The adipocyte clock controls brown adipogenesis through the TGF-β and BMP signaling pathways - PubMed (original) (raw)

. 2015 May 1;128(9):1835-47.

doi: 10.1242/jcs.167643. Epub 2015 Mar 6.

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

Deokhwa Nam et al. J Cell Sci. 2015.

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-β pathway together with reciprocally altered BMP signaling; 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 affect brown fat formation and thermogenic regulation, which could be targeted therapeutically to combat obesity.

Keywords: Adipogenesis; Brown adipocyte differentiation; Circadian rhythm; Obesity; TGF‐β signaling pathway.

© 2015. Published by The Company of Biologists Ltd.

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Figures

Fig. 1.

Fig. 1.

Bmal1 ablation in mice enhances brown fat formation and function.(A,B) Effects of the loss of Bmal1 on BAT formation as analyzed by brown adipose tissue weight (A) and its ratio to body weight (BW) (B) in 8-week-old wild-type (WT) and Bmal1−/− mice (n = 6–8/group). (C–E) RT-qPCR analysis of BAT gene expression (C), representative hematoxylin and eosin staining of BAT histology (D) and 24 hour cold-tolerance test at 4°C (E) in wild-type and Bmal1−/− mice (n = 6/group). (F–I) The 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 4°C. Immunoblot analysis of UCP-1 (H), and RT-qPCR analysis of brown fat gene expression (I) at basal ambient temperature and under cold conditions (4°C) in ap2-Cre−/BMfl/fl and ap2-Cre+/BMfl/fl mice (n = 5–6). Tissue samples were collected the previous day at 09.00 prior to (basal) or after 24-hour cold challenge (4°C cold). All quantitative data show the mean±s.e.m. *_P_≤0.05; **_P_≤0.01 _Bmal1−/_− versus wild type [Student's _t_-test or one-way ANOVA (cold tolerance test)]. Scale bar: 50 µm.

Fig. 2.

Fig. 2.

Bmal1-deficient primary brown preadipocytes display enhanced adipogenic differentiation.(A,B) Representative images of Oil Red O staining (ORO) and phase-contrast microscopy (Phase) of day-6-differentiated primary preadipocytes isolated from wild-type (WT) and Bmal1−/− mice (A) or ap2-Cre−/BMfl/fl and ap2-Cre+/BMfl/fl mice (B). Scale bars: 100 µm. (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 (D1) and day 5 (D5) of differentiation of ap2-Cre+/BMfl/fl versus ap2-Cre−/BMfl/fl preadipocytes (n = 3). Data show the mean±s.e.m. *_P_≤0.05; **_P_≤0.01 for Bmal1−/− versus wild type or for ap2-Cre+/BMfl/fl versus ap2-Cre−/BMfl/fl (Student's _t_-test).

Fig. 3.

Fig. 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, mitochondrial staining by Mitotracker and phase contrast microscopy of C3H10T1/2 cells with stable transfection of scrambled control shRNA (shSC) or Bmal1-specific shRNA (shBmal1) subjected to brown-adipocyte-specific differentiation conditions at day 9. (C) RT-qPCR analysis of brown adipogenic gene expression at day 1 (D1) and day 9 (D9) of differentiation in shSC- and shBmal1-treated cells (n = 3). Data show the mean±s.e.m. *P<0.05; **P<0.01 for shBmal1 versus shSC.

Fig. 4.

Fig. 4.

Silencing of Bmal1 promotes HIB1B brown preadipocyte terminal differentiation. (A) Immunoblot analysis of Bmal1 protein expression in HIB-1B cells with scrambled control shRNA (shSC) or stable shRNA-mediated Bmal1 knockdown (shBmal1). (B) The effect of stable Bmal1 knockdown on HIB-1B differentiation as shown by BODIPY and Mitotracker staining at day 3 of differentiation. (C) RT-qPCR analysis of brown adipocyte marker gene expression in shSC and shBmal1 cells (n = 3). Data show the mean±s.e.m. *P<0.05; **P<0.01 for shBmal1 versus shSC. (D) Immunoblot analysis of brown adipogenic markers during differentiation day 1–4 (D1–4) of shSC and shBmal1 cells, and their response to forskolin at day 4.

Fig. 5.

Fig. 5.

Forced expression of Bmal1 inhibits HIB1B brown preadipocyte terminal differentiation. (A) Immunoblot analysis of Bmal1 protein expression in HIB-1B cells with Bmal1 stable overexpression (BM cDNA) or empty vector control (pcDNA3). (B) The effect of Bmal1 overexpression on HIB-1B differentiation as shown by BODIPY and Mitotracker staining at day 3 of differentiation. (C) RT-qPCR analysis of brown adipocyte marker gene expression of day-3 differentiated HIB-1B cells expressing pcDNA3 or BM cDNA (n = 3). Data show the mean±s.e.m. *P<0.05; **P<0.01 (BM cDNA versus pcDNA3).

Fig. 6.

Fig. 6.

Bmal1 regulates the signaling activities of TGF-β and BMP cascades. (A,B) The effect of Bmal1 silencing and forced expression on TGF-β signaling as assessed by using the TGF-β-responsive luciferase reporters, 3×TP-Luc (A) and SBE4-Luc (B), under basal or TGF-β-stimulated conditions in C3H10T1/2 cells (n = 4). Ctr, control. ##P<0.01 under basal conditions; **P<0.01 under TGF-β-treated conditions (Student's _t_-test). (C) TGF-β signaling activity as assessed by Smad2 and Smad3 phosphorylation under basal conditions or in response to TGF-β1 or BMP7 ligand treatment in shSC and shBmal1 cells. (D) BMP signaling as assessed by Smad1/5 and p38 phosphorylation under basal conditions or in response to TGF-β1 or BMP7 ligand treatment in shSC and shBmal1 cells. (E) BMP signaling as assessed by a BMP-responsive luciferase reporter, BRE2-Luc, under basal or BMP4-stimulated conditions (n = 4). RLU, relative luciferase units; Ctrl, control. ##P<0.01 under basal conditions; **P<0.01 under BMP4-treated conditions (Student's _t_-test). All quantitative data show the mean±s.e.m.

Fig. 7.

Fig. 7.

TGF pathway activation or BMP pathway blockade suppresses enhanced 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 the absence or presence of TGF-β1 treatment. Ctr, control. Scale bars: 50 µm. (B,C) RT-qPCR analysis of TGF-β1, noggin or TGF-β1 plus Noggin (T+N) treatment on brown-adipocyte-specific gene expression (B) or adipogenic gene expression (C) of day-3-differentiated shSC and shBmal1 cells. Cells were treated with TGF-β1 (2 ng/ml) or noggin (100 ng/ml) for 8 hours prior to the induction of differentiation and treatment was maintained throughout differentiation (n = 3). Data show the mean±s.e.m. #P<0.05; ##P<0.01 (treatment versus non-treated control); *P<0.01; **P<0.05 (shBmal1 versus shSC).

Fig. 8.

Fig. 8.

Bmal1 exerts direct transcriptional control on genes of the TGF-β pathway. (A) Chromatin immunoprecipitation qPCR (ChIP-qPCR) analysis of Bmal1 occupancy on identified TGF-β pathway gene promoters at 8 and 20 hours after serum shock in HIB-1B cells. Bmal1 occupancy of the Rev-erbα promoter E-box (a known target) is included as a positive control and that of Tbp as a negative control. Values are presented as the fold enrichment of the percentage of total input over IgG control (n = 4). CT, circadian time, with time immediately after serum shock taken as CT0. *P<0.05; **P<0.01 (CT8 versus CT20). (B) Immunoblot analysis of Bmal1 and Smad3 protein oscillation induced by serum shock from CT8 to 32. (C–F) RT-qPCR gene expression analysis of components of TGF-β and BMP pathways in _Bmal1_-knockdown (C,E,F), or _Bmal1_-overexpressing HIB-1B cells (D). n = 3. All quantitative data show the mean±s.e.m. *P<0.05; **P<0.01 (shBmal1 versus shSC, or BM cDNA versus pcDNA3).

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