The adipocyte clock controls brown adipogenesis through the TGF-β and BMP signaling pathways.

© 2015. Published by The Company of Biologists Ltd.

The adipocyte clock controls brown adipogenesis via TGF-β/BMP signaling pathway Deokhwa Nam1, Bingyan Guo2, Somik Chatterjee1, Miao-Hsueh Chen3, David Nelson4, Vijay K. Yechoor5, Ke Ma1* 1

Center for Diabetes Research, Department of Medicine, The Methodist Hospital Research Institute, Houston, TX, 77030 2

Department of Cardiovascular Medicine, Second Affiliated Hospital, Hebei Medical University, Shijiazhuang, 050017, Hebei, China.

Journal of Cell Science

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3

Department of Pediatrics, Children’s Nutrition Research Center, 4Department of Molecular and Human Genetics, 5Diabetes and Endocrinology Research Center, Department of Medicine, Baylor College of Medicine, Houston, TX, 77030

Running Title: Bmal1 regulates brown adipogenesis

* To whom correspondence should be addressed: E-mail: [email protected] Phone: (713) 441 5084 Fax: (713) 793 7162

Key Words: brown adipocyte differentiation, circadian rhythm, adipogenesis, obesity, TGF-β signaling pathway

1 JCS Advance Online Article. Posted on 6 March 2015

Abstract The molecular clock is intimately linked with 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, the essential clock transcription activator, inhibits brown adipogenesis to adversely impact brown fat formation and thermogenic capacity. Global ablation of Bmal1 in mice increases brown fat mass and cold tolerance, while adipocyte-selective inactivation of Bmal1 recapitulates these effects

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of function studies in mesenchymal precursors and committed brown progenitors reveal that


and demonstrates its cell-autonomous role in brown adipocyte formation. Further loss- and gain-

formation and thermogenic regulation, which may be targeted therapeutically to combat obesity.

Bmal1

inhibits

brown

adipocyte

lineage

commitment

and

terminal

differentiation.

Mechanistically, Bmal1 inhibits brown adipogenesis through direct transcriptional control of key components of the TGF-β pathway together with reciprocally altered BMP signaling, and activation of TGF-β, 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 impact brown fat

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Introduction The 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 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


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machinery functions in the metabolically active brown adipose tissue (BAT), a specialized thermogenic organ (Cannon and Nedergaard, 2004), is largely unknown. In both central clock , the suprachiasmatic nuclei (SCN), and peripheral tissues, the ∼24hr daily rhythm is driven by an intricate molecular feedback loop consisting of positive and negative regulators coupled with post-transcriptional control (Dibner et al.). Brain and muscle Arnt like 1(Bmal1), 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 (CACGTG) as a heterodimer with CLOCK (Circadian Locomotor Output Cycles Kaput) and elicits transcription of the negative regulators, Periods (Per1, 2 and 3) and Cryptochromes (Cry1 and 2). These factors in turn inhibit the transcriptional activity of Bmal1/CLOCK, which coupled with temporally-controlled proteosome-mediated degradation mechanisms, leads to rhythmic oscillation of the molecular clock and thus generates the daily rhythm. Interestingly, despite studies demonstrate 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 adipocyte 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 critical role in 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., 3

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 may have therapeutic potentials in treating obesity (Nedergaard and Cannon, 2010). Due to its distinct function, formation of mature brown adipocyte, 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


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although it originates from the mesoderm similarly as 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. We recently demonstrated that the essential core clock gene, Bmal1, has a novel function in suppressing adipogenesis and WAT formation (Guo et al., 2012). On the other hand, it potently promotes myogenic progression in skeletal muscle (Chatterjee et al., 2013). As BAT shares a common mesodermal origin with WAT and skeletal muscle, in the current study, we investigated whether Bmal1 participates in brown adipocyte development. Employing various genetic approaches in animal models and cellular systems, our study uncovers the adipocyte-intrinsic Bmal1 function in suppressing brown adipocyte lineage commitment and differentiation, which consequently impacts brown fat thermogenic capacity. Results Ablation of Bmal1 promotes brown fat formation and thermogenesis in vivo We first investigated the role of Bmal1 in BAT formation in the global Bmal1-null (Bmal1-/-) mouse model (Bunger et al., 2000). Ablation of Bmal1 leads to a ∼30% increase in the amount of BAT as compared to wild-type (WT) littermate controls, as assessed by fat pad weight (Fig. 1A) or its ratio to total body weight (Fig. 1B). In Bmal1-/- BAT, mRNA expression of Bmal1 and its direct target gene in the molecular clock, Rev-erbα, are nearly absent (Fig. 1C), as expected. Notably, the absence of Bmal1 leads to marked 1.5-2-fold induction of Ucp-1, Cebpβ and Pparγ transcripts, indicating potentially increased thermogenesis and adipogenesis in BAT. In line with this finding, neonatal (P5) Bmal1-/- mice BAT or after 2-month high-fat diet-feeding exhibit stronger cytoplasmic staining typical of high mitochondrial density with less lipid accumulation 4

as compared to WT (Fig. 1D). Direct assessment of thermogenic response by cold tolerance test reveals that lack of Bmal1 significantly improves the resistance to cold, as Bmal1-/- mice maintains higher core body temperature than the WT controls at 40C (Fig. 1E). 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 adipocytespecific deleter, the Fabp4 (ap2)-Cre transgenic mice (He et al., 2003). Mice with selective adipocyte Bmal1 deletion, the ap2-Cre+/BMfl/fl, display near complete absence of Bmal1 protein


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in BAT (Fig. 1F). RT-qPCR analysis reveals ~80% reduction of Bmal1 mRNA in mature brown adipocytes, but not preadipocytes, along with down-regulation of Bmal1 target genes, Rev-erbα and Dbp (D-element binding protein, Fig. S1A). Similarly reduced Bmal1 expression is detected in white adipocytes (Fig. S1B) in these mice, likely due to ap2-Cre activity in WAT (He et al., 2003). As indicated by intact Bmal1 immunostaining in suprachiamatic nuclei of ap2Cre+/BMfl/fl mice (Fig. S1C), adipocyte-selective ablation of Bmal1 does not affect its protein expression in the central clock. Furthermore, the central clock function is preserved in these mice as compared to control ap2-Cre-/BMfl/fl littermates, since they display normal circadian behavioral rhythms as assessed by wheel-running actograms (Fig. S1D). When subjected to 40C challenge, ap2-Cre+/BMfl/fl mice exhibit enhanced tolerance to cold as compared to ap2-Cre/BMfl/fl littermates (Fig. 2G). This similarly improved thermogenic response as seen in the Bmal1/-

mice suggests that this effect is likely attributable to loss of Bmal1 function in brown

adipocytes. In line with previous observation in Bmal1-/- mice (Guo et al., 2012), the body weight (Fig. S2A) and brown adipose weight (Fig. S2C) of ap2-Cre+/BMfl/fl mice are increased as compared to the controls without cold challenge. Interestingly, 40C cold for 8 hours induced a greater weight loss in ap2-Cre+/BMfl/fl mice (Fig. S2B), suggesting higher thermogenic energy expenditure under this condition. The enhanced thermogenic ability in ap2-Cre+/BMfl/fl mice is accompanied by substantially elevated UCP-1 protein in BAT both at the basal state under ambient temperature, or 40C cold (Fig. 1H). Gene expression analysis under these conditions reveals a significant induction of the brown thermogenic program, including Prdm16, Dio2 (Deiodinase 2) and Pgc1α ap2-Cre+/BMfl/fl mice (Fig. 1I). Collectively, these findings indicate that Bmal1 inhibits brown fat formation and its thermogenic function,

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Absence of Bmal1 promote primary brown adipocyte differentiation To directly examine test the function of Bmal1 in brown adipogenesis, we isolated brown primary preadipocytes from WT and 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 display enhanced differentiation to mature brown adipocytes as compared to WT (Fig. 2A). Furthermore, not only expression of mature brown adipocyte markers (Ucp-1, Dio2) are markedly induced in Bmal1-null cells, the early brown lineage markers Prdm16 and Myf5 are robustly up-regulated as well (Fig. 2B).

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Furthermore, similar to these findings, primary brown preadipocytes from ap2-Cre+/BMfl/fl mice display enhanced brown adipogenesis upon induction, as compared to ap2-Cre-/BMfl/fl control cells (Fig. 2C). This effect is accompanied with robustly induction of brown-specific (Ucp-1) and adipogenic markers (Pparγ, Fabp4), as well as mitochondrial genes (Pgc1α, CytC1, Cox7a1) at day 1 and day 5 through the differentiation time course (Fig. 2D). Interestingly, the early brown lineage markers, Prdm16, Myf5 and Cebpβ, are not altered, likely due to the nature of ap2-Cre mediated Bmal1 deletion in these cells. Markedly elevated Ucp-1 expression along with


induction of the brown adipogenic gene program is found in ap2-Cre+/BMfl/fl BAT as well (Fig. S1E). Taken together, these findings indicate that Bmal1 inhibits brown fat formation and thermogenic function, and suggest these effects could be mediated its cell-autonomous action in suppressing brown adipogenesis.

Bmal1 inhibits brown adipocyte terminal differentiation and lineage specification Brown adipocytes arise from mesenchymal precursors that undergo brown lineage commitment and subsequent terminal differentiation (Gesta et al., 2007). To directly test cellautonomous function of Bmal1 in these distinct stages of brown adipogenesis, we created stable cell lines with genetic gain- or loss-of-functions of Bmal1 in mesenchymal precursor cells, C3H10T1/2 (10T1/2), and brown preadipocytes, HIB1B. Analysis of Bmal1 protein expression reveals that it is highly abundant in brown adipocyte cell lines than white preadipocytes, the 3T3L1, at levels comparable to that of primary myoblasts (Fig. 3D). Silencing of Bmal1 by an shRNA construct in 10T1/2 mesenchymal precursors reduced its protein level to ∼30% of that of scrambled control (SC), similar as we reported previously (Guo et al., 2012). Under a specific brown adipogenic condition that induces 10T1/2 lineage determination and differentiation into 6

mature brown adipocyte (Tseng et al., 2008), stable knockdown of Bmal1 results in an increase of cells converting to mature brown adipocytes with rounded morphology and stronger lipid staining than that of the SC, as shown in Fig. 3B by Bodipy and phase-contrast images. Oil-redO staining in day 10-differentiated shSC and shBmal1 cells further confirms these findings (Fig. S2). Moreover, MitoTracker Red staining of functional mitochondria in live cells is substantially increased in Bmal1-deficient cells than that of the shSC, indicating increased mitochondrial abundance associated with differentiation. Gene expression analysis at both early (day 1) and late (day 9) stages of differentiation demonstrates a substantially augmented brown adipogenic

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genes, Myf5 and Prdm16, and adipogenic genes, Cebpβ and Pparγ (Fig. 3C).


program with marked up-regulation of brown-specific marker Ucp-1, early brown progenitor

brown adipocytes (Fig. 4B). Analysis of brown-specific and adipogenic gene expression in the

Next, we examined the role of Bmal1 in brown adipocyte terminal differentiation using committed brown preadipocyte line HIB1B. Stable knockdown of Bmal1 (shBmal1) in these cells largely depletes Bmal1 protein as compared to scrambled controls (Fig. 4A). Assessment of mature differentiation of these cells through lipid accumulation (Bodipy) and mitochondria abundance (MitoTracker) reveals that Bmal1 depletion markedly enhances the formation of

Bmal1-deficient cells further demonstrates that Bmal1 inhibits brown preadipocyte terminal differentiation (Fig. 4C). Moreover, UCP-1 and CEBP-β protein are elevated in Bmal1-depleted cells during the differentiation time course as compared to controls (Fig. 4D). Notably, UCP-1 also exhibits augmented response to Forskolin stimulation in day-4 terminally differentiated cells. As Forskolin treatment mimics a cAMP-mediated cold-induced thermogenic response in brown adipocytes, this increased responsiveness of UCP-1 suggests a potentially augmented thermogenic response with Bmal1 depletion and is in line with the observed enhanced coldtolerance of Bmal1-null mice in vivo. On the other hand, forced overexpression of Bmal1 in brown preadipocytes by a cDNA plasmid vector (BM cDNA), which results in high expression of Bmal1 protein and transcript (Fig. 5 A & 5C) as compared to empty vector control (pcDNA3), leads to marked suppression of differentiation as demonstrated by lipid and mitochondrial staining (Fig. 5B). The significantly lower mRNA expression of brown adipogenic markers corroborates the inhibitory effect of Bmal1 overexpression on terminal differentiation of brown adipocytes (Fig. 5C). Together, these in vitro analyses further delineate the cell-intrinsic

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functions of Bmal1 to suppressed lineage commitment and terminal differentiation of brown adipocytes. The TGF-β/BMP signaling cascade mediates Bmal1 function in brown adipogenesis To investigate the underlying mechanisms responsible for Bmal1 inhibition of brown adipogenesis, we interrogated transcriptional targets of Bmal1 in Bmal1 KD 10T1/2 cells by gene expression profiling analysis, and found that the TGF-β/BMP cascade is among the top differentially regulated processes (Suppl. Table S1). As TGF-β and BMPs are key negative and


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positive signals controlling brown fat formation, respectively (Fournier et al.; Koncarevic et al.; Tseng et al., 2008; Yadav et al., 2011), we postulated that Bmal1 may alter activities of these pathways to impact brown adipocyte differentiation. We thus determined TGF-β signaling activities in Bmal1 loss- and gain-of-function cells using TGF-β-responsive luciferase reporters, 3xTP-Luc (Wrana et al., 1992) and SBE4-Luc (Zhou et al., 1998). As demonstrated in Fig. 6A and 6B, while TGF-β treatment in normal control cells leads to robust 15-fold (3XTP-Luc) or 32fold (SBE4-Luc) induction of reporter activities, depletion of Bmal1 profoundly compromised TGF-β signal transduction to ∼10% of that of controls. In contrast, forced expression of Bmal1 significantly increased TGF-β reporter activity by ∼67% under TGF-β-stimulated condition. The TGF-β signaling defect in Bmal1-deficient cells is further corroborated by blunted phosphorylation of Smad3, the ultimate signal transducer of the TGF-β pathway (Massague and Wotton, 2000), upon either TGF-β1 or BMP7 stimulation (Fig. 6C). Interestingly, total amount of Smad3 protein is also reduced in Bmal1 knockdown cells as compared to controls, likely reflecting Bmal1 transcriptional regulation on Smad3 as suggested by microarray analysis (Table S1). In contrast, Smad2 and its ligand-activated phosphorylation are not altered, suggesting potential specificity of Bmal1-regulated targets in the TGF-β signaling pathway. Surprisingly, Bmal1-deficiency also markedly increases activity of the canonical BMP signaling pathway, as indicated by much higher Smad1/5 phosphorylation at basal and ligand-stimulated state as compared to SC (Fig. 6D). In contrast, the p38 MAP kinase branch of the BMP signaling is not affected by loss of Bmal1 function. Additional assessment of BMP activity by a wellcharacterized BMP-responsive luciferase reporter, the BRE2-Luc (Korchynskyi and ten Dijke, 2002), further confirms the robustly augmented BMP signaling under basal and BMP-stimulated conditions in Bmal1-depleted brown preadipocytes (Fig. 6E).

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The finding of attenuated TGF-β pathway activity together with augmented BMP signaling in Bmal1-deficient cells suggests that these mechanisms may contribute to the enhanced differentiation observed in these cells. Therefore, we tested whether activation of TGF-β or blockade of BMP signaling can rescue, i.e., suppress, the enhanced brown adipogenic phenotype. As shown in Fig. 7A, TGF-β1 treatment of normal brown preadipocytes effectively inhibits their adipogenic differentiation as indicated by weaker lipid (BODIPY) and mitochondrial (MitoTracker) staining relative to vehicle-treated control, as expected. Notably, TGF-β1 also

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blocks the differentiation of Bmal1-deficient cells as efficiently as in the controls. Consistent with these findings on morphological differentiation, TGF-β1 robustly suppresses brown-specific marker genes induction, including Ucp-1, Deiodinase2 (Dio2) and Prdm16, in shBmal1 cells to the same degree as seen in the shSC cells (Fig. 7B), suggesting that activation of the TGF-β1 pathway can rescue the effect of Bmal1 depletion on brown adipogenesis. We found that TGF-β signaling activities of Bmal1-deficient cells are severely impaired as compared to shSC control, although TGF-β1 can still induce weak activation of 3xTP-Luc (2-fold), SBE4-Luc (6-fold) and Smad3 phosphorylation than non-stimulated condition (Fig. 6A-C). So it is possible that TGF-β1


treatment induced signaling Bmal1 KD cells, although significantly diminished, was sufficient to suppress adipogenic differentiation. Furthermore, blockade of BMP signaling cascade by Noggin, a specific BMP inhibitor, similarly inhibits the induction of brown markers in Bmal1deficient cells as the TGF-β1 treatment, suggesting that inhibiting BMP pathway activation is also sufficient to abrogate enhanced differentiation induced by Bmal1-depletion. Interestingly, the combined effects of TGF-β1 and Noggin on suppressing brown marker gene inductions are similar to TGF-β1 and Noggin alone, suggesting either a saturated response to TGF-β1 or Noggin alone in these cells, or these pathways are inter-dependent through potential cross-talk. The regulation of TGF-β1 and Noggin on adipogenic genes in Bmal1-deficient is distinct from that of the brown-specific genes, as demonstrated in Fig. 7C. The down-regulation of Cebpα in response to these agents is the same extent as seen in brown-specific markers. In contrast, Pparγ is suppressed by TGF-β1 but not Noggin in Bmal1-deficient cells, while Cebpβ display increased expression in the presence of these ligands, possibly reflecting differential ligand sensitivities of these genes in brown adipocytes. Together, these results indicate that Bmal1 regulation of TGF-β as well as BMP pathways contributes to its function in modulating brown adipogenesis.

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Bmal1 exerts direct transcriptional control of TGF-β pathway genes and confers circadian regulation Bmal1 is an essential transcriptional activator of the core clock regulatory loop, and has been implicated in transcriptional control of developmental signaling pathways including the TGF-β or BMP cascades (Janich et al., 2011). Therefore, prompted by findings from the microarray study, we examined whether components of the TGF-β or BMP pathways are direct transcriptional targets of Bmal1. Through computational screening of putative Bmal1 binding


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sites, the canonical E-box elements (CACGTG) (Rey et al., 2011), in the proximal promoter regions (-2kb + first intron) of key genes in these pathways, we identified a number of TGF-β pathway genes harboring the E-boxes, including the ligands Tgfb1 and Tgfb2, the receptor Tgfbr2, and signal transducer Smad3 (Fig. S4A). However, our computational analysis failed to identify E-box elements among the genes of the BMP pathway examined, including BMPs, Smad1 and 5. To determine Bmal1 occupancy of these potential binding sites and its potential circadian time-dependent association with target promoters, we performed chromatin immunoprecipitation (ChIP) analysis in HIB1B cells under serum shock condition (Balsalobre et al., 1998), an established method to elicit cell-intrinsic clock oscillation and synchronize circadian gene rhythmicity (Guo et al., 2012; Janich et al., 2011). As shown in Fig. 8A, robust recruitment of Bmal1 to a known target promoter, Rev-erbα, is detected at CT8 (CT: circadian time) upon serum shock, whereas this activity is nearly absent at CT20. This rhythmic Bmal1 promoter occupancy coincides with its protein peak observed at CT8 and trough at CT20 (Fig. 8B). Bmal1 enrichment on identified sites of Tgfb1 and Smad3 promoters display a circadian-dependent manner similar to that of Rev-erbα, with stronger chromatin association occurring at CT8 (Fig. 8A). In contrast, other candidate target promoters, Tgfb2 and Tgfbr2, exhibit only modestly enriched Bmal1 association. Notably, serum shock in HIB1B cells elicits oscillation of Smad3 protein with a rhythmic profile similar to that of Bmal1 (Fig. 8B), suggesting an intrinsic temporal regulation of the key signaling transducer of the TGF-β cascade. Likely due to direct Bmal1 regulation, silencing of Bmal1 blunted (Fig. 8C), whereas its forced expression augmented expression of the identified target genes in the TGF-β pathway, Tgfb1, Tgfb2, Tgfbr2 and Smad3 (Fig. 8D). In line with the results from our initial screening indicating absence of potential Bmal1 response elements on BMP signaling genes, mRNA expression of Smad1 and 10

Smad5 are not affected by Bmal1 KD (Fig. 8E). However, canonical BMP signaling targets, Id1 and Id2, are markedly up-regulated in Bmal1-deficient cells, additional evidence of augmented BMP activity in these cells as revealed by findings of Smad5 phosphorylation and BMP reporters previously. Given that intracellular BMP signal transduction is subjected to negative feedback regulations (Kavsak et al., 2000; Kawamura et al., 2012), we further analyzed whether loss of Bmal1 could affect negative regulatory mechanisms involved in BMP signaling pathway to impact its activity. Interestingly, as demonstrated by expression analysis of BMP pathway inhibitory molecules (Fig. 8F), Smad6, SnoN and Ski transcripts are significantly attenuated in

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Bmal1-deficient cells. This finding of inhibition of BMP negative regulator expression with loss of Bmal1 suggests a relief of inhibition of the BMP pathway may account for the enhanced BMP activity in these cells. Discussion The unique energy-dissipating function of brown adipose tissue and its presence in humans holds promise as a potential therapeutic target against obesity (Cypess et al., 2009; van Marken


Lichtenbelt et al., 2009; Virtanen et al., 2009). Accumulating evidence suggests that disruption of the clock mechanism leads to various metabolic abnormalities, particularly obesity and insulin resistance (Pan et al., 2011; Roenneberg et al., 2012; Sahar and Sassone-Corsi, 2012). While diverse aspects of metabolism are subjected to circadian regulation (Bass and Takahashi, 2010; Green et al., 2008), whether the adipocyte-intrinsic timekeeping mechanism participates in brown fat cell development is not known. Our study demonstrates that the essential transcription activator of the molecular clock, Bmal1, exerts direct temporal control of the TGF-β pathway, a key inhibitory signal of brown fat formation (Fournier et al., 2012; Koncarevic et al., 2012; Yadav et al., 2011). And together with reciprocally altered BMP activity, these developmental signaling mechanisms driven by Bmal1 modulate brown adipogenesis and consequently impact adaptive thermogenesis in vivo. As critical negative and positive signals involved in brown adipogenesis respectively (Fournier et al.; Koncarevic et al.; Kuo et al., 2014; Qian et al., 2013; Tseng et al., 2008; Yadav et al., 2011), our finding of circadian regulation of TGF-β and BMP activities implicates a temporal regulatory control of adipocyte lineage commitment and differentiation.

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The TGF-β 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 differentiation, respectively. BMP7 promotes both early commitment of mesenchymal precursors to the brown lineage (Tseng et al., 2008) and subsequent differentiation (Cao et al., 2004; Sellayah et al.). TGF-β signaling, on the other hand, exerts inhibitory effects on BAT development, with ablation of Smad3, the ultimate signal transducer of the TGF-β pathway, resulting in selective induction of brown-specific markers genes and enhanced mitochondrial function (Yadav et al.). In addition, inhibition of Activin receptor IIB and myostatin, both

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signaling through Smad3 (Fournier et al.; Kim et al.; Koncarevic et al.; Zhang et al.), were recently reported to promote brown adipogenesis. We have previously shown that Bmal1 suppresses white adipocyte differentiation (Guo et al., 2012). The current finding of Bmal1 inhibitory effect on brown adipogenesis is in parallel with its role in white adipocyte formation, although the molecular pathways mediating these effects are distinct. We found that Bmal1 modulates Wnt signaling in white adipose tissue (Guo et al., 2012), whereas in brown fat, this core clock regulator exerts transcriptional control on TGF-β pathway. Interestingly, recent


studies indicate that the circadian clock can act on various signaling cascades, including the TGF-β and BMP pathways, to modulate epidermal stem cell activation 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 array of developmental signals involved in transmitting a circadian timing cue to fine-tune tissue homeostasis. Findings of the Bmal1 direct promoter association with key components of TGF-β pathway, such as Tgfbr2 and Smad3, indicate that the TGF-β signaling cascade could be under a coordinated circadian control in brown preadipocytes. Likely due to this circadian element present in Smad3 promoter, a robust rhythmic oscillation of Smad3 protein in phase with Bmal1 is elicited upon serum shock. In addition, we found that Bmal1 deficiency leads to significantly attenuated Smad3 activation and reporter activity, indicating that TGF-β signaling transduction could be subjected to temporal regulation as well. Indeed, circadian expression of Smad3 was detected in human gingival fibroblasts, mesenchymal stem cells and mouse liver (Sato et al., 2012). Furthermore, in epidermal stem cells (Janich et al., 2011; Karpowicz et al., 2013) and central clock, the suprachiasmatic nuclei (Beynon et al., 2009), circadian pattern of TGF-β signaling activity as indicated by Smad3 phosphorylation has been reported. Given the 12

importance of TGF-β signaling in stem cell proliferation and differentiation (Gaarenstroom and Hill, 2014), circadian modulation of this pathway may confer appropriate response to temporal cues involved in controlling stem cell behavior and tissue homeostasis. While our current study defines a specific role of Bmal1 in brown fat differentiation program, its broader impact on various important TGF-β-regulated biological processes remains to be elucidated. An interesting observation from our study is the reciprocally altered BMP signaling with altered TGF-β pathway activity in Bmal1 loss- or gain-of-function studies. Based on several lines

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effect of Bmal1 on TGF-β pathway. First, we failed to identify canonical E-box elements in


of evidence, changes in BMP activity may have occurred secondary to the direct regulatory

TGF-β signaling can augment BMP activity. Ski (Ehnert et al., 2012; Wang et al., 2000) and

genes directly involved in BMP signal transduction through extensive promoter screening, although it is possible that there could be non-canonical elements or additional genes not screened. Secondly, searches in available Bmal1 ChIP-Seq datasets (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 relationship between TGF-β and BMP signal transduction has been well-characterized (Shi and Massague, 2003) and loss of endogenous

SnoN (Kawamura et al., 2012) are known BMP inhibitory molecules that can be induced by TGF-β to mediate the antagonism between these signaling events, while Smad6 is a ubiquitin ligase specific for BMP-activated Smad1/5/8 degradation. In Bmal1-deficient cells, as the attenuated TGF-β activity is accompanied by down-regulation of SnoN, Ski and Smad6, a relief of these inhibitory mechanisms may have contributed to enhanced BMP signaling transduction. Moreover, we observed that TGF-β alone, or BMP blockade, is sufficient to suppress adipogenic induction in these cells (Fig. 7), but these effects are not additive, further suggesting potential inter-dependence of the two pathways. These observations support a model that direct transcriptional control of the TGF-β pathway by Bmal1 may consequently alter BMP signaling, and together they 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), the precise molecular mechanisms responsible for these effects are yet to be defined. As our study demonstrates, circadianly-controlled brown fat thermogenic capacity may contribute to this 13

phenomenon. Two recent reports indicate that circadian regulators Rev-erbα (Gerhart-Hines et al., 2013) and Per2 (Chappuis et al., 2013) both contribute to regulation of brown adipose tissue function. In particular, our findings are in agreement with the study by Chappuis et al., as mice lacking Per2, a repressor of Bmal1, display sensitivity to cold, whereas Bmal1 ablation leads to resistance to cold. Based on the opposing regulations of Per2 and Bmal1 in the core clock circuit, our current study, together with these previous reports, suggests the participation of a coordinated temporal control mechanism in modulating thermogenic capacity. While previous studies mainly focus on clock genes in BAT function, our study addresses an additional layer of


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temporal control in the brown fat concerning brown adipocyte differentiation. Nonetheless, specific strategies, such as circadian shift regimens, will be needed in the future to assess the direct impact of altered clock regulation on BAT function and its contribution to metabolic diseases. Given that certain clock regulators, such as Rev-erbα, is amenable to pharmacological manipulation by synthetic ligands (Solt et al., 2012), the clock circuit represent a potential target for therapeutic interventions. Compare to our extensive knowledge of white adipocyte development, the current understanding of regulatory mechanisms governing BAT formation is only emerging. Our study elucidates a previously unappreciated temporal regulatory mechanism involved in brown adipocyte development that ultimately contributes to fine-tuning of adaptive thermogenesis. Future efforts to determine how this mechanism impacts systemic metabolic homeostasis may lead to discovery of new therapies against widespread circadian disruption-induced metabolic disorders. Materials and Methods Animals-Animals were maintained in the Methodist Hospital Research Institute mice facility under a constant 12:12 light dark cycle, with light on at 7:00 AM (ZT0). Room temperature was controlled at 220C. 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. Bmal1-/-, Bmal1fl/fl , and ap2-Cre transgenic mice were obtained from Jackson Laboratory (Storch et al., 2007). Mice are maintained ad lib on standard chow diet (AIN-76A, Research Diets) with free access to water.

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Tissue samples were obtained at ambient temperature, unless indicates otherwise, and at the same time of the day to ensure reproducible circadian gene expression. Cold tolerance test 12-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 highprecision thermometer. Body temperature was measured twice at 9AM before the mice were subjected to 40C for 24 hours, with temperature monitored at indicated times. Cell culture C3H10T1/2 and HIB1B cell lines were obtained from ATCC and maintained in

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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 similarly as described (Tseng et al., 2008) but without BMP7 pretreatment. Briefly, cells at confluency were subjected to brown induction media containing insulin (20nM), T3 (1nM), isobutylmethylxanthine (0.5 mM), dexamethasone (5mM), and rosiglitazone (1 mg/ml) for 3 days. Cells were then switched to maintenance media supplemented with insulin and T3 only for 9 days. Fully differentiated brown phenotype with


significant lipid accumulation and Ucp-1 expression occur at 9 days (D9) after induction. Differentiation of HIB-1B and primary brown adipocytes were induced using same induction media for two days and maintenance media for two to four days.

Primary brown adipocyte, preadipocyte isolation and immortalization-Primary brown adipocytes and preadipocytes were isolated from interscapular brown adipose tissue pad of 6week old mice, as described (Timmons et al., 2007). Briefly, tissues were digested by Type I collagenase in the presence of 1% BSA at 370C for 30 minutes. The suspension was filtered, centrifuged, and the top fat layer was collected as adipocytes and 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 (Tseng et al., 2008). Serum shock synchronization of cellular clock Serum shock to synchronize cells in culture was performed as previously described (Guo et al., 2012). Briefly, confluent cultures were incubated in serum-free medium overnight and subjected to 20% FBS serum shock for 1 hour. 15

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 indicated times after serum shock. RNA extraction and quantitative reverse-transcriptase PCR analysis RNeasy miniprep kits (Qiagen) were used to isolate total RNA from snap-frozen tissues or cells. Tissues samples are collected at times as indicated and cell samples were obtained at non-synchronized normal culture conditions. cDNA was generated using q-Script cDNA Supermix kit (Quanta

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Biosciences) and quantitative PCR was performed using a Roche 480 Light Cycler with Perfecta SYBR Green Supermix (Quanta Biosciences), as described (Guo et al., 2012). Relative expression levels were determined using the comparative Ct method to normalize target genes to 36B4 internal control, and compared to experimental controls as indicated. Immunoblot analysis 40-50 µg of total protein were used for the analysis, as previously described (Chatterjee et al., 2013). Smad2, 3, or 1/5 and p38 phosphorylation were assessed at 1 hour after indicated ligand treatment (TGF-β1 10ng/ml, BMP7 100ng/ml). The primary


antibodies that were used are listed in supplemental procedures. Chromatin immunoprecipitation (ChIP)-qPCR analysis-Immunoprecipitation was performed using Bmal1 (AB93806) or control rabbit IgG plus protein A/G beads, as described (Chatterjee et al., 2013). Briefly, cells were fixed by formaldehyde, lysed and sonicated to shear the chromatin. The immunoprecipitated chromatin fragments were eluted, treated with proteinase K and purified using Qiaquick PCR purification kit (Qiagen). Real-time PCR using Perfecta SYBR Green Supermix (Quanta Biosciences) was carried out with an equal volume (4ul) of each reaction with specific primers.

Negative control TBP primer was also included. Primers

sequences used are listed in Fig. S4. Values are expressed as fold enrichment of percentage of input normalized to IgG control. Oil-Red-O, BODIPY and Mitotracker staining-Oil red O staining was carried out in fixed cells using 0.5% oil-red-O for 1 hour, as described (Guo et al., 2012). BODIPY staining (Molecular Probes, Carlsbad, CA, USA) was carried out at a concentration of 1mg/ml for 30 minutes. Mitotracker (100 nM, Molecular Probes) staining was performed according to manufacturer’s protocol and applied to cells in culture at 37°C for 30 minutes before fixation. 16

Luciferase reporter assays Cells were grown to 80% confluency and transiently transfected using FuGENE 6 (Roche) in four replicates, as described (Beynon et al., 2009). TGF-β1 (2 ng/ml) or BMP4 (100 ng/ml) were added 16 hours after transfection and luciferase activity was measured using Dual-Glo luciferase assay system (Promega) 24 hours following ligand treatment. Reporter luciferase values were normalized to Renilla readings and expressed as fold of induction over controls. TGF-β luciferase reporters, 3xTP-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) is a kind gift from Dr. P. Ten Dijke (Leiden University


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Medical Center, The Netherlands). Statistical analysis Data is expressed as Mean ± SE. Difference between groups of coldtolerance tests were analyzed by one-way analysis of variance (ANOVA). Statistical differences of other experiments were assessed by Student's t test. Author Contributions K. M. and D. N. designed and performed the experiments, analyzed the data, and wrote the manuscript. B. G., S. C., R. L. and H. Y. performed experiments on adipocytes, M. C. performed experiments on brown fat development, Z. Z. and D. N. carried out circadian actogram analysis. Acknowledgements We thank The Houston Methodist Research Institute (HMRI) for start-up support and the Center for Diabetes Research for technical assistance. This project is supported by American Heart Association grant 12SDG12080076 and American Diabetes Association grant 1-13-BS-118 to KM, American Diabetes Association grant 7-12-BS-210 and National Institute of Health grant DK097160-01 to VY.

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Figure Legends Figure 1. Bmal1 ablation in mice enhances brown fat formation and function. (A-B) Effects of loss of Bmal1 on BAT formation as analyzed by brown adipose tissue weight (A) and its ratio to body weight (B) in 8-week-old mice WT and Bmal1-/- mice (n=6-8/group). RT-qPCR analysis of BAT genes expression (C), representative heamotoxylin and eosin staining of BAT histology (D), and 24 hour cold-tolerance test at 40C (E) in wild-type and Bmal1-/- mice. (n=6/group). (F-I) Effect of adipocyte-specific Bmal1 ablation on BAT. (F) Immunoblot analysis of Bmal1 protein in ap2-Cre+/BMfl/fl and ap2-Cre-/BMfl/fl littermate control mice. (G) Cold-tolerance test at 40C. Immunoblot analysis of UCP-1 (H), and RT-qPCR analysis of brown fat gene expression (I) at basal ambient temperature and 40C cold conditions in ap2-Cre-/BMfl/fl and ap2-Cre+/BMfl/fl mice (n=5-6). Tissue samples were collected at previous day 9AM prior to (basal), or after 24 hour cold challenge (40C cold). *, **: P≤ 0.05 and 0.01 Bmal1-/- vs. WT by Students t test or one-way ANOVA (cold tolerance test). Figure 2. Bmal1-deficient primary brown preadipocytes display enhanced adipogenic differentiation. (A, B) Representative images of oil-red-O staining (ORO) and phase-contrast (phase) of day 6-differentiated primary preadipocytes isolated from wild-type and Bmal1-/- mice (A) or ap2-Cre-/BMfl/fl and ap2-Cre+/BMfl/fl mice (B). (C) RT-qPCR analysis of brown adipogenic gene expression at day 5 of differentiation of wild-type and Bmal1-/- preadipocytes (n=3). (D) RT-qPCR analysis of brown adipogenic gene expression at day 1 and day 5 of differentiation of ap2-Cre+/BMfl/fl vs. ap2-Cre-/BMfl/fl preadipocytes (n=3). *, **: P≤ 0.05 and 0.01 Bmal1-/- vs. WT, or ap2-Cre+/BMfl/fl vs. ap2-Cre-/BMfl/fl by Students t test. Figure 3. Silencing of Bmal1 promotes C3H10T1/2 mesenchymal precursor differentiation to brown adipocytes. (A) Immunoblot analysis of Bmal1 protein in mesodermal lineage cell lines. (B) Representative images of Bodipy lipid staining, mitochondiral staining by Mitotracker, and phase-contrast of C3H10T1/2 cells with stable transfection of scrambled control shRNA (shSC) or Bmal1 knockdown shRNA (shBmal1) subjected to brown adipocyte-specific differentiation condition at day 9. (C) RT-qPCR analysis of brown adipogenic gene expression at day 1 and 9 of differentiation in shSC and shBmal1 cells (n=3). *, **: P

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JNK signaling axis.

β-catenin pathways.

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β-catenin signaling pathways.

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Smad signaling pathway.

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