the adipocyte clock controls brown adipogenesis through ... · inhibits brown adipocyte lineage...
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RESEARCH ARTICLE
The adipocyte clock controls brown adipogenesis through theTGF-b and BMP signaling pathways
Deokhwa Nam1, Bingyan Guo2, Somik Chatterjee1, Miao-Hsueh Chen3, David Nelson4, Vijay K. Yechoor5 andKe Ma1,*
ABSTRACT
The molecular clock is intimately linked to metabolic regulation, and
brown adipose tissue plays a key role in energy homeostasis.
However, whether the cell-intrinsic clock machinery participates in
brown adipocyte development is unknown. Here, we show that
Bmal1 (also known as ARNTL), the essential clock transcription
activator, inhibits brown adipogenesis to adversely affect brown fat
formation and thermogenic capacity. Global ablation of Bmal1 in mice
increases brown fat mass and cold tolerance, and adipocyte-
selective inactivation of Bmal1 recapitulates these effects and
demonstrates its cell-autonomous role in brown adipocyte formation.
Further loss- and gain-of-function studies in mesenchymal
precursors and committed brown progenitors reveal that Bmal1
inhibits brown adipocyte lineage commitment and terminal
differentiation. Mechanistically, Bmal1 inhibits brown adipogenesis
through direct transcriptional control of key components of the TGF-b
pathway together with reciprocally altered BMP signaling; activation
of TGF-b or blockade of BMP pathways suppresses enhanced
differentiation in Bmal1-deficient brown adipocytes. Collectively, our
study demonstrates a novel temporal regulatory mechanism in fine-
tuning brown adipocyte lineage progression to affect brown fat
formation and thermogenic regulation, which could be targeted
therapeutically to combat obesity.
KEY WORDS: Brown adipocyte differentiation, Circadian rhythm,Adipogenesis, Obesity, TGF-b signaling pathway
INTRODUCTIONThe molecular clock is an evolutionarily conserved timekeeping
mechanism that entrains diverse biological processes to timing
cues (Reppert and Weaver, 2002). Recently, it has been
recognized that through temporal regulation of key metabolic
pathways (Panda et al., 2002), the clock machinery ensures the
adaptation of nutrient metabolism and energy homeostasis with
circadian timing, and resident peripheral clocks in metabolic
tissues are key components in this regulation (Bass and
Takahashi, 2010; Green et al., 2008). However, whether the
clock machinery functions in the metabolically active brown
adipose tissue (BAT), a specialized thermogenic organ (Cannon
and Nedergaard, 2004), is largely unknown.
In both the central clock – the suprachiasmatic nuclei (SCN) –
and peripheral tissues, the ,24-hour daily rhythm is driven by anintricate molecular feedback loop consisting of positive and
negative regulators coupled with post-transcriptional control
(Dibner et al., 2010). Brain and muscle Arnt like 1 (Bmal1, also
known as ARNTL), a basic helix-loop-helix transcription factor, is
an essential positive regulator of the core molecular clock loop
(Reppert and Weaver, 2002). Bmal1 binds to conserved E-box
elements (59-CACGTG-39) as a heterodimer with CLOCK(circadian locomotor output cycles kaput) and elicits transcription
of the negative regulator period proteins (Per1, Per2 and Per3) and
cryptochromes (Cry1 and Cry2). These factors in turn inhibit the
transcriptional activity of Bmal1–CLOCK, which, coupled with
temporally controlled proteasome-mediated degradation mechanisms,
leads to rhythmic oscillation of the molecular clock and thus
generates the daily rhythm. Interestingly, despite studies
demonstrating that Bmal1 plays key roles in metabolic regulation
(Cho et al., 2012; Rudic et al., 2004; Solt et al., 2012) and
adipogenesis (Fontaine et al., 2003; Guo et al., 2012; Wang and
Lazar, 2008), its function in brown adipocytes has not been studied.
BAT possesses a unique thermogenic ability mediated by the
action of uncoupling protein-1 (UCP-1), an inner mitochondrial
membrane proton channel that dissipates the chemical gradient
derived from oxidative phosphorylation as heat instead of ATP
generation (Cannon and Nedergaard, 2004). It has been
increasingly recognized that, in addition to its crucial role in
the maintenance of body temperature under physiological
conditions and cold stress (Cannon and Nedergaard, 2004), the
energy-dissipating capacity of BAT is an important regulatory
component of whole-body energy balance (Saito et al., 2009; van
Marken Lichtenbelt et al., 2009). Functional BAT is present in
humans (Cypess et al., 2009; van Marken Lichtenbelt et al., 2009;
Virtanen et al., 2009) and its activity is inversely correlated with
body mass index in obese patients (van Marken Lichtenbelt et al.,
2009), suggesting that expanding or stimulating the thermogenic
capacity of BAT might have therapeutic potential in treating
obesity (Nedergaard and Cannon, 2010). Owing to its distinct
function, the formation of mature brown adipocytes – brown
adipogenesis – requires not only activation of the adipogenic
cascade, but also the coordination of a thermogenic program and
mitochondrial biogenesis (Peirce et al., 2014). Interestingly,
recent lineage tracing studies of BAT embryonic development
indicate that, although it originates from the mesoderm similar to
the white adipose tissue (WAT), BAT diverges from a Myf5+
myogenic lineage under the instructive signal of Prdm16 (Seale
et al., 2008), an early marker of brown adipocyte precursors.
1Center for Diabetes Research, Department of Medicine, The Methodist HospitalResearch Institute, Houston, TX, 77030, USA. 2Department of CardiovascularMedicine, Second Affiliated Hospital, Hebei Medical University, Shijiazhuang,050017, Hebei, China. 3Department of Pediatrics, Children’s Nutrition ResearchCenter, Baylor College of Medicine, Houston, TX, 77030, USA. 4Department ofMolecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030,USA. 5Diabetes and Endocrinology Research Center, Department of Medicine,Baylor College of Medicine, Houston, TX, 77030, USA.
*Author for correspondence ([email protected])
Received 13 December 2014; Accepted 25 February 2015
� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1835–1847 doi:10.1242/jcs.167643
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mailto:[email protected]
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We recently demonstrated that the essential core clock geneBmal1 has a novel function in suppressing adipogenesis and WAT
formation (Guo et al., 2012). By contrast, it potently promotesmyogenic progression in skeletal muscle (Chatterjee et al., 2013).As BAT shares a common mesodermal origin with WAT andskeletal muscle, in the current study, we investigated whether Bmal1
participates in brown adipocyte development. Employing variousgenetic approaches in animal models and cellular systems, our studyuncovers the adipocyte-intrinsic Bmal1 function in suppressing
brown adipocyte lineage commitment and differentiation, whichconsequently affects brown fat thermogenic capacity.
RESULTSAblation of Bmal1 promotes brown fat formation andthermogenesis in vivoWe first investigated the role of Bmal1 in BAT formation in theglobal Bmal1-null (Bmal12/2) mouse model (Bunger et al., 2000).
Ablation of Bmal1 leads to a ,30% increase in the amount of BATas compared to wild-type littermate controls, as assessed by fat pad
weight (Fig. 1A) or its ratio to total body weight (Fig. 1B). InBmal12/2 BAT, mRNA expression of Bmal1 and its direct targetgene in the molecular clock, Rev-erba (also known as Nr1d1), arenearly absent (Fig. 1C), as expected. Notably, the absence of
Bmal1 leads to a marked 1.5–2-fold induction of Ucp-1, Cebpb andPparg transcripts, indicating potentially increased thermogenesisand adipogenesis in BAT. In line with this finding, BAT from
neonatal [postnatal day (P)5] Bmal12/2 mice or that from miceafter 2 months of high-fat diet feeding exhibited strongercytoplasmic staining typical of high mitochondrial density with
less lipid accumulation as compared to wild type (Fig. 1D). Directassessment of thermogenic response by using a cold-tolerance testrevealed that lack of Bmal1 significantly improved the resistance to
cold, as Bmal12/2 mice maintained higher core body temperaturethan the wild-type controls at 4 C̊ (Fig. 1E).
Fig. 1. Bmal1 ablation in mice enhancesbrown fat formation and function.(A,B) Effects of the loss of Bmal1 on BATformation as analyzed by brown adipose tissueweight (A) and its ratio to body weight (BW) (B) in8-week-old wild-type (WT) and Bmal12/2 mice(n56–8/group). (C–E) RT-qPCR analysis of BATgene expression (C), representative hematoxylinand eosin staining of BAT histology (D) and24 hour cold-tolerance test at 4˚C (E) in wild-typeand Bmal12/2 mice (n56/group). (F–I) The effectof adipocyte-specific Bmal1 ablation on BAT. (F)Immunoblot analysis of Bmal1 protein in ap2-Cre+/BMfl/fl and ap2-Cre2/BMfl/fl littermate controlmice. (G) Cold-tolerance test at 4˚C. Immunoblotanalysis of UCP-1 (H), and RT-qPCR analysis ofbrown fat gene expression (I) at basal ambienttemperature and under cold conditions (4˚C) inap2-Cre2/BMfl/fl and ap2-Cre+/BMfl/fl mice (n55–6). Tissue samples were collected the previousday at 09.00 prior to (basal) or after 24-hour coldchallenge (4˚C cold). All quantitative data showthe mean6s.e.m. *P#0.05; **P#0.01 Bmal12/2
versus wild type [Student’s t-test or one-wayANOVA (cold tolerance test)]. Scale bar: 50 mm.
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To further test cell-autonomous functions of Bmal1 in brown fat,we selectively ablated Bmal1 in adipocytes by crossing Bmal1fl/fl
mice (BMfl/fl) (Storch et al., 2007) with an adipocyte-specificdeleter, the ap2-Cre (Ap2 is also known as Fabp4) transgenic mice(He et al., 2003). Mice with selective adipocyte Bmal1 deletion,ap2-Cre+/BMfl/fl, display near complete absence of Bmal1 protein in
BAT (Fig. 1F). RT-qPCR analysis reveals an ,80% reduction inBmal1 mRNA in mature brown adipocytes, but not in preadipocytes,along with downregulation of the Bmal1 target genes Rev-erba andDbp (D-element binding protein, supplementary material Fig. S1A).Similarly, reduced Bmal1 expression is detected in white adipocytes(supplementary material Fig. S1B) in these mice, likely due to ap2-
Cre activity in WAT (He et al., 2003). As indicated by intact Bmal1immunostaining in suprachiasmatic nuclei of ap2-Cre+/BMfl/fl
mice (supplementary material Fig. S1C), adipocyte-selective
ablation of Bmal1 does not affect its protein expression in thecentral clock. Furthermore, the central clock function is preserved inthese mice as compared to control ap2-Cre2/BMfl/fl littermates,because they display normal circadian behavioral rhythms as
assessed by wheel-running actograms (supplementary material Fig.S1D). When subjected to 4 C̊ challenge, ap2-Cre+/BMfl/fl miceexhibit enhanced tolerance to cold as compared to ap2-Cre2/BMfl/fl
littermates (Fig. 2G). This similarly improved thermogenic
response as seen in the Bmal12/2 mice suggests that this effect islikely attributable to the loss of Bmal1 function in brown adipocytes.
In line with previous observations in Bmal12/2 mice (Guo et al.,2012), the body weight (supplementary material Fig. S2A) andbrown adipose weight (supplementary material Fig. S2C) of ap2-Cre+/BMfl/fl mice are increased as compared to the controls without
cold challenge. Interestingly, exposure to cold (4 C̊) for 8 hoursinduced a greater weight loss in ap2-Cre+/BMfl/fl mice (supplementarymaterial Fig. S2B), suggesting higher thermogenic energy
expenditure under this condition. The enhanced thermogenic abilityof ap2-Cre+/BMfl/fl mice is accompanied by substantially elevatedUCP-1 protein in BAT both at the basal state under ambient
temperature and at 4 C̊ (Fig. 1H). Gene expression analysis underthese conditions revealed a significant induction of genes involved inthe brown thermogenic program, including Prdm16, Dio2 (deiodinase
2) and Pgc1a (also known as Ppargc1a) in ap2-Cre+/BMfl/fl mice(Fig. 1I). Collectively, these findings indicate that Bmal1 inhibitsbrown fat formation and its thermogenic function.
The absence of Bmal1 promotes primary brownadipocyte differentiationTo directly test the function of Bmal1 in brown adipogenesis,
we isolated brown primary preadipocytes from wild-type and
Fig. 2. Bmal1-deficient primary brownpreadipocytes display enhanced adipogenicdifferentiation. (A,B) Representative images of OilRed O staining (ORO) and phase-contrast microscopy(Phase) of day-6-differentiated primary preadipocytesisolated from wild-type (WT) and Bmal12/2 mice (A)or ap2-Cre2/BMfl/fl and ap2-Cre+/BMfl/fl mice (B).Scale bars: 100 mm. (C) RT-qPCR analysis of brownadipogenic gene expression at day 5 of differentiationof wild-type and Bmal12/2 preadipocytes (n53). (D)RT-qPCR analysis of brown adipogenic geneexpression at day 1 (D1) and day 5 (D5) ofdifferentiation of ap2-Cre+/BMfl/fl versus ap2-Cre2/BMfl/fl preadipocytes (n53). Data show themean6s.e.m. *P#0.05; **P#0.01 for Bmal12/2
versus wild type or for ap2-Cre+/BMfl/fl versusap2-Cre2/BMfl/fl (Student’s t-test).
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Bmal1-null mice and induced differentiation using a brown-adipocyte-specific induction cocktail. Using Oil Red O staining
of lipid accumulation to label differentiated brown adipocytes,preadipocytes devoid of Bmal1 were found to display enhanceddifferentiation to mature brown adipocytes as compared to wild-type preadipocytes (Fig. 2A). Furthermore, not only was the
expression of mature brown adipocyte markers (Ucp-1, Dio2)markedly induced in Bmal1-null cells, the early brown lineagemarkers Prdm16 and Myf5 were robustly upregulated as well
(Fig. 2C). Furthermore, similar to these findings, primary brownpreadipocytes from ap2-Cre+/BMfl/fl mice displayed enhancedbrown adipogenesis upon induction, as compared to ap2-Cre2/
BMfl/fl control cells (Fig. 2B). This effect was accompanied byrobust induction of brown-specific (Ucp-1) and adipogenicmarkers (Pparg, Fabp4), as well as mitochondrial genes
[Pgc1a, CytC1 (also known as Cyc1), Cox7a1] at day 1 andday 5 through the differentiation timecourse (Fig. 2D).Interestingly, the early brown lineage markers Prdm16, Myf5and Cebpb are not altered, likely owing to the nature of ap2-Cre-
mediated Bmal1 deletion in these cells. Markedly elevated Ucp-1expression along with induction of the brown adipogenic gene
program is found in ap2-Cre+/BMfl/fl BAT as well (supplementarymaterial Fig. S1E). Taken together, these findings indicate that
Bmal1 inhibits brown fat formation and thermogenic function, andsuggest that these effects could be mediated by its cell-autonomousaction in suppressing brown adipogenesis.
Bmal1 inhibits brown adipocyte terminal differentiation andlineage specificationBrown adipocytes arise from mesenchymal precursors that
undergo brown lineage commitment and subsequent terminaldifferentiation (Gesta et al., 2007). To directly test the cell-autonomous function of Bmal1 in these distinct stages of brown
adipogenesis, we created stable cell lines with genetic gain or lossof function of Bmal1 in mesenchymal precursor cells, C3H10T1/2 (10T1/2), and brown preadipocytes, HIB1B. Analysis of Bmal1
protein expression reveals that it is more highly abundant inbrown adipocyte cell lines than in white preadipocytes, 3T3-L1cells, at levels comparable to those of primary myoblasts(Fig. 3A). Silencing of Bmal1 by a short hairpin RNA (shRNA)
construct in 10T1/2 mesenchymal precursors reduced itsprotein level to ,30% of that of scrambled control shRNA
Fig. 3. Silencing of Bmal1 promotesC3H10T1/2 mesenchymal precursordifferentiation to brown adipocytes.(A) Immunoblot analysis of Bmal1protein in mesodermal lineage celllines. (B) Representative images ofBODIPY lipid staining, mitochondrialstaining by Mitotracker and phasecontrast microscopy of C3H10T1/2cells with stable transfection ofscrambled control shRNA (shSC) orBmal1-specific shRNA (shBmal1)subjected to brown-adipocyte-specificdifferentiation conditions at day 9.(C) RT-qPCR analysis of brownadipogenic gene expression at day 1(D1) and day 9 (D9) of differentiation inshSC- and shBmal1-treated cells(n53). Data show the mean6s.e.m.*P,0.05; **P,0.01 for shBmal1versus shSC.
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(shSC)-treated cells, similar to what we have reported previously(Guo et al., 2012). Under a specific brown adipogenic condition
that induces 10T1/2 lineage determination and differentiation intomature brown adipocytes (Tseng et al., 2008), stable knockdownof Bmal1 resulted in an increase in the number of cells convertingto mature brown adipocytes with rounded morphology and
stronger lipid staining than that of the shSC-treated cells, asshown in Fig. 3B by BODIPY and phase-contrast images. OilRed O staining in day 10 differentiated shSC- and shBmal1-
treated cells further confirms these findings (supplementarymaterial Fig. S3). Moreover, MitoTracker Red staining offunctional mitochondria in live cells is substantially increased
in Bmal1-deficient cells compared to that of the shSC-treatedcells, indicating increased mitochondrial abundance associatedwith differentiation. Gene expression analysis at both early
(day 1) and late (day 9) stages of differentiation demonstrated a
substantially augmented brown adipogenic program with markedupregulation of the brown-adipocyte-specific marker Ucp-1, early
brown progenitor genes, Myf5 and Prdm16, and adipogenicgenes, Cebpb and Pparg (Fig. 3C).
Next, we examined the role of Bmal1 in brown adipocyteterminal differentiation using the committed brown preadipocyte
line HIB1B. Stable knockdown of Bmal1 (using shBmal1) inthese cells largely depletes Bmal1 protein as compared toscrambled controls (Fig. 4A). Assessment of mature differentiation
of these cells through lipid accumulation (BODIPY) andmitochondrial abundance (MitoTracker) reveals that Bmal1depletion markedly enhances the formation of brown
adipocytes (Fig. 4B). Analysis of brown-adipocyte-specific andadipogenic gene expression in the Bmal1-deficient cells furtherdemonstrated that Bmal1 inhibits brown preadipocyte terminal
differentiation (Fig. 4C). Moreover, UCP-1 and CEBP-b (also
Fig. 4. Silencing of Bmal1 promotesHIB1B brown preadipocyte terminaldifferentiation. (A) Immunoblotanalysis of Bmal1 protein expression inHIB-1B cells with scrambled controlshRNA (shSC) or stable shRNA-mediated Bmal1 knockdown(shBmal1). (B) The effect of stableBmal1 knockdown on HIB-1Bdifferentiation as shown by BODIPYand Mitotracker staining at day 3 ofdifferentiation. (C) RT-qPCR analysis ofbrown adipocyte marker geneexpression in shSC and shBmal1 cells(n53). Data show the mean6s.e.m.*P,0.05; **P,0.01 for shBmal1 versusshSC. (D) Immunoblot analysis ofbrown adipogenic markers duringdifferentiation day 1–4 (D1–4) of shSCand shBmal1 cells, and their responseto forskolin at day 4.
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known as CEBPB) protein levels were elevated in Bmal1-depleted cells during the differentiation timecourse as compared
to those of controls (Fig. 4D). Notably, UCP-1 also exhibited anaugmented response to forskolin stimulation in day-4 terminallydifferentiated cells. As forskolin treatment mimics a cAMP-mediated cold-induced thermogenic response in brown
adipocytes, this increased responsiveness of UCP-1 suggests apotentially augmented thermogenic response with Bmal1depletion and is in line with the observed enhanced cold
tolerance of Bmal1-null mice in vivo. In contrast, forcedoverexpression of Bmal1 in brown preadipocytes by a cDNAplasmid vector (BM cDNA), which results in high expression of
Bmal1 protein and transcript (Fig. 5A,C) as compared to emptyvector control (pcDNA3), leads to marked suppression ofdifferentiation as demonstrated by lipid and mitochondrial
staining (Fig. 5B). The significantly lower mRNA expressionof brown adipogenic markers corroborates the inhibitory effect ofBmal1 overexpression on terminal differentiation of brownadipocytes (Fig. 5C). Taken together, these in vitro analyses
further delineate the cell-intrinsic functions of Bmal1 insuppressing lineage commitment and terminal differentiation ofbrown adipocytes.
The TGF-b and BMP signaling cascade mediates Bmal1function in brown adipogenesisTo investigate the underlying mechanisms responsible for Bmal1inhibition of brown adipogenesis, we interrogated transcriptionaltargets of Bmal1 in Bmal1-knockdown 10T1/2 cells by geneexpression profiling analysis, and found that the TGF-b and BMPcascade is among the top differentially regulated processes(supplementary material Table S1). As TGF-b and BMPs are keynegative and positive signals controlling brown fat formation,
respectively (Fournier et al., 2012; Koncarevic et al., 2012; Tsenget al., 2008; Yadav et al., 2011), we postulated that Bmal1 mightalter the activities of these pathways to affect brown adipocyte
differentiation. We thus determined TGF-b signaling activitiesin Bmal1 loss- and gain-of-function cells using the TGF-b-responsive luciferase reporters 36TP-Luc (Wrana et al., 1992)and SBE4-Luc (Zhou et al., 1998). As demonstrated in Fig. 6A,B,whereas TGF-b treatment in normal control cells leads to robust15-fold (3XTP-Luc) or 32-fold (SBE4-Luc) induction of reporteractivities, depletion of Bmal1 profoundly compromised TGF-bsignal transduction to ,10% of that of controls. In contrast,forced expression of Bmal1 significantly increased TGF-breporter activity by ,67% under TGF-b-stimulated conditions.
Fig. 5. Forced expression of Bmal1inhibits HIB1B brown preadipocyteterminal differentiation.(A) Immunoblot analysis of Bmal1protein expression in HIB-1B cells withBmal1 stable overexpression (BMcDNA) or empty vector control(pcDNA3). (B) The effect of Bmal1overexpression on HIB-1Bdifferentiation as shown by BODIPYand Mitotracker staining at day 3 ofdifferentiation. (C) RT-qPCR analysisof brown adipocyte marker geneexpression of day-3 differentiated HIB-1B cells expressing pcDNA3 or BMcDNA (n53). Data show themean6s.e.m. *P,0.05; **P,0.01 (BMcDNA versus pcDNA3).
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The TGF-b signaling defect in Bmal1-deficient cells was furthercorroborated by blunted phosphorylation of Smad3, the ultimate
signal transducer of the TGF-b pathway (Massagué and Wotton,2000), upon either TGF-b1 or BMP7 stimulation (Fig. 6C).Interestingly, the total amount of Smad3 protein was also reducedin Bmal1-knockdown cells as compared to controls, likely
reflecting Bmal1 transcriptional regulation of Smad3 assuggested by microarray analysis (supplementary material TableS1). In contrast, Smad2 and its ligand-activated phosphorylation
are not altered, suggesting potential specificity of Bmal1-regulated targets in the TGF-b signaling pathway. Surprisingly,Bmal1 deficiency also markedly increases the activity of the
canonical BMP signaling pathway, as indicated by much higherSmad1 and Smad5 phosphorylation at basal and ligand-stimulatedstate as compared to shSC-treated cells (Fig. 6D). In contrast, the
p38 MAP kinase (MAPK11–MAPK14) branch of the BMPsignaling is not affected by loss of Bmal1 function. Additionalassessment of BMP activity by using a well-characterized BMP-responsive luciferase reporter, BRE2-Luc (Korchynskyi and ten
Dijke, 2002), further confirmed the robustly augmented BMPsignaling under basal and BMP-stimulated conditions in Bmal1-depleted brown preadipocytes (Fig. 6E).
The finding of attenuated TGF-b pathway activity togetherwith augmented BMP signaling in Bmal1-deficient cells suggeststhat these mechanisms might contribute to the enhanced
differentiation observed in these cells. Therefore, we testedwhether activation of TGF-b or blockade of BMP signalingcan rescue (i.e. suppress) the enhanced brown adipogenicphenotype of Bmal1-deficient cells. As shown in Fig. 7A, TGF-b1 treatment of normal brown preadipocytes effectively inhibitedtheir adipogenic differentiation as indicated by weaker lipid
(BODIPY) and mitochondrial (MitoTracker) staining relative tovehicle-treated controls, as expected. Notably, TGF-b1 alsoblocked the differentiation of Bmal1-deficient cells as efficiently
as for the controls. Consistent with these findings onmorphological differentiation, TGF-b1 robustly suppressed theinduction of brown-adipocyte-specific marker genes, including
Ucp-1, Dio2 and Prdm16, in shBmal1 cells to the same degree asseen in the shSC cells (Fig. 7B), suggesting that activation of theTGF-b1 pathway can rescue the effect of Bmal1 depletion onbrown adipogenesis. We found that TGF-b signaling activities ofBmal1-deficient cells were severely impaired as compared toshSC-treated controls, although TGF-b1 can still induce weakactivation of 36TP-Luc (twofold), SBE4-Luc (sixfold) andSmad3 phosphorylation compared to the non-stimulatedcondition (Fig. 6A–C). So it is possible that TGF-b1-inducedsignaling in Bmal1-knockdown cells, although significantly
diminished, is sufficient to suppress adipogenic differentiation.Furthermore, blockade of the BMP signaling cascade by noggin, aspecific BMP inhibitor, inhibits the induction of brown adipocyte
Fig. 6. Bmal1 regulates the signaling activitiesof TGF-b and BMP cascades. (A,B) The effect ofBmal1 silencing and forced expression on TGF-bsignaling as assessed by using the TGF-b-responsive luciferase reporters, 36TP-Luc (A) andSBE4-Luc (B), under basal or TGF-b-stimulatedconditions in C3H10T1/2 cells (n54). Ctr, control.##P,0.01 under basal conditions; **P,0.01 underTGF-b-treated conditions (Student’s t-test).(C) TGF-b signaling activity as assessed by Smad2and Smad3 phosphorylation under basal conditionsor in response to TGF-b1 or BMP7 ligand treatmentin shSC and shBmal1 cells. (D) BMP signaling asassessed by Smad1/5 and p38 phosphorylationunder basal conditions or in response to TGF-b1 orBMP7 ligand treatment in shSC and shBmal1 cells.(E) BMP signaling as assessed by a BMP-responsive luciferase reporter, BRE2-Luc, underbasal or BMP4-stimulated conditions (n54). RLU,relative luciferase units; Ctrl, control. ##P,0.01under basal conditions; **P,0.01 under BMP4-treated conditions (Student’s t-test). All quantitativedata show the mean6s.e.m.
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markers in Bmal1-deficient cells similarly to the TGF-b1treatment, suggesting that inhibiting BMP pathway activation isalso sufficient to abrogate enhanced differentiation induced byBmal1 depletion. Interestingly, the combined effects of TGF-b1and noggin on suppressing the induction of brown adipocytemarker genes are similar to those of TGF-b1 or noggin alone,suggesting either a saturated response to TGF-b1 or noggin alonein these cells, or that these pathways are interdependent throughpotential crosstalk. The regulation by TGF-b1 and noggin ofadipogenic genes in Bmal1-deficient cells is distinct from that ofthe brown-adipocyte-specific genes, as demonstrated in Fig. 7C.
The downregulation of Cebpa in response to these agents occursto the same extent as that seen for brown-adipocyte-specificmarkers. In contrast, Pparg is suppressed by TGF-b1 but notnoggin in Bmal1-deficient cells, whereas Cebpb displaysincreased expression in the presence of these ligands, possiblyreflecting differential ligand sensitivities of these genes in brown
adipocytes. Taken together, these results indicate that Bmal1-mediated regulation of TGF-b as well as BMP pathwayscontributes to its function in modulating brown adipogenesis.
Bmal1 exerts direct transcriptional control of TGF-b pathwaygenes and confers circadian regulationBmal1 is an essential transcriptional activator of the core clock
regulatory loop, and has been implicated in transcriptional controlof developmental signaling pathways including the TGF-b or
BMP cascades (Janich et al., 2011). Therefore, prompted by
findings from the microarray study, we examined whethercomponents of the TGF-b or BMP pathways are directtranscriptional targets of Bmal1. Through computational
screening of putative Bmal1-binding sites, the canonical E-boxelements (59-CACGTG-39) (Rey et al., 2011), in the proximalpromoter regions (22 kb+first intron) of key genes in thesepathways, we identified a number of TGF-b pathway genesharboring the E-boxes, including the ligands Tgfb1 and Tgfb2, thereceptor Tgfbr2 and the signal transducer Smad3 (supplementarymaterial Table S2). However, our computational analysis failed to
identify E-box elements among the genes of the BMP pathwayexamined, including BMPs, Smad1 and Smad5.
To determine Bmal1 occupancy of these potential binding sites
and its potential circadian time-dependent association with targetpromoters, we performed chromatin immunoprecipitation (ChIP)analysis in HIB1B cells under serum shock conditions
(Balsalobre et al., 1998), an established method to elicit cell-intrinsic clock oscillation and synchronize circadian generhythmicity (Guo et al., 2012; Janich et al., 2011). As shown inFig. 8A, robust recruitment of Bmal1 to a known target promoter,
Rev-erba, is detected at circadian time (CT)8 upon serum shock,whereas this activity is nearly absent at CT20. This rhythmicpromoter occupancy by Bmal1 coincides with its protein peak
observed at CT8 and trough at CT20 (Fig. 8B). Bmal1enrichment on identified sites of Tgfb1 and Smad3 promoters
Fig. 7. TGF pathway activation orBMP pathway blockade suppressesenhanced adipogenesis of Bmal1-deficient brown preadipocytes.(A) Representative images of lipid(BODIPY) and mitochondrial(Mitotracker) staining of day-3-differentiated shSC- and shBmal1-expressing HIB-1B cells in theabsence or presence of TGF-b1treatment. Ctr, control. Scale bars:50 mm. (B,C) RT-qPCR analysis ofTGF-b1, noggin or TGF-b1 plusNoggin (T+N) treatment on brown-adipocyte-specific gene expression(B) or adipogenic gene expression(C) of day-3-differentiated shSC andshBmal1 cells. Cells were treated withTGF-b1 (2 ng/ml) or noggin (100 ng/ml) for 8 hours prior to the induction ofdifferentiation and treatment wasmaintained throughout differentiation(n53). Data show the mean6s.e.m.#P,0.05; ##P,0.01 (treatment versusnon-treated control); *P,0.01;**P,0.05 (shBmal1 versus shSC).
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occurred in a circadian-dependent manner similar to that
occurring at Rev-erba, with stronger chromatin associationoccurring at CT8 (Fig. 8A). In contrast, other candidate targetpromoters, Tgfb2 and Tgfbr2, exhibited only modestly enriched
Bmal1 association. Notably, serum shock in HIB1B cells elicitsoscillation of Smad3 protein with a rhythmic profile similar tothat of Bmal1 (Fig. 8B), suggesting an intrinsic temporal
regulation of the key signaling transducer of the TGF-bcascade. Likely due to direct Bmal1-mediated regulation,silencing of Bmal1 blunted (Fig. 8C), whereas its forcedexpression augmented the expression of the identified target
genes in the TGF-b pathway – Tgfb1, Tgfb2, Tgfbr2 and Smad3(Fig. 8D). In line with the results from our initial screeningindicating the absence of potential Bmal1-response elements on
BMP signaling genes, mRNA expression of Smad1 and Smad5was not affected by Bmal1 knockdown (Fig. 8E). However, thecanonical BMP signaling targets Id1 and Id2 were markedly
upregulated in Bmal1-deficient cells, additional evidence foraugmented BMP activity in these cells as revealed by the findingsof Smad5 phosphorylation and BMP reporter activity discussedabove. Given that intracellular BMP signal transduction is
subjected to negative-feedback regulation (Kavsak et al., 2000;Kawamura et al., 2012), we further analyzed whether loss ofBmal1 could affect negative regulatory mechanisms involved in
the BMP signaling pathway to affect its activity. Interestingly, asdemonstrated by expression analysis of BMP pathway inhibitory
molecules (Fig. 8F), the levels of Smad6, SnoN (also known as
Skil) and Ski transcripts are significantly reduced in Bmal1-deficient cells. This finding of inhibition of the expression ofBMP negative regulators with loss of Bmal1 suggests that a relief
of inhibition of the BMP pathway might account for the enhancedBMP activity in these cells.
DISCUSSIONThe unique energy-dissipating function of brown adipose tissueand its presence in humans holds promise as a potentialtherapeutic target against obesity (Cypess et al., 2009; van
Marken Lichtenbelt et al., 2009; Virtanen et al., 2009).Accumulating evidence suggests that disruption of the clockmechanism leads to various metabolic abnormalities, particularly
obesity and insulin resistance (Pan et al., 2011; Roenneberg et al.,2012; Sahar and Sassone-Corsi, 2012). Although diverse aspectsof metabolism are subjected to circadian regulation (Bass and
Takahashi, 2010; Green et al., 2008), whether the adipocyte-intrinsic timekeeping mechanism participates in brown fat celldevelopment is not known. Our study demonstrates that theessential transcription activator of the molecular clock, Bmal1,
exerts direct temporal control of the TGF-b pathway, a keyinhibitory signal of brown fat formation (Fournier et al., 2012;Koncarevic et al., 2012; Yadav et al., 2011). Together with
reciprocally altered BMP activity, these developmental signalingmechanisms driven by Bmal1 modulate brown adipogenesis and
Fig. 8. Bmal1 exerts direct transcriptionalcontrol on genes of the TGF-b pathway.(A) Chromatin immunoprecipitation qPCR (ChIP-qPCR) analysis of Bmal1 occupancy on identifiedTGF-b pathway gene promoters at 8 and20 hours after serum shock in HIB-1B cells.Bmal1 occupancy of the Rev-erba promoter E-box (a known target) is included as a positivecontrol and that of Tbp as a negative control.Values are presented as the fold enrichment ofthe percentage of total input over IgG control(n54). CT, circadian time, with time immediatelyafter serum shock taken as CT0. *P,0.05;**P,0.01 (CT8 versus CT20). (B) Immunoblotanalysis of Bmal1 and Smad3 protein oscillationinduced by serum shock from CT8 to 32.(C–F) RT-qPCR gene expression analysis ofcomponents of TGF-b and BMP pathways inBmal1-knockdown (C,E,F), or Bmal1-overexpressing HIB-1B cells (D). n53. Allquantitative data show the mean6s.e.m.*P,0.05; **P,0.01 (shBmal1 versus shSC, orBM cDNA versus pcDNA3).
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consequently affect adaptive thermogenesis in vivo. As crucialnegative and positive signals involved in brown adipogenesis,
respectively (Fournier et al., 2012; Koncarevic et al., 2012; Kuoet al., 2014; Qian et al., 2013; Tseng et al., 2008; Yadav et al.,2011), our finding of circadian regulation of TGF-b and BMPactivities implicates a temporal regulatory control of adipocyte
lineage commitment and differentiation.The TGF-b and BMP pathways are known to suppress
(Fournier et al., 2012; Koncarevic et al., 2012; Yadav et al.,
2011) and drive (Kuo et al., 2014; Qian et al., 2013; Tseng et al.,2008) brown adipocyte differentiation, respectively. BMP7promotes both early commitment of mesenchymal precursors to
the brown adipocyte lineage (Tseng et al., 2008) and subsequentdifferentiation (Cao et al., 2004; Sellayah et al., 2011). Incontrast, TGF-b signaling exerts inhibitory effects on BATdevelopment, with ablation of Smad3, the ultimate signaltransducer of the TGF-b pathway, resulting in selectiveinduction of brown-adipocyte-specific marker genes andenhanced mitochondrial function (Yadav et al., 2011). In
addition, inhibition of activin receptor IIB and myostatin, bothsignaling through Smad3 (Fournier et al., 2012; Kim et al., 2012;Koncarevic et al., 2012; Zhang et al., 2012), were recently
reported to promote brown adipogenesis. We have previouslyshown that Bmal1 suppresses white adipocyte differentiation(Guo et al., 2012). The current finding of a Bmal1 inhibitory
effect on brown adipogenesis is in parallel with its role in whiteadipocyte formation, although the molecular pathways mediatingthese effects are distinct. We found that Bmal1 modulates Wnt
signaling in white adipose tissue (Guo et al., 2012), whereas inbrown fat, this core clock regulator exerts transcriptional controlon the TGF-b pathway. Interestingly, recent studies indicate thatthe circadian clock can act on various signaling cascades, including
the TGF-b and BMP pathways, to modulate epidermal stem cellactivation in accordance with environmental stimulatory cues(Janich et al., 2011; Karpowicz et al., 2013). These findings, in
aggregate, likely reflect the tissue specificity and the diverse arrayof developmental signals involved in transmitting a circadiantiming cue to fine-tune tissue homeostasis.
Findings of the direct association of Bmal1 with the promotersof key components of the TGF-b pathway, such as Tgfbr2 andSmad3, indicate that the TGF-b signaling cascade could be undera coordinated circadian control in brown preadipocytes. Likely
owing to this circadian element present in the Smad3 promoter, arobust rhythmic oscillation of Smad3 protein in phase with Bmal1is elicited upon serum shock. In addition, we found that Bmal1
deficiency leads to significantly attenuated Smad3 activation andreporter activity, indicating that TGF-b signaling transductioncould be subjected to temporal regulation as well. Indeed,
circadian expression of Smad3 was detected in human gingivalfibroblasts, mesenchymal stem cells and mouse liver (Sato et al.,2012). Furthermore, in epidermal stem cells (Janich et al., 2011;
Karpowicz et al., 2013) and the central clock (the suprachiasmaticnuclei; Beynon et al., 2009), a circadian pattern of TGF-bsignaling activity as indicated by Smad3 phosphorylation hasbeen reported. Given the importance of TGF-b signaling in stemcell proliferation and differentiation (Gaarenstroom and Hill,2014), circadian modulation of this pathway might conferappropriate responses to temporal cues involved in controlling
stem cell behavior and tissue homeostasis. Although our currentstudy defines a specific role of Bmal1 in the brown fatdifferentiation program, its broader impact on various important
TGF-b-regulated biological processes remains to be elucidated.
An interesting observation from our study is the reciprocallyaltered BMP signaling with altered TGF-b pathway activity inBmal1 loss- or gain-of-function studies. Based on several lines ofevidence, changes in BMP activity might have occurredsecondary to the direct regulatory effect of Bmal1 on TGF-bpathway. First, we failed to identify canonical E-box elements in
genes directly involved in BMP signal transduction throughextensive promoter screening, although it is possible that therecould be non-canonical elements or additional genes not
screened. Secondly, searches in available Bmal1 ChIP-Seqdatasets (Cho et al., 2012; Koike et al., 2012; Rey et al., 2011)for binding peaks among BMP pathway genes also yield negative
findings. Most importantly, a mutually antagonistic relationshipbetween TGF-b and BMP signal transduction has been well-characterized (Shi and Massagué, 2003), and loss of endogenous
TGF-b signaling can augment BMP activity. Ski (Ehnert et al.,2012; Wang et al., 2000) and SnoN (Kawamura et al., 2012) areknown BMP inhibitory molecules that can be induced by TGF-bto mediate the antagonism between these signaling events,
whereas Smad6 is a ubiquitin ligase specific for BMP-activatedSmad1/5/8 degradation. In Bmal1-deficient cells, as theattenuated TGF-b activity is accompanied by downregulation ofSnoN, Ski and Smad6, a relief of these inhibitory mechanismsmight have contributed to enhanced BMP signaling transduction.Moreover, we observed that TGF-b alone, or BMP blockade, issufficient to suppress adipogenic induction in these cells (Fig. 7),but these effects are not additive, further suggesting potentialinterdependence of the two pathways. These observations support
a model that direct transcriptional control of the TGF-b pathwayby Bmal1 might consequently alter BMP signaling, and togetherthey exert concerted actions to drive brown adipogenesis.
Despite the established link between circadian disruption and
metabolic abnormalities such as obesity and insulin resistance(Roenneberg et al., 2012; Sahar and Sassone-Corsi, 2012), theprecise molecular mechanisms responsible for these effects are
yet to be defined. As our study demonstrates, circadian-controlledbrown fat thermogenic capacity might contribute to thisphenomenon. Two recent reports indicate that circadian
regulators Rev-erba (Gerhart-Hines et al., 2013) and Per2(Chappuis et al., 2013) both contribute to the regulation ofbrown adipose tissue function. In particular, our findings are inagreement with the study by Chappuis et al., as mice lacking
Per2, a repressor of Bmal1, display sensitivity to cold, whereasBmal1 ablation leads to resistance to cold. Based on the opposingregulation of Per2 and Bmal1 in the core clock circuit, our current
study, together with these previous reports, suggests theparticipation of a coordinated temporal control mechanism inmodulating thermogenic capacity. Whereas previous studies
mainly focus on clock genes in BAT function, our studyaddresses an additional layer of temporal control in the brownfat concerning brown adipocyte differentiation. Nonetheless,
specific strategies, such as circadian shift regimens, will beneeded in the future to assess the direct impact of altered clockregulation on BAT function and its contribution to metabolicdiseases. Given that certain clock regulators, such as Rev-erba,are amenable to pharmacological manipulation by syntheticligands (Solt et al., 2012), the clock circuit represents a potentialtarget for therapeutic interventions.
Compared to our extensive knowledge of white adipocytedevelopment, the current understanding of regulatorymechanisms governing BAT formation is only emerging. Our
study elucidates a previously unappreciated temporal regulatory
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mechanism involved in brown adipocyte development thatultimately contributes to fine-tuning of adaptive thermogenesis.
Future efforts to determine how this mechanism affectssystemic metabolic homeostasis might lead to the discovery ofnew therapies against widespread circadian-disruption-inducedmetabolic disorders.
MATERIALS AND METHODSAnimalsAnimals were maintained in the Methodist Hospital Research Institute
mice facility under a constant 12:12 light-dark cycle, with light on at 7:00
(ZT0). Room temperature was controlled at 22 C̊. All experimental
protocols were approved by the IACUC animal care research committee
of the Houston Methodist Research Institute, and carried out in
accordance with the NIH Guidelines for the Care and Use of
Laboratory Animals. Bmal12/2, Bmal1fl/fl and ap2-Cre transgenic mice
were obtained from the Jackson Laboratory (Storch et al., 2007). Mice
were fed ad lib on standard chow diet (AIN-76A, Research Diets) with
free access to water. Tissue samples were obtained at ambient
temperature, unless indicated otherwise, and at the same time of the
day to ensure reproducible circadian gene expression.
Cold-tolerance test12-week-old mice were placed in individual cages with free access to
water without food or sedation. Rectal temperature was measured with a
probe connected to a high-precision thermometer. Body temperature was
measured twice at 09.00 before the mice were subjected to 4 C̊ for
24 hours, with temperature monitored at the indicated times.
Cell cultureC3H10T1/2 and HIB1B cell lines were obtained from ATCC and
maintained in 10% FBS DMEM. Stable cell lines were constructed and
selected as described previously (Guo et al., 2012) using shRNA
constructs from Open Biosystems. C3H10T1/2 differentiation to brown
adipocytes was conducted as described previously (Tseng et al., 2008)
but without BMP7 pretreatment. Briefly, cells at confluency were
cultured in brown induction medium containing insulin (20 nM), T3
(1 nM), isobutylmethylxanthine (0.5 mM), dexamethasone (5 mM) and
rosiglitazone (1 mg/ml) for 3 days. Cells were then switched to
maintenance medium supplemented with insulin and T3 only for
9 days. The fully differentiated brown adipocyte phenotype with
significant lipid accumulation and Ucp-1 expression occurs at 9 days
(D9) after induction. Differentiation of HIB-1B cells and primary brown
adipocytes was induced using the same induction medium for 2 days and
maintenance medium for 2–4 days.
Primary brown adipocyte and preadipocyte isolationand immortalizationPrimary brown adipocytes and preadipocytes were isolated from the
interscapular brown adipose tissue pad of 6-week-old mice as described
previously (Timmons et al., 2007). Briefly, tissues were digested with
type I collagenase in the presence of 1% BSA at 37 C̊ for 30 minutes.
The suspension was filtered and centrifuged, and the top fat layer was
collected as adipocytes and the pellet containing the stromal vascular
fraction was resuspended and plated. Preadipocytes were passaged once
prior to adipogenic differentiation. Immortalization of isolated primary
preadipocytes was performed using retroviral SV-40 Large T antigen
transformation and puromycin selection as described previously (Tseng
et al., 2008).
Serum shock synchronization of the cellular clockSerum shock to synchronize cells in culture was performed as described
previously (Guo et al., 2012). Briefly, confluent cultures were incubated
in serum-free medium overnight and subjected to 20% FBS serum shock
for 1 hour. The medium was then removed and replaced with 10% serum
normal culture medium. This was considered circadian time (CT) 0, and
samples were collected at the indicated times after serum shock.
RNA extraction and quantitative reverse-transcriptasePCR analysisRNeasy miniprep kits (Qiagen) were used to isolate total RNA from
snap-frozen tissues or cells. Tissue samples were collected at the times
indicated, and cell samples were obtained under non-synchronized
normal culture conditions. cDNA was generated using q-Script cDNA
Supermix kit (Quanta Biosciences), and quantitative PCR was performed
using a Roche 480 Light Cycler with Perfecta SYBR Green Supermix
(Quanta Biosciences) as described previously (Guo et al., 2012). Relative
expression levels were determined using the comparative Ct method to
normalize target genes to the 36B4 (also known as Rplp0) internal
control, and compared to experimental controls as indicated.
Immunoblot analysisTotal protein (40–50 mg) was used for the analysis as describedpreviously (Chatterjee et al., 2013). Smad2, Smad3 or Smad1/5 and
p38 phosphorylation were assessed at 1 hour after the indicated ligand
treatment (TGF-b1, 10 ng/ml; BMP7 100 ng/ml). The primary antibodiesthat were used are listed in supplementary material Table S4.
ChIP-qPCR analysisImmunoprecipitation was performed using Bmal1 (AB93806) or
control rabbit IgG plus Protein A/G beads as described previously
(Chatterjee et al., 2013). Briefly, cells were fixed with formaldehyde,
lysed and sonicated to shear the chromatin. The immunoprecipitated
chromatin fragments were eluted, treated with proteinase K and
purified using the Qiaquick PCR purification kit (Qiagen). Real-time
PCR using Perfecta SYBR Green Supermix (Quanta Biosciences) was
carried out with an equal volume (4 ml) of each reaction with specificprimers. Negative control primers for TBP were also included. The
primer sequences used are listed in supplementary material Table S3.
Values are expressed as the fold enrichment of the percentage of input
normalized to IgG control.
Oil Red O, BODIPY and Mitotracker stainingOil Red O staining was carried out in fixed cells using 0.5% Oil Red O
for 1 hour as described previously (Guo et al., 2012). BODIPY staining
(Molecular Probes, Carlsbad, CA) was carried out at a concentration of
1 mg/ml for 30 minutes. Mitotracker (100 nM, Molecular Probes)
staining was performed according to the manufacturer’s protocol and
applied to cells in culture at 37 C̊ for 30 minutes before fixation.
Microscopy images were captured on a Nikon 80i microscope with a
color camera and processed using Nikon NIS Elements acqusition
software. BODIPY and Mitotracker images were captured using
excitation/emission wavelength at 590-650/700 and 465-495/535 nm,
respectively using appropriate exposure times.
Luciferase reporter assaysCells were grown to 80% confluency and transiently transfected using
FuGENE 6 (Roche) in four replicates as described previously (Beynon
et al., 2009). TGF-b1 (2 ng/ml) or BMP4 (100 ng/ml) were added16 hours after transfection and luciferase activity was measured using the
Dual-Glo luciferase assay system (Promega) 24 hours following ligand
treatment. Reporter luciferase values were normalized to Renilla readings
and expressed as the fold induction over controls. TGF-b luciferasereporters, 36TP-Luc (Wrana et al., 1992) and SBE4-Luc (Zhou et al.,1998) were obtained from Addgene, and the BRE2-Luc reporter
(Korchynskyi and ten Dijke, 2002) was a kind gift from Dr Peter Ten
Dijke (Leiden University Medical Center, The Netherlands).
Statistical analysisData are expressed as the mean6s.e.m. Differences between groups inthe cold-tolerance tests were analyzed by one-way analysis of variance
(ANOVA). Statistical differences of other experiments were assessed by
using Student’s t-test.
Competing interestsThe authors declare no competing or financial interests.
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Author contributionsK.M. and D.N. designed and performed the experiments, analyzed the data andwrote the manuscript. B.G., S.C., R.L. and H.Y. performed experiments onadipocytes, M.C. performed experiments on brown fat development, Z.Z. andD.N. carried out circadian actogram analysis.
FundingWe thank The Houston Methodist Research Institute (HMRI) for start-up supportand the Center for Diabetes Research for technical assistance. This project issupported by the American Heart Association [grant number 12SDG12080076];and the American Diabetes Association [grant numbers 1-13-BS-118 to K.M. and7-12-BS-210]; and the National Institutes of Health [grant number DK097160-01]to V.Y. Deposited in PMC for release after 12 months.
Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.167643/-/DC1
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