Obesity-Associated miR-199a/214 Cluster InhibitsAdipose Browning via PRDM16–PGC-1a TranscriptionalNetworkLinyun He,1 Mowei Tang,1 Ting Xiao,1 Hailan Liu,1 Wei Liu,2 Guangdi Li,1 Feng Zhang,1 Yalun Xiao,1

Zhiguang Zhou,1 Feng Liu,1,3 and Fang Hu1

Diabetes 2018;67:2585–2600 | https://doi.org/10.2337/db18-0626

miRNAs are important regulators of differentiation, de-velopment, and function of brown and beige fat cells. Inthis study, we identify the role of the miR-199a/214cluster in the regulation of brown and beige adipocytedevelopment and thermogenesis in vitro and in vivo. Weshow that expression of the miR-199a/214 cluster isdramatically decreased during brown and beige adipo-cyte differentiation and in response to cold exposure orb-adrenergic receptor activation. The cluster levels aresignificantly upregulated in the adipose tissues of obesemice and human subjects. Overexpression of the miR-199a/214 cluster suppresses brown adipocyte differen-tiation and inhibits thermogenic gene expression andmitochondrial respiration, whereas knockdown of thecluster increases thermogenic gene expression andmitochondrial function in beige adipocytes. In addition,inhibition of the miR-199a/214 cluster promotes beigingeffects in vivo. We further show that miR-199a/214 sup-presses brown adipocyte differentiation and beige fatdevelopment by directly targeting PRDM16 and peroxi-some PGC-1a, two key transcriptional regulators of ad-ipose browning. Together, these observations reveal thatthe miR-199a/214 cluster is a key negative regulator ofbrown and beige fat development and thermogenesis.

The global incidence of obesity and obesity-related disor-ders, including metabolic syndrome and diabetes, gives riseto a demand for effective therapeutic interventions. Brownand brown-like adipocytes (called beige adipocytes) are

emerging as potential targets for the treatment of obesityand related metabolic diseases (1–4). Both brown and beigeadipocytes trigger a program of mitochondrial respirationand thermogenesis through induction of UCP1 expressionand dissipation of chemical energy to produce heat (5,6).Stimulation of brown and beige fat development leads toincreased energy expenditure and a lean, healthy pheno-type in neonatal mammals, hibernators, rodents, and adulthumans (7,8). However, the mechanisms regulating ther-mogenic fat cells still need to be further elucidated.

The differentiation and development of brown andbeige adipocytes are regulated by multiple transcriptionalfactors and cofactors such as PRD1-BF1-RIZ1 homologousdomain-containing 16 (PRDM16) and peroxisome prolif-erator–activated receptor a and g (PPARa and PPARg)coactivator-1a (PGC-1a) (9–11). PRDM16 is a criticaldeterminant of the brown fat lineage and a key transcrip-tional regulator of brown fat differentiation. It stimulatesdifferentiation of Myf5-positive myogenic precursor cellsinto brown fat cells while prohibiting myogenic differen-tiation by robustly inducing expression of brown adiposetissue (BAT)–selective genes such as UCP1 and PGC-1a(12,13). Increased PRDM16 expression can drive the ex-pression of BAT-selective genes in beige fat cells (14–16).PGC-1a is an important transcriptional coactivator thatregulates brown fat thermogenesis (17,18). It interactswith several transcriptional factors and nuclear receptors,thus controlling the entire program of thermogenesisincluding the transcription of UCP1 and mitochondrial

1Department of Metabolism and Endocrinology, Metabolic Syndrome ResearchCenter, The Second Xiangya Hospital of Central South University, Changsha,Hunan, China2Minimally Invasive Surgery Center, The Second Xiangya Hospital of Central SouthUniversity, Changsha, Hunan, China3Department of Pharmacology, University of Texas Health Science Center at SanAntonio, San Antonio, TX

Corresponding author: Fang Hu, hu_fang98@csu.edu.cn.

Received 6 June 2018 and accepted 20 September 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0626/-/DC1.

L.H. and M.T. contributed equally to the work.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 67, December 2018 2585

OBESITY

STUDIES

biogenesis (17–19). PGC-1a itself is regulated by a num-ber of well-known factors related to cellular energy andmitochondrial homeostasis, including PPARa and PPARg(20). PRDM16 positively regulates PGC-1a expressionby promoting induction of PGC-1a gene transcriptionthrough PPARa (21). However, factors that enable andcoordinate the program of concerted cooperation be-tween the transcriptional factors and coregulators nec-essary for brown and beige adipogenesis remain largelyunknown.

miRNAs are small noncoding RNAs that regulate geneexpression at the posttranscriptional level. Recent studiessuggest that miRNAs are important factors regulatingdifferentiation, development, and function of brownand beige fat cells (for reviews, see refs. 4 and 22). SomemiRNAs, including miR-193b/365 (23), miR-196a (24),miR-133 (25), miR-155 (26), and miR-455 (27), havebeen reported to modulate brown adipocyte differentia-tion by targeting adipogenic regulators. Additionally, miR-328 mediates the action of Dicer1 to control brownadipocyte differentiation and function (28). Very recently,we reported that miR-30 family members are required tomaintain brown adipocyte function and beige fat devel-opment (29). However, it remains unclear how miRNAsmodulate and integrate multiple signaling networksregulating mitochondrial biogenesis and UCP1 expression,the defining signature of brown adipogenesis.

To identify miRNAs that are important for brown adi-pocyte differentiation, we performed a miRNA microarrayanalysis and identified a group of miRNAs that are signif-icantly up- or downregulated during brown adipocyte dif-ferentiation. In this study, we show that expression of themiR-199a/214 cluster is dramatically decreased duringbrown adipocyte differentiation. Reduced expression ofthe miR-199a/214 cluster is also observed in response tocold exposure or treatment with a b-adrenergic receptoractivator. In contrast, adipose miR-199a/214 expression ispositively correlated with obesity in mice and humans.Overexpression of the miR-199a/214 cluster suppressesbrown adipogenesis and decreases thermogenic gene ex-pression in brown adipocytes. Conversely, knockdown ofthe cluster increases the expression of Ucp1 and thermo-genic genes in cultured beige adipocytes and in subcutane-ous white adipose tissue (sWAT) in vivo. We further showthat miR-199a/214 directly targets PRDM16 and PGC-1a,demonstrating a mechanism by which this miRNA clusternegatively regulates beiging and thermogenesis. This studyhighlights the inhibitory roles of the miR-199a/214 clusterin gene cascades involved in brown adipogenesis and theregulation of BAT and beige fat development, thereforeuncovering a potential therapeutic target against obesity.

RESEARCH DESIGN AND METHODS

Cell Culture, Oil Red O Staining, Transfection, andTreatmentThe isolation and culture of the stromal vascular fraction(SVF) from interscapular BAT and inguinal sWAT were

performed as previously described (29,30). The BATs orsWATs from 3-week-old male C57BL/6 mice were slicedand digested in HEPES buffer containing type II collage-nase (Sigma-Aldrich) and BSA (Gibco). The preadipocyteswere plated, cultured, and induced to differentiate accord-ing to the procedure previously reported (18,29).

For Oil Red O staining, mature adipocytes were washedtwice with PBS, fixed in 4% paraformaldehyde for 1 h, andstained with Oil Red O for 1 h. The cells were visualizedand photographed using a light microscope (Olympus,Tokyo, Japan).

For transfection, the duplex oligonucleotide (mimic) orsingle-stain antisense (inhibitor) designed formiR-199a,miR-214, or the respective nonspecific control (NC) (GenePharma,Shanghai, China) was added to 70–80% confluent culturedcells at a final concentration of 100 nmol/L. A pCDNA3.1vector expressing N-terminally Flag-tagged PRDM16 wasa gift from Dr. J. Lin at the University of Michigan (AnnArbor, MI). Transfections were performed using Lipofect-amine 2000 according to the manufacturer’s protocol.

For treatment, the preadipocytes incubated with freshDMEM were treated with or without CL-316243 (CL;20 mmol/L), a selective b3-adrenergic receptor activator;isoproterenol (8 mmol/L), a nonselective b-adrenergic re-ceptor activator; or forskolin (20 mmol/L), a cellular cAMPinducer. Cells were collected 24 h after the treatment andstored at 280°C for further analyses.

Mitochondrial Respiration AssayTo determine mitochondrial respiration activity, the O2

concentration was measured using an XF24 extracellularflux analyzer (Seahorse Bioscience, Billerica, MA). Basalmitochondrial respiration was measured in untreated cells.The cells were then treated with oligomycin (OL; Sigma-Aldrich), carbonyl cyanide 4-trifluoromethoxy phenylhy-drazone (FCCP; Sigma-Aldrich), or rotenone and antimycinA (Sigma-Aldrich). The oxygen consumption rate (OCR)was calculated by plotting the O2 concentration in themedium as a function of time and protein mass (picomolesper minute per microgram protein).

Measurement of mtDNA ContentTo determine mtDNA content, we extracted total cellularDNA according to a previously described method (31).Quantitative PCR was performed with primers targeting16s rRNA (mtDNA) or hexokinase 2 (a nuclear gene).Relative mtDNA levels were calculated based on the ratioof mtDNA to the nuclear gene hexokinase 2 (32).

AnimalsSix-week-old male C57BL/6J mice were purchased fromShanghai Laboratory Animal Co. Ltd. (Shanghai, China)and housed in a temperature-controlled environment witha 12:12-h light/dark cycle. The mice were allowed access tofood and water ad libitum. After a 1-week acclimationperiod, the mice were randomly distributed into weight-matched groups and fed either a normal chow diet (ND) ora high-fat diet (HFD; 60 kcal% fat) (Research Diets Inc,

2586 miR-199a/214 Cluster Inhibits Browning Diabetes Volume 67, December 2018

New Brunswick, NJ). After 16 weeks of HFD feeding,animals were sacrificed, and fat depots from interscapularBAT, inguinal sWAT, and perigonadal visceral white adi-pose tissues (vWATs) were rapidly removed, immediatelyfrozen in liquid nitrogen, and stored at 280°C untilfurther analysis.

For cold stress studies, 8-week-old C57BL/6 male micewere kept at room temperature (25°C) or cold temperature(6°C). In both groups, each mouse was maintained ina single cage on a 12:12-h light/dark cycle with free accessto water and food. After 24 h or 7 days, fat depots wereisolated and subjected to further analyses.

miRNA Antagomir Treatment In VivomiRNA antagomirs are chemically modified, cholesterolconjugated, stable miRNA inhibitors. In vivo delivery ofmiRNA antagomirs is capable of specifically silencingendogenous miRNAs (29,33). To determine the role ofmiR-199a/214 in vivo, we used mixture of miRNA-199aand -214 antagomirs to repress the expression of miR-199a/214 in fat tissues. Briefly, the 8-week-old male ob/obmice purchased from the National Resource Center forMutant Mice (Nanjing, China) were inguinal sWATinjected with antagomirs miR-199a/214 (20 nmol/mice)or scrambled negative controls (Ribobio, Guangzhou,China). The injections were performed four times at 3-dayintervals.

After the final injection, the mice were housed ina temperature-controlled environment with a 12:12-hlight/dark cycle with access to food and water ad libitum.Body weight, food intake, and blood glucose levels weremonitored weekly. Four weeks after the last injection, thebody composition of mice was monitored by magnetnuclear magnetic resonance minispec (Bruker’s minispecLF50 Body Composition Analyzer; Bruker Daltonics, Ham-burg, Germany), and the mice were placed in the Compre-hensive Lab Animal Monitoring System (ColumbusInstruments) to evaluate whole-body energy metabolism.Mice were acclimated in individual metabolic chamberswith free access to food and water. After a 24-h acclimationperiod, the CO2 and O2 levels, total and wheel activities,and food intake weremonitored and recorded over a periodof 48 h.

Human SubjectsWe recruited 33 Chinese people (average age: 49.096 2.55years; 21 females and 12 males) from the Minimally In-vasive Surgery Center, The Second Xiangya Hospital ofCentral South University (Hunan, China). All subjects wereundergoing abdominal surgery for benign hepatobiliaryconditions, such as cholecystitis or gallstones. PreoperativeBMI (calculated as body weight in kilograms over squaredheight in meters) and blood pressure were measured, andserum levels of triglycerides, cholesterol, HDL cholesterol,LDL cholesterol, and fasting glucose were determined. Themetabolic characteristics of the recruited human subjectsare shown in Supplementary Table 1. During the opera-tion, abdominal sWAT and intra-abdominal WAT (iWAT)

adipose tissues (�2 cc each) were collected, snap-frozen,and stored at 280°C before RNA extraction.

miRNA Gene Chip AssayTotal adipocyte miRNA was extracted using the mirVanamiRNA Isolation Kit (catalog number AM1560; AppliedBiosystems). Five micrograms of total miRNA was usedfor miRNA chip analysis using the TaqMan Array Ro-dent MiRNA A+B Cards Set v3.0 (catalog number4444909; Applied Biosystems). Briefly, following re-verse transcription of miRNA targets using MegaplexRT Primers (#4444746; Applied Biosystems), a TaqManUniversal PCR Master Mix (#4324018) was combinedwith each reaction and pipetted into each sample load-ing port in the TaqMan array card. The real-time PCRreaction was performed on an Applied Biosystems 7900HTsystem.

To analyze the data, we screened miRNAs for whichthreshold cycle (Ct) values in the miRNA assay were alldetermined at days 0, 4, and 8 of brown adipocytesdifferentiation and were between 20 and 30 for the qualitycontrol. To identify the expression difference of miRNAsbetween baseline (day 0) and days 4 or 8, we measured thelog fold-change (ΔΔCt) using average DCt values (ΔΔCt =ΔCt at day 4 or 82 ΔCt at day 0). If ΔΔCt.0, miRNA wasset as upregulated; if ΔΔCt ,0, miRNA was downregu-lated; and if ΔΔCt = 0, there was no regulation.

RNA Isolation and Real-time Quantitative RT-PCRTotal RNA was extracted using the TRIzol Reagent (Invi-trogen, Life Technologies, Grand Island, NY) following themanufacturer’s instructions. mRNAwas reverse-transcribed,and amplification was carried out on a 7900HT FastReal-Time PCR System (Applied Biosystems). The primersequences for the genes are shown in SupplementaryTable 2.

For miRNA analysis, cDNA was synthesized from 1 mgof RNA using the PrimeScript One Step miRNA cDNASynthesis Kit (Takara Bio, Tokyo, Japan) and subjected toreal-time PCR. The miRNA sequences are shown in Sup-plementary Table 3, and the relative expression levels ofmiRNAs were quantified by the SYBR PrimeScript miRNART-PCR kit (Takara Bio) and normalized to U6 expression.To estimate the absolute abundance of the miRNAs, weisolated total RNA from fat tissues, performed miRNAreal-time quantitative RT-PCR (qRT-PCR), and calculatedthe copy numbers of miR-199a and miR-214 by comparingthe Ct values of standards (Ribobio).

Western BlottingThe primary antibodies were: anti–b-actin (A5441; Sigma-Aldrich), anti-UCP1 (U6382; Sigma-Aldrich), anti–PGC-1a(ab54481; Abcam), or anti-PRDM16 (ab106410; Abcam).Signals were detected using the ChemiDOC XRS+ and theImage Lab system (Bio-Rad).

Luciferase Constructs and Reporter AssayThe 39-untranslated regions (39-UTRs) from the mousePrdm16 and Ppargc1a genes were PCR-amplified from

diabetes.diabetesjournals.org He and Associates 2587

cDNA using specific primers (Supplementary Table 4). Thewild-type (WT) or mutant 39-UTRs were cloned into thepmiR-RB-Report vector (Ribobio) between the XhoI andNotI sites downstream of the Renilla luciferase gene. Togenerate the mutant Prdm16 39-UTR reporter, thenucleotides containing the miR-199a seed sequence(59-CACTGGA-39) were point-mutated (Mut) to 59-GTGACCT-39. For Ppargc1a, the nucleotides (1,519–1,526) of the 39-UTR containing the miR-199a seed

sequence (59-ACACTGG-39) were point-mutated to 59-TGTGACC-39. Because there are two sites containing themiR-214 seed sequence (59-CTGCTG-39) within thePpargc1a 39-UTR, we made single site mutations (Mut1: 217–223 and Mut 2: 568–574) and double site muta-tions (Mut 3: 217–223/568–574) by changing the seedsequences to 59-GACGAC-39. The primer sequences foramplification of the WT and Mut 39-UTRs are shown inSupplement Table 4. All mutations were generated using

Figure 1—Expression profiles of miR-199a/214 cluster in the brown and beige adipocyte differentiation and in response to cold stress. A:Heat map of miRNA expression in brown adipocyte differentiation. Brown adipocyteswere cultured and collected at differentiation day 0, day4, and day 8, miRNA chip assays were performed, and genome-wide miRNA expression patterns were analyzed. The subfigure at left showsthe expression levels of 77 miRNAs in ascending order according to the log fold-change (ΔΔCt) of average DCt values. The subfigure at rightshows the corresponding fold changes of miRNA expression with regard to day 4 vs. day 0 (red) or day 8 vs. day 0 (cyan). B: Relativeexpression levels of miR-199a, miR-214, host gene Dnm3os, and thermogenic genes Prdm16, Ppargc1a, and Ucp1 during brown and beigeadipocyte differentiation. Brown and SVF-derived beige adipocytes were cultured and induced to differentiation. Cells were collected at day0 and days 2, 4, or 8 of postinduction (n = 4 independent experiments; *P, 0.05; **P, 0.01; ***P, 0.001 vs. day 0). C: Relative expressionlevels of miR-199a and miR-214 in mouse fat tissues (n = 4; **P, 0.01; ***P, 0.001 vs. BAT). D: Relative expression levels of miR-199a andmiR-214 in mouse fat tissues in response to cold stress. C57BL/6 mice were maintained at control temperature (25°C) or exposed to cold(6°C) for 24 h or 7 days (n = 6; *P, 0.05; **P, 0.01; ***P, 0.001 vs. control). E: SVF-derived brown preadipocytes were cultured and inducedto differentiation. On the day 4 postinduction, cells were treated with 20 mmol/L b3-adrenergic receptor activator CL, 8 mmol/L nonselectiveb-adrenergic receptor activator isoproterenol, 20 mmol/L cellular cAMP inducer forskolin, or vehicle control for 24 h. Relative miR-199a and-214 levels were determined by real-time qRT-PCR (n = 6; *P , 0.05 vs. control).

2588 miR-199a/214 Cluster Inhibits Browning Diabetes Volume 67, December 2018

the Site-Directed Gene Mutagenesis Kit (Beyotime Instituteof Biotechnology, Suzou, China), and both the WT andmutated constructs were verified by sequencing.

For luciferase assays, HEK293T cells were transfectedwith the 39-UTR WT or mutant reporters with or

without the miR-199a or miR-214 mimic using Lipo-fectamine 2000 (Invitrogen, Carlsbad, CA). Luciferaseactivity in the cell lysate was measured using theDual-Glo Luciferase Assay System (Promega, Madison,WI).

Figure 1—Continued.

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Figure 2—Expression of the miR-199/214 cluster in adipose tissues is positively correlated with obesity in mice and humans. A: Eight-week-old ob/ob and littermate WT mice were sacrificed, and expression levels of miR-199a/214 cluster in the adipose tissues were determined byreal-time qRT-PCR (n = 6; *P, 0.05; **P, 0.01 vs.WTmice).B: C57BL/6micewere fedwith HFD andND for 16weeks, and expression levelsof miR-199a/214 cluster in the adipose tissues were determined by real-time qRT-PCR (n = 6; *P , 0.05; **P , 0.01 vs. ND mice). C:Expression levels of miR-199a in the sWAT of lean (BMI ,25 kg/m2), overweight (BMI .25 kg/m2), and obese (BMI .30 kg/m2) Chinesepeople (lean: n = 10; overweight: n = 13; obese: n = 10; *P, 0.05; ***P, 0.001). Left panel: mean6 SEM of miR-199a; right panel: correlationof miR-199a levels with BMI (n = 33, Pearson correlation analysis). D: Expression levels of miR-214 in the sWAT of lean, overweight, andobese Chinese people (lean: n = 10; overweight: n = 13; obese: n = 10; **P, 0.01). Left panel: mean6 SEM; right panel: correlation of miR-199a levels with BMI (n = 33, Pearson correlation analysis). E: Expression levels of miR-214 in the iWAT of lean, overweight, and obeseChinese people (lean: n = 10; overweight: n = 13; obese: n = 10; **P, 0.01). Left panel: mean6 SEM; and right panel: correlation of miR-199alevels with BMI (n = 33, Pearson correlation analysis).

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Statistical AnalysesAll results were presented as the mean 6 SEM. Compar-isons between two groups were assessed using a Stu-dent two-tailed t test for independent samples, andmultiple groups were analyzed by one-way ANOVA. Thelinear correlation between two variables was analyzed byPearson correlation coefficient. Statistical analyses werecarried out using SPSS statistics software (v19.0; SPSS Inc.,Chicago, IL). Statistical analysis and plotting for metabolicstudies was performed in the R programming languagewith CalR, a web-based analysis tool for indirect calorim-etry experiments (34). The ANCOVA was performed toanalyze differences in oxygen consumption (VO2) andrespiratory exchange ratio between the control and exper-imental groups while statistically controlling for the effectsof covariate lean mass. Values of P, 0.05 were consideredto be statistically significant.

Study ApprovalAll procedures involving animals were conducted in accor-dance with the guidelines set forth by the UniversityCommittee on the Care and Use of Animals of the CentralSouth University. All recruited human participants were

given written informed consent forms, and the protocolwas approved by the Ethics Committee of the SecondXiangya Hospital of Central South University. A writteninformed consent was received from participants prior toinclusion in the study.

RESULTS

Expression Profiles of miR-199a/214 Cluster in theBrown and Beige Adipocyte Differentiation and inResponse to Cold StressTo uncover miRNAs that are important for brown adipocytedifferentiation and maturation, we performed miRNA chipassays and compared genome-wide miRNA expression pat-terns of brown adipocytes collected at differentiation day 0,day 4, and day 8. Using the criteria described in RESEARCH

DESIGN AND METHODS, among 768 detected miRNAs, we iden-tified 77 miRNAs for which Ct values were all between20 and 30 and were differentially expressed during brownadipocyte differentiation (Fig. 1A). Among these miRNAs,themiR-199a andmiR-214 gene cluster was one of the mostsignificantly downregulated (Fig. 1A). By qRT-PCR, weanalyzed expression levels of miR-199a and miR-214 duringBAT- and sWAT-derived SVF or beige adipogenesis. We

Figure 2—Continued.

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Figure 3—miR-199a/214 negatively regulates brown adipocyte differentiation, thermogenic gene expression, and mitochondrial respiration.A: Representative whole wells and images of Oil RedO staining of mature brown adipocytes. Brown adipocytes transfected withmiR-199a ormiR-214mimic along or together were cultured and induced to differentiation. Mature adipocyteswere fixed and stainedwithOil RedO (scalebar, 50mm).B: Effects of overexpression of miR-199a/214 cluster on genes related to thermogenesis and adipogenesis in brown adipocytes.Brown preadipocytes were transfected with miR-199a and miR-214 mimics or their respective NC for 48 h and then subjected to

2592 miR-199a/214 Cluster Inhibits Browning Diabetes Volume 67, December 2018

found that the expression levels of both miR-199a andmiR-214 were significantly decreased during brown andbeige adipocyte differentiation (Fig. 1B). Additionally, thedecreased miRNA levels were negatively correlated withthe expression of Ucp1, Ppargc1a, and Prdm16 (Fig. 1B).Dnm3os, a noncoding transcript that harbors the miR-199a andmiR-214 gene cluster, showed a similar expressionpattern to that of the miRNAs during brown and beigeadipocyte differentiation (Fig. 1B).

We analyzed expression levels of miR-199a and miR-214 in three fat depots (including BAT, sWAT, andperigonadal vWAT). Compared with those in BAT, therelative miR-199a and miR-214 levels are higher in vWAT(Fig. 1C). We also calculated the absolute abundance ofmiR-199a and miR-214 in the above three fat depots(Supplementary Table 5). Consistently, the absolute abun-dance of the miRNAs in white tissues is also higher thanthat in BAT.

To determine the expression of the miR-199a/214cluster in response to cold stress, we maintained themice at room (25°C) or cold (6°C) temperature for 24 hor 7 days. The levels of both miR-199a and miR-214decreased significantly in BAT and sWAT, whereas onlymiR-199a levels decreased in vWAT in response to short-(24-h) or long-term (7-d) cold exposure (Fig. 1D). Toconfirm these results in vitro, we treated brown adipocyteswith the selective b3-adrenergic receptor activator CL, thenonselective b-adrenergic receptor activator isoproterenol,or the cellular cAMP inducer forskolin. Treating the cellswith these chemicals significantly downregulated miR-199a and miR-214 expression (Fig. 1E), indicating theinvolvement of the b-adrenergic receptor signaling path-way in the regulation of miR-199a/214 expression.

Expression of the miR-199a/214 Cluster in AdiposeTissues Is Positively Correlated With Obesity in Miceand HumansTo determine the potential effect of obesity on theexpression of the miR-199a/214 cluster, we examinedmiR-199a and miR-214 levels in adipose tissue of HFD-fed C57BL/6 mice and ob/ob mice. The expression levelsof both miR-199a and miR-214 were significantly in-creased in interscapular BAT and inguinal sWAT ofboth ob/ob mice (Fig. 2A) and HFD-induced obese mice(Fig. 2B) compared with their respective control mice. Wealso examined the expression of the miR-199a/214 clus-ter in sWAT and iWAT of obese and/or overweighthumans. The human study group included 10 lean(BMI ,25 kg/m2), 13 overweight (BMI .25 kg/m2),

and 10 obese individuals (BMI .30 kg/m2). As therewere no differences between male and female subjects, alldata were pooled and analyzed. Expression levels of miR-199a in sWAT were significantly increased in obesepeople compared with the lean and overweight subjects(Fig. 2C), and there was a positive correlation betweenmiR-199a levels and BMI (Fig. 2C). The miR-214 levels insWAT of both overweight and obese subjects were sig-nificantly higher than those in the lean control subjects(Fig. 2D) and also significantly increased in iWAT of obesepeople (Fig. 2E). Similarly, miR-214 levels in both sWATand iWAT were positively correlated with BMI of testedhuman subjects (Fig. 2D and E). These data stronglysuggest dysregulation of the miR-199a/214 cluster inoverweight or obesity.

miR-199a/214 Negatively Regulates AdipocyteDifferentiation and Thermogenic Gene Expression inBrown AdipocytesTo determine the role of the miR-199a/214 cluster inbrown adipocytes, we transfected brown preadipocyteswith miR-199a, miR-214 mimics, or a nonspecific oligo-nucleotide control (miR-ctrl). Differentiation was inducedin the preadipocytes, and the effect of miR-199a and miR-214 on lipid accumulation was examined by Oil Red Ostaining. The results showed that transfection of miR-199a, but not miR-214, dramatically reduced the forma-tion of lipid droplets in mature adipocytes (Fig. 3A),suggesting distinct effects of the miR-199a on lipid accu-mulation in brown adipocytes.

Overexpression of miR-199a and miR-214 cluster sig-nificantly decreased mRNA expression of the reportedtarget gene Ppard (PPARd) (35) and thermogenic genesincluding Ucp1, Cebpb (C/EBPb), Cidea, Dio2, Elvol3, andIrf4 (Fig. 3B) in brown adipocytes. In addition, over-expression of the cluster significantly decreased the ex-pression of adipogenesis-related genes including Pparg(PPARg), Cebpa (C/EBPa), Adipoq (Adiponectin), andFabp4 (Fig. 3B). Conversely, suppression of the miR-199a/214 cluster significantly increased the expressionof thermogenic genes in brown adipocytes (Fig. 3C). Theseresults strongly suggest an inhibitory role of the miR-199a/214 cluster on brown adipocyte differentiation andthermogenic gene expression.

To determine the effect of the cluster on cellularrespiration, we treated brown adipocytes with miR-199a/214 mimics and analyzed O2 consumption. Al-though there were no differences between the mimicand control groups in response to OL treatment, the

differentiation. Cells were harvested, and relativemRNA levels of tested geneswere determined by real-time qRT-PCR (n = 3; *P, 0.05; **P,0.01 vs. miR-ctrl). C: Effects of miR-199a/214 cluster inhibition on thermogenic gene expression in brown adipocytes. Brown preadipocyteswere transfected with miR-199a and miR-214 inhibitors or their respective NC for 48 h and then subjected to differentiation. Cells wereharvested, and relative mRNA levels of tested genes were determined by real-time qRT-PCR (n = 3; *P , 0.05; **P , 0.01; ***P , 0.001 vs.miR-ctrl). D and E: The OCRs of brown adipocytes. Brown preadipocytes were transfected with miR-199a and miR-214 mimics (D) orinhibitors (E) for 48 h. The OCRs of basal level and in the presence of ATP synthase inhibitor OL, uncoupler FCCP, or rotenone/antimycin A(Rot/Anti) were determined using a Seahorse Bioscience XF24 respirometry analyzer (n = 4; *P , 0.05; **P , 0.01 vs. miR-ctrl).

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Figure 4—Suppression of miR-199a/214 induces thermogenic gene expression and mitochondrial respiration and promotes beige fatdevelopment in vitro and in vivo. A: Relative mRNA levels of genes related to thermogenesis and beigeing in white adipocytes. SVF-derivedwhite adipocytes were transfected with miR-199a and miR-214 inhibitors (Anti-miR-199a/214) or NC for 48 h and then subjected todifferentiation. Cells were harvested at day 6 postinduction, and relativemRNA levels of tested geneswere determined by real-time qRT-PCR(n = 3; *P, 0.05; **P, 0.01; ***P, 0.001 vs. controls). B: Relative mRNA levels of genes related to mitochondrial metabolism (Cox7a1,Nrf1,and Tfam), fatty acid oxidation (CD36 and Acadm), and lipolysis (Atgl and Hsl) in white adipocytes transfected with miR-199a and miR-214inhibitors (n = 3; **P, 0.01; ***P, 0.001 vs. controls).C: mtDNA copy numbers in brown and beige adipocytes. SVF-derived brown andwhiteadipocytes were transfected with miR-199a and miR-214 inhibitors, total cellular DNA was extracted, and quantitative PCR was performed.Relative mtDNA levels were calculated based on the ratio of mtDNA (16s RNA) to the nuclear gene hexokinase 2 (HEK2) (n = 3; *P , 0.05;**P, 0.01 vs. controls). D: The OCR of SVF-derived white adipocytes transfected with miR-199a and miR-214 inhibitors. The OCRs of basallevels and in the presence of OL, FCCP, or rotenone/antimycin A (Rot/Anti) were determined using a Seahorse Bioscience XF24respirometry analyzer (n = 4; *P , 0.05; **P , 0.01vs. controls). E: The expression of miRNAs in sWAT of ob/ob mice inguinal sWATinjected with miR-199a/214 antagomir or their respective scrambled controls was verified by real-time qRT-PCR (n = 6/group; ***P ,0.001 vs. controls). F: Changes of body weight of the mice that received antagomir miR-199a/214 or scrambled controls through inguinal

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basal respiration and FCCP-induced uncoupled respira-tion were significantly lower in brown adipocytes withmiR-199a/214 overexpression compared with the con-trol (Fig. 3D), suggesting that miR-199a/214 might im-pair mitochondrial electron transport capacity, but notATP production. Conversely, suppression of the miR-199a/214 cluster significantly increased basal andFCCP-induced maximal respiration in brown adipocytes(Fig. 3E).

Suppression of miR-199a/214 Cluster InducesThermogenic Gene Expression and MitochondrialRespiration and Promotes Beige Fat DevelopmentIn Vitro and In VivoTo determine the role of miR-199a/214 in beige adipo-cytes, we transfected sWAT-derived beige adipocytes withmiR-199a and miR-214 antisense oligonucleotides. Sup-pression of the miR-199a/214 cluster significantly in-creased the expression of both thermogenic and beigecell genes, including Tbx1 and Tmem26 (Fig. 4A). miR-199a/214 suppression also increased the expression of

genes involved in mitochondrial metabolism (Tfam,Cox7a1, and Nrf1), fatty acid oxidation (CD36 and Acadm),and lipolysis (Atgl and Hsl) (Fig. 4B). In addition, suppres-sion of miR-199a/214 in brown and beige adipocytessignificantly increased mtDNA copy number (as indicatedby the ratio of the mtDNA [16s rRNA] to the nuclear genehexokinase 2) (Fig. 4C). Consistently, the basal and chem-ical-induced OCRs were significantly higher in beige adi-pocytes in which miR-199a/214 was suppressed (Fig.4D). Together, these data suggest that decreased expres-sion of the miR-199a/214 cluster could enhance mito-chondrial biogenesis and respiration activity and promotebeige fat development.

To determine the role of miR-199a/214 in beigingeffects in vivo, we delivered miR-199a/214 antagomirsor their respective NC to the sWAT of ob/ob mice throughinguinal subcutaneous fat pad injection. We found thatexpression of the miR-199a/214 cluster, but not othermiRNAs (miR-27, miR-133, and miR-155), in sWAT couldbe efficiently decreased by antagomir injection (Fig. 4E).We also confirmed that expression of miRNA cluster in

sWAT injection (n = 6/group). G: The basal levels of OCRs of the sWAT of ob/ob mice. After 4 weeks of miR-199a/214 antagomir or controlinjection, the ob/ob mice were sacrificed, fresh sWAT was collected, and OCRs were determined by a Seahorse Bioscience XF24 res-pirometry analyzer (n = 5; **P , 0.01 vs. controls). H: The mRNA levels of thermogenic genes in the sWAT of ob/obmice that received miR-199a/214 antagomir or control injection (n = 6; *P , 0.05; **P , 0.01 vs. controls). I: The protein expressions of PGC-1a and UCP1 in thesWAT of ob/ob mice that received miR-199a/214 antagomir or control injection. J: UCP1 immunohistochemistry staining of the sWAT ofob/ob mice that received miR-199a/214 antagomir or control injection (scale bars, 50 mm).

Figure 4—Continued.

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other tissues, including BAT, vWAT, and muscle, was notchanged by injection (data not shown). Four weeks afterinjection, although there were no significant differences inbody composition, food intake, locomotive activity, oxygenconsumption, and respiratory exchange ratio (Supplemen-tary Figs. 1–3), ob/ob mice that received antagomir in-jection in the sWAT had a relatively lower postinjectionbody weight (Fig. 4F). In addition, antagomir-based knock-down of miR-199a/214 significantly increased basal OCRlevels as measured by respirometry analyzer (Fig. 4G) andmRNA levels of thermogenic genes, including Ppargc1a,Pparg, Ucp1, Cidea, Dio2, and Elvol3 as well as the beigegenes Tmem26 and Tbx1 in the sWAT of obese mice (Fig.4H). The increased expression of PGC-1a and UCP1 wasalso confirmed by Western blotting (Fig. 4I) and UCP1immunohistochemistry analyses (Fig. 4J). Together, theseresults, which were consistent with observations in cul-tured beige adipocytes, suggest that suppression of miR-199a/214 could promote thermogenic gene expression andmitochondrial respiration that might contribute to bodyweight loss in obese mice.

miR-199a/214 Directly Targets PRDM16 and PGC-1aIn silico analysis using the online programs TargetScan-Mouse 6.2 (http://www.targetscan.org/) and miRDB(http://www.mirdb.org/) identified Prdm16, a critical de-terminant of the brown fat lineage, as a predicted target ofmiR-199a (Fig. 5A). To determine whether miR-199a candirectly targetPrdm16, we cloned the Prdm1639-UTR segmentcontaining the predictedmiR-199a seed sites or amutatedsequence. Luciferase reporter assays showed that overexpres-sion ofmiR-199a reduced the luciferase activity of theWT butnot the mutant Prdm16 39-UTR segment reporter (Fig. 5B),demonstrating that miR-199a directly interacts with thepredicted target sites in the Prdm16 transcript.

To confirm these results, we transfected brown preadi-pocytes with miR-199a and miR-214 mimics alone ortogether and induced differentiation of the cells. Over-expression of miR-199a (but not miR-214) signifi-cantly decreased Prdm16 mRNA expression during brownadipogenesis (Fig. 5C). Conversely, the suppression ofmiR-199a expression induced Prdm16mRNA levels (Fig. 5C).

To determine whether the effects of miR-199a onbrown fat–selective genes occurred via targeting ofPrdm16, we overexpressed miR-199a with or withoutPrdm16-expressing plasmid containing miR-199a targetsequence. Our data showed that overexpression of miR-199a decreased, whereas overexpression of Prdm16 inducedthermogenic gene expression in the brown adipocytes,and the suppressing effects of miR-199a on gene expres-sion were rescued by coexpression of Prdm16 (Fig. 5D).These results suggest that the inhibitory effect of miR-199aon brown adipocyte thermogenic gene expression is medi-ated by directly targeting Prdm16.

Bioinformatic analysis also predicts PGC-1a (genePpargc1a) as a potential target of both miR-199a andmiR-214. There is one seed site for miR-199a and two

sites for miR-214 within the Ppargc1a 39-UTR (Fig. 5E).Luciferase reporter assays showed that overexpression ofeither miR-199a or miR-214 significantly reduced theluciferase activity of the Ppargc1a 39-UTR WT construct.This effect was enhanced by overexpression of miR-199a and miR-214 together. However, such effects wereabolished when miR-199a binding sites or miR-214double binding sites were mutated (Fig. 5F), indicatingthat both miR-199a and miR-214 could directly target thePpargc1a 39-UTR, and double targeting sites are requiredfor miR-214 to bind with the 39-UTR of Ppargc1a.

The qRT-PCR analyses confirmed that mRNA levels ofPpargc1a were significantly downregulated throughoutbrown adipocyte differentiation when either miR-199a ormiR-214 was overexpressed. These effects could be en-hanced by cotransfection of two miRNAs (Fig. 5G). Con-versely, knockdown of miR-199a and miR-214 expressionby inhibitors induced Ppargc1a mRNA levels in brownadipocytes (Fig. 5G). Consistent with the above findings(Fig. 5C and G), overexpressing or suppressing miR-199a/214 expression inhibited or induced, respectively, proteinexpression of PRDM16, PGC-1a, and UCP1 (Fig. 5H).

DISCUSSION

Brown and beige fat development and thermogenic func-tion are involved in several essential transcriptional andepigenetic regulators, and among them, miRNAs playunique roles by targeting multiple regulatory factors(11,36). In this study, we identified the miR-199a/214cluster as a key negative regulator of brown adipogenesisand brown/beige fat thermogenesis. Overexpression of themiR-199a/214 cluster inhibited thermogenic gene expres-sion and mitochondrial respiration in brown adipocytes,whereas knockdown of the cluster increased these param-eters in both brown and beige adipocytes. Antagomir-based knockdown of miR-199a/214 in sWAT promotedthermogenic gene expression and body weight loss inobese mice. We also demonstrated that the miR-199a/214 cluster suppresses brown adipogenesis and thermo-genic gene expression by directly targeting PRDM16 andPGC-1a. Our study demonstrates for the first time thatthe obesity-associated miR-199a/214 cluster plays a keynegative role in brown adipogenesis and the beige fatdevelopment.

Previous studies showed that miR-199a is one of themembers of a muscle miRNA (or myomiR) family regu-lated by serum response factor (14). Expression of themiR-199a increases during myogenic differentiation andpromotes normal myogenesis (14). Our study found thatexpression of the miR-199a is downregulated during thebrown and beige adipocyte differentiation, and miR-199aplays a negative role in brown adipocyte differentiation.As brown adipocytes and skeletal myocytes have beenindicated to originate from somite-derived lineage cellsexpressing Pax7 and Myf5 (13,37), from which PRDM16controls the switch between brown and skeletal musclecells in the lineages (10,38,39), it is reasonable to

2596 miR-199a/214 Cluster Inhibits Browning Diabetes Volume 67, December 2018

Figure 5—miR-199a/214 directly target PRDM16 and PGC-1a.A: The conserved 39-UTR sequences of Prdm16 gene in the vertebrates. Redletters demonstrate the miR-199a targeting sequences of the 39-UTR. B: Luciferase activity of Prdm16 39-UTR. HEK293T cells weretransfected with or without miR-199a mimic, together with WT or mutant (MUT) Prdm16 39-UTR inserted downstream of the firefly luciferasegene construct (n = 4; *P, 0.05).C: Relative mRNA levels of Prdm16 in the course of brown adipocytes differentiation. Brown preadipocyteswere transfected with miR-199a and miR-214 mimics (left panel) or inhibitors (right panel) alone or together for 48 h and then subjected todifferentiation (n = 3; **P , 0.01; ***P , 0.001 miR-199a or miR-199a/214 cotransfection vs. controls). D: Relative mRNA levels ofthermogenic genes. Brown preadipocytes were transfected with miR-199a with or without Prdm16 expression plasmid containing miR-199atarget or with Prdm16 plasmid along and induced to differentiation. Cells were harvested, and relative mRNA levels of tested genes weredetermined by real-time qRT-PCR (n = 3; *P, 0.05; **P, 0.01; ***P, 0.001). E: The position of miR-199a (pink) andmiR-214 (cyan) genes inDnm3os locus and predicted binding sites on the 39-UTR of Ppargc1a gene. F: Luciferase activity of Ppargc1a 39-UTR in HEK293T cellstransfected with or without miR-199a or miR-214 mimic, together with WT or mutant Ppargc1a 39-UTR constructs (n = 4; *P , 0.05; **P ,0.01; ***P , 0.001). G: Relative mRNA levels of Ppargc1a in the course of brown adipocyte differentiation. Brown preadipocytes weretransfected with miR-199a and miR-214 mimics (left panel) or inhibitors (right panel) alone or together for 48 h and then subjected todifferentiation (n = 3; #P, 0.05; ##P, 0.01: miR-199a or miR-214 mimics or inhibitors, respectively, vs. controls; **P , 0.01; ***P , 0.001:miR-199a andmiR-214mimics or inhibitors cotransfection vs. controls).H: Western blot analyses of thermogenic genes in brown adipocytestransfected with miR-199a and miR-214 mimics (left panel) or inhibitors (right panel). MUT, miR-199a binding sites mutant; MUT1, miR-214single binding sites mutant (position 217–223); MUT2, miR-214 single binding sites mutant (position 568–574); MUT3, miR-214 doublebinding sites mutant (positions 217–223 and 568–574).

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propose that miR-199a might be an essential lineageregulator of BAT and muscle cells by functioning upstreamof PRDM16.

miRNAs play multiple important roles in maintainingmetabolic homeostasis. Dysfunction or dysregulation ofmiRNAs is associated with various disorders, includingobesity and diabetes (35,40). The miR-199a/214 is

a hypoxia-induced cluster implicated as a mediator in-tegrating hypoxic signaling in the heart (41,42). Induc-tion of the cluster through hypoxic conditions reducesmitochondrial fatty acid oxidative capacity and cardiaccontractility resulting in heart failure (41). We foundthat miR-199a/214 levels in adipose tissues were dra-matically increased in obese mice and in overweight/

Figure 5—Continued.

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obese humans. Although it is not clear whether obesity-associated upregulation of miR-199a/214 is caused byadipose tissue hypoxia, our data showed that upregula-tion of this gene cluster leads to reduced mitochondrialactivity in the brown adipocytes, suggesting that dysre-gulation of this cluster might result in dysfunction of fattissues contributing to development of obesity and re-lated metabolic disorders.

Importantly, our studies demonstrate that suppres-sion of miR-199a/214 expression strongly promotesbeige fat development in vitro and in vivo. Knockdownof the cluster increased thermogenic gene expression andmitochondrial respiration in both brown and beige adi-pocytes. In addition to Ucp1 and other thermogenicgenes, we observed increased expression of genes in-volved in fatty acid oxidation, lipolysis, and mito-chondrial function in the miR-199a/214-suppressedadipocytes. Antagomir-based knockdown of the clusterin sWAT increased thermogenic gene expression and mi-tochondrial respiration and had potential to lower bodyweight in obese mice. In the future, genetically modifiedmouse models with fat tissue–specific manipulation of thisgene cluster will help to identify a better therapeuticintervention for obesity in vivo.

PRDM16 is a key transcriptional regulator that de-termines the commitment, differentiation, and developmentof brown/beige adipocytes (8,15). We demonstratedthat miR-199a directly targets the 39-UTR of Prdm16and that the inhibitory effect of miR-199a on brownadipogenesis could be recovered by coexpression ofPrdm16, suggesting that Prdm16 mediates the effect ofmiR-199a on brown adipocyte differentiation. Previousstudies showed that miR-199a affects myogenic cell pro-liferation and differentiation by targeting the WNT sig-naling pathway (9). Our study unveils a novel role of thismiRNA in brown adipocytes. Several miRNAs have beenreported to be involved in brown adipogenesis (22). Ofthese, miR-133a/b and miR-27 are known to play inhib-itory roles via targeting of Prdm16 (25,43–45). Theseparallel findings suggest that several miRNAs could cotar-get the same key regulatory molecules and coordinate toregulate biological processes, such as cold exposure–in-duced thermogenesis and brown and beige adipogenesis(27,46).

Our study also demonstrated that both miR-199a andmiR-214 directly target the 39-UTR of Ppargc1a. PGC-1a isa master regulator of thermogenesis and mitochondrialbiogenesis (22). Increased PGC-1a expression could con-tribute to increased mitochondrial biogenesis, expressionof genes involved in fatty acid mobilization and lipolysis,and mitochondrial respiration in beige adipocytes withsuppression of the miR-199a/214 cluster. Previous studiesusing Ppargc1a-null mice show that PGC-1a is not nec-essary for brown fat cell differentiation per se (4,18). Inagreement with this, we found that brown adipogenesiswas impaired bymiR-199a (which targets both Prdm16 andPpargc1a) but not by miR-214 (which targets Ppargc1a

only). Both miR-199a and miR-214 target the Ppargc1a39-UTR, resulting in an additive suppressive effect onPpargc1a expression. As Prdm16 is a transcriptionalregulator of PGC-1a, the inhibitory effects of the miR-199a/214 on PGC-1a expression could also be caused, inpart, by targeting Prdm16. Thus, the miR-199a/214 clusteracts as a key negative regulator of brown and beige adipo-cyte development and function by simultaneously targetinga regulatory gene cascade involved in beige and brownadipogenesis and thermogenesis.

Like many miRNA clusters, the synergistic action ofthe miR-199a/214 cluster on PGC-1a and other genesmight be related tomRNA secondary structures. Secondarystructures surrounding seed sequences can affect RNAdeadenylation or sequestration, both of which are impor-tant for miRNA–mRNA interactions and gene repression(25). It has been suggested that, due to sequence homol-ogy, miRNAs in a cluster act as a family and have bothcommon and unique mRNA targets that lie within thesame pathway, thereby allowing these miRNAs to playregulatory roles in several components of a cellularprocess (12,47).

In summary, we revealed the obesity-associatedmiR-199a/214 cluster as a novel gene cluster that negativelyregulates brown and beige adipogenesis, thermogenesis,and mitochondrial respiration by targeting the majorbrown transcriptional regulators PRDM16 and PGC-1a.This study provides new insights into the mecha-nisms coordinating the gene cascades that regulatethermogenesis and energy metabolism and provides a po-tential therapeutic target against obesity.

Funding. This work was supported by grants from the National Natural ScienceFoundation of China (31471131 and 31871180) and the International Scienceand Technology Cooperation Program of China (2014DFG32490) to F.H., theNational Basic Research Program of China (2014CB910501) to F.L., and theNational Natural Science Foundation of China (31571368) and the Project ofInnovation-Driven Plan of Central South University (2016CX031) to G.L.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. L.H. and M.T. designed the study, carried out theresearch, and analyzed the results. T.X., H.L., F.Z., and Y.X. conducted theexperiments. W.L. recruited and conducted the human studies. G.L. conductedbioinformatics analysis. Z.Z. and F.L. reviewed and edited the manuscript. F.H.supervised experiments, analyzed data, and wrote and revised the manuscript.F.H. is the guarantor of this work and, as such, had full access to all of the data inthe study and takes responsibility for the integrity of the data and the accuracy ofthe data analysis.

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