Essential Role for miR-196a in Brown Adipogenesis of
White Fat Progenitor Cells
Masaki Mori1, Hironori Nakagami1,2*, Gerardo Rodriguez-Araujo1, Keisuke Nimura1, Yasufumi Kaneda1*
1Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan, 2Division of Vascular Medicine and Epigenetics, United Graduate School
of Child Development, Osaka University, Osaka, Japan
The recent discovery of functional brown adipocytes in adult humans illuminates the potential of these cells in the
treatment of obesity and its associated diseases. In rodents, brown adipocyte-like cells are known to be recruited in white
adipose tissue (WAT) by cold exposure or b-adrenergic stimulation, but the molecular machinery underlying this
phenomenon is not fully understood. Here, we show that inducible brown adipogenesis is mediated by the microRNA miR-
196a. We found that miR-196a suppresses the expression of the white-fat gene Hoxc8 post-transcriptionally during the
brown adipogenesis of white fat progenitor cells. In mice, miR-196a is induced in the WAT-progenitor cells after cold
exposure or b-adrenergic stimulation. The fat-specific forced expression of miR-196a in mice induces the recruitment of
brown adipocyte-like cells in WAT. The miR-196a transgenic mice exhibit enhanced energy expenditure and resistance to
obesity, indicating the induced brown adipocyte-like cells are metabolically functional. Mechanistically, Hoxc8 targets and
represses C/EBPb, a master switch of brown-fat gene program, in cooperation with histone deacetylase 3 (HDAC3) through
the C/EBPb 39 regulatory sequence. Thus, miR-196a induces functional brown adipocytes in WAT through the suppression of
Hoxc8, which functions as a gatekeeper of the inducible brown adipogenesis. The miR-196a-Hoxc8-C/EBPb signaling
pathway may be a therapeutic target for inducing brown adipogenesis to combat obesity and type 2 diabetes.
Citation: Mori M, Nakagami H, Rodriguez-Araujo G, Nimura K, Kaneda Y (2012) Essential Role for miR-196a in Brown Adipogenesis of White Fat Progenitor
Cells. PLoS Biol 10(4): e1001314. doi:10.1371/journal.pbio.1001314
Academic Editor: Antonio J. Vidal-Puig, University of Cambridge, United Kingdom
Received December 13, 2011; Accepted March 16, 2012; Published April 24, 2012
Copyright: ? 2012 Mori 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: This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology; the Japan Heart Association; and the
Miyata Heart Foundation. M.M. is supported by a Research Fellowship from the Japan Society for the Promotion of Science (JSPS) for Young Scientists. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: ASO, antisense oligonucleotide; BAT, brown adipose tissue; epiWAT, epididymal adipose tissue; iBAT, interscapular brown adipose tissue;
ingWAT, inguinal white adipose tissue; SVF, stromal vascular fraction; WAT, white adipose tissue
* E-mail: email@example.com (HN); firstname.lastname@example.org (YK)
Brown adipose tissue (BAT) combusts excess energy through
mitochondrial energy uncoupling mediated by Uncoupling protein-
.Recent discoveries ofmetabolically active BATinadult humans
[2–6] have highlighted BAT as a new therapeutic target for treating
obesity and its associated diseases, such as type 2 diabetes mellitus
. The activity of BAT is inversely correlated with body mass
index in humans [3–4], implying a significant role for BAT in the
development of obesity. Importantly, the brown adipocyte-like cells
in white adipose tissue (WAT) can be generated by cold exposure or
b3-adrenergic stimulation in rodents [8–9], and the activity of BAT
can be increased by cold exposure or b3-adrenergic stimulation in
humans . The molecular mechanisms underlying this inducible
brown adipogenesis have not been fully elucidated.
The expression patterns of the Hox family of homeobox genes
(Hox genes) are characteristically distinct between BAT and WAT
[10–12], which implies a significant role of Hox genes in the
determination of two fat types. But its significance has not been fully
understood. Hox genes are representative of developmental genes
and confer an anteroposterior positional identity during embryo-
genesis. Several Hox genes have roles in differentiation systems,
such as hematopoiesis , myogenesis , and cardiogenesis
, but relatively less is known about their roles in adipogenesis.
Among the differentially expressed Hox genes, Hoxc8 is more highly
expressed in WAT than in BAT and is categorized as a white-fat
gene [11,16]. These observations imply that Hoxc8 may have an
unknown role in the determination of the two fat types.
microRNAs (miRNAs) are important regulators of the gene
small, non-coding RNAs that base pair with specific mRNAs and
suppress gene expression post-transcriptionally . miRNAs con-
stitute an essential regulatory layer at the level of the transcriptional
network . Through their regulatory capacity, miRNAs affect the
output of signaling networks by fine-tuning or switching output levels
 and promote or redirect dynamic flow in genetic circuits and
affect differentiation . The roles of miRNAs in the inducible
brown adipogenesis in WAT are not well understood.
We here show that single miRNA miR-196a is capable of
recruitingthe metabolically functional brown adipocytes in WAT in
mice. The miR-196a expression is induced in the WAT-progenitor
cells in mice exposed to cold or b3-adrenergic stimulation. The
induction of miR-196a is required for the brown fat gene expression
and is sufficient to generate the metabolically functional brown
adipocyte-likecells inWAT in mice. The target gene of miR-196a is
white-fat gene Hoxc8, which directly represses the expression of C/
EBPb, a master regulator of brown adipogenesis.
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HOXC8 Represses Brown-Fat Genes and Is Down-
Regulated During Brown Adipogenesis of Human WAT-
Recent reports have shown that the WAT-derived progenitor
cells undergo brown adipogenesis in vitro in both mice [16,21] and
humans [16,22]. Consistently, the human fat progenitor cells
derived from flank subcutaneous WAT (hereafter, WAT-progen-
itor cells) exhibited increased brown-fat gene expression after
differentiation (Figure S1A and S1B). HOXC8 is categorized as a
white-fat gene  and RNA-seq analysis revealed that HOXC8
was most highly expressed among the clustered HOX genes in the
human WAT-progenitor cells (Figure 1A and 1B). We noticed that
HOXC8 was down-regulated in the differentiated adipocytes
(Figure 2A and 2B). Contrarily, the expression of HOXC6 did not
change after differentiation (Figure S1C) and was not particularly
high in WAT (Figure S1D), though HOXC6 is located adjacent to
HOXC8 in HOXC cluster and was the second most highly
expressed gene (Figure 1A and 1B). These results implied the
existence of specific regulatory machinery for HOXC8 expression.
Down-regulation of HOXC8 was observed at the protein level
(Figure 2C) but not at the mRNA level (Figure 2D). These results
implied that HOXC8 might be regulated post-transcriptionally.
Transduction of HOXC8 in the human WAT-progenitor cells
suppressed the brown-fat genes including C/EBPb , UCP1
, and ADIPSIN (also known as CFD) (Figure 2E) . In
contrast, HOXC8 did not suppress the white-fat genes including
leptin , CD24 , HMGA2 , and ADIPOQ (also called
adiponectin) (Figure 2E). These results suggested that HOXC8 might
regulate the brown-fat genes and that HOXC8 might be an
important regulator for brown adipogenesis of the WAT-
Hoxc8 Is Down-Regulated During Brown Adipogenesis In
To extend our findings in vitro to in vivo, we proceeded to
a mouse model of brown adipogenesis. In mice, the Hoxc8
expression was higher in WAT than BAT and other tissues (Figure
S2). Stromal vascular fraction (SVF) of fat depots contains fat
progenitor cells (hereafter, SVF cells). The Hoxc8 expression was
suppressed after the SVF cells were induced to undergo brown
adipogenesis (Figure 3A and 3B) and expressed Ucp1 (Figure 3C),
Pgc-1a, and C/EBPb (Figure 3D). In mice, brown adipogenesis can
be induced in WAT by administering a b3-adrenergic agonist,
CL-316,243, or by exposing mice to cold environment. After
administration of CL-316,243, the expression of Hoxc8 was down-
regulated prominently in inguinal WAT (ingWAT) (Figure 3E).
The down-regulation of Hoxc8 was relatively modest in epi-
didymal WAT (epiWAT) and interscapular BAT (iBAT) than in
ingWAT (Figure 3E). To delineate the Hoxc8 expression changes
during white and brown adipogenesis, the Hoxc8 expression levels
were compared between the progenitor cell fraction (SVF) and
tissue fraction mainly composed of mature adipocytes. As a result,
the Hoxc8 expression is slightly increased in saline-treated WAT
than in SVF and is down-regulated in CL-316,243-treated fat that
underwent brown adipogenesis, indicating that Hoxc8 is down-
regulated specifically during brown adipogenesis, but not during
white adipogenesis (Figure 3F). Thus, the down-regulation of
Hoxc8 is observed during brown adipogenesis both in vitro and in
miR-196a Regulates Hoxc8 Expression in Brown
Adipogenesis of WAT-Progenitor Cells
We next sought to identify the mechanism underlying the down-
regulation of Hoxc8 during brown adipogenesis. Post-transcrip-
tional regulation of Hoxc8 was suggested by the in vitro exper-
iments. Characteristically, a number of Hox genes are regulated
by miRNAs [14,27–29] and the Hoxc8 expression can be down-
regulated by evolutionally conserved miR-196a via translational
inhibition during vertebrate development . There are two
genes encoding miR-196a (miR-196a-1 and miR-196a-2) located
within the Hox gene clusters . Based on the hypothesis that
Hoxc8 might be regulated by miR-196a, we investigated the miR-
196a expression during the brown adipogenesis in mice. We found
that the miR-196a expression was induced in WAT depots of
mice exposed to cold environment or b3-adrenergic stimulations
(Figure 4A). More specifically, miR-196a was more highly induced
in the SVF cells (Figure 4B) than in mature adipocytes (Figure S3).
Thus, miR-196a expression is induced in the SVF cells in mice
exposed to b3-adrenergic stimulation or cold exposure. The in situ
hybridization analysis of miR-196a showed the induction of miR-
196a in WAT after CL-316,243 administration (Figure 4C). Based
on the finding that the miR-196a expression is induced during
the brown adipogenesis in WAT in mice, we next investigated
whether the miR-196a induction is required for the induction of
brown adipogenesis and Hoxc8 suppression. In vitro, the miR-
196a expression is induced during the differentiation of WAT-
progenitor cells derived from both mice (Figure 4D) and humans
(Figures S4A). More detailed analyses showed that miR-196a was
induced by forskolin, an adenylyl cyclase activator, implying the
significant role of cyclic AMP pathway to regulate miR-196a
expression (Figure S4B). To address the necessity of miR-196a in
the brown adipogenesis, antisense oligonucleotide (ASO) against
mR-196a was transfected to the mouse SVF cells. The miR-196a
expression was suppressed in the transfected cells (Figure 4E) and
the Hoxc8 expression was recovered in the transfected adipocytes
Obesity is caused by the accumulation of surplus energy
in a fatty tissue called white adipose tissue (WAT) and
can lead to important health problems such as diabetes.
Mammals additionally possess brown adipose tissue
(BAT), which serves to generate body heat to stabilize
body temperature under exposure to cold, and is
abundant in hibernating animals and human neonates.
In performing its function BAT consumes energy,
thereby reducing WAT fat accumulation. Recent studies
have shown that exposure to a cold environment
stimulates the partial conversion of WAT to BAT in mice,
and given that human adults have a limited amount of
BAT, such a conversion has the potential to afford a
novel method of obesity control. Here, we analyze the
molecular mechanism of this conversion using geneti-
cally manipulated mice and cells isolated from human
adipose tissue. We find that the expression levels of
a microRNA, miR-196a, positively correlate with the
conversion of WAT to BAT under cold exposure con-
ditions. We show that forced expression of miR-196a in
mouse adipose tissue increases BAT content and energy
expenditure, thereby rendering the animals resistant to
obesity and diabetes. Mechanistically, we observe that
miR-196a acts by inhibiting the expression of the
homeotic gene Hoxc8, a repressor of brown adipogen-
esis. These findings introduce the therapeutic possibility
of using microRNAs to control obesity and its associated
diseases in humans.
Brown Adipogenesis Induced by miR-196a
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(Figure 4F), indicating that Hoxc8 suppression was mediated by
miR-196a. The ASO against miR-196a suppressed the expression
of Ucp1 (Figure 4G and 4H) and other brown-fat genes (Figure 4H),
but not the leptin expression, indicating that miR-196a is necessary
for the brown fat gene expression. Thus, the upregulation of miR-
196a is required for the induction of brown fat gene expression
during the differentiation of WAT-progenitor cells.
We next sought whether the findings above are possible to be
generalized to the conventional brown adipogenesis, which occurs
in the iBAT. The miR-196a expression level was significantly
Figure 1. HOXC8 is most highly expressed among clustered HOX genes in human WAT-progenitor cells. (A) Strand-specific RNA-seq
results showing the expression levels of clustered HOX genes in undifferentiated human white fat (WAT) progenitor cells. The results with the clusters
of HOXA, HOXB, HOXC, and HOXD are shown. The position of RefSeq genes are shown below. (B) The expression levels of clustered Hox genes from
two biological replicates. FPKM, fragments per kilobase of exon per million mapped fragments.
Brown Adipogenesis Induced by miR-196a
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lower in iBAT than WAT (Figure S4C) and was not altered during
the differentiation of the iBAT-SVF cells (Figure S4D), suggesting
that miR-196a might not be involved in conventional brown
adipogenesis in iBAT. Furthermore, endogenous expression of
Hoxc8 was not detected in iBAT-SVF cells (Figure S5). Taken
together, miR-196a is upregulated in the WAT-progenitor cells
during the inducible brown adipogenesis in mice and is required
for the induction of brown fat gene expression.
miR-196a Induces Brown-Fat Genes Through Hoxc8
We next asked whether Hoxc8 was an essential target of miR-
196a for the induction of brown-fat genes. We cloned the wild-type
(Hoxc8-wt39UTR) and miR-196a-binding site-deleted (Hoxc8-
DmiR-196-BS) Hoxc8-39UTR into a pCX4 retroviral vector and
transduced these constructs into human WAT-progenitor cells
(Figure S6A). The exogenous expression levels were comparable
among the constructs (Figure S6A). After the adipogenic induction,
the protein expression of Hoxc8 was suppressed in the Hoxc8-
wt39UTR-transduced cells than in Hoxc8-DmiR-196-BS- or
Hoxc8-transduced cells (Figure S6B), suggesting that the suppres-
sion of Hoxc8 was dependent on the miR-196a-binding site in the
Hoxc8 39UTR. The brown fat gene expression was specifically high
in the Hoxc8-wt39UTR-tranduced cells (Figure S6C), indicating
that the induction of brown-fat genes was regulated in a manner
dependent on the miR-196a-binding site of Hoxc8 mRNA. These
results suggest that miR-196a regulates brown-fat genes through
suppression of Hoxc8. To further corroborate that Hoxc8
suppression is an important step, Hoxc8 was knocked down using
Hoxc8 shRNA (Figure S7). As a result, the brown-fat genes
including C/EBPb and Ucp1 were induced (Figure S7A and S7B),
indicating that the suppression of Hoxc8 is a critical step for the
induction of brown-fat genes.
miR-196a Induces Brown Adipocyte-Like Cells in WAT
Based on the finding that miR-196a is required for the inducible
brown adipogenesis, we next addressed whether miR-196a is
capable of inducing brown adipogenesis in mice. We created
transgenic mice in which miR-196a and EGFP were expressed
under the control of the aP2 promoter/enhancer, which is
exclusively active in adipose tissues . The transgenic mice
(hereafter, the miR-196a mice) were born in a Mendelian ratio
Figure 2. HOXC8 represses brown-fat genes and is down-regulated during brown adipogenesis of human WAT-progenitor cells. (A)
The immunofluoresence analysis of HOXC8 in human WAT-progenitor cells. Arrowheads indicate differentiated adipocytes that exhibit multiple
vesicles. The cells were counterstained with CellTracker and DAPI. The scale bars indicate 30 mm. (B) The percentage of HOXC8-expressing cells
among undifferentiated cells (Undiff) and differentiated cells (Diff). (C) The immunoblots for HOXC8 in human WAT-progenitor cells treated with an
adipogenic induction medium (Induction) or left untreated. b-actin was used as a loading control. (D) The qRT-PCR of HOXC8 mRNA expression levels
in human WAT-progenitor cells left untreated or induced to undergo differentiation. The data were normalized to 18S. (E) The qRT-PCR analysis of
genes in human WAT-progenitor cells transduced with Hoxc8 or control vector and induced to undergo differentiation. The results are normalized to
18S. All data are presented as means 6 SEM; * p,0.05 versus untreated. n.s., not significant.
Brown Adipogenesis Induced by miR-196a
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and were viable. The SVF cells isolated from the miR-196a mice
were EGFP-negative immediately upon isolation, but they became
EGFP-positive while they were kept in culture (Figure S8A) and
expressed miR-196a (Figure S8B), resulting in Hoxc8 suppression
(Figure S8C and S8D). After differentiation induction, the cells
expressed more intense EGFP and underwent adipogenesis. The
aP2 promoter activity was observed in the fibroblast-like cells in
ingWAT depots (Figure S8E), which might represent the fat
progenitor cells undergoing adipogenesis. The SVF cells isolated
from the miR-196a mice expressed brown-fat genes more highly
than the cells from wild-type (WT) mice after differentiation in
vitro (Figure S8F), indicating that miR-196a promotes brown
adipocyte differentiation of the WAT-progenitor cells. To ask
whether the miR-196a function is cell-autonomous, the human
WAT-progenitor cells were transduced with lentivirus expressing
miR-196a. As a result, miR-196a enhanced the brown fat gene
expression during differentiation, indicating the cell-autonomous
function of miR-196a (Figure S9).
In vivo, the gene-expression analysis revealed an induction of
brown-fat genes, including C/EBPb, Prdm16, and Ucp1 in ingWAT
(Figure 5A), and the histological analysis revealed clusters of
multilocular cells with Ucp1 expression (Figure 5B). It is known
that different WAT depots respond to brown fat-inducing
stimulations to different extents , and we therefore addressed
the responses to the miR-196a expression in different fat depots.
The miR-196a expression levels were comparable among the
different fat depots in the miR-196a mice (Figures 5C and S10).
The induction of C/EBPb, Ucp1, and Pgc-1a was more
prominent in the ingWAT than in the epiWAT (Figure 5D and
5E) and was further augmented after CL-316,243 treatment
(Figure 5D and 5E). In the iBAT, no appreciable influence of miR-
196a was observed (Figure 5D and 5E). Thus, miR-196a induces
the brown adipocyte-like cells with characteristic appearance and
gene expression profile of brown adipocytes in WAT.
The miR-196a Mice Show Resistance to Obesity and
Improved Glucose Metabolism
Based on the finding that miR-196a is capable of inducing the
brown adipocyte-like cells, we next addressed whether they were
metabolically functional. The miR-196a mice showed a tendency
to be leaner than WT mice (Figure 6B), and even when fed a high-
fat diet, they exhibited resistance to obesity (Figure 6A and 6B),
despite the fact that their food intake tended to be increased
compared with that of the WT littermates (Figure 6C). The weight
reduction was attributable to a reduced fat accumulation (Figure
S11). To interrogate the mechanism behind the obesity resistance
of the miR-196a mice, indirect calorimetry was performed. We
used mice with similar body weight under a normal diet. As a
result, the oxygen consumption (Figure 6D) and the energy
Figure 3. Hoxc8 is down-regulated during brown adipogenesis in vivo. (A) Immunofluorescence analysis of Hoxc8 in the mouse SVF cells
derived from inguinal WAT. The cells were untreated (Undifferentiated) or induced to undergo differentiation (Adipogenic induction). The lipid
droplets and nuclei were counterstained with Bodipy and DAPI, respectively. Scale bars indicate 30 mm. (B) Immunoblots of Hoxc8 in the mouse SVF
cells left untreated or induced to undergo differentiation. b-actin served as a loading control. (C) Upper, the UCP1 expression in the differentiated
mouse SVF cells. Scale bars indicate 30 mm. Lower, the fold increase of mRNA expression levels of Ucp1 and Ucp2 in the mouse WAT-SVF cells induced
to undergo differentiation. The results were normalized to b-actin. (D) The expression of Pgc-1a and C/EBPb induced during the differentiation of
mouse SVF cells. The results were normalized to b-actin. The data are presented as means 6 SEM; * p,0.05. (E) Western blot analysis in different fat
depots from mice treated with or without CL-316,243, a b3-adrenergic receptor agonist. ingWAT, epiWAT, and iBAT denote inguinal WAT, epididymal
WAT, and interscapular BAT, respectively. (F) Western blot analysis of Hoxc8 in SVF cells and ingWAT of mice treated with CL-316,243 (CL) or saline.
Brown Adipogenesis Induced by miR-196a
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Figure 4. miR-196a is induced in SVF cells during brown adipogenesis and is required for UCP1 expression. (A) The expression of miR-
196a in the ingWAT depots of mice housed at 4uC (Cold) or at ambient temperature for 5 h (n=6), and of mice treated with CL-316,243 (CL) or saline
for 7 consecutive days (n=6). The results are normalized to U6. (B) The expression of miR-196a in WAT-SVF cells of the mice exposed to cold
environment or CL-316,243 (CL, n=6). The data are normalized to U6. (C) The in situ hybridization of miR-196a in the ingWAT depots of mice treated
with CL-316,243 or saline. The sections were probed with a miR-196a-antisense (AS) probe or control (Ctrl) probe. Size bars indicate 50 mm. All data
are presented by means 6 SEM; * p,0.05. (D) The miR-196a expression levels in mouse SVF cells during differentiation in vitro. (E) The miR-196a
expression level in the mouse SVF cells transfected with antisense oligonucleotides (ASO) against miR-196a. (F) The immunoblots for Hoxc8 in mouse
SVF cells transfected with ASO against miR-196a or with control (Ctrl) oligonucleotides after differentiation. b-actin served as a loading control. (G)
The immunoblots for Ucp1 in mouse SVF cells transfected with ASO against miR-196a or with Ctrl oligo followed by adipogenic induction. b-actin
served as a loading control. (H) The mRNA expression levels in the mouse SVF cells transfected with ASO against miR-196a or control (Ctrl)
oligonucleotides followed by differentiation induction. The results were normalized to b-actin. All data are presented by means 6 SEM; * p,0.05.
Brown Adipogenesis Induced by miR-196a
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expenditure (Figure 6E and Table S1) were enhanced during both
the light and dark phases in the miR-196a mice compared to the
WT mice, indicating the accelerated energy metabolism. The
difference of the oxygen consumption and the energy expenditure
was even enlarged when the mice were fed a high-fat diet (Figure
S12). The core body temperature was higher in the miR-196a
mice than in the WT mice (Figure 6F). These findings suggest that
miR-196a boosted the cellular energy combustion through the
induction of brown adipocyte-like cells. We next analyzed impacts
of miR-196a on glucose metabolism in the miR-196a mice. In the
glucose tolerance tests, the miR-196a mice showed lower blood
glucose (Figure 6G) and insulin levels (Figure 6H). After insulin
administration, they exhibited more pronounced declines in their
blood glucose levels (Figure 6I). These results imply that miR-196a
prevented the mice from developing insulin resistance, the
premorbid condition of type 2 diabetes. Taken together, these
findings suggest that the miR-196a-induced brown adipocyte-like
cells are metabolically functional and have favorable impacts on
glucose metabolism in mice.
Hoxc8 Targets C/EBPb in Cooperation With HDAC3 to
Regulate Brown-Fat Genes
The concept that miR-196a induces brown adipogenesis
through the suppression of Hoxc8, which might function as a
gatekeeper of brown adipogenesis in WAT, facilitated us to
investigate the target gene of Hoxc8 transcription factor. The
chromatin immunoprecipitation (ChIP) assays among the candi-
date genes revealed a significant enrichment of Hoxc8 in the C/
EBPb locus in the mouse genome (Figure 7A). C/EBPb is a crucial
regulator of brown adipogenesis, which is highly expressed in BAT
compared to WAT . The enrichment was found in the 39
region, which harbors high interspecies conservation (Figure 7B,
Figure 5. miR-196a induces brown adipocyte-like cells in WAT. (A) The gene-expression analysis in the ingWAT of the wild-type (WT) and the
aP2-miR-196a transgenic mice. The results are normalized to b-actin. The values for the WT mice are set to 1. (B) Left, Hematoxylin and eosin staining
of an ingWAT section from a miR-196a mouse showing clusters of multilocular cells. Right, the corresponding section subjected to
immunofluorescence staining with a UCP1 antibody. The insets show the multilocular appearance of the induced brown adipocyte-like cells. The
scale bar indicates 100 mm. (C) The miR-196a expression levels in different fat pads of the WT and miR-196a mice. The results are normalized to U6. (D)
The protein expression in the different fat depots of the WT and miR-196a mice treated with or without CL-316,243. (E) The densitometric analysis of
(D). CL, CL-316,243. All data are presented as means 6 SEM; * p,0.05.
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‘‘4’’). In human WAT-progenitor cells, too, the enrichment of
HOXC8 was observed in the C/EBPb 39 region (Figure 7C and
7D). The enrichment of HOXC8 was also observed in the
promoter of osteopontin (OPN) gene used as a positive control
(Figure 7C) . To ask whether the binding of Hoxc8 in the 39 of
C/EBPb has a regulatory role, we performed the reporter assay by
replacing the C/EBPb coding region with luciferase gene. Indeed,
the C/EBPb 39 sequence induced luciferase activity, which was
further augmented by adipogenic stimulation (Figure 7E). This
luciferase expression was suppressed by concomitant transfection
of Hoxc8 but not by that of Hoxc8 with a mutated homeodomain
(HDm) lacking DNA-binding capacity (Figure 7F) . These
results implied that Hoxc8 regulates the C/EBPb expression via
the C/EBPb 39 regulatory sequence. Furthermore, the suppressive
effect of Hoxc8 was abolished by trichostatin A, a histone
deacetylase (HDAC) inhibitor, indicating that the suppressive
effect involves histone deacetylation (Figure 7G). In this regard,
Hoxc8 interacted with HDAC3 (Figure 7H) [34–35], but not with
HDAC1 or HDAC2. The interaction was independent of the
DNA binding capacity of Hoxc8 (Figure 7I). To further
corroborate that HDAC3 cooperates with Hoxc8, HDAC3 was
suppressed using siRNA (Figure 7J), resulting in partial elimination
of the suppressive effects of Hoxc8 (Figure 7K). To demonstrate
that C/EBPb is an essential target of Hoxc8, C/EBPb was
Figure 6. The miR-196a mice show resistance to obesity and improved glucose metabolism. (A) The appearance of the WT and miR-196a
mice fed a high-fat diet for 16 wk. (B) The body weights of the WT and miR-196a mice (n=8) fed a high-fat diet (HFD) or normal diet (ND) after 8 wk
old. (C) The daily food intake of the WT and miR-196a mice (n=8). (D) Oxygen consumption rates (V ˙O2) in the WT and miR-196a mice fed a normal
diet (n=6). Measurements were performed on 3- to 4-mo-old mice with similar body weight that were given a standard diet. (E) The energy
expenditure in the WT and miR-196a mice fed a normal diet (n=6) calculated based on V ˙O2 and V ˙CO2 values and averaged separately for the light
and dark phases. Measurements were performed on 3- to 4-mo–old mice with similar body weight that were given a standard diet. (F) The core body
temperatures of the WT and miR-196a mice (n=6). (G) The glucose tolerance test results for the WT and miR-196a mice (n=10). (H) The plasma
insulin concentrations after glucose injection in the WT (n=8) and miR-196a (n=10) mice. (I) The insulin tolerance test for the WT and miR-196a mice
(n=10). All data are presented as means 6 SEM; * p,0.05.
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Figure 7. HOXC8 targets C/EBPb in cooperation with HDAC3 to regulate brown-fat genes. (A) The ChIP analysis in 3T3-L1 preadipocytes
expressing Flag-Hoxc8 in mouse C/EBPb locus. H1foo is an oocyte-specific gene and served as a negative control. The numbers 1–4 in C/EBPb
correspond to 1–4 in (B), respectively. (B) The interspecies conservation of the mouse C/EBPb 39. The data obtained from the UCSC Genome Browser
map. (C) The ChIP analysis in the human WAT-progenitor cells in human C/EBPb locus. Osteopontin (OPN) served as a positive control. (D) The
interspecies conservation and location of ChIP primers used in (C) in the human C/EBPb locus. The data obtained from the UCSC Genome Browser
map. (E) Luciferase reporter assay to assess the transcriptional activity of C/EBPb 39 sequence inserted into the 39 end of the luciferase gene. The
activity was measured in 3T3-L1 preadipocytes left untreated (Untreated) or induced to undergo adipogenesis (induction). * p,0.05.¡p,0.05. (F)
Luciferase reporter activity of C/EBPb 39 sequence measured in 3T3-L1 preadipocytes transfected with Hoxc8, homeodomain-mutated Hoxc8 (HDm),
or control vector. (G) Luciferase reporter activity in 3T3-L1 preadipocytes transfected with Hoxc8, HDm, or control vector in the presence of
trichostatin A, a histone deacetylase (HDAC) inhibitor. (H) Immunoprecipitation in 3T3-L1 preadipocytes stably expressing Flag-Hoxc8. The
immunoblot analysis was performed after immunoprecipitation with anti-Flag antibody. The white dot indicates a non-specific band. (I)
Immunoprecipitation in 3T3-L1 preadipocytes stably expressing Flag-HDm. The immunoblot analysis was performed after immunoprecipitation with
anti-Flag antibody. The white dot indicates a non-specific band. (J) The immunoblot of HDAC3 in the 3T3-L1 preadipocytes transfected with siRNA
against HDAC3. (K) Luciferase reporter activity in 3T3-L1 preadipocytes transfected with Hoxc8 and siRNA against HDAC3. (L) The mRNA expression
levels in the human WAT-progenitor cells stably expressing HOXC8 followed by transfection with C/EBPb or EGFP and adipogenic induction. All data
are presented as means 6 SEM. * p,0.05. n.s., not significant.
Brown Adipogenesis Induced by miR-196a
PLoS Biology | www.plosbiology.org9 April 2012 | Volume 10 | Issue 4 | e1001314
transfected into the human WAT-progenitor cells that stably
expressed human HOXC8, resulting in restoration of the brown-fat
gene expression that had been suppressed by HOXC8 (Figure 7L).
Thus, Hoxc8 targets and represses C/EBPb in an HDAC3-
In summary, during the brown adipogenesis induced by cold
exposure or b3-adrenergic stimulations, miR-196a is induced in
WAT-progenitor cells and suppresses Hoxc8, which targets C/
EBPb, an essential regulator of brown adipogenesis. The miR-
196a expression is required for the brown-fat gene expression and
sufficient to induce metabolically functional brown adipocyte-like
cells in WAT in mice. Our findings imply the therapeutic potential
of targeting the miR-196a-Hoxc8-C/EBPb signaling pathway that
induces metabolically functional brown adipocytes in WAT to
treat obesity and its associated diseases.
Recent discoveries of metabolically active BAT in adult
humans have highlighted BAT as a therapeutic target for treating
obesity and its associated diseases. The brown adipocyte-like cells
in WAT can be generated by cold exposure or b-adrenergic
stimulation in rodents, but the molecular mechanisms underlying
these phenomena have not been fully elucidated. In this work,
we elucidated that miR-196a induces functional brown adipo-
cytes in WAT in mice. miR-196a is upregulated in WAT-
progenitor cells during brown adipogenesis induced by cold or
b-adrenergic stimulations. miR-196a is required for the brown
fat gene expression and is sufficient to induce metabolically
functional brown adipocyte-like cells in mice. The target gene
of miR-196a is Hoxc8, which is categorized as a white-fat gene
with a previously undermined role in adipogenesis. Hoxc8
directly targets and represses C/EBPb, a master switch of
brown adipogenesis. Thus, the miR-196a-Hoxc8-C/EBPb path-
way underlies the brown adipogenesis in WAT (Figure 8) and
might be a therapeutic target for the treatment of obesity and
type 2 diabetes.
Elucidation of the molecular mechanism regulating the brown
adipogenesis in WAT is important from both a biological and
clinical viewpoint. Recent studies uncovered the existence of
WAT-progenitor cells that harbor a potential to differentiate to
brown adipocytes [16,21–22,36]. The molecular mechanism
behind the inducible brown adipogenesis in WAT is relatively
unknown, but recent studies elucidated the importance of
cyclooxygenase-2 [36–37] and Prdm16 . C/EBPb is an essential
regulator of brown fat gene program [23,39–41], but whether C/
EBPb has a significant role in the inducible brown adipogenesis
was not fully understood. We found that miR-196a suppresses
Hoxc8, thereby derepressing C/EBPb, which leads to the activation
of the brown fat gene program. Our findings imply the relevance
of C/EBPb not only in the conventional brown adipogenesis but
also in the inducible brown adipogenesis in WAT.
The cellular origin of the inducible brown adipocyte-like cells in
WAT is an important question. Transdifferentiation is a significant
mechanism that has been reported to contribute to brown
adipocyte recruitment in WAT [42–43]. Because the increase in
Ucp1 mRNA is detectable within a few hours after cold stimulation
[1,31], and in vitro SVF cell differentiation is a longer process,
transdifferentiation might have a significant role in the rapid
response to stimulation. The important questions include the
relative contribution of transdifferentiation and the progenitor cell-
mediated mechanism in brown adipocyte recruitment throughout
the different phases upon exposure to a cold environment and
physiological energy regulation.
miRNAs regulate the gene networks underlying various
physiological and pathological phenomena and might be thera-
peutic targets [18–19,44–46]. miR-196a has been implicated in
the in vitro osteoblast differentiation of human fat progenitor cells,
where miR-196a suppresses Hoxc8 , but the in vivo relevance
remains unknown. We elucidated that miR-196a is induced in the
WAT-progenitor cells after the induction of brown adipogenesis, is
required for the induction of brown fat gene expression, and is
sufficient to induce the metabolically functional brown adipocyte-
like cells in WAT.
Our observations indicate that miR-196a has only a modest, if
any, effect on iBAT. The endogenous expression of Hoxc8 and
miR-196a was much lower in iBAT than in ingWAT and
epiWAT. The forced expression of miR-196a in mice did not
yield appreciable effects in iBAT. Treatment of mice with b3-
adrenergic receptor agonists usually leads to a much more
moderate induction of Ucp1 expression in iBAT than in WAT
depots. Although the primary cultures of brown adipocytes from
iBAT are highly sensitive to b3-adrenergic activation , a
moderate but significant induction of Ucp1 was reported in iBAT in
response to b3-adrenoreceptor agonists in vivo . A relatively
modest response from iBAT to the b3-adrenergic receptor agonist
compared with subcutaneous and visceral WAT has also been
reported in other studies [16,43,49]. These results imply that
distinct machinery regulates brown adipocyte recruitment in
iBAT, which was previously suggested by Petrovic et al. .
A number of miRNAs function as a molecular switch [46,50–
53], and further elucidating how the miRNAs influence the
physiological output will enable better understanding and clinical
use of miRNAs.
The significance of the distinct expression patterns of Hox genes
between BAT and WAT has been unknown [10–12]. We here
demonstrate that Hoxc8 functions as an important determinant
of white fat lineage and negatively regulates the induction of
brown adipogenesis in WAT-progenitor cells by repressing C/
EBPb, which is a master switch of brown adipogenesis [39–41].
Mechanistically, Hoxc8 directly represses the C/EBPb expression
Figure 8. A schematic of miR-196a-regulated brown adipogen-
esis of WAT-progenitor cells. Cold temperatures or b3-adrenergic
stimulations induce miR-196a in the WAT-resident progenitor cells in
mice. miR-196a post-transcriptionally suppress Hoxc8, which is one of
the white-fat genes. The direct target of Hoxc8 is C/EBPb, a master
switch of brown adipogenesis that provokes brown fat gene program in
the WAT-progenitor cells.
Brown Adipogenesis Induced by miR-196a
PLoS Biology | www.plosbiology.org 10 April 2012 | Volume 10 | Issue 4 | e1001314
through the 39 regulatory sequence. Similar conserved non-coding
regulatory elements have been reported for the Foxp3 gene ,
and previous studies suggested that the majority of transcription
factors bind to sites other than the promoter [20,55]. Hoxc8
recruits HDAC3, which is implicated in the regulation of
metabolic genes [34,35]. Since the HDAC proteins lack DNA-
binding activity, they are recruited to target genes via association
with transcriptional factors . Our findings imply the possible
therapeutic efficacy of HDAC inhibitors for obesity through
inducing brown adipogenesis, but further study is required to
address the possibility.
The induction of brown adipogenesis in WAT has great
therapeutic potential. Our findings suggest that the miR-196a-
Hoxc8-C/EBPb pathway may constitute a promising strategy for
addressing the social and health problems caused by obesity and its
Materials and Methods
Mice were handled in accordance with protocols approved by
the Ethics Committee for Animal Experiments of the Osaka
University Graduate School of Medicine.
The coding sequence of human Hoxc8 (Gene ID: 3224) was
cloned into pCX4-puro  and pCAGIP vector . The
pCX4-Hoxc8 retroviral vector was used to generate human WAT-
progenitor cells stably expressing Hoxc8. Human C/EBPb was
cloned into the pCAGIP vector. The homeodomain mutant
(I195A/Q198A/N199A/M202A)  of Hoxc8 (HDm) was
created by site-directed mutagenesis. For lentivirus-mediated
shRNA expression, pLenti6-miR-196a, -shHoxc8, and -shLacZ
were generated from pcDNA6.2 constructs by Gateway reactions.
Lentivirus was generated by cotransfection of the pLenti6
construct with packaging plasmids into 293FT cells according to
the manufacturer’s instruction (Invitrogen). For Hoxc8 39UTR
analysis, human Hoxc8 39UTR sequence was cloned and inserted
to the 39 end of Hoxc8 cDNA. The miR-196a binding site
(CCCAACAACTGAGACTGCCTA) was deleted to generate
Gene Expression Analysis
Total RNA was isolated using the RNeasy Lipid Tissue Mini
Kit (QIAGEN, CA). Reverse transcription and quantitative PCR
were performed as previously described . For microRNA
quantification, total RNA was isolated using a mirVana miRNA
isolation kit (Applied Biosystems). Reverse transcription and
quantitative PCR were performed according to the manufacturer’s
instructions. A list of probes is provided in Text S1.
RNA from human white fat (WAT) progenitor cells was
extracted with RNeasy (QIAGEN) following the manufacturer’s
instructions. 12.5 mg of total RNA were subjected to two rounds of
oligo-dT purification using Ambion MicroPoly(A) Purist Kit
(Ambion). 50 ng of the fragmented poly(A) RNA by using
RNaseIII were ligated to SOLiD Adaptor Mix and were
reverse-transcribed by using SOLiD Total RNA-Seq Kit (Life
Technologies). First-strand cDNA from 100 bp to 150 bp was
selected by using Agencourt AMPure XP reagent (Beckman
Coulter Genomics) and was amplified by SOLiD 59 PCR primer
and barcoded SOLiD 39 PCR primers (Life Technologies).
Sequencing libraries were prepared according to Life Technolo-
gies’ protocol. RNA-seq libraries were sequenced with SOLiD 4.
Mapping of resulting reads was performed by Bioscope (Life
Technologies), and analysis of mapped reads (31,825,850 reads in
hADSC_1 and 42,009,231 reads in hADSC_2) was performed by
Human WAT-progenitor cells were isolated from human flank
subcutaneous fat lipoaspirate (Lonza, Switzerland) and maintained
in mesenchymal stem cell growth medium (Lonza). For adipogen-
esis, 2-d post-confluent cells were treated with an induction
medium containing 0.5 mM IBMX, 10 mg/ml insulin, and 1 mM
dexamethasone (MDI). The induction medium was changed every
2 d. Forskolin (40 mM, Sigma-Aldrich) was added to the medium
as noted. Antisense oligonucleotide against miR-196a (Anti-miR
miRNA inhibitor, AM10068, Ambion) was transfected according
to the manufacturer’s instruction. The fat progenitor cells were
isolated from inguinal white adipose tissue (WAT) or interscapular
BAT (iBAT) of C57Bl/6 mice using a standard method .
Adipogenic induction was performed by treating the cells with the
induction medium for 2 d.
Western Blot Analysis
Western blotting was performed with antibodies against Hoxc8
(1:1,000, ab86236, abcam), C/EBPb (1:200, sc-150, Santa Cruz
Biotechnology, CA), UCP1 (1:1,000, U6382, Sigma-Aldrich),
PGC-1a (1:1,000, ab54481, abcam), b-actin (1:5,000, AC-15,
Sigma-Aldrich), and GAPDH (1:5,000, ab8245, abcam). The
secondary antibodies (GE Healthcare) were used at a 1:1,000
dilution ratio. Immunoreactive bands were detected with Chemi-
LumiOne L (Nacalai Tesque) or ECL plus (GE Healthcare).
Densitometry was performed with the ImageJ software (NIH;
Immunocytochemistry was performed using antibodies against
Hoxc8 (1:200, MMS-286R, Covance), Hoxc6 (1:200, ab41587,
Abcam), Pgc-1a (1:300, ab54481, Abcam), or UCP1 (1:500,
ab10983, Abcam) as previously described . The primary
antibodies were detected using anti-mouse-Alexa Fluor 546, anti-
mouse-Alexa Fluor 488, or anti-rabbit-Alexa Fluor 546 (1:1,000,
Invitrogen). Cells were counterstained with CellTracker Green
Bodipy (Invitrogen), Bodipy 493/503 (D3922, Invitrogen), and 49-
6-diamidino-2-phenylindole (DAPI, Invitrogen).
These experiments were approved by the Ethics Committee for
Animal Experiments of the Osaka University Graduate School of
Medicine. Male outbred C57Bl/6 mice were used. For acute cold-
exposure studies, 3- to 4-mo-old male mice were housed at 4uC for
5 h. For b3-adrenaline receptor stimulation, CL-316,243 (Sigma),
at 0.5 mg/kg, was injected intraperitoneally once daily for 7 d.
Transgenic mice with fat-specific forced expression of miR-196a
were generated using a transgene encoding miR-196a driven by
the enhancer/promoter of the aP2 gene , and littermates were
used as the wild-type controls.
Inguinal fat sections were fixed in 10% buffered formalin and
paraffin-embedded sections were incubated with antibodies
against UCP1 (1:1,000, ab10983, Abcam) followed by detection
using ABC Vectastain-Elite kit (Vector Labs). Nuclei were
Brown Adipogenesis Induced by miR-196a
PLoS Biology | www.plosbiology.org 11 April 2012 | Volume 10 | Issue 4 | e1001314
counterstained with modified Mayer’s hematoxylin (Diagnostic
miRNA In Situ Hybridization
Inguinal WAT depots of mice were dissected after perfusion and
fixation with Tissue Fixative (Genostaff), embedded in paraffin,
and sectioned at 6 mm. The sections were de-waxed with xylene
and rehydrated. The sections were fixed with 4% paraformalde-
hyde (PFA) for 15 min, treated with 8 mg/ml proteinase K for
30 min at 37uC, re-fixed with 4% PFA, and placed in 0.2 N HCl
for 10 min. The sections were acetylated with 0.1 M tri-
ethanolamine-HCl, pH 8.0, and 0.25% acetic anhydride for
10 min. After being washed with PBS, the sections were treated
with PBS at 80uC for 5 min. The sections were hybridized with 39-
digoxygenated probes (18 pmol/ml, miR-196a-AS-LNA1: cCcaA-
caAcaTgaAacTacCta, Control (Ctrl)-LNA1: cGacTacAcaAat-
CagCgaTtt, capitals denote LNA) in Probe Diluent-1 (Genostaff)
at 50uC for 16 h and washed in 56 HybriWash (Genostaff) at
50uC for 20 min, 50% formamide in 26HybriWash at 50uC for
20 min, twice in 26HybriWash at 50uC for 20 min, and twice in
0.26 HybriWash at 50uC for 20 min. The sections were treated
with 0.5% blocking reagent (Roche) in TBST for 30 min and
incubated with anti-DIG AP conjugate (1:1,000, Roche) for 2 h at
RT. The sections were washed twice with TBST and incubated in
a solution with a composition of 1,000 mM NaCl, 50 mM MgCl2,
0.1% Tween-20, 100 mM Tris-HCl, pH 9.5. Coloring reactions
were performed with NBT/BCIP solution (Sigma) overnight
followed by counterstaining with Kernechtrot stain solution
Mice were given a standard diet or a high-fat diet (20.4%
protein, 33.2% fat, 46.4% carbohydrates by calories; MF+;
Oriental Yeast Co., Japan). Metabolic measurements were
performed on 3- to 4-mo-old mice with similar body weight that
were given a standard diet. Food intake and body weight were
measured daily and weekly, respectively. For glucose tolerance
tests, the mice were deprived of food for 16 h and were injected
intraperitoneally with glucose (2 g/kg). For insulin tolerance tests,
the mice were allowed ad libitum access to food followed by an
intraperitoneal injection of human insulin (0.75 U/kg, Eli Lilly).
The plasma concentration of glucose was measured with a
Glucometer (Sanwa Kagaku Kenkyusho, Japan), and insulin was
measured with an ELISA (Morinaga Institute of Biological
Science, Japan). Indirect calorimetry was performed under 12 h
light and dark cycles beginning at 8:00 a.m. and 8:00 p.m.,
respectively. After 1 d of acclimation, V ˙O2 and V ˙CO2 were
recorded every 3 min over 3 d using the Metabolism Measure-
ment System (MK-5000, Muromachi Kikai, Japan). Energy
expenditure (EE) was calculated using the equation of Weir: EE
(kcal/kg/h)=(3.8156V ˙O2)+(1.2326V ˙CO2). For body tempera-
ture measurement, mice were housed singly and unrestrained and
had free access to food and water. Body temperature was
measured using a rectal probe (Perimed, Sweden).
Native ChIP Assays
Chromatin immunoprecipitation was performed as previously
described  with 3T3-L1 preadipocytes expressing Flag-tagged
human Hoxc8. Primer sequences are listed in Text S1.
The C/EBPb39-luciferase constructs (C/EBPb-Luc) were gen-
erated by cloning the 39 sequence of the human C/EBPb gene
(+1,021 to +1,837) into the downstream of luciferase gene in pGL3
promoter plasmid (Promega). Dual luciferase assays were per-
formed as previously described  with 3T3-L1 preadipocytes.
Trichostatin A (330 nM, Sigma-Aldrich) was added 4 h after
transfection as indicated. Mission siRNA (Sigma) for HDAC3
(sense: 59GUAUCCUGGAGCUGCUUAATT, antisense: 59UU
AAGCAGCUCCAGGAUACTT) was transfected using Neon
transfection system (Invitrogen).
Nuclear extracts were prepared as previously described 
from 3T3-L1 preadipocytes transfected with Flag-Hoxc8, pretreat-
ed with Protein G Sepharose beads (Amersham Bioscience), and
incubated with anti-Flag M2 Affinity Gel (A2220, Sigma-Aldrich)
or control mouse IgG AC (Santa Cruz) overnight at 4uC. The
beads were washed 3 times with nuclear isolation buffer containing
500 mM NaCl and 0.15% NP-40. Purified proteins were
subjected to immunoblotting using antibodies against HDAC1
(3:1,000, Millipore), HDAC2 (1:2,000, H3159, Sigma), and
HDAC3 (1:500, ab16047, Abcam).
The statistical analysis was performed with StatView 5.0
software, JMP8 (SAS Institute, NC) and SPSS (IBM). All results
are expressed as mean 6 SEM. The data were compared using
ANOVA, followed by Dunnett’s test for pairwise comparisons
against controls and by Tukey’s test for multiple comparisons. For
the analysis of energy expenditure, a one-way analysis of covariance
(ANCOVA) was conducted. The body weight was used as the
covariate. Statistical significance was defined as p,0.05.
The RNA-seq data have been submitted to the NCBI Sequence
Read Archive (SRA). The accession number is SRA048274.1.
progenitor cells. (A) The summary of the microarray results from
human WAT-progenitor cells transduced with Hoxc8 or control
vector followed by adipogenic induction for 14 d. The expression
levels were compared to those in the untreated cells and the fold
changes in the expression levels are shown. (B) The immunoflu-
orescence analysis of HOXC8 and PGC-1a in human WAT-
progenitor cells induced to undergo differentiation for 14 d. The
nuclei are stained with DAPI. The scale bar indicates 100 mm. (C)
The immunofluorescence analysis of HOXC6 in human fat
progenitor cells left untreated (Undifferentiated) or induced to
undergo differentiation for 14 d (Adipogenic induction). The
HOXC6 expression is maintained in the differentiated cells
(arrowheads) that exhibit multiple vesicles. The nuclei are stained
with DAPI. DIC, differential interference contrast. The scale bar
indicates 50 mm. (D) The tissue distribution of HOXC6 expression
in mice. The data are normalized to 18S. All data are presented as
means 6 SEM.
The gene-expression analysis in human WAT-
is higher in white adipose tissue (WAT) than in brown adipose
tissue (BAT) and other tissues. The real-time PCR results are
normalized to b-actin.
Hoxc8 expression in mouse tissues. Hoxc8 expression
adipocyte fraction. (A) miR-196a expression levels in SVF and
The expression of miR-196a and Hoxc8 in SVF and
Brown Adipogenesis Induced by miR-196a
PLoS Biology | www.plosbiology.org 12April 2012 | Volume 10 | Issue 4 | e1001314
adipocyte fraction from mice treated with CL-316,243 or saline.
The results are normalized to U6. (B) Western blot analysis of
Hoxc8 in SVF and adipocyte fraction from mice treated with CL-
316,243 or saline.
expression is upregulated during differentiation in the human
WAT-progenitor cells. The results are normalized to U6. (B) The
miR-196a expression is upregulated by treatment with forskolin in
human WAT-progenitor cells. The results are normalized to U6.
(C) The miR-196a expression levels in different tissues of the wild-
type mice. The results are normalized to U6. (D) The miR-196a
expression is not altered significantly during the differentiation of
iBAT-derived SVF cells (conventional brown adipogenesis). The
results are normalized to U6. All data are presented as means 6
SEM. * p,0.05.
The expression analysis for miR-196a. (A) miR-196a
SVF cells. (A) Immunoblots of Hoxc8 in iBAT-SVF cells treated
with or without adipogenic induction cocktail. The results of
WAT-SVF cells were shown for comparison. b-actin served as a
loading control. (B) Immunofluorescence analysis of Hoxc8 in the
undifferentiated iBAT-SVF cells. The nuclei are stained with
DAPI. The scale bar indicates 50 mm.
The expression analysis of Hoxc8 in iBAT-derived
scheme of constructs. Human HOXC8 39UTR sequence was
inserted to the 39 end of HOXC8 cDNA to generate pCX4-
HOXC8-wild-type (wt) 39UTR.ThemiR-196a complementary site
was deleted to generate pCX4-HOXC8-DmiR-196-BS (binding
site). (B) Immunoblots of HOXC8 in human WAT-SVF cells
transduced with retroviral vector-encoded HOXC8, HOXC8-
wt39UTR, HOXC8-DmiR-196-BS, or control EGFP. The trans-
duced cells were treated with or without adipogenic induction
cocktail. b-actin served as a loading control. (C) The qRT-PCR
analysis of brown-fat genes in the transduced cells induced to
undergo differentiation for 14 d. The results are normalized to 18S.
All data are presented as means 6 SEM; * p,0.05.
The analysis of human HOXC8 39UTR. (A) The
brown fat genes. (A) The qRT-PCR analysis of adipogenesis-related
genes in mouse WAT-SVF cells transduced with control shRNA or
shRNA against Hoxc8 followed by adipogenic induction. The
results were normalized to b-actin. The data are presented as means
with control shRNA, shRNA against Hoxc8, or miR-196a encoded
by lentiviral vectors. b-actin served as a loading control.
The effects of Hoxc8 knockdown on the expression of
derived from the miR-196a mice. (A) The fluorescent microscopic
view of the SVF cells derived from the aP2-miR-196a mice
maintained without adipogenic induction. The scale bar indicates
100 mm. (B) The miR-196a expression levels in the WAT-
progenitor cells derived from inguinal WAT of the WT and
miR-196a mice. Data were normalized to U6. (C,D) The Western
blot (C) and immunofluorescence (D) analysis of Hoxc8 in the
WAT-progenitor cells. The scale bars indicate 30 mm. (E) A
confocal 3-D image of an inguinal WAT from a miR-196a mouse.
The vasculature and nuclei were visualized using anti-CD31
antibody and DAPI, respectively. V, vasculature; F, fat cells. (F)
The gene expression analysis in WAT-progenitor cells induced to
Gene expression analysis in the WAT-progenitor cells
undergo adipogenesis for 14 d. Data are presented as the mean 6
SEM. * p,0.05, ** p,0.01 versus WT.
The qRT-PCR analysis of miR-196a in human WAT-SVF cells
transduced with lentiviral vector-encoded miR-196a. The results
are normalized to U6. (B) Immunoblots of HOXC8 in human
WAT-SVF cells transduced with miR-196a or control miR-LacZ.
b-actin served as a loading control. (C) The qRT-PCR analysis of
brown fat genes in human WAT-SVF cells transduced with miR-
196a or control miR-LacZ followed by adipogenic induction. All
data are presented as means 6 SEM. * p,0.05.
miR-196a functions in a cell-autonomous manner. (A)
miR-196a mice. The qRT-PCR analysis of miR-196a in tissues of
the miR-196a mice. The results are normalized to U6. All data are
presented as means 6 SEM.
The miR-196a expression levels in tissues of the
attributable to a reduced fat accumulation. (A) Body length does
not differ significantly between the WT and miR-196a mice
(n=6). (B) The organ weights for the WT and miR-196a mice fed
a high-fat diet for 16 wk (n=3). The weight of the inguinal fat,
epididymal WAT and liver is significantly lower in the miR-196a
mice than in the WT mice. The WT mice exhibit more severe
fatty livers than the miR-196a mice. All data are presented as
means 6 SEM. * p,0.05. (C) The appearance of the organs from
the WT and miR-196a mice fed a high-fat diet for 16 wk.
The weight reduction in the miR-196a mice is
in the WT and miR-196a mice fed a high-fat diet. (A) Oxygen
consumption rates (V ˙O2) in the WT and miR-196a mice (n=6)
under a high-fat diet. (B) The energy expenditure in the WT and
miR-196a mice (n=6) under a high-fat diet calculated based on
V ˙O2 and V ˙CO2 values and averaged separately for the light and
dark phases (n=6).
Oxygen consumption rates and energy expenditure
ANCOVA analysis for energy expenditure by body
TaqMan probes and ChIP primers.
We thank the members of the Kaneda laboratory for their support and
helpful suggestions. We also thank Dr. Kazuhisa Maeda (Department of
Complementary and Alternative Medicine, Osaka University Graduate
School of Medicine) for the aP2 promoter/enhancer plasmid and Dr.
Tsuyoshi Akagi (KAN Research Institute) for providing the pCX4
retroviral vectors. We are grateful to Dr. Juro Sakai (Research Center
for Advanced Science and Technology, University of Tokyo) for thoughtful
advice regarding the design of the study.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: MM HN GR
KN YK. Performed the experiments: MM HN GR KN. Analyzed the
data: MM HN GR KN YK. Contributed reagents/materials/analysis
tools: MM HN GR KN YK. Wrote the paper: MM HN GR KN YK.
Brown Adipogenesis Induced by miR-196a
PLoS Biology | www.plosbiology.org13 April 2012 | Volume 10 | Issue 4 | e1001314
1.Cannon B, Nedergaard J (2004) Brown adipose tissue: function and
physiological significance. Physiol Rev 84: 277–359.
Nedergaard J, Bengtsson T, Cannon B (2007) Unexpected evidence for active
brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab 293:
Cypess AM, Lehman S, Williams G, Tal I, Rodman D, et al. (2009)
Identification and importance of brown adipose tissue in adult humans.
N Engl J Med 360: 1509–1517.
van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM,
Kemerink GJ, et al. (2009) Cold-activated brown adipose tissue in healthy men.
N Engl J Med 360: 1500–1508.
Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, et al. (2009)
Functional brown adipose tissue in healthy adults. N Engl J Med 360:
Zingaretti MC, Crosta F, Vitali A, Guerrieri M, Frontini A, et al. (2009) The
presence of UCP1 demonstrates that metabolically active adipose tissue in the
neck of adult humans truly represents brown adipose tissue. Faseb J 23:
Nedergaard J, Cannon B (2010) The changed metabolic world with human
brown adipose tissue: therapeutic visions. Cell Metab 11: 268–272.
Cousin B, Cinti S, Morroni M, Raimbault S, Ricquier D, et al. (1992)
Occurrence of brown adipocytes in rat white adipose tissue: molecular and
morphological characterization. J Cell Sci 103(Pt. 4): 931–942.
Cinti S (2009) Transdifferentiation properties of adipocytes in the Adipose
Organ. Am J Physiol Endocrinol Metab.
10. Cantile M, Procino A, D’Armiento M, Cindolo L, Cillo C (2003) HOX gene
network is involved in the transcriptional regulation of in vivo human
adipogenesis. J Cell Physiol 194: 225–236.
11. Gesta S, Tseng YH, Kahn CR (2007) Developmental origin of fat: tracking
obesity to its source. Cell 131: 242–256.
12. Timmons JA, Wennmalm K, Larsson O, Walden TB, Lassmann T, et al. (2007)
Myogenic gene expression signature establishes that brown and white adipocytes
originate from distinct cell lineages. Proc Natl Acad Sci U S A 104: 4401–4406.
13. Argiropoulos B, Humphries RK (2007) Hox genes in hematopoiesis and
leukemogenesis. Oncogene 26: 6766–6776.
14. Naguibneva I, Ameyar-Zazoua M, Polesskaya A, Ait-Si-Ali S, Groisman R, et al.
(2006) The microRNA miR-181 targets the homeobox protein Hox-A11 during
mammalian myoblast differentiation. Nat Cell Biol 8: 278–284.
15. Waxman JS, Keegan BR, Roberts RW, Poss KD, Yelon D (2008) Hoxb5b acts
downstream of retinoic acid signaling in the forelimb field to restrict heart field
potential in zebrafish. Dev Cell 15: 923–934.
16. Schulz TJ, Huang TL, Tran TT, Zhang H, Townsend KL, et al. (2011)
Identification of inducible brown adipocyte progenitors residing in skeletal
muscle and white fat. Proc Natl Acad Sci U S A 108: 143–148.
17. Gangaraju VK, Lin H (2009) MicroRNAs: key regulators of stem cells. Nat Rev
Mol Cell Biol 10: 116–125.
18. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function.
Cell 116: 281–297.
19. Herranz H, Cohen SM (2010) MicroRNAs and gene regulatory networks:
managing the impact of noise in biological systems. Genes Dev 24: 1339–1344.
20. Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, et al. (2008)
Connecting microRNA genes to the core transcriptional regulatory circuitry of
embryonic stem cells. Cell 134: 521–533.
21. Petrovic N, Walden TB, Shabalina IG, Timmons JA, Cannon B, Nedergaard J
(2010) Chronic peroxisome proliferator-activated receptor gamma (PPAR-
gamma) activation of epididymally derived white adipocyte cultures reveals a
population of thermogenically competent, UCP1-containing adipocytes molec-
ularly distinct from classic brown adipocytes. J Biol Chem 285: 7153–7164.
22. Elabd C, Chiellini C, Carmona M, Galitzky J, Cochet O, et al. (2009) Human
multipotent adipose-derived stem cells differentiate into functional brown
adipocytes. Stem Cells 27: 2753–2760.
23. Kajimura S, Seale P, Kubota K, Lunsford E, Frangioni JV, et al. (2009)
Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta
transcriptional complex. Nature 460: 1154–1158.
24. Feldmann HM, Golozoubova V, Cannon B, Nedergaard J (2009) UCP1
ablation induces obesity and abolishes diet-induced thermogenesis in mice
exempt from thermal stress by living at thermoneutrality. Cell Metab 9:
25. Rodeheffer MS, Birsoy K, Friedman JM (2008) Identification of white adipocyte
progenitor cells in vivo. Cell 135: 240–249.
26. Anand A, Chada K (2000) In vivo modulation of Hmgic reduces obesity. Nat
Genet 24: 377–380.
27. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS (2004) Human
MicroRNA targets. PLoS Biol 2: e363. doi:10.1371/journal.pbio.0020363.
28. Yekta S, Shih IH, Bartel DP (2004) MicroRNA-directed cleavage of HOXB8
mRNA. Science 304: 594–596.
29. Ma L, Teruya-Feldstein J, Weinberg RA (2007) Tumour invasion and metastasis
initiated by microRNA-10b in breast cancer. Nature 449: 682–688.
30. Ross SR, Graves RA, Greenstein A, Platt KA, Shyu HL, et al. (1990) A fat-
specific enhancer is the primary determinant of gene expression for adipocyte P2
in vivo. Proc Natl Acad Sci U S A 87: 9590–9594.
31. Guerra C, Koza RA, Yamashita H, Walsh K, Kozak LP (1998) Emergence of
brown adipocytes in white fat in mice is under genetic control. Effects on body
weight and adiposity. J Clin Invest 102: 412–420.
32. Lei H, Wang H, Juan AH, Ruddle FH (2005) The identification of Hoxc8 target
genes. Proc Natl Acad Sci U S A 102: 2420–2424.
33. LaRonde-LeBlanc NA, Wolberger C (2003) Structure of HoxA9 and Pbx1
bound to DNA: Hox hexapeptide and DNA recognition anterior to posterior.
Genes Dev 17: 2060–2072.
34. Knutson SK, Chyla BJ, Amann JM, Bhaskara S, Huppert SS, Hiebert SW
(2008) Liver-specific deletion of histone deacetylase 3 disrupts metabolic
transcriptional networks. Embo J 27: 1017–1028.
35. Montgomery RL, Potthoff MJ, Haberland M, Qi X, Matsuzaki S, et al. (2008)
Maintenance of cardiac energy metabolism by histone deacetylase 3 in mice.
J Clin Invest 118: 3588–3597.
36. Vegiopoulos A, Muller-Decker K, Strzoda D, Schmitt I, Chichelnitskiy E, et al.
(2010) Cyclooxygenase-2 controls energy homeostasis in mice by de novo
recruitment of brown adipocytes. Science 328: 1158–1161.
37. Madsen L, Pedersen LM, Lillefosse HH, Fjaere E, Bronstad I, et al. (2010)
UCP1 induction during recruitment of brown adipocytes in white adipose tissue
is dependent on cyclooxygenase activity. PLoS One 5: e11391. doi:10.1371/
38. Seale P, Conroe HM, Estall J, Kajimura S, Frontini A, et al. (2011) Prdm16
determines the thermogenic program of subcutaneous white adipose tissue in
mice. J Clin Invest 121: 96–105.
39. Wu Z, Xie Y, Bucher NL, Farmer SR (1995) Conditional ectopic expression of
C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates
adipogenesis. Genes Dev 9: 2350–2363.
40. Karamanlidis G, Karamitri A, Docherty K, Hazlerigg DG, Lomax MA (2007)
C/EBPbeta reprograms white 3T3-L1 preadipocytes to a Brown adipocyte
pattern of gene expression. J Biol Chem 282: 24660–24669.
41. Kajimura S, Seale P, Spiegelman BM (2010) Transcriptional control of brown
fat development. Cell Metab 11: 257–262.
42. Himms-Hagen J, Melnyk A, Zingaretti MC, Ceresi E, Barbatelli G, Cinti S
(2000) Multilocular fat cells in WAT of CL-316243-treated rats derive directly
from white adipocytes. Am J Physiol Cell Physiol 279: C670–C681.
43. Barbatelli G, Murano I, Madsen L, Hao Q, Jimenez M, et al. (2010) The
emergence of cold-induced brown adipocytes in mouse white fat depots is
determined predominantly by white to brown adipocyte transdifferentiation.
Am J Physiol Endocrinol Metab 298: E1244–E1253.
44. van Rooij E, Olson EN (2007) MicroRNAs: powerful new regulators of heart
disease and provocative therapeutic targets. J Clin Invest 117: 2369–2376.
45. Li X, Cassidy JJ, Reinke CA, Fischboeck S, Carthew RW (2009) A microRNA
imparts robustness against environmental fluctuation during development. Cell
46. Liu N, Olson EN (2010) MicroRNA regulatory networks in cardiovascular
development. Dev Cell 18: 510–525.
47. Kim YJ, Bae SW, Yu SS, Bae YC, Jung JS (2009) miR-196a regulates
proliferation and osteogenic differentiation in mesenchymal stem cells derived
from human adipose tissue. J Bone Miner Res 24: 816–825.
48. Inokuma K, Okamatsu-Ogura Y, Omachi A, Matsushita Y, Kimura K, et al.
(2006) Indispensable role of mitochondrial UCP1 for antiobesity effect of beta3-
adrenergic stimulation. Am J Physiol Endocrinol Metab 290: E1014–E1021.
49. Jimenez M, Barbatelli G, Allevi R, Cinti S, Seydoux J, et al. (2003) Beta 3-
adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown
adipocytes in white fat. Eur J Biochem 270: 699–705.
50. Schratt GM, Tuebing F, Nigh EA, Kane CG, Sabatini ME, et al. (2006) A
brain-specific microRNA regulates dendritic spine development. Nature 439:
51. van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN (2007)
Control of stress-dependent cardiac growth and gene expression by a
microRNA. Science 316: 575–579.
52. Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M, et al. (2007)
Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice
lacking miRNA-1-2. Cell 129: 303–317.
53. Nicoli S, Standley C, Walker P, Hurlstone A, Fogarty KE, Lawson ND (2010)
MicroRNA-mediated integration of haemodynamics and Vegf signalling during
angiogenesis. Nature 464: 1196–1200.
54. Zheng Y, Josefowicz S, Chaudhry A, Peng XP, Forbush K, Rudensky AY (2010)
Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-
cell fate. Nature 463: 808–812.
55. Stitzel ML, Sethupathy P, Pearson DS, Chines PS, Song L, et al. (2010) Global
epigenomic analysis of primary human pancreatic islets provides insights into
type 2 diabetes susceptibility loci. Cell Metab 12: 443–455.
56. Haberland M, Montgomery RL, Olson EN (2009) The many roles of histone
deacetylases in development and physiology: implications for disease and
therapy. Nat Rev Genet 10: 32–42.
57. Akagi T, Sasai K, Hanafusa H (2003) Refractory nature of normal human
diploid fibroblasts with respect to oncogene-mediated transformation. Proc Natl
Acad Sci U S A 100: 13567–13572.
Brown Adipogenesis Induced by miR-196a
PLoS Biology | www.plosbiology.org14 April 2012 | Volume 10 | Issue 4 | e1001314
58. Hayashi M, Nimura K, Kashiwagi K, Harada T, Takaoka K, et al. (2007)
Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP
signaling. J Cell Sci 120: 1350–1357.
59. Mori M, Nakagami H, Koibuchi N, Miura K, Takami Y, et al. (2009) Zyxin
mediates actin fiber reorganization in epithelial-mesenchymal transition and
contributes to endocardial morphogenesis. Mol Biol Cell 20: 3115–3124.
60. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, et al. (2010)
Transcript assembly and quantification by RNA-Seq reveals unannotated
transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:
61. Nakagami H, Morishita R, Maeda K, Kikuchi Y, Ogihara T, Kaneda Y (2006)
Adipose tissue-derived stromal cells as a novel option for regenerative cell
therapy. J Atheroscler Thromb 13: 77–81.
62. Nimura K, Ura K, Shiratori H, Ikawa M, Okabe M, et al. (2009) A histone H3
lysine 36 trimethyltransferase links Nkx2-5 to Wolf-Hirschhorn syndrome.
Nature 460: 287–291.
Brown Adipogenesis Induced by miR-196a
PLoS Biology | www.plosbiology.org15 April 2012 | Volume 10 | Issue 4 | e1001314