti S. 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Recruited vs. nonrecruited molecular signatures of brown, “brite,” and white adipose tissues. Am J Physiol Endocrinol Metab . 2012; 302( 1): E19– E31. Google Scholar CrossRef Search ADS PubMed  19. Ueta CB, Fernandes GW, Capelo LP, Fonseca TL, Maculan FD, Gouveia CH, Brum PC, Christoffolete MA, Aoki MS, Lancellotti CL, Kim B, Bianco AC, Ribeiro MO. β(1) Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice. J Endocrinol . 2012; 214( 3): 359– 365. Google Scholar CrossRef Search ADS PubMed  20. Lee YH, Petkova AP, Konkar AA, Granneman JG. Cellular origins of cold-induced brown adipocytes in adult mice. FASEB J . 2015; 29( 1): 286– 299. Google Scholar CrossRef Search ADS PubMed  21. Mattsson CL, Csikasz RI, Chernogubova E, Yamamoto DL, Hogberg HT, Amri EZ, Hutchinson DS, Bengtsson T. β1-Adrenergic receptors increase UCP1 in human MADS brown adipocytes and rescue cold-acclimated β3-adrenergic receptor-knockout mice via nonshivering thermogenesis. 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Glucocorticoids Suppress the Browning of Adipose Tissue via miR-19b in Male Mice

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2017-00566
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Abstract

Abstract Physiological levels of glucocorticoids (GCs) are required for proper metabolic control, and excessive GC action has been linked to a variety of pandemic metabolic diseases. MicroRNA (miRNA)-19b plays a critical role in the pathogenesis of GC-induced metabolic diseases. This study explored the potential of miRNA-based therapeutics targeting adipose tissue. Our results showed that overexpressed miR-19b in stromal vascular fraction (SVF) cells derived from subcutaneous adipose tissue had the same effects as dexamethasone (DEX) treatment on the inhibition of adipose browning and oxygen consumption rate. The inhibition of miR-19b blocked DEX-mediated suppression of the expression of browning marker genes as well as the oxygen consumption rate in differentiated SVF cells derived from subcutaneous and brown adipose tissue. Overexpressed miR-19b in SVF cells derived from brown adipose tissue had the same effects as DEX treatment on the inhibition of brown adipose differentiation and energy expenditure. Glucocorticoids transcriptionally regulate the expression of miR-19b via a GC receptor–mediated direct DNA binding mechanism. This study confirmed that miR-19b is an essential target for GC-mediated control of adipose tissue browning. It is hoped that the plasticity of the adipose organ can be exploited in the next generation of therapeutic strategies to combat the increasing incidence of metabolic diseases, including obesity and diabetes. Since the discovery of the beneficial effects of adrenocortical extract in treating adrenal insufficiency more than 80 years ago, glucocorticoids (GCs) and their cognate intracellular receptor, the GC receptor (GR), have been shown to be critical components of the delicate hormonal control system that regulates energy homeostasis in mammals. Although physiological levels of GCs are required for normal metabolic control, excessive GC activity has been linked to a variety of pervasive metabolic diseases, such as type 2 diabetes and obesity (1). Given its importance in human health, the molecular mechanisms of GC action have been extensively studied in biomedical research. In particular, elucidation of the tissue-specific functions of the GC pathway has identified therapeutic strategies for the treatment of severe metabolic disorders (2). Adipose tissue is made up of multiple depots located in two compartments of the body: some are below the skin (subcutaneous depots), and some are in the trunk (visceral depots). Adipose tissues thus make up a multi-depot organ that is involved in many critical survival functions, facilitating thermogenesis, lactation, and immune responses and serving as a fuel for metabolism. Traditionally, adipocytes have been divided into two types. White adipocytes compose the bulk of adipose tissue in most animals; they can be found in the marbling in steaks and in abdominal fat after weight gain (3). Brown adipocytes, in contrast, are highly specialized cells that dissipate stored chemical energy in the form of heat. This is mediated through the actions of uncoupling protein-1 (Ucp1), a brown adipose tissue (BAT)-specific protein located within the mitochondria, which are densely packed in these cells (4, 5). Numerous studies have demonstrated that Ucp1-expressing thermogenic adipocytes, so-called “brite” or “beige” adipocytes, can also be activated in white adipose tissue (WAT), thus resulting in WAT “browning,” which contributes to increased energy expenditure (6, 7). Recently, large depots of genuine BAT in adult humans have been identified on the basis of radiological observations of symmetrical [18F]-2-fluoro-D-2-deoxy-d-glucose positron emission tomography–positive loci in the supraclavicular and spinal regions (8, 9). The plasticity of the adipose organ might be exploited in the next generation of therapeutic strategies to combat the increasing incidence of metabolic diseases, including obesity and type 2 diabetes mellitus (10). MicroRNAs (miRNAs) are small (∼22 nucleotides) noncoding RNAs that are important regulators of mRNA expression. Recent findings have indicated that miRNAs are involved in the networks regulating many biological processes, including cell differentiation, animal development, metabolism, tumorigenesis, and other diseases, by regulating transcription factors and/or other genes (11, 12). Several miRNAs expressed in the adipocytes of mammals have been shown to play a role in adipogenesis and may affect adipogenesis dysfunction (13). In addition, many miRNAs are dysregulated in the metabolic tissues of obese animals and humans, thus potentially contributing to the pathogenesis of obesity-associated complications (14). In a previous study, the expression of more than 20 miRNAs regulated by GCs in human adipocytes has been examined (15). miR-19b is the most upregulated miRNA in response to GC treatment (15). Here, we found that the repression of BAT function, as well as browning in subcutaneous WAT by GCs, can be mediated by miR-19b. These results provide a basis for future research on miRNA-based therapeutics to target adipose tissue, which may lead to new strategies in the treatment of obesity and its associated complications. Materials and Methods Adipocyte culture and differentiation Inguinal, epididymal, and interscapular adipose tissues from 3-week-old male C57BL/6J mice were removed and minced with a scalpel. After digestion with collagenase and centrifugation, adipocyte precursors derived from the stromal vascular fraction (SVF) were cultured in growth medium [Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS)]. After 48 hours, cells were cultured in differentiation medium [DMEM containing 10% FBS, 1 µM dexamethasone (DEX), 0.5 mmol/L 3-isobutyl-1-methylxanthine, and 20 nM insulin] for 4 days. Cells were grown in maintenance medium containing 10% FBS and 20 nM insulin for the remainder of the culture period. For BAT in vitro differentiation, the cells were treated with growth medium containing 20 nM insulin and 1 nM tri-iodothyronine for 48 hours. After this induction period, the cells were washed and incubated in differentiation medium with an additional 1 µM DEX. The culture medium was changed every 2 days. Full differentiation was achieved after 6 days. The methods were carried out in accordance with the approved guidelines of the Animal Care and Use Committee of Nanjing Medical University. The experimental protocols were approved by the Animal Care and Use Committee of Nanjing Medical University. Murine adipocyte transfection SVF cells derived from subcutaneous adipose tissue (SAT)/visceral adipose tissue (VAT)/BAT were cultured in growth medium as described earlier. After the cells had grown to 80% confluence, they were transfected with either a mimic–miR-19b or mimic–miR–control transfection complex consisting of 50 nM miRNA and 1 μL of siPORT NeoFX transfection agent (Ambion, Austin, TX) in 50 μL of Opti-MEM. To inhibit miR-19b expression, the cells were transfected with 2 nM locked nucleic acid–modified anti–miR-19b oligonucleotides (methylene bridge between the 2′-O and the 4′-C atoms) (Exiqon, Copenhagen, Denmark) or locked nucleic acid–scrambled miR-19b control (anti–miR-control). RNA preparation and quantitative real-time polymerase chain reaction of mRNA Total RNA was extracted from adipose tissues or adipocytes by using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA (2 µg) was reverse-transcribed to cDNA using 200 U M-MLV reverse transcription (Promega, Madison, WI) in the presence of 0.5 mM deoxynucleotide triphosphate, 25 U RNase inhibitor, and 0.5 μg N15 random primers in a total volume of 25 μL. The sequences of the primers are shown in Supplemental Table 1. Each quantitative real-time polymerase chain reaction (PCR) reaction was carried out in triplicate in a 25 μL volume using SYBR Green Real-time PCR Master Mix (Roche, Basel, Switzerland). β-actin was used as an internal reference as described previously. The expression of each gene was arbitrarily set at 1 to facilitate comparison between several treatment groups. For miRNA quantitative real-time PCR, a miRNA-specific stem-loop reverse transcription primer was hybridized to the miRNA and then reverse-transcribed. Then, the reverse transcription product was amplified and monitored in real time using a miRNA-specific upstream primer and a universal downstream primer (Supplemental Table 1; universal downstream primer: 5′-GTGCAGGGTCCGAGGT-3′). The expression levels were normalized to that of small noncoding RNA U6. Western blotting The cell extracts were lysed in radio-immunoprecipitation assay buffer [0.5% NP-40, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, and 50 mM Tris-Cl, pH 7.5]. The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA), and probed with anti-Ucp1 (1:1000, Abcam, Cambridge, MA), anti-Adrb1 (1:1000, Sigma-Aldrich, Munich, Germany), and anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Abcam, Cambridge, MA) antibodies overnight at 4°C. The blots were treated with horseradish peroxidase–conjugated anti-rabbit IgG (1:10,000) (Vector Laboratories, Burlingame, CA) in Tris-buffered saline with Tween 20 containing 1% (w/v) bicinchoninic acid for 60 minutes, and immune complexes were detected using an enhanced chemiluminescence plus detection kit (Cell Signaling Technology, Danvers, MA). The bands were quantified using densitometric image analysis software (Quantity One; Bio-Rad, Hercules, CA). The relative expression of Ucp1 and Adrb1 was normalized to that of GAPDH. Chromatin immunoprecipitation assay Chromatin in the control and treated cells was cross-linked with 1% formaldehyde. The cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris, pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablets and phenylmethylsulfonyl fluoride. DNA was fragmented (∼500 bp) using a Branson 250 sonicator (Branson Ultrasonics Corp., Danbury, MA). Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-GR antibody (Cell Signaling Technology). The EpiQuik chromatin immunoprecipitation (ChIP) kit (Epigentek Group Inc., Farmingdale, NY) was used to measure DNA precipitated by particular antibodies. Precipitated genomic DNA was amplified by real-time PCR. Plasmid construction The plasmids comprising the full-length GR, the miR-19b promoter (sequence shown in Supplemental Material), or the miR-19b promoter with mutations in putative glucocorticoid response element (GRE) motifs (see Supplemental Material) were synthesized by Integrated DNA Technologies, annealed, and cloned into the pGL3 vector (Promega) for use in luciferase reporter assays. Site-directed mutagenesis The oligonucleotides comprising the wild-type or mutated miR-19b target sequence were synthesized by Integrated DNA Technologies, annealed, and cloned into the pMiRGLO vector (Promega) for luciferase reporter assays. The miR-19b target sequences within the 3′-untranslated regions (3′-UTRs) of Adrb1 were predicted by TargetScan. The sequence of Adrb1 wild-type was 5′-UGGUUGGAAGGCCAUUUUGCACA-3′, and the mutated sequence was 5′-UGGUUGGAAGGCCAUAAACGUGU-3′. Luciferase assay 3T3-L1 cells (RRID:CVCL_0123) were differentiated for 5 days by culturing of the cells in differentiation medium (DMEM containing 10% FBS, 1 µM DEX, 0.5 mmol/L 3-isobutyl-1-methylxanthine, and 20 nM insulin) for 3 days and in maintenance medium containing 10% FBS and 20 nM insulin for 2 days. Luciferase assays for the GR segment and miR-19b promoter were performed using 3T3-L1 cells after the completion of differentiation. Cells were transfected with luciferase reporter constructs containing the miR-19b promoter or the miR-19b promoter with mutations in GRE sites (mut-miR-19b) and cotransfected with or without GR constructs. Twenty-four hours after transfection, cells were incubated with or without DEX (1 µM), and 24 hours later, the cells were lysed, and extracts were prepared and analyzed using a luciferase reporter assay system (Promega). Luciferase assays for miR-19b and Adrb1 3′-UTR were performed using HEK293T cells (RRID:CVCL_0063). Cells were seeded in 96-well plates, and a plasmid containing the 3′UTR of the murine Adrb1 gene or a mutated version (mut 3′-UTR) was transfected together with mimic–miR-19b or mimic–miR–control using Lipofectamine 2000 (Invitrogen). Luciferase activities were assayed after transfection for 24 hours using a luciferase reporter assay system (Promega). Measurement of O2 consumption SVF cells were cultured in 24-well plates and differentiated as indicated (Seahorse Bioscience, North Billerica, MA). The medium was replaced with prewarmed unbuffered DMEM (DMEM basal medium supplemented with 25 mM glucose, 2 mM sodium pyruvate, 31 mM NaCl, 2 mM GlutaMax, 15 mg/L phenol red, and 2% essentially fatty acid-free bovine serum albumin, pH 7.4) and incubated at 37°C in a non-CO2 incubator for 1.5 hours. The oxygen consumption rate (OCR) was measured at basal glucose levels with oligomycin (ATP synthase inhibitor, 1 µM) (Sigma-Aldrich), which disrupts the respiratory chain. Ucp1-mediated uncoupling respiration was determined after isoproterenol (0.5 μM) stimulation (Sigma-Aldrich). The maximum respiratory capacity was assessed after carbonylcyanide-p-trifluorophenylhydrazone (FCCP) stimulation (1 μM) (Sigma-Aldrich). Finally, the mitochondrial respiration was blocked by 1 µM rotenone (Sigma-Aldrich). The residual OCR was considered nonmitochondrial respiration. Immunofluorescence staining Cells were fixed in 4% paraformaldehyde solution and incubated with primary antibody against Ucp1 (1:100, Abcam, Cambridge, MA) at 4°C overnight, followed by the FITC-conjugated secondary antibody (1:100, Jackson ImmunoResearch Inc., West Grove, PA) for 60 minutes at room temperature. To counterstain the nuclei, 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, Danvers, MA) was used. Cells were visualized under ×20 magnification by fluorescence microscopy (Olympus, Tokyo, Japan). Statistical analysis The results are presented as the mean ± standard error of the mean (SEM). Statistically significant differences were calculated using Student t test. A value of P < 0.05 was considered significant. Results GCs transcriptionally upregulate miR-19b in murine adipocytes We have previously demonstrated that GCs inhibit the function of brown fat and the browning activity of white fat (15, 16). A previous miRNA microarray analysis and other studies have shown that GCs transcriptionally regulate miR-27b expression, thereby promoting body fat accumulation by suppressing the browning of SAT and VAT. In this study, we focused on miR-19b because we have previously found it to be strongly upregulated by 1 µM DEX treatment in differentiated SVF cells derived from human SAT or VAT (15). SVF cells were derived from the SAT or VAT of 3-week-old male C57BL/6J mice in the presence of DEX at a concentration of 0 to 1 µM. Without DEX treatment, the constitutive expression level of miR-19b was significantly higher (1.3-fold) in 4-day differentiated SVF cells derived from VAT than cells derived from SAT [Fig. 1(a)]. DEX treatment elevated miR-19b expression in SAT- and VAT-derived differentiated cells in a dose-dependent manner, and peak levels were observed at 1 µM [Fig. 1(a)]. In contrast, the constitutive expression level of Ucp1 was higher in 4-day differentiated SVF cells derived from SAT (2.4-fold) than in cells derived from VAT. However, compared with that of SAT, the browning ability of VAT was weaker. Ucp1 mRNA expression was still significantly inhibited by DEX in a dose-dependent manner [Fig. 1(b)]. Reverse regulation of Ucp1 and miR-19b by DEX in murine primary adipocyte cultures suggested that DEX suppression of Ucp1 expression and the browning of SAT and VAT may be mediated by miR-19b. Figure 1. View largeDownload slide GCs transcriptionally upregulate miR-19b in murine adipocytes. (a, b) Relative miR-19b and Ucp1 mRNA levels in SVF cells derived from murine SAT or VAT and differentiated for 4 days in the presence of DEX at doses of 0, 0.1, and 1 µM. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05, **P < 0.01, DEX-treated SAT vs control; #P < 0.05, DEX-treated VAT vs control; $P < 0.05, $$P < 0.01, SAT vs VAT. (c) The 3T3-L1 cells were transfected with plasmids containing the miR-19b promoter (miR-19b) or the miR-19b promoter with mutations GRE motifs (mut-miR-19b) and cotransfected with or without GR constructs. Twenty-four hours after transfection, cells were incubated with or without DEX (1 µM) and, 24 hours later, cells were lysed and extracted for analysis of luciferase activity. The experiments were performed in triplicate wells and repeated three times. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (d) Differentiated SVF cells derived from SAT (s) or VAT (v) were treated with or without 1 µM DEX for 24 hours. ChIP assays were performed by using antibodies against GR (GR), RNA-pol II (pol II), and IgG (IgG). Quantitative PCR using primers of miR-19b promoter region (miR-19b) were performed on the chromatin precipitated with anti-GR, anti–RNA-pol II, and anti-IgG antibodies. Quantitative PCR using primers of Gilz and GAPDH were performed on the chromatin precipitated with anti-GR antibodies. Data are expressed as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01, con vs DEX. Figure 1. View largeDownload slide GCs transcriptionally upregulate miR-19b in murine adipocytes. (a, b) Relative miR-19b and Ucp1 mRNA levels in SVF cells derived from murine SAT or VAT and differentiated for 4 days in the presence of DEX at doses of 0, 0.1, and 1 µM. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05, **P < 0.01, DEX-treated SAT vs control; #P < 0.05, DEX-treated VAT vs control; $P < 0.05, $$P < 0.01, SAT vs VAT. (c) The 3T3-L1 cells were transfected with plasmids containing the miR-19b promoter (miR-19b) or the miR-19b promoter with mutations GRE motifs (mut-miR-19b) and cotransfected with or without GR constructs. Twenty-four hours after transfection, cells were incubated with or without DEX (1 µM) and, 24 hours later, cells were lysed and extracted for analysis of luciferase activity. The experiments were performed in triplicate wells and repeated three times. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (d) Differentiated SVF cells derived from SAT (s) or VAT (v) were treated with or without 1 µM DEX for 24 hours. ChIP assays were performed by using antibodies against GR (GR), RNA-pol II (pol II), and IgG (IgG). Quantitative PCR using primers of miR-19b promoter region (miR-19b) were performed on the chromatin precipitated with anti-GR, anti–RNA-pol II, and anti-IgG antibodies. Quantitative PCR using primers of Gilz and GAPDH were performed on the chromatin precipitated with anti-GR antibodies. Data are expressed as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01, con vs DEX. The effects of GCs are mediated through the GR, which activates the transcription of genes by binding to a consensus GRE. To determine whether GCs transcriptionally regulate miR-19b expression by binding to GRE sites in vitro, we performed luciferase reporter assays. We generated luciferase reporter constructs containing the miR-19b promoter region (miR-19b) or mutant miR-19b promoters with mutations of putative GRE motifs (mut–miR-19b). Then, 1 µM DEX was added to induce miR-19b activity in 3T3-L1 cells cotransfected with GR construct to more than 1.5-fold higher than that of cells in the absence of DEX, whereas there was about 20% activity induction by DEX observed in the cells without GR cotransfection compared with the untreated cells, thus indicating endogenous GR activity in 3T3-L1 cells. Additionally, mutations in the GREs resulted in the complete abrogation of miR-19b promoter activation by DEX, indicating that liganded GR bound to GRE motifs on the promoter regions of miR-19b [Fig. 1(c)]. To further verify that liganded GR transcriptionally regulated the expression of miR-19b, ChIP assay was performed. Our observation revealed that 1 µM DEX treatment led to more then 3.5-fold enhancements in GR binding to the miR-19b promoter in the 4-day differentiated SVF cells derived from SAT and VAT compared with the cells without DEX treatment, which were consistent with our luciferase assay. We detected a >2.5-fold elevation in RNA–pol II binding to the miR-19b promoter region with 1 µM DEX treatment, indicating the elevation of miR-19b’s transcription. Also, we examined a known GR target locus (Gilz) as positive control by PCR on the chromatin precipitated with anti-GR antibody. Treatment with 1 µM DEX led to more than twofold enhancement in GR binding to the Gilz promoter region. In addition, we detected GAPDH on the chromatin precipitated with anti-GR antibody. As a negative control of ChIP assay, GR barely bound to GAPDH promoter region, which obviously did not bring any significant change in GAPDH promoter region compared with the cells without DEX treatment [Fig. 1(d)]. These results indicated that GCs transcriptionally upregulate miR-19b expression via a GR-mediated DNA binding mechanism. miR-19b negatively regulates brown marker genes in differentiated SVF cells derived from SAT We next examined the expression of miR-19b in the primary adipocyte cultures derived from SAT or VAT over a 6-day time course of differentiation. During differentiation, the expression of miR-19b decreased and reached a minimum on day 4 (35% lower than on day 0) and then increased to the baseline level on day 6. In contrast, the Ucp1 mRNA expression increased during differentiation, peaking on day 4 (5.7-fold higher than on day 0) [Fig. 2(a) and 2(b)]. This inverse relationship between the mRNA expression of Ucp1 and miR-19b suggested that miR-19b might negatively regulate Ucp1 expression. Figure 2. View largeDownload slide Overexpression of miR-19b suppresses the browning effect on adipocytes. (a, b) Relative expression of miR-19b and Ucp1 mRNA during SVF cells differentiation under adipogenic conditions, which were isolated from SAT or VAT (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, SAT; #P < 0.05, VAT, compared with day 0. (c, d) miR-19b mRNA expression and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells from SAT by standard differentiation (Vehicle) or transfecting with either mimic-control (Vehicle + mimic-con) or mimic–miR-19b (Vehicle + miR-19b) after 4 days differentiation. A concentration of 1 mmol/L DEX (DEX + mimic-con) presents as positive control (n = 4). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con. (e) Representative images of Western blots of Ucp1 protein of whole cell lysates from differentiated SVF cells isolated from SAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). (f) Bands were quantified using densitometric image analysis software. The relative expression of Ucp1 was normalized to that of GAPDH (mean ± SEM; n = 3). **P < 0.01, SAT mimic-con vs mimic–miR-19b. (g, h) Immunofluorescence staining of Ucp1 (green) in 4 days differentiated SVF cells isolated from SAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity were quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, SAT mimic-con vs mimic–miR-19b. (i) OCRs of SVF cells derived from SAT were quantified in the cell cultures under the same conditions as in (c) and (d) under basal conditions (Basal) or with the following drugs disrupting the respiratory chain: OL, FCCP (a mitochondrial uncoupler), and Rot (mean ± SEM; n = 5). **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05, Vehicle + mimic-con vs DEX + mimic–miR-con. Figure 2. View largeDownload slide Overexpression of miR-19b suppresses the browning effect on adipocytes. (a, b) Relative expression of miR-19b and Ucp1 mRNA during SVF cells differentiation under adipogenic conditions, which were isolated from SAT or VAT (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, SAT; #P < 0.05, VAT, compared with day 0. (c, d) miR-19b mRNA expression and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells from SAT by standard differentiation (Vehicle) or transfecting with either mimic-control (Vehicle + mimic-con) or mimic–miR-19b (Vehicle + miR-19b) after 4 days differentiation. A concentration of 1 mmol/L DEX (DEX + mimic-con) presents as positive control (n = 4). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con. (e) Representative images of Western blots of Ucp1 protein of whole cell lysates from differentiated SVF cells isolated from SAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). (f) Bands were quantified using densitometric image analysis software. The relative expression of Ucp1 was normalized to that of GAPDH (mean ± SEM; n = 3). **P < 0.01, SAT mimic-con vs mimic–miR-19b. (g, h) Immunofluorescence staining of Ucp1 (green) in 4 days differentiated SVF cells isolated from SAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity were quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, SAT mimic-con vs mimic–miR-19b. (i) OCRs of SVF cells derived from SAT were quantified in the cell cultures under the same conditions as in (c) and (d) under basal conditions (Basal) or with the following drugs disrupting the respiratory chain: OL, FCCP (a mitochondrial uncoupler), and Rot (mean ± SEM; n = 5). **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05, Vehicle + mimic-con vs DEX + mimic–miR-con. The constitutive Ucp1 expression level was extremely low in VAT, which was considered to be classic white adipose and highly resistant to browning. It is widely accepted that subcutaneous fat (but not visceral fat) is highly susceptible to browning in rodents (17, 18). We therefore use SAT to clarify the function of miR-19b in regulating the browning activity of adipocytes. Mimic–miR-19b was transfected into SVF cells derived from SAT in the presence or absence of 1 µM DEX. Compared with expression in the vector control cells (mimic-con), DEX treatment led to a threefold increase in miR-19b expression, and expression increased by 18-fold in cells treated with mimic–miR-19b [Fig. 2(c)]. Accordingly, DEX-treated cells showed a substantial reduction in the expression of browning marker genes, including Ucp1, Cidea, Cox7a1, and Cox8b. Overexpression of miR-19b also led to a decrease in these genes [Fig. 2(d)]. Notably, the mRNA expression of Fabp4, a marker of mature white adipocytes, was not altered by mimic–miR-19b transfection. Western blot analyses also indicated that the Ucp1 protein level was decreased in SVF cells transfected with mimic–miR-19b- derived from SAT after 4 days of differentiation [Fig. 2(e) and 2(f)]. Additionally, immunofluorescence staining of Ucp1 showed a significant decrease in the overexpression of miR-19b (mimic–miR-19b) compared with the vector control cells (mimic-con) [Fig. 2(g) and 2(h)]. Consistent with the inhibited expression of genes for browning, the oxygen consumption rate (OCR) in basal condition (Basal) and the maximal respiration stimulated by the mitochondrial uncoupler FCCP of cells transfected with mimic–miR-19b and cultured under the same conditions as described for Fig. 2(c) were markedly inhibited to levels similar to those observed after DEX treatment. However, there were no significant differences in coupled respiration [oligomycin (OL)] and nonmitochondrial respiration [rotenone (Rot)] OCR [Fig. 2(i)]. We then transfected anti–miR-19b to induce cells’ browning abilities. Scrambled miR-19b oligonucleotides (scr-miR) were used as a control. After 4 days of differentiation of SVF cells derived from SAT in the presence or absence of 1 µM DEX, anti–miR-19b transfection successfully inhibited miR-19b expression in both DEX-treated (86%) and untreated cells (90%) [Fig. 3(a)]. Silencing of miR-19b led to a significant increase in the mRNA expression of Ucp1, Cidea, Cox7a1, and Cox8b and blocked the effect of DEX on the mRNA expression of these browning marker genes [Fig. 3(b)]. Fabp4 mRNA was not altered by either DEX or anti–miR-19b treatment. Western blot analyses indicated that the Ucp1 protein level was also substantially elevated in SVF cells transfected with anti–miR-19b derived from SAT after 4 days of differentiation [Fig. 3(c) and 3(d)]. Immunofluorescence staining of Ucp1 also markedly raised in the silence of miR-19b (anti–miR-19b) compared with the vector control cells (scr-miR) [Fig. 3(e) and 3(f)]. DEX treatment led to a significant decrease of OCR in basal condition (Basal), coupled respiration (OL), and cells stimulated by the FCCP. Furthermore, silencing of miR-19b blocked the inhibitory effects of DEX on OCR in basal conditions, coupled respiration (OL), and FCCP stimulation. There were no differences in nonmitochondrial respiration (Rot) OCR [Fig. 3(g)]. Figure 3. View largeDownload slide Inhibition of miR-19b promotes the browning effect in adipocytes. (a, b) Relative expression of miR-19b and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in differentiated SVF cells from SAT after transfection with scrambled (scr) control (Vehicle + scr-miR), anti–miR-19b (Vehicle + anti-miR-19b), or 1 µM DEX treatment (DEX + scr-miR, DEX + anti-miR-19b) after 4 days of differentiation (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05; ##P < 0.01; ###P < 0.001, Vehicle + scr-miR vs DEX + scr-miR. $P < 0.05; $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b). Experiments were repeated three times. (c) Representative images of Western blots of Ucp1 protein of whole cell lysates from differentiated SVF cells isolated from SAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti-miR-19b). (d) Bands were quantified using densitometric image analysis software. The relative expression of Ucp1 was normalized to that of GAPDH (mean ± SEM; n = 3). *P < 0.05, SAT scr-miR vs anti–miR-19b). (e, f) Immunofluorescence staining of Ucp1 in 4-day differentiated SVF cells isolated from SAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, SAT scr-miR vs anti–miR-19b. (g) OCRs were quantified in the cell cultures under the same conditions as in (a) and (b) under basal conditions (Basal) or with the following drugs disrupting the respiratory chain: OL, FCCP, and Rot (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05, Vehicle + scr-miR vs DEX + scr-miR. $P < 0.05, DEX + scr-miR vs DEX + anti–miR-19b. Figure 3. View largeDownload slide Inhibition of miR-19b promotes the browning effect in adipocytes. (a, b) Relative expression of miR-19b and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in differentiated SVF cells from SAT after transfection with scrambled (scr) control (Vehicle + scr-miR), anti–miR-19b (Vehicle + anti-miR-19b), or 1 µM DEX treatment (DEX + scr-miR, DEX + anti-miR-19b) after 4 days of differentiation (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05; ##P < 0.01; ###P < 0.001, Vehicle + scr-miR vs DEX + scr-miR. $P < 0.05; $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b). Experiments were repeated three times. (c) Representative images of Western blots of Ucp1 protein of whole cell lysates from differentiated SVF cells isolated from SAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti-miR-19b). (d) Bands were quantified using densitometric image analysis software. The relative expression of Ucp1 was normalized to that of GAPDH (mean ± SEM; n = 3). *P < 0.05, SAT scr-miR vs anti–miR-19b). (e, f) Immunofluorescence staining of Ucp1 in 4-day differentiated SVF cells isolated from SAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, SAT scr-miR vs anti–miR-19b. (g) OCRs were quantified in the cell cultures under the same conditions as in (a) and (b) under basal conditions (Basal) or with the following drugs disrupting the respiratory chain: OL, FCCP, and Rot (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05, Vehicle + scr-miR vs DEX + scr-miR. $P < 0.05, DEX + scr-miR vs DEX + anti–miR-19b. miR-19b negatively regulates the function of BAT A recent investigation has confirmed that GCs inhibit the function of BAT (16). The current study investigated the underlying mechanism by validating the expression of miR-19b in SVF cells derived from BAT. During brown adipocyte differentiation, the expression of miR-19b decreased and reached a minimum on day 6 (2.7-fold lower than day 0) [Fig. 4(a)]. In contrast, the expression of Ucp1 mRNA increased and reached a maximum on day 4 (2.1-fold higher than day 0) and remained at that level on day 6 (1.81-fold higher than day 0) during differentiation [Fig. 4(b)]. We then overexpressed miR-19b (mimic–miR-19b) in SVF cells derived from BAT and differentiated the cells for 4 days in the presence or absence of 1 µM DEX. Compared with expression in the vector control cells (mimic-con), DEX treatment led to a threefold increase in miR-19b expression, and expression increased by eightfold in cells treated with mimic–miR-19b [Fig. 4(c)]. Accordingly, DEX-treated cells showed a substantial reduction in the expression of brown adipocyte marker genes, including Ucp1, Cidea, Cox7a1, and Cox8b. Overexpression of miR-19b led to a decrease in these genes [Fig. 4(d)]. In addition, immunofluorescence staining showed that overexpression of miR-19b (mimic–miR-19b) dramatically repressed Ucp1 expression compared with the vector control cells (mimic-con) [Fig. 4(e) and 4(f)]. Importantly, the OCR of cells transfected with mimic–miR-19b in basal conditions and after stimulation with OL and FCCP was markedly inhibited to levels similar to those observed after DEX treatment [Fig. 4(g)]. We then silenced miR-19b in SVF cells derived from BAT. Cells transfected with either anti–miR-19b or scrambled miR-19b oligonucleotide control (scr-miR) were differentiated for 4 days in the presence or absence of 1 µM DEX. Anti–miR-19b transfection successfully inhibited miR-19b expression in both DEX-treated (61%) and untreated cells (66%) [Fig. 4(h)]. Compared with the expression in anti-control cells (scr-miR), anti–miR-19b led to a notable increase in the mRNA expression of Ucp1, Cidea, Cox7a1, and Cox8b and blunted the effect of DEX on the mRNA expression of these genes [Fig. 4(i)]. Accordingly, miR-19b deficiency group (anti–miR-19b) displayed a remarkable increase in the immunofluorescence staining of Ucp1 compared with the vector control group (scr-miR) [Fig. 4(j) and 4(k)]. Silencing of miR-19b significantly blocked the inhibitory effects of DEX on the OCR in basal conditions and after stimulation by OL and FCCP [Fig. 4(l)]. Together, these data indicated that GCs inhibit the function of BAT by activating the expression of miR-19b in vitro. Figure 4. View largeDownload slide miR-19b is a potent negative regulator of the functions of brown adipocytes. (a, b) Relative expression of miR-19b and Ucp1 mRNA during differentiation of SVF cells, which were isolated from BAT and differentiated under adipogenic conditions. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, compared with day 0. (c, d) miR-19b mRNA expression and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells from BAT by standard differentiation (Vehicle) or transfecting with either mimic-control (Vehicle + mimic-con) or mimic–miR-19b (Vehicle + miR-19b) after 4 days of differentiation. A concentration of 1 mmol/L DEX (DEX + mimic-con) presents as positive control (mean ± SEM; n = 4). **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con). (e, f) Immunofluorescence staining of Ucp1 in 4-day differentiated SVF cells isolated from BAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, BAT mimic-con vs mimic–miR-19b. (g) OCRs of SVF cells derived from BAT were quantified under basal conditions and after treatment with drugs disrupting the respiratory chain: OL, FCCP, and Rot (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con. (h, i) Relative expression of miR-19b and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells derived from BAT after transfection with scrambled (scr) control (Vehicle + scr-miR), anti–miR-19b (Vehicle + antimiR-19b), or 1 µM DEX treatment (DEX + scr-miR and DEX + anti–miR-19b) after 4 days of differentiation. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + scr-miR vs DEX + scr-miR. $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b. (j, k) Immunofluorescence staining of Ucp1 in 4 days differentiated SVF cells isolated from BAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti–miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity were quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). *P < 0.05, BAT scr-miR vs anti–miR-19b). (l) OCRs were quantified in the cells described in (h) (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. ##P < 0.01, Vehicle + scr-miR vs DEX + scr-miR. $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b. Figure 4. View largeDownload slide miR-19b is a potent negative regulator of the functions of brown adipocytes. (a, b) Relative expression of miR-19b and Ucp1 mRNA during differentiation of SVF cells, which were isolated from BAT and differentiated under adipogenic conditions. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, compared with day 0. (c, d) miR-19b mRNA expression and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells from BAT by standard differentiation (Vehicle) or transfecting with either mimic-control (Vehicle + mimic-con) or mimic–miR-19b (Vehicle + miR-19b) after 4 days of differentiation. A concentration of 1 mmol/L DEX (DEX + mimic-con) presents as positive control (mean ± SEM; n = 4). **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con). (e, f) Immunofluorescence staining of Ucp1 in 4-day differentiated SVF cells isolated from BAT after transfection with mimic-control (mimic-con) or mimic–miR-19b (mimic-miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity was quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). **P < 0.01, BAT mimic-con vs mimic–miR-19b. (g) OCRs of SVF cells derived from BAT were quantified under basal conditions and after treatment with drugs disrupting the respiratory chain: OL, FCCP, and Rot (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + mimic-con vs Vehicle + mimic–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + mimic-con vs DEX + mimic-con. (h, i) Relative expression of miR-19b and browning signature genes (Ucp1, Cidea, Cox7a1, Cox8b) in SVF cells derived from BAT after transfection with scrambled (scr) control (Vehicle + scr-miR), anti–miR-19b (Vehicle + antimiR-19b), or 1 µM DEX treatment (DEX + scr-miR and DEX + anti–miR-19b) after 4 days of differentiation. The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05; ##P < 0.01, Vehicle + scr-miR vs DEX + scr-miR. $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b. (j, k) Immunofluorescence staining of Ucp1 in 4 days differentiated SVF cells isolated from BAT after transfection with anticontrol (scr-miR) or anti–miR-19b (anti–miR-19b). Nuclei were counterstained with DAPI. Fluorescence intensity were quantified using densitometric image analysis software with cell quantity adjustment (mean ± SEM; n = 5). *P < 0.05, BAT scr-miR vs anti–miR-19b). (l) OCRs were quantified in the cells described in (h) (mean ± SEM; n = 5). *P < 0.05; **P < 0.01, Vehicle + scr-miR vs Vehicle + anti–miR-19b. ##P < 0.01, Vehicle + scr-miR vs DEX + scr-miR. $$P < 0.01, DEX + scr-miR vs DEX + anti–miR-19b. miR-19b directly targets Adrb1 β-Adrenergic receptors are the major mediators of the facultative thermogenesis activated by the sympathetic nervous system and have a fundamental role in regulating energy expenditure (19). β-Adrenergic receptor-1 (Adrb1) knockout mice are cold intolerant and obese. Additionally, they are more prone to developing obesity when they are fed a high-fat diet (20). In humans, Adrb1 plays an important role in regulating nonshivering thermogenesis. The activation of Adrb1 increases Ucp1 mRNA and protein levels (21). According to miRNA target prediction analyses [TargetScan, miRanda, and Diana MicroT-CDS (22)], Adrb1 was a predicated target gene of miR-19b, and a putative miR-19b target site is present at a highly conserved octamer seed motif within the 3′-untranslated region (3′-UTR) of Adrb1 [Fig. 5(a)]. We therefore performed luciferase assays to investigate the direct targeting of the Adrb1 3′-UTR by miR-19b. HEK293T cells transfected with reporter plasmids containing the Adrb1 3′-UTR showed significantly decreased luciferase activity in the presence of miR-19b. Mutation of the conserved seed sequence (Mut 3′-UTR) abrogated the miRNA-induced repression of the Adrb1 3′UTR [Fig. 5(b)]. To verify the interaction between miR-19b and Adrb1 in adipose tissue, we examined the Adrb1 mRNA expression levels in response to the miR-19b mimics and inhibitors in the presence or absence of DEX in the 4-day differentiated SVF cells isolated from SAT. Overexpression of miR-19b significantly suppressed Adrb1 mRNA expression levels [Fig. 5(c)]. Silencing of miR-19b stimulated the expression of Adrb1 mRNA and substantially blunted the effect of DEX on the expression of Adrb1 mRNA [Fig. 5(d)], thus indicating that DEX suppressed Adrb1 mRNA expression via miR-19b. Additionally, Western blot analysis revealed an elevation of Adrb1 protein levels in SVF cells transfected with anti–miR-19b derived from SAT on day 4 after induction of differentiation [Fig. 5(e) and 5(f)]. Figure 5. View largeDownload slide miR-19b directly targets Adrb1. (a) The miRNA target prediction analyses from TargetScan and Diana microT-CDS. (b) The 3′-UTR of the Adrb1 gene contains the predicted binding site for miR-19b, as shown by the colored sequences, and was cloned into the pMiRGLO vector (Adrb1 3′UTR). In the mutant construct (mut 3′UTR of Adrb1), the corresponding binding sequences that were mutated are indicated in black. Relative luciferase activity in HEK293T cells transfected with plasmid reporter constructs containing the 3′-UTR (3′UTR) or mutated 3′-UTR (Mut 3′UTR) of Adrb1 is shown. The experiments were performed in triplicate wells and repeated three times (mean ± SEM). *P < 0.05, con vs miR-19b. (c) Relative Adrb1 mRNA expression in 4-day differentiated SVF cells derived from SAT after no treatment (Vehicle) or transfection with mimic-control (Vehicle + mimic-con), mimic-control with 1 µM DEX treatment (DEX + mimic-con), or mimic–miR-19b (Vehicle + mimic–miR-19b) (mean ± SEM; n = 4). **P < 0.01, Vehicle + mimic-con vs Vehicle + miR-19b. #P < 0.05, Vehicle + mimic-con vs DEX + mimic-con. (d) Relative Adrb1 mRNA expression in 4-day differentiated SVF cells derived from SAT or VAT after transfection with scrambled control (Vehicle + scr-miR), anti–miR-19b (Vehicle + anti–miR-19b), or 1 µM DEX treatment (DEX + scr-miR and DEX + anti-miR-19b). The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05, Vehicle + scr-miR vs DEX + scr-miR. $$$P < 0.001, DEX + scr-miR vs DEX + anti–miR-19b. (e) Representative images of Western blots of Adrb1 protein of whole cell lysates from 4-day differentiated SVF cells isolated from SAT after transfection with anti-control (scr-miR) or anti–miR-19b (antimiR-19b). (f) Bands were quantified using densitometric image analysis software. The relative expression of Adrb1 was normalized to that of GAPDH (mean ± SEM; n = 3). **P < 0.01, scr-miR vs anti–miR-19b. Figure 5. View largeDownload slide miR-19b directly targets Adrb1. (a) The miRNA target prediction analyses from TargetScan and Diana microT-CDS. (b) The 3′-UTR of the Adrb1 gene contains the predicted binding site for miR-19b, as shown by the colored sequences, and was cloned into the pMiRGLO vector (Adrb1 3′UTR). In the mutant construct (mut 3′UTR of Adrb1), the corresponding binding sequences that were mutated are indicated in black. Relative luciferase activity in HEK293T cells transfected with plasmid reporter constructs containing the 3′-UTR (3′UTR) or mutated 3′-UTR (Mut 3′UTR) of Adrb1 is shown. The experiments were performed in triplicate wells and repeated three times (mean ± SEM). *P < 0.05, con vs miR-19b. (c) Relative Adrb1 mRNA expression in 4-day differentiated SVF cells derived from SAT after no treatment (Vehicle) or transfection with mimic-control (Vehicle + mimic-con), mimic-control with 1 µM DEX treatment (DEX + mimic-con), or mimic–miR-19b (Vehicle + mimic–miR-19b) (mean ± SEM; n = 4). **P < 0.01, Vehicle + mimic-con vs Vehicle + miR-19b. #P < 0.05, Vehicle + mimic-con vs DEX + mimic-con. (d) Relative Adrb1 mRNA expression in 4-day differentiated SVF cells derived from SAT or VAT after transfection with scrambled control (Vehicle + scr-miR), anti–miR-19b (Vehicle + anti–miR-19b), or 1 µM DEX treatment (DEX + scr-miR and DEX + anti-miR-19b). The experiments were repeated three times (mean ± SEM; n = 4). *P < 0.05, Vehicle + scr-miR vs Vehicle + anti–miR-19b. #P < 0.05, Vehicle + scr-miR vs DEX + scr-miR. $$$P < 0.001, DEX + scr-miR vs DEX + anti–miR-19b. (e) Representative images of Western blots of Adrb1 protein of whole cell lysates from 4-day differentiated SVF cells isolated from SAT after transfection with anti-control (scr-miR) or anti–miR-19b (antimiR-19b). (f) Bands were quantified using densitometric image analysis software. The relative expression of Adrb1 was normalized to that of GAPDH (mean ± SEM; n = 3). **P < 0.01, scr-miR vs anti–miR-19b. Discussion Although physiological levels of GCs are required for normal metabolic control, aberrant GC activity has been linked to a variety of common metabolic diseases. Given the importance of GC signaling pathways in human health, studies of the molecular mechanisms underlying these pathways have become a major focus in biomedical research. Therefore, the current study investigated the effects of GC modulation on metabolic homeostasis, focusing on the tissue-specific contributions of the glucocorticoid axis to the control of energy metabolism. Our current study demonstrated that miR-19b plays a crucial role in the pathogenesis of the detrimental effects of high-dose GCs in adipose tissues and energy metabolism and consequently may be a potential target to prevent GC-induced obesity and metabolic syndromes (Fig. 6). Figure 6. View largeDownload slide Schematic model of the effects of GCs on adipose tissue function in mice. GCs transcriptionally upregulate miR-19b expression. The GC-induced miR-19b then suppresses the expression of its target gene Adrb1 and further inhibits the browning signature genes like Ucp1, Cidea, Cox7a1, and Cox8b. The inhibition of the browning of SAT and the function of BAT results in low energy expenditure. The low energy expenditure consequently causes or at least partly contributes to GC-induced fat accumulation and insulin resistance. Figure 6. View largeDownload slide Schematic model of the effects of GCs on adipose tissue function in mice. GCs transcriptionally upregulate miR-19b expression. The GC-induced miR-19b then suppresses the expression of its target gene Adrb1 and further inhibits the browning signature genes like Ucp1, Cidea, Cox7a1, and Cox8b. The inhibition of the browning of SAT and the function of BAT results in low energy expenditure. The low energy expenditure consequently causes or at least partly contributes to GC-induced fat accumulation and insulin resistance. We have recently reported that GCs inhibit the browning of SAT and VAT. A previous miRNA microarray analysis has found that miR-19b is the miRNA most strongly upregulated by 1 µM DEX treatment in differentiated SVF cells derived from human SAT and VAT (15). Therefore, we focused on the regulation of miR-19b in murine adipocytes. In the current study, we discovered that miR-19b is a GC-target miRNA. DEX treatment elevated miR-19b expression in 4-day differentiated SVF cells derived from SAT or VAT in a dose-dependent manner. Using luciferase assay, we proved that liganded GR regulated miR-19b through binding GRE motifs. We then performed ChIP assay to verify that GRs transcriptionally regulate the expression of miR-19b. By ChIP assay, we detected increased GR binding on miR-19b promoter region as well as an enhancement of RNA–pol II recruitment. Using luciferase assays and ChIP analyses, we eventually confirmed that miR-19b is transcriptionally upregulated by GCs via a GC receptor–mediated DNA binding mechanism. miR-19b is constitutively expressed in murine adipose tissue but was lower in SAT than VAT. In contrast, the gene expression of Ucp1 was higher in SAT than in VAT. Because VAT, the typical white adipose, was considered to be resistant to browning, mimic–miR-19b or anti–miR-19b was only transfected into SVF cells isolated from SAT. According to our studies, DEX treatment led to a decrease in BAT-specific gene expression and OCR, whereas overexpression of miR-19b further suppressed the expression of these genes. Silencing of miR-19b significantly blocked the inhibitory effect of DEX on the expression of Ucp1, Cidea, Cox7a1, and Cox8b and the OCR at day 4 of differentiation, thus indicating that miR-19b mediates the adverse effects of DEX through its inhibitory action on browning and oxygen consumption. The presence of BAT has been reported in adult humans, thus making this tissue a potential target for the treatment of obesity and metabolic syndromes (23). We have recently shown that the expression of BAT-specific genes, including Ucp1, Cidea, Cox7a1, and Cox8b, is significantly decreased in the BAT of DEX-treated mice (16). We therefore hypothesized that miR-19b may mediate GC regulation of BAT functions. Consistent with our observations in SAT, DEX treatment led to a decrease in BAT-specific gene expression and OCR, whereas overexpression of miR-19b further suppressed the expression of these genes. In contrast, anti–miR-19b significantly blocked the effect of DEX on the mRNA expression of BAT-specific genes and OCR. Thus, we confirmed that GCs inhibit the function of BAT by activating the expression of miR-19b in vitro. There is mounting evidence indicating miRNAs play essential roles in regulation of target genes. Using miRNA target prediction analyses, we identified a putative miR-19b target site in the 3′−UTR of Adrb1, a regulator of both the BAT function and the browning of white fat. Using luciferase reporter assays, we confirmed a role for miR-19b in the regulation of Adrb1. Mutation of the conserved target site abrogated the miRNA-induced repression of the Adrb1 3′-UTR. DEX treatment led to a decrease in Adrb1 mRNA expression, and overexpression of miR-19b further suppressed the expression of Adrb1 mRNA, whereas anti–miR-19b substantially blocked the effect of DEX on the mRNA expression of Adrb1 in differentiated SVF cells derived from SAT. As expected, a significant increase in the Adrb1 protein level was observed in SVF cells transfected with anti–miR-19b derived from SAT after 4 days of differentiation. These results indicate that DEX suppressed Adrb1 mRNA expression via miR-19b. The increase in Adrb1 by inhibition of GC-induced miR-19b may be essential for the observed effects, which requires further investigation. Overall, our data showing that miR-19b is upregulated by GCs provide a mechanism underlying browning in subcutaneous adipose tissue as well as BAT function. Owing to the increased importance of adipose functions, it would be interesting to investigate whether miR-19b is a potent therapeutic target for clinical purposes. Appendix. Antibody Table Peptide/Protein Target  Name of Antibody  Manufacturer, Catalog No.  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  Ucp1  AntiUcp1  Abcam, ab10983  Rabbit; polyclonal  1:1000  AB_2241462  Adrb1  Anti-Adrb1  Sigma-Aldrich, SAB4500573  Rabbit; polyclonal  1:1000  AB_10745010  GAPDH  Anti-GAPDH  Abcam, ab9485  Rabbit; polyclonal  1:1000  AB_307275  GR  Anti-GR  Cell Signaling Technology, 12041  Human; monoclonal  1:50  AB_2631286  Peptide/Protein Target  Name of Antibody  Manufacturer, Catalog No.  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  Ucp1  AntiUcp1  Abcam, ab10983  Rabbit; polyclonal  1:1000  AB_2241462  Adrb1  Anti-Adrb1  Sigma-Aldrich, SAB4500573  Rabbit; polyclonal  1:1000  AB_10745010  GAPDH  Anti-GAPDH  Abcam, ab9485  Rabbit; polyclonal  1:1000  AB_307275  GR  Anti-GR  Cell Signaling Technology, 12041  Human; monoclonal  1:50  AB_2631286  Abbreviation: RRID, Research Resource Identifier. View Large Abbreviations: 3′-UTR 3′-untranslated region BAT brown adipose tissue ChIP chromatin immunoprecipitation DAPI 4′,6-diamidino-2-phenylindole DEX dexamethasone DMEM Dulbecco's modified Eagle medium FBS fetal bovine serum GAPDH glyceraldehyde 3-phosphate dehydrogenase GC glucocorticoid GR glucocorticoid receptor GRE glucocorticoid response element miRNA microRNA OCR oxygen consumption rate OL oligomycin PCR polymerase chain reaction Rot rotenone SAT subcutaneous adipose tissue scr-miR scrambled miR-19b oligonucleotides SDS sodium dodecyl sulfate SEM standard error of the mean SVF stromal vascular fraction Ucp1 uncoupling protein-1 VAT visceral adipose tissue WAT white adipose tissue. Acknowledgments Financial Support: This work was supported by National Natural Science Foundation of China Grants 81370950 and 81170796 (to G.-X.D.) and by the Natural Science Foundation of Jiangsu Province Youth Project, Grant BK20151036 (to J.Y.). Author Contributions: Y.-F.L. and J.Y. wrote the main manuscript and performed the majority of the experiments. Y.-L.S. and M.H. collected the samples. X.-C.K., W.-J.D., and J.L provided oversight for the project and participated in editing of the manuscript. H.Z. participated in analysis and interpretation of data and editing the manuscript. G.-X.D. and H. L. conceived and designed the study. All authors reviewed the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Anagnostis P, Athyros VG, Tziomalos K, Karagiannis A, Mikhailidis DP. Clinical review. The pathogenetic role of cortisol in the metabolic syndrome: a hypothesis. J Clin Endocrinol Metab . 2009; 94( 8): 2692– 2701. Google Scholar CrossRef Search ADS PubMed  2. Buttgereit F, Burmester GR, Lipworth BJ. Optimised glucocorticoid therapy: the sharpening of an old spear. Lancet . 2005; 365( 9461): 801– 803. Google Scholar CrossRef Search ADS PubMed  3. Cinti S. 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EndocrinologyOxford University Press

Published: Jan 1, 2018

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