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miR-133a Regulates Adipocyte Browning In Vivo
Weiyi Liu
1.
, Pengpeng Bi
1.
, Tizhong Shan
1
, Xin Yang
1
, Hang Yin
2
, Yong-Xu Wang
3
, Ning Liu
4
,
Michael A. Rudnicki
2
, Shihuan Kuang
1,5
*
1 Department of Animal Sciences, Purdue University, West Lafayette, Indiana, United States of America, 2 Regenerative Medicine Program, Ottawa Hospital Research
Institute, Ottawa, Ontario, Canada, 3 Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Mass achusetts, United States of
America, 4 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 5 Purdue University Center for
Cancer Research, Purdue University, West Lafayette, Indiana, United States of America
Abstract
Prdm16 determines the bidirectional fate switch of skeletal muscle/brown adipose tissue (BAT) and regulates the
thermogenic gene program of subcutaneous white adipose tissue (SAT) in mice. Here we show that miR-133a, a microRNA
that is expressed in both BAT and SATs, directly targets the 39 UTR of Prdm16. The expression of miR-133a dramatically
decreases along the commitment and differentiation of brown preadipocytes, accompanied by the upregulation of Prdm16.
Overexpression of miR-133a in BAT and SAT cells significantly inhibits, and conversely inhibition of miR-133a upregulates,
Prdm16 and brown adipogenesis. More importantly, double knockout of miR-133a1 and miR-133a2 in mice leads to
elevations of the brown and thermogenic gene programs in SAT. Even 75% deletion of miR-133a (a1
2/2
a2
+/2
) genes results
in browning of SAT, manifested by the appearance of numerous multilocular UCP1-expressing adipocytes within SAT.
Additionally, compared to wildtype mice, miR-133a1
2/2
a2
+/2
mice exhibit increased insulin sensitivity and glucose
tolerance, and activate the thermogenic gene program more robustly upon cold exposure. These results together elucidate
a crucial role of miR-133a in the regulation of adipocyte browning in vivo.
Citation: Liu W, Bi P, Shan T, Yang X, Yin H, et al. (2013) miR-133a Regulate s Adipocyte Browning In Vivo. PLoS Genet 9(7): e1003626. doi:10.1371/
journal.pgen.1003626
Editor: Shingo Kajimura, University of California San Francisco, United States of America
Received December 21, 2012; Accepted May 30, 2013; Published July 11, 2013
Copyright: ß 2013 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by funding from the National Institute of Health (NIH R01AR060652) and United States Department of Agriculture (USDA
2009-35206-05218) to SK. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interest s: The authors have declared that no competing interests exist.
* E-mail: skuang@purdue.edu
. These authors contributed equally to this work.
Introduction
Adipose tissues are classified as brown (BAT) and white (WAT),
and an intermediate category of ‘‘brite’’ or ‘‘beige’’ adipocytes
exist within subcutaneous WAT [1,2]. WAT are located in
multiple subcutaneous or visceral locations of body, in the form of
distinct fat depots, and contribute to overweight, obesity, insulin
resistance and Type 2 diabetes. Brown adipocytes contain more
mitochondria and express high levels of Ucp1, a mitochondria
inner-membrane channel uncoupling ATP production with
oxidative phosphorylation, therefore producing heat and dissipat-
ing chemical energy. Due to the global concern of obesity,
browning of the white adipocytes, the induction of white
adipocytes into beige adipocytes, has become a research focus.
The signaling pathway that determines the developmental
commitment and differentiation of brown and beige adipocytes
is therefore crucial for understanding the process and importance
of adipose browning.
Prdm16 is a critical regulator of brown adipocyte development
and determines the thermogenic gene program in SAT. Down-
regulation of Prdm16 in brown adipocytes promotes their fate
switch to myoblasts [3]. Conversely, ectopic overexpression of
Prdm16 and its co-activator C/EBPb in myoblasts or fibroblasts
transdifferentiated them into brown adipocytes [3,4]. Similarly,
overexpression of Prdm16 in the stromal vascular fraction (SVF)
cells of SAT led to the browning of white adipocytes [5].
Mechanistic studies have shown that Type 2 diabetic drug
Rosiglitazone can stabilize Prdm16 protein, which activates
PPARc2 and initiates a brown adipocyte gene program that
converts white adipocytes to beige adipocytes [6]. The signals that
regulate Prdm16 transcription and post-transcriptional modifica-
tion may offer new strategies for clinical applications and drug
discoveries.
MicroRNAs are small non-coding RNAs that negatively
regulate mRNA stability or protein translation through targeting
the 39untranslated regions (UTR) of mature mRNA. Previous
studies have demonstrated that several myogenic microRNAs (i.e.
miR-1, miR-206 and miR-133) are enriched in BAT in relative to
WAT [7]. In addition, the cluster of miR-193b and miR-365,
downstream signals of Prdm16, are required for brown adipocyte
differentiation [8]. Moreover, miR-196a mediates the browning of
white adipocytes through targeting Hoxc8, a repressor of brown
adipogenic marker C/EBPb [9]. These studies indicate that
microRNAs play important roles in brown adipose development
and the browning of white adipocytes.
In the present study, we examined the expression of over 30
microRNAs in the anterior subcutaneous WAT (asWAT) and
inguinal WAT (ingWAT) that expressed relatively low and high
levels of Prdm16, respectively. We identified several microRNAs
whose expression is inversely correlated to Prdm16 expression.
Based on this discovery, we conducted gain- and loss- of function
studies to demonstrate that miR-133a regulates brown adipocyte
PLOS Genetics | www.plosgenetics.org 1 July 2013 | Volume 9 | Issue 7 | e1003626
biogenesis and browning of white adipocytes through the
repression of Prdm16. Analysis of miR-133a knockout mice
confirmed the in vivo function of this microRNA in regulating the
adaptive plasticity of white adipocytes. We conclude that miR-
133a plays a repressive role in adipocyte browning.
Results
miR-133a targets the 39 UTR of Prdm16
In the course of adipogenic marker screening among SAT
depots, we found that Prdm16 is expressed at much higher levels in
the ingWAT compared to the asWAT (Fig. 1A). Interestingly, we
identified four miRNAs (miR-1, miR-206, miR-133a and miR-
128) that are expressed at significantly lower levels in the ingWAT
compared to the asWAT (Fig. 1B, Fig. S1). The strong inverse
correlation between the expression of Prdm16 and miRNAs
implies that Prdm16 may be regulated by these miRNAs. Within
the 39 UTR of Prdm16, there are putative target sites for miR-1,
miR-206, miR-133a and miR-128 (Fig. 1C), raising the possibility
that these miRNAs target Prdm16 mRNA. Using classical
luciferase assay in HEK293 cells, we found that miR-133a indeed
repressed the luciferase activity by 20% at 1 nM and over 50% at
10–100 nM (Fig. 1D). Mutation of the miR-133a target sequence
in the 39 UTR of Prdm16 totally abolished the repression of
luciferase activity by miR-133a (data not shown). miR-128 also
repressed the luciferase activity by ,50% at 10–100 nM (Fig. 1E).
Both miR-1 and miR-206 failed to repress the luciferase activity.
These results suggest that miR-133a and miR-128 targets the 39
UTR of Prdm16.
Downregulation of miR-133a is accompanied by
upregulation of Prdm16 during brown adipocyte
commitment and differentiation
Prdm16 is a transcriptional regulator that controls brown
adipocyte fate determination [3]. As miR-133a targets the 39 UTR
of Prdm16, we sought to examine if miR-133a is involved in
Prdm16-mediated brown adipocyte commitment and differentia-
tion. To separate committed preadipocytes from more primitive
progenitors in the SVF of BAT, we used the aP2-Cre/mTmG
mouse model, in which aP2 lineage cells show green fluorescence
(mG
+
) and non-aP2 derived cells exhibit red fluorescence (mT
+
).
Previous studies reported that aP2 expression marks adipocyte
progenitors but not bipotential stem cells [10,11,12]. The SVF
cells of BAT was sorted based on mT and mG fluorescence and
subjected to gene expression analyses (Fig. 2A).
Compared to the non-committed (mT
+
) cells, committed (mG
+
)
preadipocytes completely lost the expression of Pax7 and MyoD,
two myogenic genes expressed by BAT, though the expression of
dual BAT/muscle marker Myf5 was the same in both populations
(Fig. 2B). These data indicate that committed preadipocytes
concomitantly downregulate the expression of myogenic genes.
The adipogenic commitment of mG
+
cells is further supported by
the elevated expression of the adipogenic markers, including
Pparc2, Prdm16, Ucp1 and Cidea, compared to the mT
+
cells
(Fig. 2C). Notably, qPCR results demonstrate that miR-133a is
decreased by 80%, but miR-128 is not significantly altered, in the
mG
+
cells (Fig. 2D), suggesting that miR-133a is more likely to
target Prdm16 in vivo. By contrast, the expression of miR-193b
and miR-365, previously shown to be direct downstream targets of
Prdm16 and required for BAT differentiation [8], were increased
in mG
+
compared to mT
+
cells (Fig. 2D). The mG
+
cells also
expressed higher levels of miR-143, miR-145 and miR-455
(Fig. 2D), known as adipogenic miRNAs [7]. We further
compared the relative expression of miRNAs and BAT related
genes in adipose progenitor cells (APC, Fig. 2E) and mature
adipocytes collected from the floating fractions of enzymatically
digested BAT. Consistently, miR-133a but not miR-128 was
significantly downregulated in the differentiated mature adipocytes
compared to APC (Fig. 2F). By striking contrast, Prdm16 and other
adipogenic markers including aP2, Pgc1a, Cidea and Ucp1 were all
dramatically upregulated in mature adipocytes (Fig. 2G–K).
Together, these data indicate that miR-133a downregulation
along the commitment and differentiation of brown adipocytes
might play a role in Prdm16 upregulation during brown
adipogenesis.
miR-133a inhibits brown adipocyte biogenesis in BAT
and SAT
To examine if miR-133a directly regulates Prdm16 and plays a
role in BAT adipogenesis, we overexpressed miR-133a in cultured
BAT APCs (Fig. 3A). Electroporation-mediated gene transfer
resulted in 213-fold increase in the expression of miR-133a
(Fig. 3B). As a consequence, Prdm16 mRNA was downregulated by
48% (Fig. 3C), and the BAT markers Ucp1 and Cidea were
downregulated by ,70% (Fig. 3D). Other adipogenic genes
Pparc2 and Pgc1a were moderately decreased, by 25%,30%
(Fig. 3D). Importantly, the effects of miR-133a overexpression
were totally reversed by concomitant overexpression of Prdm16,
and even led to ,3 fold increase (overshoot) in adipogenic marker
expression (Fig. 3D–E). This complete reversal and overshoot can
be explained as overexpression of the miR-133a insensitive Prdm16
cDNA (lacking 39 UTR) overrides the repression of miR-133a on
endogenous Prdm16. Conversely, we used antisense oligonucleo-
tide LNAs to specifically inhibit miR-133a in BAT APCs.
Downregulation of miR-133a led to 40% upregulation of Pparc 2
and ,3-fold increases of Prdm16, Ucp1, and Cidea (Fig. 3G). Our
data suggest that BAT adipogenesis is inhibited by overexpression,
and promoted by inhibition, of miR-133a.
Similarly, we overexpressed miR-133a in SAT preadipocytes
(Fig. S2A). A 95-fold overexpression of miR-133a led to 38%
downregulation of Prdm16 (Fig. S2B), accompanied by 50%
downregulation of Pparc2 and ,70% downregulation of Ucp1,
Author Summary
Global obesity and associated health issues have raised the
significance of adipocyte biology. Adipose tissues are
classified as brown and white adipose. White adipose
tissues store lipids, leading to overweight, obesity, insulin
resistance and Type2 diabetes. In contrast, brown adipose
tissues use lipid storage to generate heat, increase insulin
sensitivity, and are negatively correlated with the inci-
dence of Type2 diabetes. Recent studies indicate that
white adipose is plastic and contains an intermediate type
of adaptive adipocytes (so-called beige/brite adipocytes)
that have the energy-dissipating properties of brown
adipocytes. Prdm16 is a key molecule that determines the
development of both brown and beige adipocytes. Thus,
Prdm16 represents a novel molecular switch that expands
brown/beige adipocytes and increases energy expendi-
tures. However, how Prdm16 is regulated has been
unclear. Here we report that the microRNA miR-133a
specifically targets Prdm16 at the posttranscriptional level.
Inhibition or knockout of miR-133a significantly increases
Prdm16 expression and the thermogenic gene program in
white adipose tissues, resulting in dramatically enhanced
insulin sensitivity in animals. Our results suggest that miR-
133a represents a potential drug target against obesity
and Type2 diabetes.
miR-133a Regulates Browning
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Cidea and Lhx8 (Fig. S2C). Our results together suggest that miR-
133a represses BAT adipogenesis and WAT browning through
targeting Prdm16.
Genetic ablation of miR-133a upregulates the
thermogenic gene program in SAT
miR-133a has t wo alleles, miR-133a1 and miR-133a2, which
have identical sequences and are located in different chromo-
somes. To investigate the function of miR-133a in BAT and
SAT, we examined miR-133a double knockout mice (dKO,
miR-133a1
2/2
a2
2/2
), generated by inte rcrossing mice with the
genotype of miR-133a 1
2/2
a2
+/2
. Previous study indicates t hat
compared to wildtype mice, knockout of either miR-133a1 or
miR-133a2 le d to a 40%–50% downregulat ion of miR-133a in
skeletal and cardiac muscles [13,14]. W e examined the
expression of vario us BAT an d mit ochondria markers in th e
dKO mice using miR-133a1
2/2
a2
+/+
littermatesasthecontrol.
As expected , miR -133a levels were reduced b y 80%–9 8% in
BAT, asWAT and ingWAT in the dKO compared to the
controls (Fig. 4A, 4D, 4G).
Surprisingly, neither BAT markers ( Prdm16 , Ucp1 and Cidea) nor
mitochondria and lipolysis markers (Cox8a, Hsl, Atgl and Cpt2) were
significantly affected in BAT tissue of miR-133a dKO mice
(Fig. 4B–C). In striking contrast, the dKO mice had ,1.5–2 fold
elevated expression of the brown adipose markers including
Prdm16, Ucp1 and Cidea, and the mitochondria/lipolysis markers
including Cox8b, Hsl, Atgl and Cpt2, both in asWAT (Fig. 4E and
4F) and ingWAT (Fig. 4H and 4I). As SAT is responsible for cold-
and hormone-induced browning, these data suggest that miR-
133a represses browning of white adipocytes in vivo under
physiological conditions.
Reduced level of miR-133a leads to browning of WAT
and improves body insulin sensitivity in vivo
Due to high perinatal lethality (76%) and cardiac myopathy-
related postnatal sudden death of the few surviving miR-133a dKO
Figure 1. miR-133a and miR-128 target the 39 UTR of Prdm16 in HEK293 cells. (A–B) qPCR analysis of miR-1, miR-206, miR-133a, miR-128
and Prdm16, for asWAT and ingWAT of wildtype mice. (C) Putative miRNA target sites within the 3 9UTR of Prdm16. (D–E) Luciferase assay. Plasmids
carrying luciferase gene linked to 39 UTR of Prdm16 were cotransfected to HEK293 cells, along with control miRNA, miR-133a (D) or miR-128 (E) at
indicated doses. Luciferase activity was measure at 48h post-transfection and normalized. N = 4, *P,0.05, **P,0.01.
doi:10.1371/journal.pgen.1003626.g001
miR-133a Regulates Browning
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mice [13,14], we used in the subsequent studies miR-133a1
2/2
a2
+/2
mice that had three out of the four miR-133a alleles knocked
out but had normal cardiac and skeletal muscles. We used age,
gender and genetic background matched WT mice (miR-133a1
+/+
a2
+/+
) as control. We reasoned that if we can detect phenotype in
mice with 75% reduction of miR-133a, then there should be even
more robust effects if miR-133a is completely knocked out.
To examine if the observed upregulation of brown adipose and
mitochondrial specific genes in SAT of miR-133a mutants are
associated with browning of white adipose, we conducted
histological analysis. The ingWAT of miR-133a1
2/2
a2
+/2
mice
appeared to be browner than that of wildtype mice (Fig. 5A).
Western blots confirm that UCP1 protein is indeed upregulated in
the ingWAT of miR-133a1
2/2
a2
+/2
mice compared to the
wildtype mice (Fig. 5B). H&E staining reveals the appearance of
numerous multilocular brown adipocyte-like cells in the ingWAT
of miR-133a1
2/2
a2
+/2
mice, but not wildtype mice (Fig. 5C–D).
Immunohistochemical staining with brown adipocyte specific
UCP1 antibody indicates that these multilocular brown adipocytes
are UCP1
+
(brown signal), and the UCP1 immunoreactivity is
much more abundant in the ingWAT of miR-133a1
2/2
a2
+/2
mice
compared to the wildtype mice (Fig. 5E–F).
To directly test how miR-133a affects insulin sensitivity and
glucose metabolism, we conducted glucose tolerance test (GTT) and
insulin tolerance test (ITT). Strikingly, GTT indicates that the miR-
133a mutants had ,50% lower overnight fasting glucose levels than
WT mice (Fig. 5G). The mutants also had much improved glucose
tolerance at all the time points examined (Fig. 5G). Similar results
were observed by ITT. Upon I.P. administration of insulin (0.75 U/
Kg BW), blood sugar dropped much more rapidly and remained
lower during 2 h examination period in the miR-133a1
2/2
a2
+/2
compared to the wildtype mice (Fig. 5H). These results indicate that
reduced miR-133a level is associated with improved insulin
sensitivity and glucose disposal in vivo.
Reduced level of miR-133a promotes the activity of cold-
inducible thermogenesis gene program in vivo
Subcutaneous white adipose is capable of thermogenesis under
cold exposure. The adaptive thermogenesis capacity is correlated
to the level of Prdm16 expression. To investigate if inhibition of
Figure 2. Downregulation of miR-133a with upregulation of
Prdm16
along brown adipocyte commitment and differentiation. (A) In
aP2-Cre/mTmG mouse model, aP2 derived cells show green fluorescence (mG
+
) and non-aP2 derived cells show red fluorescence (mT
+
). The stromal-
vascular fraction (SVF) of BAT was sorted based on fluorescence and the freshly sorted cells were used for qPCR analysis of (B) myogenic markers, (C)
adipogenic markers and (D) adipogenic miRNAs. (E) Strategy for isolating adipose progenitor cells (APC) by FACS. (F) Relative expression of miR-113a,
miR-206 and miR-128 in freshly sorted APC and mature adipocytes collected from the floating fraction of collagenase digested BAT. (G–K) Relative
expression of BAT related genes in freshly sorted APC and mature adipocytes. N = 3–5, *P,0.05, **P,0.01.
doi:10.1371/journal.pgen.1003626.g002
miR-133a Regulates Browning
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miR-133a promotes the adaptive thermogenesis of white adipose,
we exposed miR-133a1
2/2
a2
+/2
and wildtype mice to cold
environment. After 5 d exposure at 4uC, the level of miR-133a
in miR-133a1
2/2
a2
+/2
ingWAT is about 2% of that in wildtype
ingWAT (Fig. 6A), but the level of Ucp1 is about 130 times higher
in miR-133a1
2/2
a2
+/2
ingWAT (Fig. 6B). The expression levels of
Pparc2, Prdm16, Pgc1a, and Cidea genes were 1.6-, 2.5-, 5-, and 10-
fold higher in the ingWAT of miR-133a1
2/2
a2
+/2
mice compared
to wildtype mice (Fig. 6C). Accordingly, genes related to
mitochondrial function (Cox8b and Cpt2) and lipolysis (Hsl and
Atgl) were also upregulated in the miR-133a1
2/2
a2
+/2
ingWAT
(Fig. 6D). Consistent with the relative mRNA levels, UCP1 protein
levels in asWAT and ingWAT of the miR-133a1
2/2
a2
+/2
mice
are obviously higher than those of the wildtype mice at room
temperature and after cold exposure (Fig. S3). Therefore, reduced
level of miR-133a promotes the activity of cold-inducible
thermogenesis gene program in vivo.
Reduced level of miR-133a predispose white
preadipocytes to become adaptive beige adipocytes
upon differentiation
The adaptive thermogenesis of subcutaneous WAT has been
shown to be mediated by a population of beige adipocytes [15].
Figure 3. miR-133a inhibits brown adipocyte biogenesis of BAT progenitors. (A) Strategies for electroporation of miRNAs or LNAs to
cultured BAT APCs, followed by induction and differentiation for 4 days. (B–D) qPCR analysis of miR-133a and the brown markers in the control and
miR-133 overexpressed BAT adipocytes. (E–F) qPCR analyses of Prdm16 and BAT specific genes in adipocytes overexpressing control miRNA, miR-
133a with control retrovirus and miR-133a with Prdm16-overexpressing retrovirus. (G) qPCR analysis of BAT marker gene expression after miR-133a
was inhibited by LNAs. N = 3, *P,0.05, **P,0.01, ***P,0.001.
doi:10.1371/journal.pgen.1003626.g003
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We examined the ingWA T of miR-133a1
2/2
a2
+/2
and wildtype
mice to address if reduc tion of miR-133a predisposes white
preadipocytes to become beige cells that express unique beige
markers and common BAT markers [15]. The expression level of
miR-133a is r educed by 95% in the ingWAT of miR-133a1
2/2
a2
+/2
mice compared to the wildtype mice (Fig. 7A). Accord-
ingly, the BAT-specif ic genes Prdm16, Cidea and Ucp1 were
expressed at 2.5-, 5.5- and 8.4-fold in the ingWAT of miR-
133a1
2/2
a2
+/2
mice compared to the wildtype mice (Fig. 7B).
Importantly, beige adipocyte specific CD137 and Tmem26 genes
were also expressed at higher levels (, 6times)inthemiR-
133a1
2/2
a2
+/2
ingWAT compared to the wildtype (Fig. 7C). W e
further isolated and differentiated stromal vascular preadipocytes
from subcutaneous WAT of mi R-133a1
2/2
a2
+/2
and wildtype
mice. Adipocytes differentiated from the miR-133a1
2/2
a2
+/2
preadipocytes expressed 23 times more Ucp1 than wildtype
adipocytes (Fig. 7D). Accordingly, expression of other BAT-
specific genes Prdm16, Pgc1a, Ppara, Pparc2, and Cpt2 were als o
upregulated in the in vitro differentiated miR-133a1
2/2
a2
+/2
adipocytes (Fig. 7E). Together, the in vivo and in vitro gene
expression analysis demonstrate that inhibition of miR-133a
predispose white preadipocytes to become adaptive beige
adipocytes upon differentiation.
Discussion
We identified miR-133a as a regulator of Prdm16 in vivo. Based
on the mutual exclusion model of miRNA-mRNA interactions, the
cells that express high levels of miRNAs should have less
expression of their targets, and vice versa [16]. However, we
found both miR-133a and Prdm16 are expressed at very high
levels in BAT compared to WAT. This paradox led us to
hypothesize that within the BAT, there are different populations of
cells that express high levels of miR-133a or Prdm16, respectively,
with the notion that miR-133a is highly expressed in cells
expressing low levels of Prdm16, and vice versa. In the course of
brown adipocyte commitment and differentiation, aP2 expression
marks more committed progenitors and preadipocytes, whereas
aP2
2
cells contain more primitive adipocyte progenitors, mesen-
chymal stem cells and other cell types [12]. Compared to the more
primitive cells (aP2
2
), aP2
+
cells express increased Prdm16 and
decreased miR-133a. Orchestrated with this notion is the
observation that compared to APCs, differentiated brown
adipocytes nearly lost the expression of miR-133a. These data
imply that miR-133a-mediated Prdm16 repression occurs mainly
in uncommitted stem cells to restrict their differentiation towards
brown fat, and maintain their multipotency. The luciferase
reporter assay, gain- and loss-of-function studies provide direct
evidence that miR-133a target Prdm16. In consistency with our
study, two recent studies demonstrated that miR-133 can target
Prdm16 in both satellite cells and brown adipose cell lines [17,18].
Interestingly, miR-133a dKO mouse has adipocyte browning in
SAT but has no overt phenotype in BAT. Several possibilities
might have led to this observation. First, miR-133b, another miR-
133 family member, is also highly expressed in BAT (than in
WAT) and maintains its expression in the miR-133a dKO BAT.
Notably, miR-133b is dramatically downregulated in the asWAT
and ingWAT of miR-133a dKO mice for unknown reasons. The
loss of both miR-133a and miR-133b in SAT might have led to
the upregulation of Prdm16 and activation of the BAT and
thermogenic gene program. Second, the loss of miR-133a may be
insufficient to further upregulate Prdm16, which is already highly
Figure 4. Genetic ablation of
miR-133a
promotes the browning and thermogenic gene program in SAT but not BAT. miR-133a has two
alleles, miR-133a1 and miR-133a2. The miR-133a-dKO mice (miR-133a1
2/2
; miR-133a2
2/2
) were obtained by intercrossing mice with the genotype of
miR-133a1
2/2
; miR-133a2
+/2
. The mice (miR-133a1
2/2
; miR-133a2
2/2
) were used as a control for the qPCR analysis of BAT, asWAT and ingWAT. (A)
miR-133a levels in BAT, (B) brown marker expression in BAT, (C) thermogenic gene program in BAT. (D) miR-133a levels in asWAT, (E) brown marker
expression in asWAT, (F) thermogenic gene program in asWAT. (G) miR-133a levels in ingWAT, (H) brown marker expression in ingWAT, (I)
thermogenic gene program in ingWAT. The brown markers include Prdm16, Ucp1 and Cidea; the thermogenic genes include Cox8b, Hsl, Atgl and Cpt2.
N = 3, *P,0.05, **P,0.01.
doi:10.1371/journal.pgen.1003626.g004
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expressed in the BAT. By contrast, Prdm16 is expressed at levels
several fold lower in the SAT and the loss of miR-133a can
therefore lead to an upregulation of Prdm16.
It has been reported that 76% of miR-133a1
2/2
a2
2/2
dKO
mice die prior to P10 and the few surviving mice are subjected to
sudden death due to cardiomyopathy [13]. Due to the extremely
low survival rate of the miR-133a1
2/2
a2
2/2
dKO mice [13,14],
we used miR-133a1
2/2
a2
+/2
that had three out of the four miR-
133a alleles knocked for most in vivo studies. We found that the
ingWAT of miR-133a1
2/2
a2
+/2
mice contains numerous multi-
locular adipocytes that express UCP1. Our observation that beige
adipocyte specific CD137 and Tmem26 genes are highly upregu-
lated in the miR-133a1
2/2
a2
+/2
compared to the wildtype
ingWAT suggests that the multilocular adipocytes in the miR-
133a1
2/2
a2
+/2
WAT are probably the cold-inducible adaptive
beige adipocytes. Consistent with this notion, we found that miR-
133a1
2/2
a2
+/2
mice activate the thermogenic gene program
much more robustly than the wildtype mice upon cold exposure.
More importantly, ITT and GTT demonstrate that the miR-
133a1
2/2
a2
+/2
mice exhibit increased sensitivity to insulin and
glucose tolerance compared to WT controls. These results
together provide strong in vivo evidence that miR-133a regulates
the normal physiological function of adipose tissues.
Upon catecholamine hormone stimulations, WAT depots
undergo lipid mobilization, a lipolysis process that hydrolyzes
triglycerides of white adipocytes [19]. Free fatty acids released
from white adipocyte lipolysis undergo beta-oxidation in the
mitochondria of brown adipose tissue and provide energy. The
Fatty acids also activate special Pparc2 complex which directly
activates Ucp1 expression and dissipates chemical energy [20].
Prdm16 is a Pparc2 coactivator that drives the brown adipocyte
gene program [3]. Here we showed that miR-133a inhibits white
adipocyte browning, it would be interesting to study if miR-133a is
involved in the repression of hormone stimulated adipocyte
browning process.
Obesity has disrupted catecholamine signals, leading to excess
fat accumulation and multiple metabolic diseases [19]. Prdm16
drives express ion of the browning and thermogenic gene program
[2,5]. Overexpression of Prdm16 in adipose lineage resu lted in
large number of beige cell formation in SAT and more energy
expenditure, which improved glucose metabol ism and enhanced
insulin sensitivity [2]. However, there is no report to show the
anti-obese and anti-di abetic role of Prdm16, or adipocyte
browning, in obese and diabetic backgro und. It would be
interesting to examine if overexpression of Prdm16 in the ob/
ob mice or the db/db mice can ameliorate excessive fat
accumulation and improve system insulin sensitivity. In this
study we showed that miR-133a negative ly regulates Prdm16 and
miR-133a KO mice have dramatic phenotype including adipo-
cyte browning, improved glucose metabolism and insulin
sensitivity. Consistent with our observation, blockage of endog-
enous miR133 by antisense nucleotides in mice can greatly lower
blood glucose lev els [18]. It rema ins to be investigated if
inhibition of miR-133a can inc rease system energy expenditure
in the ob/ob and db/db background.
Interscapular brown adipose is detectable in the newborn
humans to maintain body temperature but its mass gradually
decreases in the postnatal life [21]. Recent studies have
demonstrated that adult humans develop active brown adipo-
cytes in response to cold exposure and the amount of BAT is
inversely correlated with body weight [22,23,24]. Detailed
molecular signature analys is suggested that the adult human
brown adipocytes are more similar to murine beige cells, but not
the classical interscapular BAT cells [15,25]. Our study revealed
that miR-133a represses white adipocyte browning and beige
adipocyte formation in th e mouse model. It remains to be
investigated if miR-133a also plays a crucial role in the naturally
occurred adipocyte browning in humans. As the increased beige
adipocytes absorb more glucose and increase insulin sensitivity,
it will be interesting to investigate if miR-133a could be a potent
drug target for clinical purposes.
Materials and Methods
Animals
All procedures involving the use of animals were performed in
accordance with the guidelines presented by Purdue University’s
Animal Care and Use Committee. mTmG and aP2-Cre mice were
from Jackson Lab under stock# 007576 and 005069, respectively.
miR-133a knockout mice were previously described [13]. For
glucose tolerance test (GTT), 7-week-old mice were fasted
overnight and injected with 1.5 mg/g glucose/body weight. For
insulin tolerance test (ITT), 2–3 month old mice were fasted for
4 hours and injected with 0.75 U/Kg insulin/body weight. The
blood glucose levels were monitored at 30 min intervals for 2 h
with an ACCU-CHEK Active Blood Glucose System (Roche)
using tail tip blood samples. For cold exposure, mice in their
regular filter-top cages with double bedding and nesting materials
were placed in ventilated plastic bins and housed in a 4uC room
for 5 days.
Primary adipocyte cultures
Primary adipocyte cultures were performed as previously
reported [26]. Interscapular BAT and various WAT depots were
collected, minced and digested with isolation buffer for proper
time at 37uC on a shaker. The isolation buffer contains 123 mM
NaCl, 5 mM KCl, 1.3 mM CaCl
2
, 5 mM Glucose, 100 mM
HEPES, 4% BSA, 1%P/S and 1.5 mg/ml Collagenase I. The
digestion was stopped with DMEM containing 2%FBS and 1%
HEPES, filtered through 100
mm filters, and cells were pelleted at
4506 g for 5 min. The cells were cultured in growth medium
containing DMEM, 20% FBS, 2% HEPES and 1% P/S at 37uC
with 5% CO2 for 3 days, and then fresh media was changed every
2 days. Upon confluence, cells were exposed to induction medium
for 4 days and then differentiation medium for several days until
adipocytes mature. The induction medium contains DMEM, 10%
FBS, 2.85
mM insulin, 0.3 mM dexamethasone (DEXA) and
0.63 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma), and the
differentiation medium contains DMEM, 10% FBS, 200 nM
insulin and 10 nM T3.
Figure 5. Knockdown of miR-133a leads to browning of WAT and improves body insulin sensitivity in vivo. Wildtype (WT; miR-133a1
+/+
a2
+/+
) and miR-133a knockdown (KO; miR-133a1
2/2
a2
+/2
) mice were used. (A) Representative image of inguinal WAT isolated from WT and KO mice.
(B) Western blot image showing relative expression of UCP1 and b-actin in WT and KO ingWAT. (C–D) H&E staining of WT (C) and KO (D) ingWAT. (E–
F) immunohistostaining of UCP1 of WT (E) and KO (F) ingWAT. UCP1 signal is in brown and cell membrane is counter stained in blue. Scale
bar = 50
mm. (G) Blood glucose levels during IP-GTT test (n = 4 pairs of mice). (H) Blood glucose levels after IP insulin injection, normalized to initial
glucose measurement as 100% (n = 6 pairs of mice). *P,0.05, **P,0.01.
doi:10.1371/journal.pgen.1003626.g005
miR-133a Regulates Browning
PLOS Genetics | www.plosgenetics.org 8 July 2013 | Volume 9 | Issue 7 | e1003626
Luciferase assay
Plasmids carrying Renila luciferase gene linked to a fragment of
Prdm16-39UTR harboring miR-133a putative binding sites were
cotransfected to HEK293 cells, along with control miRNA or
miR-133a mimic (Invitrogen). The mutant 39 UTR of Prdm16
was performed by mutagenesis of the miR-133a recognized
sequences from GGACCAA into TTGGTCC. Samples were
collected at 48 h post-transfection. Luciferase activity was
measured with the use of the Dual Luciferase Assay System
(Promega), and the relative luciferase activities were normalized to
firefly luciferase. Plasmids carrying firefly luciferase gene linked to
fragments of Prdm16-39UTRs harboring the putative target sites
of miR-206, miR-1, or miR-128 were co-transfected to HEK293
cells along with control miRNA, miR-206 mimic, miR-1 mimic, or
miR-128 mimic (Invitrogen). The relative luciferase activities were
normalized to Renila luciferase.
Fluorescent activated cell sorting (FACS)
The stromal vascular fraction of adipose tissues was isolated as
described above and cells were filtered through 30
mm sterile
nylon mesh. The BAT SVF cells from aP2-mTmG are selected on
the basis of fluorescence characteristics. mTmG adipocytes were
used as a positive control for gating RFP+ cells. Cell debris and
dead cells were removed by staining of dead cell dye. The adipose
progenitor cells were sorted out from SVF cells of wildtype mice by
antibodies against CD31-PE-Cy7, CD45-PE-Cy7, Ter119-PE-
Cy7, CD34-FITC, and Sca1-Pacific blue (eBioscience). After
sorting, cells were collected for RNA extraction right away or
cultured in CO2 incubator at 37uC for differentiation or
transfection experiments.
microRNA transfection of BAT APCs and SAT SVF cells
The transfection was performed by Neon electroporation
system (Invitrogen). Final concentration of 500 nM miRNA
mimics or miRNA LNAs were incubated with the indicated cells
(50,000–100,000 cells) on ice for 5 min and the electroporation is
performed under 1150 voltage, 20 msec intervals and 2 pulses.
The cells were then seed on 12-well plates. After 12 hours the
transfection complex was replaced with fresh adipogenic induction
medium. After 4 days of induction, the medium was replaced with
adipogenic differentiation medium and the cells were collected for
RNA analysis after an additional 4 day differentiation.
Retrovirus production and infection
The plasmids pMSCV-Prdm16 or the empty vector was
transfected by lipofactamine 2000, along with the packing vector
pEco to 10-cm Hek293 cells. Freshly isolated 48 h supernatants
containing retrovirus particles were filtered and mixed with 4 ug/ml
Polybrene. The mixtures (1 ml) were added to each well of BAT
APCs, which have been recovered for 8–12 hours after the
electroporation of miR-133a. Fresh culture medium was added
12 hours later and was replaced by BAT induction medium after
additional 12 hours. Induction and differentiation were same as
described above.
Quantitative realtime PCR (qPCR)
RNA was extracted and purified from mature adipocytes of
adipose tissues or cell cultures with Trizol and contaminating
DNA was removed with DNase I. Random hexamer primers were
used to convert RNA into cDNA. For microRNA qPCR, multiple
adenosine nucleotides were added to 39 end of RNAs by E. coli
DNA polymerase and cDNAs were synthesized with a specific RT
primer [27]. QPCR was performed by using a light cycler 480
(Roche) machine for 40 cycles and the fold change for all the
samples was calculated by 2
2DDct
methods. 18s was used as
housekeeping gene for mRNA expression analysis. 18s and U6
mRNA was used as housekeeping gene for microRNA expression
analysis.
Figure 6. Knockdown of miR-133a promotes the activity of
cold-inducible thermogenesis gene program in vivo. qPCR
analysis of relative expression of genes in inguinal WAT tissue after 5
day exposure of wildtype (WT) and miR-133a knockdown (KO; miR-
133a1
2/2
a2
+/2
) mice at 4uC. (A) miR-133a, (B) Ucp1, (C) other brown
adipocyte markers, Prdm16, Pgc1a, Pparc2, Cidea, (D) mitochondria
genes Cox8b and Cpt2, and lipolysis genes Hsl and Atgl. N = 4–6,
*P,0.05, **P,0.01.
doi:10.1371/journal.pgen.1003626.g006
miR-133a Regulates Browning
PLOS Genetics | www.plosgenetics.org 9 July 2013 | Volume 9 | Issue 7 | e1003626
Histology and immunohistochemist ry
Serial sections of white fat were cut at 4 mm thick, de-
paraffinized, and rehydrated through xylene, ethanol, and water
by standard methods. For antigen retrieval, slides were submerged
in 0.01 mol/L sodium citrate (pH 6.0) and heated to 96uC for
20 minutes in a laboratory microwave (PELCO). Immunohisto-
chemistry was performed on a Dako Autostainer (Dako,
Carpinteria, CA). Slides were incubated with 3% hydrogen
peroxide and 2.5% normal horse serum (S-2012, Vector), followed
by incubation with rabbit polyclonal anti-UCP-1 primary
Figure 7. Mutation of miR-133a predispose white preadipocytes to become adaptive beige adipocytes upon differentiation. qPCR
analysis of relative gene expression in inguinal WAT tissue (n = 3 pairs) and cultured WAT adipocytes from SVF (n = 4 pairs) of wildtype (WT) and miR-
133a knockdown (KO; miR-133a1
2/2
a2
+/2
) mice. (A) miR-133a in ingWAT, (B) BAT marker genes in ingWAT, (C) beige adipocyte marker genes, CD137,
Tmem26 and Tbx1, in ingWAT. (D) Ucp1 and (E) other BAT marker gene expression in adipocytes differentiated from stromal vascular cells of ingWAT.
*P,0.05, **P,0.01.
doi:10.1371/journal.pgen.1003626.g007
miR-133a Regulates Browning
PLOS Genetics | www.plosgenetics.org 10 July 2013 | Volume 9 | Issue 7 | e1003626
antibody (ab23841, Abcam) diluted 1:200 in 2.5% normal horse
serum (Vector, S-2012) for 60 minutes. Primary antibody binding
was detected with an anti-rabbit horseradish peroxidase (HRP)–
ImmPRESS Anti-Rabbit Ig (peroxidase) Polymer Detection Kit
(MP-7401, Vector). Labeling was visualized with 3, 39-diamino-
benzidine (DAB) as the chromogen (SK-4105, Vector). Slides were
counterstained with Harris hematoxylin (EK Industries, Joliet, IL)
and whole slide digital images were collected at 206 magnification
with an Aperio ScanScope slide scanner (Aperio, Vista, CA).
Statistical analysis
The data are presented with mean 6 standard error of the
mean (SEM). P-values were calculated using two-tailed student’s t-
test. The ones with P-value less than 0.05 were considered as
statistic significant.
Supporting Information
Figure S1 Relative expression of miroRNAs in various subcu-
taneous WAT depots. asWAT, anterior subcutaneous WAT;
bsWAT, back subcutaneous WAT; ingWAT, inguinal WAT. The
expression of bsWAT is normalized to 1. N = 3. *P,0.05,
**P,0.01.
(TIF)
Figure S2 miR-133a inhibits adipocyte browning in SAT. SAT
SVFs were transfected with synthetic miRNA133a by electropo-
ration and cultured to confluence, followed by adipogenic
induction and differentiation for 4 days each. (A–C) qPCR
analysis of miR-133a and the brown markers after cells were
differentiated. N = 3, *P,0.05, **P,0.01.
(TIF)
Figure S3 Knockdown of miR-133a upregulates UCP1 expres-
sion. Depots of asWAT and ingWAT were harvested from
widltype (WT) and miR-133a knockdown (KO; miR-133a1
2/2
a2
+/2
) mice that were housed at room temperature or at 4uC for 5
days. Pictured are representative Western Blot images showing the
relative expression levels of UCP1. Beta-Actin is used as internal
control for protein input.
(TIF)
Acknowledgments
We thank Tracy Lynn Wiegand and Carol Bain (Purdue University) for
assistance with histology, immunohistochemistry and image scanning Dr.
Eric Olson (University of Texas Southwestern Medical Center) for
providing the miR-133a knockout mice, and Jun Wu for mouse colony
maintenance and technical assistance.
Author Contributions
Conceived and designed the experiments: WL PB SK. Performed the
experiments: WL PB TS XY. Analyzed the data: WL PB SK. Contributed
reagents/materials/analysis tools: HY Y-XW NL MAR. Wrote the paper:
WL SK.
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