1 mir-30 promotes thermogenesis and the development of ... · 4 fang hu1,2, min wang1, ting xiao1,...

1

miR-30 Promotes Thermogenesis and the Development of Beige Fat by Targeting 1

RIP140 2

3

Fang Hu1,2

, Min Wang1, Ting Xiao

1, 2, Bangqi Yin

1, Linyun He

1, 2,Wen Men

1, 4

Meijuan Dong1, 2

, Feng Liu1, 2, 3 *

5

1Metabolic Syndrome Research Center, the Second Xiangya Hospital, Central South 6

University, Changsha, Hunan 410011, China 7

2Key Laboratory of Diabetes Immunology, Ministry of Education, Institute of Metabolism 8

and Endocrinology, the Second Xiangya Hospital, Central South University, Changsha, 9

Hunan 410011, China 10

3Department of Pharmacology, University of Texas Health Science Center at San Antonio 11

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Running title: miR-30 Promotes Beige Fat Development and Thermogenesis 13

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*Corresponding to: [email protected] 15

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Word counts: 3923 17

Figure numbers: 718

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Diabetes Publish Ahead of Print, published online January 9, 2015

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Abstract 1

2

Members of the microRNA (miR)-30 family have been reported to promote 3

adipogenesis and inhibit osteogenesis, yet their role in the regulation of thermogenesis 4

remains unknown. In this study, we show that miR-30b/c levels are greatly increased during 5

adipocytes differentiation and are stimulated by cold exposure or the β-adrenergic receptor 6

activator. Over-expression and knockdown of miR-30b and -30c induced and suppressed, 7

respectively, the expression of thermogenic genes such as UCP1 and Cidea in brown 8

adipocytes. Forced expression of miR-30b/c also significantly increased thermogenic gene 9

expression and mitochondrial respiration in primary adipocytes derived from subcutaneous 10

white adipose tissue, demonstrating a promoting effect of the miRNAs on the development of 11

beige fat. In addition, knockdown of miR-30b/c repressed UCP1 expression in brown adipose 12

tissue in vivo. miR-30b/c targets the 3’UTR of the receptor-interacting protein 140 (RIP140) 13

and over-expression of miR-30b/c significantly reduced RIP140 expression. Consistent with 14

the review that RIP140 is a target of miR-30b/c in regulating thermogenic gene expression, 15

over-expression of RIP140 greatly suppressed the promoting effect of miR-30b/c on the 16

expression of UCP1 and Cidea in brown adipocytes. Taken together, our study identifies 17

miR-30b/c as a key regulator of thermogenesis and uncovers a new mechanism underlying 18

the regulation of brown adipose tissue function and the development of beige fat. 19

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Key words: miRNA, brown adipocytes, white adipocytes, beige fat, thermogenesis 21

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INTRODUCTION 1

Brown adipose tissue (BAT) plays a major role in energy expenditure and non-shivering 2

thermogenesis and impairment in BAT function is associated with obesity and metabolic 3

disorders (1). Deletion of BAT-specific uncoupling protein 1 (UCP1) causes increased body 4

weight gain under the thermoneutral condition (2). In contrast, an increase in BAT mass or 5

enhanced BAT function is associated with a lean and healthy phenotype in animals due to 6

increased energy expenditure (3,4), suggesting that improving BAT function could be a 7

promising therapeutic strategy to treat obesity and related metabolic diseases. 8

Very recently, the discovery of inducible brown fat cells, known as ‘beige’ cells, in 9

subcutaneous white adipose tissue (sWAT) indicates the existence of a distinct type of 10

thermogenic fat cells (5). Beige cells are capable of triggering a program of respiration and 11

energy expenditure through inducing the expression of UCP1 (6,7). Indeed, the presence of 12

UCP1-positive cells has been found not only in sWAT of rodents, but also in the neck and 13

upper-chest region of humans (8). The induction of UCP1 expression and thermogenic 14

program are under the control of several key positive transcriptional regulators, including 15

peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), the peroxisome 16

proliferator-activated receptor- γ (PPARγ) , CCAAT/ enhancer-binding proteins β (C/EBPβ) , 17

and PRD1– BF1–RIZ1 homologous domain-containing 16 (PRDM16) (9-12). 18

Receptor-interacting protein 140 (RIP140), also known as nuclear receptor- interacting 19

protein 1 (NRIP1), is a co-repressor of genes implicated in glucose uptake, glycolysis, 20

tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis, and oxidative 21

phosphorylation in major metabolic tissues such as fat, muscle, liver, and heart (13,14). 22

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RIP140-null mice are leaner and exhibit resistance to high-fat diet (HFD)-induced obesity 1

(15). RIP140-deficiency also leads to increased UCP-1 gene expression in WAT of mice (15). 2

As a transcriptional corepressor of UCP1, RIP140 functions through histone and DNA 3

methylation by recruitment of DNA methyltransferase (Dnmt), the COOH-terminal binding 4

protein (CtBP), histone methyltransferase (HMT), and histone deacetylase (HDAC) on UCP1 5

promoter (16,17). Very recently, RIP140 has been shown to block the beigeing program in 6

WAT by preventing the expression of brown fat genes and inhibiting a triacylglycerol futile 7

cycle (18). However, how RIP140 is regulated in cells remains elusive. 8

MicroRNAs (miRNAs) are a class of short non-coding RNAs (22-24 nucleotides) that 9

regulate mRNA translation and stability by binding to the complementary sequences in the 3' 10

UTR of target genes. Recently, several miRNAs are identified in BAT that play important 11

roles in regulating differentiation and metabolism of brown adipocytes (19). MiR-193b-365, 12

a brown fat enriched miRNA gene cluster, upregulates the expression of PRDM16 and 13

PPARγ and promotes brown fat differentiation by directly targeting negative regulators of 14

brown adipogenesis (20). MiR-133, on the other hand, negatively regulates brown 15

adipogenesis and thermogenesis by repressing the expression of PRMD16 (21). Very recently, 16

we identified miR-106b/93 cluster as a negative regulator of brown adipocyte differentiation 17

(22). 18

In this study, we investigated the roles of miR-30 family members in the regulation of 19

thermogenesis. We found that the expression of miR-30 family members is greatly increased 20

during brown adipocyte differentiation and the expression of these miRNAs are induced by 21

cold exposure or the β-adrenergic receptor activator. In addition, overexpression of miR-30b 22

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and -30c induced thermogenesis in the BAT and increased UCP1 expression in sWAT. On 1

the other hand, knockdown miR-30b/c decreased UCP1 expression in BAT in vitro and in 2

vivo. We found that miR-30b/c suppresses the expression levels of RIP140, suggesting a 3

potential involvement of RIP140 in miR-30b/c-mediated regulation of thermogenic gene 4

expression. Our study highlights an important role of miR-30 family members in regulating 5

BAT function and uncovers a potential new mechanism regulating the browning/beigeing 6

process in adipose tissues. 7

8

RESEARCH DESIGN AND METHODS 9

Cell culture and transfection 10

The brown preadipocyte cell line, which was kindly provided by Dr. J.D. Lin 11

(University of Michigan, Ann Arbor, USA; (23)), were maintained in DMEM (Gibco, USA) 12

containing fetal bovine serum (FBS) and penicillin and streptomycin. To induce preadipocyte 13

differentiation, confluent cells were exposed to differentiation cocktail containing 14

isobutylmethyl- xanthine (IBMX), dexamethasone (Dex), and insulin (IDI; Sigma-Aldrich, 15

USA). After 2 days of induction, differentiation culture medium was replaced and cells were 16

incubated in fresh DMEM containing 20nM insulin and 1nM 3, 5, 3’-triiodo- L- thyronine 17

(T3) for additional 5-6 days until multiple small lipid droplets accumulated in the cytoplasma. 18

The isolation and culture of stromal vascular fraction (SVF) from interscapular BAT and 19

inguinal sWAT were performed as previously described (24). Briefly, BAT or sWAT from 20

3-week-old male C57BL/6 mice were quickly removed and minced. The tissue pieces were 21

digested in HEPES buffer containing type II collagenase (Sigma-Aldrich) and BSA with 22

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shaking. After centrifugation and washing with PBS, the preadipocytes were seeded on a 1

culture plate and induced to differentiation with the methods described above. 2

To transfect miRNA mimics or inhibitors the designed duplex oligonucleotide (miRNA 3

mimics) or single stain antisense (miRNA inhibitors) for miR-30 family members or 4

non-specific control oligos (NC) purchased from GenePharma, Inc (Shanghai, China) were 5

transfected into the cells at a final concentration of 100 nM using GM siRNA-mate 6

(GenePharma, China) according to manufacturer's instructions. Forty-eight hours after 7

transfection, cells were induced to differentiation using the standard protocol. 8

For treatment, the preadipocytes incubated with fresh DMEM were treated with or 9

without CL-316,243 (CL), a selective β3-adrenergic receptor activator, isoproterenol, a 10

non-selective β-adrenergic receptor activator, or Forskolin, a cellular cAMP inducer. Cells 11

were collected 24 hours after the treatment and stored at -80°C for further analyses. 12

13

Cold temperature exposure 14

Eight-week-old C57BL/6 male mice were kept at room temperature (25°C) or cold 15

temperature (4°C). In both groups, each mouse was maintained in a single cage on a 12 h/12 16

h light/dark cycle with free access to water and food. After seven days, interscapular BAT and 17

inguinal sWAT were isolated and subjected to the further analyses. 18

19

miRNA agomir/antagomir treatment in vivo 20

miRNA agomirs and antagomirs are chemically modified and cholesterol conjugated 21

stable miRNA mimics or inhibitors and in vivo delivery of these molecules have been shown 22

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to result in target gene silencing or up-regulation (25,26). We used a mixture of miRNA-30b 1

and -30c agomir or antagomir to activate or repress, respectively, the expression of 2

miR-30b/c in vivo. Briefly, 8-week-old male C57BJ6 mice were received agomir (10 nmol) 3

or antagomir-30b/c (20 nmol) or their respective negative controls (Ribobio, Guangzhou, 4

China) through subcutaneous injection. Three days after injection, mice were sacrificed and 5

tissues were collected. The expression of miRNAs was verified by real-time QRT-PCR and 6

the expressions of target proteins were determined by western blot. 7

Mitochondrial respiration assay 8

To determine the mitochondrial respiration activities, the O2 concentration in the cells 9

was measured using XF24 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, 10

USA). Briefly, cells were seeded in a 24-well cell culture microplate (Seahorse Bioscience). 11

The mitochondrial basal respiration was assessed in untreated cells. The cells were then 12

treated with oligomycin (Sigma-Aldrich, USA) to measure the ATP turnover. The maximum 13

respiratory capacity was assessed by addition of carbonyl cyanide 4- trifluoromethoxy 14

phenylhydrazone (FCCP, Sigma-Aldrich), a chemical uncoupler of electron transport and 15

oxidative phosphorylation, and, in the end, the mitochondrial respiration was blocked by 16

rotenone and antimysin A (Sigma-Aldrich). The oxygen consumption rate (OCR) was 17

calculated by plotting the O2 tension of the medium in the microenvironment above the cells 18

as a function of time (picomoles per minute). 19

To determine the effect of miR-30b/c suppression or up-regulation in vivo, adipose 20

tissues were isolated from mice treated with miRNA agomirs/antagomirs or respective 21

scrambled controls. OCR was determined according to the procedure as reported by Kiefer et 22

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al (27). In brief, freshly isolated BAT was rinsed with Krebs Henseleit buffer (KHB) and the 1

tissues in the absence of large vessels were cut into small pieces and washed extensively. 2

Approximately 10 mg of tissue piece was placed in each well of an XF24-well Islet Flux 3

plate (Seahorse Bioscience) and covered with a customized screen that allows free perfusion 4

while minimizing tissue movement. Basal OCRs were measured in all wells by a XF24 5

extracellular flux analyzer and the data were normalized to tissue weight. 6

7

Construction of RIP140 expression vectors 8

Complementary DNAs for the mouse RIP140 gene were obtained from total RNAs 9

prepared from mouse tissues by an RT-PCR technique using Reverse Aid First Strand cDNA 10

Synthesis Kit (Thermo Scientific, USA). The forward primer sequence was 5’-CGAGGTA 11

CCGAACGGAAGCCGAGCCCCTGTGAG-3’ and reverse sequence 5’- GCGGATATCC 12

CAAACTGGACTGGCAGGTACAC-3’. The PCR products were digested with Kpn I and 13

EcoR V (TaKaRa, Japan), purified from agarose gels, and subcloned into pcDNA3.1+ 14

(Invitrogen, USA). The PmeI (NEB) and In-Fusion HD Liquid Kits (Clontech, Takara 15

Biomedical Technology, Beijing, China) were used to construct the pWPI+RIP140 and 16

pWPI+RIP140 +3’-UTR plasmids (Addgene, Cambridge, MA, USA). All constructs were 17

confirmed by DNA sequencing. The cells were transfected with expression plasmids using 18

Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. 19

20

Luciferase reporter assay 21

The 3’-UTR sequence for RIP140 gene was PCR-amplified with specific primers: 22

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forward 5' -AGCTTTGTTTAAACATGTGTACCTGCCATACCACTTTG-3', reverse 5'-GA 1

ATGCGGCCGCCTGGTGAAGTTGTGCATTT-3'. The PCR product was cloned into a 2

pmiR-RB-REPORT vector (RiboBio, Guangzhou, China) between Xhol and Notl sites 3

downstream of the Renilla luciferase gene. To generate the mutant construct, four nucleotides 4

in the target site (TGTTTAC) of the miRNA-30 b/c seed region were mutated (TCTATTG). 5

HEK 293T cells were transfected with RIP140 3’UTR luciferase reporter or mutant or blank 6

vector alone with miR-30b/c mimic mixture or mimic controls using Lipofectamine 2000. 7

Forty-eight hours after co-transfection, cells were collected and firefly and Renilla luciferase 8

activities were measured with a Dual-Glo luciferase assay system (Promega, USA) according 9

to the manufacturer's instructions. Luciferase activities were calculated as the ratio of firefly: 10

renilla luminescence and normalized to the average ratio of the blank control. 11

12

RNA isolation and real-time quantitative RT-PCR 13

Total RNAs were isolated and prepared using TRIzol reagent (Invitrogen) following 14

manufactory’s instruction. Reverse transcription of mRNA was conducted by using 15

RevertAid First Strand cDNA Synthesis Kit (ThermoScientific, USA) and real-time PCRs 16

using FastStart Universal SYBR Green Master (Roche, USA) were carried out on a 7900HT 17

Fast Real-Time PCR System (Applied Biosystem, USA). The primer sequences for the genes 18

tested were as follows: UCP1 forward: 5’-TACCAAGCTGTGCGATGTCCA-3’, reverse:5’- 19

GCACACAAACATGATGA CGTTCC-3’; RIP140 forward: 5’-TAAACAGCCCTCTGC 20

TCTCAA-3’, reverse: 5’-TTCTTCTCATTCTTCCCTTTGC-3’; Cidea forward: 5’-AAA 21

GGGACAGAAATGGACACC-3’, reverse: 5’-TACATCGTGGCTTTGACATTG-3’; β-actin 22

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forward: 5’-CAACGAGCGGTTCCGATG- 3’, reverse: 5’-GCCACAGGATTCCATA 1

CCCA-3’. The relative levels of mRNA transcripts to control β-actin were determined by 2

2-∆∆CT

. For miRNA analysis, complementary DNAs (cDNAs) were synthesized from 1 µg of 3

RNA with the One-Step-PrimeScript miRNA cDNA Synthesis Kit (TaKaRa), and subjected 4

to real-time PCR using the SYBR Premix Ex Taq™ II (TaKaRa) following the 5

manufacturer’s protocols. Mature miRNA sequence for mouse miR-30b-5p 6

(MIMAT0000130): UGUAAACAUCCUACACUCAGCU, and mouse miR-30c-5p 7

(MIMAT0000514): UGUAAACAUCCUACACUCUCAGC. The qPCR primers against 8

mature miRNAs (Cat# MmiRQP0393, MmiRQP0396) were purchased from GeneCopoeia 9

(Rockville, MD, USA), and the expression levels of microRNAs were normalized to the U6 10

expression. 11

12

Western blotting 13

To determine protein levels, cells or tissues were lysed in RIPA lysis buffer containing 14

protease inhibitor cocktail (Roche, USA). Proteins were separated by SDS- PAGE gel 15

electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated 16

at 4°C overnight with one of the following primary antibodies: anti-β actin (Cell Signaling 17

Technology Inc, USA), anti-UCP1 (Santa Cruz Biotech Inc, USA), anti-RIP140 (Abcam, 18

USA), and followed by incubation with HRP conjugated secondary antibodies. Signals were 19

detected by using the ChemiDOCTMXRS+ and the Image LabTM system (BIO-RAD, USA). 20

21

Statistical analysis 22

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Statistical analysis was performed using SPSS 19.0 software (SPSS Inc, Chicago, IL, 1

USA). All data were expressed as means ± standard error of mean (SEM). Statistical 2

significance was determined by Student’s t-test when two groups were compared or one-way 3

ANOVA when more than two groups were compared. Value of p < 0.05 was set as 4

statistically significant. 5

6

RESULTS 7

miR-30b and miR-30c are abundantly expressed in BAT and are up-regulated during 8

brown adipocyte differentiation 9

To study the potential role of miRNAs in regulating brown adipocyte function, we 10

examined miRNA expression during brown adipocytes differentiation by microarray 11

experiments. This study led to the identification of several miRNAs whose expression levels 12

are dynamically altered during adipocyte differentiation, including two members of the 13

miR-30 family, miR-30b and -30c. By real-time qRT-PCR, we found that both of these 14

miR-30 family members were significantly up-regulated during brown adipocyte 15

differentiation (Fig 1A), which was in parallel with the expression pattern of UCP1 (Fig 1B). 16

To further confirm this result, we examined the expression of miR-30b and miR-30c in 17

BAT-derived primary adipocytes. We found that the expression levels of both miR-30b and 18

miR-30c were significantly induced during the primary brown adipocyte differentiation (Fig. 19

1C), providing further evidence on a potential role of these miRNAs in regulating adipocyte 20

function. To further test this, we examined the expression levels of miR-30 family members 21

in three different mouse fat depots including inguinal sWAT, perigonadal visceral adipose 22

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tissue (vWAT), and interscapular BAT. The expression levels of both miR-30b and -30c were 1

significantly higher in BAT compared to those in either sWAT or vWAT (Fig 1D). Taken 2

together, these results strongly suggest that miR-30b and miR-30c may play critical roles in 3

regulating brown adipocyte function and energy homeostasis. Consistent with this, the 4

expression levels of miR-30b and miR-30c were dramatically decreased in BAT of ob/ob 5

mice compared with wide type (WT) littermates (Fig. 1E). 6

miR-30b and -30c are required for thermogenic gene expression and mitochondrial 7

respiration in brown adipocytes 8

To determine the roles of miR-30b and -30c in brown adipocytes, we transfected brown 9

preadipocytes with miR-30b/c mimic mixtures or non-specific oilgonucleotide controls (NC). 10

Over-expression of the miR-30b/c mimics dramatically up-regulated miR-30b and -30c (Fig. 11

2A) but not other miR-30 family members including miR-30a, -30d, -30e, and -384 (data not 12

shown). Treating brown adipocytes with the miR-30b/c mimics dramatically induced mRNA 13

levels of UCP1 and Cidea, two abundantly expressed thermogenic genes in mouse BAT (28) 14

(Fig. 2A). Oil-red O staining showed that treating brown adipocytes with antisense 15

oligonucleotides that specifically target both miR-30b and miR-30c had no significant effects 16

on triglyceride accumulation in brown adipocytes (data not shown), but significantly 17

suppressed the mRNA levels of UCP1 and Cidea (Fig 2B). Over-expression or inhibition of 18

miR-30b and miR-30c had no significantly effects on the expression of other genes examined, 19

including PRDM16, PGC-1α, PPARγ, C/EBPα, C/EBPβ, ELVOL3, AP2, and adiponectin 20

(data not shown). Interestingly, over-expression or inhibition of miR-30b or -30c alone had 21

no significant effects on UCP1 or Cidea expression (data not shown). 22

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As UCP1 is a hallmark of brown adipocytes that dissipates mitochondrial proton 1

gradients to generate heat, we evaluated respiration of cells treated with miR-30b and -30c 2

inhibitors using the Seahorse Bioscience XF24 respirometry analyzer. Suppression of 3

miR-30b/c led to a marked decrease in basal OCR (Fig. 2C). In addition, treatment of brown 4

adipocytes with an ATP synthase inhibitor (oligomycin) or a chemical uncoupler (FCCP) 5

significantly lowered OCRs (Fig. 2C), suggesting a causative role of miR-30b and miR-30c 6

in suppressing mitochondrial respiration. 7

miR-30b and -30c induce thermogenic gene expression and mitochondrial respiration in 8

sWAT 9

To investigate the potential role of miR-30b/c in the beigeing effect of WAT, we isolated 10

and cultured sWAT-derived stromal vascular fractions (SVFs) and analyzed the expression 11

pattern of miRNAs during adipocyte differentiation. Consistent with the results found in 12

brown adipocytes, the expression levels of both miR-30b and -30c were up-regulated during 13

adipocyte differentiation (Fig 3A). In addition, over-expression of miR-30b and -30c 14

significantly induced UCP1 and Cidea expression in SVFs derived from sWAT (Fig 3B), 15

indicating that miR-30b/c could induce the beigeing effect of sWAT. Consistent with this, the 16

basal and oligomycin-treated OCRs were significantly high in miR-30b/c over-expressed 17

sWAT (Fig 3C), suggesting an increase of mitochondrial activity. 18

The expression of miR-30b and -30c is induced by cold exposure in vivo and by the 19

β-adrenergic receptor activator and cAMP inducer in primary adipocytes 20

To provide further evidence on the role of miR-30b/c in thermogenesis, we examined 21

miRNA expression in mice maintained at room temperature (25°C) or exposed to cold (4°C). 22

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The expression levels of miR-30b/c were significantly increased in BAT (Fig. 4A) and in 1

sWAT (Fig. 4B) in response to cold exposure. Treatment of preadipocytes with selective 2

β3-adrenergic receptor activator CL-316,243, non-selective β-adrenergic receptor activator 3

isoproterenol, or the cAMP inducer Forskolin significantly up-regulated miR-30b and -30c 4

expression (Fig. 4C), indicating a cell autonomous effect of the β-adrenergic receptor 5

signaling pathway on miR-30b and -30c expression. Over-expression of miR-30b/c further 6

enhanced CL-316,243-induced gene expression of UCP1 and Cidea (Fig. 4D-E), suggesting a 7

β-adrenergic receptor-independent function of miR-30b/c in the regulation of thermogenesis. 8

Identification of nuclear corepressor RIP 140 as a target of miR-30b/c 9

In silico analysis using the online programs including TargetScan 6.2 10

(http://www.targetscan.org/) and miRanda (http://www.microrna.org/microrna/) predicated a 11

potential miR-30 target, RIP140 (gene Nrip1), which has been shown to function as a 12

corepressor of thermogenic genes including UCP1 and Cidea (13,14). Bioinformatic analysis 13

showed that the miR-30 targeting sites on the RIP140 3’UTR sequence are highly conserved 14

in the vertebrates, including mouse, human, chimpanzee, and rat (Fig. 5A). RT-PCR and 15

western blot experiments showed that both the mRNA and protein levels of RIP140 were 16

significantly up-regulated at days 2 and 4 post-differentiation induction, but markedly 17

reduced at the later stage (days 6 and 8) of the brown adipocyte differentiation process (Fig 18

5B). The expression pattern of RIP140 is negatively correlated with those of miR-30b, -30c 19

and UCP1 during brown adipocyte differentiation (Figs 1A and 1B). By luciferase report 20

assays, we found that over-expression of the miR-30 mimics significantly reduced RIP140 21

3’-UTR report gene activity (Fig. 5C), indicating that miR-30b/c could directly target 3’-UTR 22

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sequence of RIP140. To determine whether targeting 3’UTR influences the protein expression 1

of RIP140, we co-transfected HEK293T cells with a plasmid encoding wild-type (WT) or 2

mutant (MUT) RIP140 3’UTR or an empty plasmid, together with the miR-30b and -30c 3

mimics. Over-expression of WT but not the mutant miR-30 targeting sequence significantly 4

reduced RIP140 protein levels (Fig. 5D), providing further evidence that miR-30 family 5

members could down-regulate the expression of RIP140 by directly targeting 3’UTR 6

sequence. 7

RIP140 mediates the effects of miR-30 on UCP1 expression in adipocytes 8

It has been reported that RIP140 could negatively regulate thermogenic gene expression 9

and represses the beigeing program in WAT (13,14,18). To further investigate the role of 10

miR-30 in the regulation of RIP140 expression, we transfected brown adipocytes with 11

mimics or inhibitor of miR-30b/c. RT-PCR and western blot analyses showed that 12

over-expression of miR-30b/c significantly reduced both the mRNA (Fig. 6A) and protein 13

(Fig. 6B) levels of RIP140. On the other hand, inhibition of miR-30b/c significantly increased 14

the mRNA (Fig. 6C) and protein (Fig. 6D) levels of RIP140. To determine if RIP140 plays a 15

role in the regulation of thermogenic gene expression by miR-30b/c, we transfected 16

preadipocytes with RIP140 expression plasmid together with or without miR-30b/c mimics. 17

Over-expression of miR-30b/c mimics significantly increased the expression of UCP1 and 18

Cidea (Fig. 6E). The stimulatory effect of miR-30b/c, however, was significantly suppressed 19

by co-expression of RIP140 (Fig. 6E), indicating that RIP140 acts downstream of miR-30b/c. 20

Together, these findings reveal that miR-30b/c regulates thermogenic gene expression by 21

suppressing RIP140 in the brown adipocytes. 22

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Knockdown of miR-30b/c decreases UCP1 expression and mitochondrial respiration in 1

BAT in vivo 2

To determine whether miR-30b/c are involved in the thermogenesis in vivo, we 3

administrated miR-30b/c agomir or antagomir, or their respective controls to mice through 4

subcutaneous injection. We found that the expression of miR-30b/c in mouse BAT was 5

efficiently increased or decreased by agomir or antagomir, respectively (Fig. 7A and C). 6

Consistent with the findings observed in cultured cells, administration of the miR-30b/c 7

antagomir significantly up-regulated the expression of RIP140 and greatly down-regulated 8

the UCP1 expression in the BAT (Fig 7D), Suppressing miR-30b/c in vivo also significantly 9

decreased tissue respiration (Fig 7E), further demonstrating an important role of miR-30b/c in 10

the regulation of thermogenic gene expression in vivo. However, agomir injection had no 11

obvious effects on the expression of UCP1 (Fig. 7B), probably due to the fact that, under this 12

condition, RIP140 is already low and further suppressing its levels will not increase UCP1 13

expression. 14

15

DISCUSSION 16

In this study, we identified miR-30b and -30c as key regulators of brown adipocyte 17

function. The expression levels of miR-30b/c were greatly increased during brown adipocyte 18

differentiation. In addition, over-expression of miR-30b and -30c dramatically increased 19

UCP1 expression in brown adipocytes and in primary white adipocytes. Furthermore, 20

suppression of miR-30 expression down-regulated UCP1 levels both in vitro and in vivo. 21

Finally, we have identified RIP140 as a direct target of miR-30b/c and over-expression of 22

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RIP140 alleviated the promoting effects of miR-30b/c on thermogenic gene expression. 1

Taken together, our study demonstrates for the first time that miR-30 family members are key 2

positive regulators of thermogenesis and the beigeing process of WAT. 3

Although members of the miR-30 family share an identical seed sequence and have 4

common predicted targets (29,30), they exert distinct functions in various cells and tissues. 5

For examples, miR-30a is important for kidney development in Xenopus (31), miR-30c 6

promotes adipocyte differentiation (32) and reduces hyperlipidemia and atherosclerosis (33), 7

and miR-30e is involved in the reciprocal regulation of osteoblast and adipocyte 8

differentiation (34). However, miRNAs of the same family also exhibit similar regulatory 9

modes and tend to work coordinately to regulate target gene expression. Bridge et al (2012) 10

showed that miR-30b and miR-30c target Delta-like 4 (DLL4), a membrane-bound ligand of 11

Notch signaling, in vitro and in vivo, and regulate angiogenesis in endothelial cells (35). The 12

synergic action of miR-30b and -30c on the thermogenic gene expression might be related to 13

the secondary structures of the mRNA in which the target sites are located and/or required for 14

RNA deadenylation or sequestration, which is important for miRNA–mRNA interactions and 15

gene repression (36). 16

In the present study, we found that the expression levels of miR-30b and miR-30c were 17

greatly stimulated by cold stress and by the β-adrenergic receptor activators and a cAMP 18

inducer. Cold exposure-induced non-shivering thermogenesis is activated by the sympathetic 19

nervous system (SNS), which stimulates the release of norepinephrine (NE) and increases 20

intracellular cAMP levels, leading to the activation of the protein kinase A (PKA) and 21

downstream pathways. However, recent studies suggest that cold temperature could stimulate 22

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UCP1 expression and thermogenesis by activating multiple signaling and β-AR–cAMP/ 1

CREB-dependent and -independent pathways (37,38). Increasing miR-30 expression might 2

be one of the responses that coordinate with other factors to regulate UCP1 expression in 3

response to cold. Interestingly, we found that overexpression of miR-30b and -30c potentiated 4

β-adrenergic receptor activator-induced thermogenic gene expression, suggesting a positive 5

feedback loop of miR-30 family members on the β-adrenergic receptor signaling and action. 6

Our data strongly suggest that suppressing RIP140 is an important mechanism by which 7

miR-30c and -30b regulate thermogenic gene expression. First, luciferase report assays 8

showed that miR-30 directly targets the 3’UTR of RIP140 gene. Secondly, over-expression 9

and knockdown of miR-30b and miR-30c decreased and increased, respectively, both mRNA 10

and protein levels of RIP140 in adipocytes as well in the BAT in vivo. Thirdly, the promoting 11

effects of miR-30b/c on thermogenic gene expression could be suppressed by overexpression 12

of RIP140. Consistent with these findings, RIP140 has been shown to play essential roles in 13

regulating metabolic function (14) and disrupting the expression of this protein increases 14

resistance against high-fat diet-induced obesity in mice (15). A very recent study 15

demonstrated that a large set of brown fat-associated genes was up-regulated in the absence 16

of RIP140 in white adipocytes (18). In line with these studies, we found that over-expression 17

of RIP140 down-regulated the expression of UCP1, Cidea and PGC-1α, and the suppressing 18

effects could be reversed by the presence of miR-30 b/c. Interestingly, RIP140 may use 19

different mode of actions to suppress Cidea and UCP1 expression. RIP140 negatively 20

regulates Cidea expression by suppressing the expression and activity of PGC-1α (39). On the 21

other hand, the protein directly targets the UCP1 promoter, leading to histone and DNA 22

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19

methylation of the promoter and thus silencing UCP1 expression in adipocytes (16). In our 1

study, we found that over-expression or knockdown miR-30 had no effects on PGC-1α 2

expression (data not shown) indicating that other factors, but not PGC-1α, might involve in 3

the miR-30 regulated thermogenic gene expression. Further investigations will be needed to 4

address this question. 5

In summary, we have discovered miR-30 family members as positive regulators of 6

thermogenic gene expression and mitochondrial respiration. In addition, we have shown that 7

miR-30b and miR-30c promote thermogenic events by targeting RIP140 transcription. The 8

study provides a new insight into the mechanisms regulating thermogenesis and energy 9

metabolism. 10

11

ACKNOWLEDGMENTS 12

This work was supported by grants from the National Science Foundation of China 13

(#31471131 to F. H.), the International Science & Technology Cooperation Program of China 14

(#2014DFG32490 to F.H.), and the National Basic Research Program of China 15

(#2012CB524903 to Y.L./F.H.; #2014CB910501 to F.L.). 16

No conflicts of interest, financial or otherwise, are declared by the author(s). 17

Author contributions: F. H. designed experiments, analyzed data, and drafted manuscript; 18

F.L. supervised experiments, analyzed data, edited and revised manuscript; M.W., T.X., B.Y., 19

and W. M. performed experiments; L.H., and M.D. prepared figures. F.L. is the guarantor of 20

this work and, as such, has full access to all the data in the study and takes responsibility for 21

the integrity of the data and the accuracy of the data analysis. 22

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The authors thank Dr Yan Wu in the Metabolic Syndrome Research Center of CSU for 1

technique advising and helping in the in vivo studies. 2

3

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26

27

28

29

30

Figure Legends 31

Fig1: Expression profiles of miR-30 family members in the brown adipocytes and fat tissues. 32

A. Relative expression levels of miR-30b and -30c during brown adipocyte differentiation 33

(n=3 independent experiments; *p< 0.05; ** p <0.01; *** p <0.001). B. Relative UCP1 34

mRNA levels during brown adipocyte differentiation (n=3; ** p <0.01; *** p <0.001 vs day 35

0). C. Relative expression levels of miR-30b and -30c during the differentiation of 36

SVF-derived brown adipocytes (n=3; ** p <0.01; *** p <0.001). D. Relative expression 37

levels of miR-30b and -30c in fat tissues of C57BL/6 mice (n=4; * p < 0.05; ** p <0.01). E. 38

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Expression levels of miR-30b and -30c in BAT of ob/ob and WT mice (n=6; * p < 0.05; ** p 1

<0.01 vs WT mice). BAT: brown adipose tissue; sWAT: subcutaneous white adipose tissue; 2

SVF: stromal vascular fraction; vWAT: visceral white adipose tissue; WT: wide-type. 3

4

Fig 2: miR-30b/c modulates the expression of thermogenic genes and mitochondrial 5

respiration in the brown adipocytes. A. Brown preadipocytes were transfected with miR-30b 6

and -30c mimics (miR-30b/c) or non-specific controls (miR-NC) for 48 h, followed by 7

induction of differentiation for 3 days. At day 4 post-induction, cells were harvested and the 8

levels of miR-30b, -30c, as well as mRNA levels of UCP1 and Cidea were determined by 9

real-time (RT)-PCR (n =3. * p < 0.05; *** p <0.001). B. Brown preadipocytes were 10

transfected with miR-30b and -30c inhibitors (anti-miR-30b/c) or non-specific controls 11

(anti-miR-NC) for 48 h and then induced to differentiation. The relative levels of miR-30b 12

and -30c as well as mRNA levels of UCP1 and Cidea were analyzed by RT-PCR (n =3. ** p 13

< 0.01; *** p <0.001). C. Brown preadipocytes were transfected with miR-30b and -30c 14

inhibitors or controls for 48 h. The cell oxygen consumption rates (OCR) of basal levels and 15

in the presence of ATP synthase inhibitor oligomycin (A), or uncoupler FCCP (B) or 16

rotenone/antimycin A (C) were determined using a Seahorse Bioscience XF24 respirometry 17

analyzer (n=4; *p< 0.05; ** p<0.01; ***p<0.001). 18

19

Fig 3. Overexpression of miR-30b and -30c induces thermogenic gene expression and 20

mitochondrial respiration in white adipocytes. A. Relative expression levels of miR-30b and 21

-30c during the differentiation of SVF-derived white adipocytes (n=3; *p< 0.05; ** p<0.01). 22

B. SVF-derived preadipocytes were transfected with miR-30b and -30c mimics (miR-30b/c) 23

or non-specific controls (miR-NC) for 48 h and then subjected to differentiation. At day 4 24

post-induction of differentiation, cells were harvested and relative levels of miRNA (miR-30b, 25

-30c) and mRNA (UCP1, Cidea) were determined by real-time PCR (n =3. *p< 0.05; 26

***p<0.001). C. SVF-derived preadipocytes were transfected with miR-30b/c or miR-NC for 27

48 h. The cell oxygen consumption rates (OCR) of basal levels and in the presence of ATP 28

synthase inhibitor oligomycin (A), or uncoupler FCCP (B) or rotenone/antimycin A (C) were 29

determined using a Seahorse Bioscience XF24 respirometry analyzer (n=4; ** p <0.01 vs 30

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miR-NC). 1

2

Fig4. Up-regulation of miR-30b and -30c by cold exposure and by β-adrenergic receptor 3

signaling. C57BL/6 mice were maintained at room temperature (25°C) or exposed to cold 4

(4°C) for one week. The relative levels of miRNAs and UCP1 in BAT (A) and sWAT (B) of 5

the mice were analyzed (n=4-5; * p < 0.05; ** p <0.01, *** p <0.001 vs 25°C ). C. 6

SVFs-derived preadipocytes were cultured and induced to differentiation. On day 4 7

post-induction of differentiation, cells were treated with β3-adrenergic receptor activator 8

CL-316,243, the non-selective β-adrenergic receptor activator isoproterenol, the cAMP 9

inducer Forskolin, or a vehicle control for 24 hrs. Relative miR-30b and -30c levels were 10

determined by RT-PCR (n =6. * p < 0.05). SVFs-derived preadipocytes were transfected with 11

miR-30b and -30c mimics (miR-30b/c) or non-specific controls (miR-NC) for 48 h, and then 12

subjected to differentiation. At day 4 post-induction, cells were treated with β-adrenergic 13

receptor activator CL-316,243 or vehicle control for 24 hrs and relative mRNA levels of 14

UCP1 (D) and Cidea (E) were determined by real-time PCR (n =6. * p < 0.05; ** p < 0.01; 15

*** p <0.001). 16

17

Fig 5. miR-30b and -30c target RIP140. (A) The 3’UTR sequence of RIP140 gene is 18

conserved in the vertebrates, including mouse, human, chimpanzee, and rat. Grey block 19

demonstrates the miR-30 targeting sites of the 3’UTR and asterisks show the mutant points. 20

(B) Expression profiles of RIP140 mRNA and protein during the differentiation of brown 21

adipocytes (n=3; * p < 0.05; ** p <0.01vs day 0). (C) HEK293T cells were co-transfected 22

with or without miR-30 mimics, together with wild-type (WT) or mutant (Mut) RIP140 23

3’UTR inserted downstream of the firefly luciferase gene construct. Forty-eight hrs after 24

transfection, cells were collected and relative luciferase activity was determined with a 25

Dual-Glo luciferase assay system (n=4; ** p <0.01vs blank and Mut). (D) HEK293T cells 26

were co-transfected with miR-30 b/c mimics (miR-30) and blank or 3’UTR-RIP140 27

expression construct (WT) or 3’UTR mutant-RIP140 construct (MUT). Forty-eight hrs after 28

transfection, cells were collected and RIP140 expression was analyzed by western blot. 29

30

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Fig 6. RIP140 mediates the effects of miR-30 on the thermogenic gene expression in the 1

brown adipocytes. Preadipocytes were transfected with miR-30 mimics (A-B) or inhibitors 2

(C-D) and stimulated to undergo differentiation. Differentiating adipocytes were collected for 3

either RT-PCR or western blot analyses to determine expression levels of mRNAs (A&C) or 4

proteins (B&D), respectively (n=3; * p < 0.05; ** p <0.01vs respective controls). (E) Brown 5

adipose cells were transfected with blank or RIP140 expression construct or co-transfected 6

with or without miR-30 mimics. Forty-eight hrs after transfection, cells were collected and 7

gene expression was analyzed by RT-PCR (n=4; * p < 0.05; ** p <0.01; *** p <0.001). 8

9

Fig 7. Knockdown of miR-30b/c decreases UCP1 expression and mitochondrial respiration in 10

mouse BAT in vivo. Eight-week-old male C57BJ6 mice were received agomir, 11

antagomir-30b/c, or their respective scrambled negative control through subcutaneous 12

injection. Three days after injection, BATs were collected and the expression of miRNAs was 13

verified by real-time QRT-PCR (Fig 7A & C; n=6/group; * p < 0.05; ** p <0.01; *** p 14

<0.001 vs respective negative controls). (B) The expressions of RIP140 and UCP1 in BAT of 15

mice injected with miRNA agomir or control (n=4; * p < 0.05). (D) The expressions of 16

RIP140 and UCP1 in BAT of mice injected with miRNA antagomir or control (n=6; * p < 17

0.05). (E) The basal levels of oxygen consumption rates (OCR) of BAT from mice injected 18

with miRNA antagomir or control were determined by a Seahorse Bioscience XF24 19

respirometry analyzer (n=5; * p< 0.05). 20

21

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Brown adipocytes

Day of Differentiation0 2 4 6 8

Rela

tive m

iRN

A level

0

10

20

30

40

50

miR-30b

miR-30c

**

***

**

Fig 1

A.

C. SVF-derived adipocytes

Day of Differentiation0 2 4 6

Rela

tive m

iRN

A l

evel

.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

miR-30b

miR-30c

**

**

***

BAT

miR-30b miR-30c

Re

lati

ve

miR

NA

le

ve

l

0.0

.2

.4

.6

.8

1.0

WT

ob/ob

*** ***

E.

UCP1

Day of differentiation

0 2 4 6 8

Rela

tive m

RN

A level

0

20

40

60

80

100

120

140

160

180

200

**

**

**

***

B.

D. Adipose tissues

miR-30b miR-30c

Rela

tive m

iRN

A level (f

old

ch

an

ge)

0.0

.2

.4

.6

.8

1.0

1.2BAT

sWAT

vWAT

**

*

**

*

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miR-30b miR-30c UCP1 Cidea

Rela

tive l

evel

(fo

ld c

ha

ng

e)

0.0

.2

.4

.6

.8

1.0

1.2

1.4 anti-miR-NC

anti-miR-30b/c

** ***** ***

Fig 2

A.

B.

C.

miR-30b miR-30c Ucp1 Cidea

Rela

tive

lev

el

(fo

ld c

ha

ng

e)

0

10

20

30

40

50

60

70

miR-NC

miR-30b/c***

***

***

*

Oligomycin FCCP Rotenone/

Antimycin A

**

*

***

*

anti-miR-NC

anti-miR-30b/c

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Day of Differentiation0 2 4 6 8

Rela

tive m

iRN

A l

evel

0

2

4

6

8

10

12

miR-30b

miR-30c

*

**

miR-30b miR-30c UCP1 Cidea

Rela

tive l

evel

(fo

ld c

han

ge)

0

20

40

60

80

100

120

miR-NC

miR-30b/c

***

**

***

Fig 3

A.

B.

C. Oligomycin FCCP

Rotenone/

Antimycin A

**

**

miR-NC

miR-30b/c

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Ucp1

Control CL-316,243

Rela

tive

mR

NA

lev

el

0

50

100

150

200

250

miR-NC

miR-30b/c

***

*

Fig 4

D. C.

E.

A.

BAT

miR-30b miR-30c UCP1

Rela

tive l

evel

(fo

ld c

han

ge)

0

2

4

6

8

10

12

14

16

25℃

4℃

** **

***sWAT

miR-30b miR-30c UCP1R

ela

tive level (f

old

ch

an

ge)

0

2

4

6

8

25℃

4℃

* *

***

B.

Cidea

Control CL-316,243

Re

lati

ve

mR

NA

le

ve

l

0

1

2

3

4

miR-NC

miR-30b/c

*

*

miR-30b miR-30c

Rela

tive

miR

NA

lev

el

0

1

2

3

4

5

Control

CL-316,243

Isoproterenol

Forskolin

****

*

***

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Fig 5

A.

B.

C.

Blank + miR-30

WT + miR-30

Mut + miR-30

Re

lati

ve

lu

cif

era

se

acti

vit

y

0.0

.2

.4

.6

.8

1.0

1.2

**

D.

Day of differentiation0 2 4 6 8 10

Re

lati

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mR

NA

le

ve

l

0

1

2

3

4

**

*

* *

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C.

Fig 6

E.

B.

D.

RIP140 UCP1 CIDEA PGC-1a

Rela

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RN

A level

(fo

ld c

ha

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e)

0

1

2

3

4

5

6miR-NC + blank

miR-30b/c + blank

miR-NC + RIP140

miR-30b/c + RIP140

*

*** **

***

*

*

**

** ***

*

RIP140 mRNA

anti-miR-NCanti-miR-30b/c

Rela

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lev

el

(fo

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0

1

2

3

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5

**

RIP140 mRNA

miR-NC miR-30b/c

Rela

tive level (f

old

ch

an

ge)

0.0

.2

.4

.6

.8

1.0

1.2

*

RIP140


Pro

tein

/be

ta-a

ctin

0.0

.1

.2

.3

.4

.5

.6

**

Col 10

UCP1


Pro

tein

/be

ta-a

ctin

0.0

.2

.4

.6

.8

1.0

*

UCP1

miR-NCmiR-30b/c

Pro

tein

/be

ta-a

ctin

0.0

.2

.4

.6

.8

1.0

1.2

1.4

1.6

**RIP140

miR-NCmiR-30b/c

Pro

tein

/beta

-actin

0.0

.1

.2

.3

.4

*

of 32 Diabetes

A.

Fig 7

B.

C. D.

sWAT

Antagomir NC

Antagomir-30b/c

OC

R (

pM

ole

s/m

in/m

g)

0

100

200

300

400

500

600

700

*

miR-30b miR-30c

Re

lati

ve

miR

NA

le

ve

l (f

old

ch

an

ge

)

0

5

10

15

20

25

30

35

agomir-NC

agomir-30b/c

**

**

miR-30b miR-30c

Rela

tiv

e m

iRN

A l

eve

l (f

old

ch

an

ge

)

0.0

.2

.4

.6

.8

1.0

1.2

1.4

antagomir-NC

antagomir-30b/c

** *

E.

UCP1 RIP140

Pro

tein

/be

ta-a

cti

n

0.0

.2

.4

.6

.8

1.0

1.2

1.4

agomir NC

agomir-30b/c

*

UCP1 RIP140

Pro

tein

/beta

-ac

tin

0.0

.2

.4

.6

.8

1.0

1.2

1.4

1.6

1.8

antagomir-NC

antagomir-30b/c

* *

of 32Diabetes

1 mir-30 promotes thermogenesis and the development of ... · 4 fang hu1,2, min wang1, ting xiao1,...

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