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. 2009 Aug 27;460(7259):1154-8.
doi: 10.1038/nature08262. Epub 2009 Jul 29.

Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta transcriptional complex

Shingo Kajimura  1 , Patrick Seale, Kazuishi Kubota, Elaine Lunsford, John V Frangioni, Steven P Gygi, Bruce M Spiegelman
Affiliations

Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta transcriptional complex

Shingo Kajimura et al. Nature. .

Abstract

Brown adipose cells are specialized to dissipate chemical energy in the form of heat, as a physiological defence against cold and obesity. PRDM16 (PR domain containing 16) is a 140 kDa zinc finger protein that robustly induces brown fat determination and differentiation. Recent data suggests that brown fat cells arise in vivo from a Myf5-positive, myoblastic lineage by the action of PRDM16 (ref. 3); however, the molecular mechanisms responsible for this developmental switch is unclear. Here we show that PRDM16 forms a transcriptional complex with the active form of C/EBP-beta (also known as LAP), acting as a critical molecular unit that controls the cell fate switch from myoblastic precursors to brown fat cells. Forced expression of PRDM16 and C/EBP-beta is sufficient to induce a fully functional brown fat program in naive fibroblastic cells, including skin fibroblasts from mouse and man. Transplantation of fibroblasts expressing these two factors into mice gives rise to an ectopic fat pad with the morphological and biochemical characteristics of brown fat. Like endogenous brown fat, this synthetic brown fat tissue acts as a sink for glucose uptake, as determined by positron emission tomography with fluorodeoxyglucose. These data indicate that the PRDM16-C/EBP-beta complex initiates brown fat formation from myoblastic precursors, and may provide opportunities for the development of new therapeutics for obesity and type-2 diabetes.

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Figures

Figure 1
Figure 1. Identification of C/EBP-β as a critical binding partner in the PRDM16 transcriptional complex
a, Strategy to identify key PRDM16 binding partners. b, C2C12 myoblasts expressing indicated viral vectors were stained with Oil-Red-O 6 days after inducing adipocyte differentiation. c, PRDM16 transcriptional complex was immunopurified from brown fat cells expressing full-length or deletion mutants of PRDM16. d, Gene expression of known or predicted transcription factors identified in the PRDM16 complex in BAT and WAT. n=6. e, Endogenous C/EBP-β was detected in the PRDM16 complex by Western blotting. Input was shown in left. f, Transcriptional activity of PGC-1α promoter in response to PRDM16 and/or C/EBP-β. n=3; error bars are s.e.m.; *P<0.05, **P<0.01.
Figure 2
Figure 2. C/EBP-β is required for the initiation of the myoblast to brown fat conversion through PRDM16
a, Western blot analysis for C/EBP-β and PRDM16 in C2C12 myoblasts expressing scr or sh-C/EBP-β with PRDM16 or vector. b, Pparg2 gene expression. n=3. c, These cells were stained with Oil-Red-O 6 days after inducing adipocyte differentiation. d, BAT-selective gene expression. n=4. e, Microarray analysis of undifferentiated C2C12 myoblasts expressing scr or sh-C/EBP-β with PRDM16 or vector. n=3. f, Top: H&E staining of BAT from WT and C/EBP-β KO mice. Bottom: Immunohistochemistry to detect UCP1 expression. Scale bar, 20 µm. g, mRNA expression of BAT and skeletal-muscle-selective genes in BAT from E17.5 embryos. n=5–8; error bars are s.e.m.; *P<0.05, **P<0.01.
Figure 3
Figure 3. Reconstitution of the brown fat gene program in fibroblasts through PRDM16 and C/EBP-β
a, Pparγ2 expression in undifferentiated MEFs expressing indicated viral vectors. n=3. b, Immortalized MEFs or skin fibroblasts expressing indicated viral vectors were stained with Oil-Red-O 6–8 days after inducing adipocyte differentiation. c, BAT-selective gene expression. d, Thermogenic gene expression. The cells were treated with cAMP for 4 hours. n=4. e, BAT-selective gene expression in primary skin fibroblasts expressing vector or PRDM16 and C/EBP-β. n=3. f, Total and uncoupled cellular respiration in differentiated brown fat cells and the MEFs expressing vector or PRDM16 and C/EBP-β. The cells were treated with dbcAMP for 12 hours. n=3; error bars are s.e.m.; *P<0.05. **P<0.01.
Figure 4
Figure 4. Generation of functional brown adipose tissue in vivo through expression of PRDM16 and C/EBP-β
a, Fat pads from transplanted MEFs expressing indicated viral vectors were stained by H&E. Scale bar, 500 µm. b, High magnification images of H&E staining in the transplants expressing PRDM16 and C/EBP-β, and endogenous BAT. Arrow-heads show multilocular fat cells. Scale bar, 50 µm. c, Immunohistochemistry to detect UCP1 expression in the transplant (left, anti-UCP1; middle, negative control) and BAT (right). Scale bar, 50 µm. d, PET/CT image of mice with engineered BAT (eBAT) and engineered WAT (eWAT). e, CT image (left) and PET image (right) of mouse skin with the eBAT and eWAT.
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