Sirt1 promotes fat mobilization in white adipocytes by repressing ...

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Sirt1 promotes fat mobilization in white adipocytes by repressing ...

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• Publié par: olonilux
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letters to nature .............................................................. methylxanthine, we observed that Sirt1 protein levels increased and peaked at day 5 after hormonal stimulation (Fig. 1a).Sirt1 Sirt1 promotes fat mobilization expression in 3T3-L1 cells was then modified through retroviral infection with either pBABE–Sirt1or pSUPER–Sirt1RNA inter- in white adipocytes by ference (RNAi) for overexpression (tenfold) or downregulation (sevenfold) of theSirt1gene, respectively (Fig. 1b). 3T3-L1 cells repressing PPARg undergo one or two mitotic divisions after induction as a prelude to 9,10 1 11 . This occurred normally in cells thatterminal differentiation Fr ´ ede ´ ricPicard,MartinKurtev,NamjinChung, 2 2 overexpressed or underexpressed Sirt1 as evaluated by 5-bromo- Acharawan ToparkNgarm , Thanaset Senawong , 1,3 24 deoxyuridine (BrdU) incorporation (data not shown). However, Rita Machado de Oliveira, Mark Leid , Michael W. McBurney compared with cells infected with the control vector, stable 3T3-L1 1 & Leonard Guarente cells overexpressing Sirt1 accumulated much less fat as determined 1 Department of Biology, Massachusetts Institute of Technology, Cambridge, by Oil red O staining after 7 days of differentiation (Fig. 1c) or direct Massachusetts 02139, USA measurement of intracellular triglyceride content (Fig. 1d). In 2 Laboratory of Molecular Pharmacology, Department of Pharmaceutical Sciences, contrast, downregulation of Sirt1 expression resulted in a significant College of Pharmacy and Environmental Health Science Center, Oregon State increase in triglyceride accumulation after differentiation University, Corvallis, Oregon 97331-3507, USA (Fig. 1c, d). These results indicate that Sirt1 acts a negative 3 Graduate Program in Basic and Applied Biology, ICBAS, University of Porto, modulator of adipogenesis in the 3T3-L1 model. 4099-003, Portugal Insulin/insulin-like growth factor (IGF)-1 signalling helps to 4 Ottawa Regional Cancer Centre and Department of Medicine, University of 11 regulate adipogenesis, and this pathway determines lifespan in Ottawa, Ontario K1H 1C4, Canada 12 nematode worms. To evaluate the possible interaction of Sirt1 ............................................................................................................................................................................. with the insulin/IGF-1 signalling cascade in adipocytes, we differ- Calorie restriction extends lifespan in organisms ranging from 1 entiated virally transduced 3T3-L1 cells in the absence of insulin but yeast to mammals . In yeast, theSIR2gene mediates the life 2 with rosiglitazone, a very potent, selective PPAR-gagonist that acts extending effects of calorie restriction . Here we show that the downstream of the insulin pathway. Although rosiglitazone treat- mammalianSIR2orthologue,Sirt1(sirtuin 1), activates a critical ment promoted adipogenesis to a greater extent than the regular component of calorie restriction in mammals; that is, fat mobil differentiation cocktail (Fig. 1c), it did not alter the phenotypes of ization in white adipocytes. Upon food withdrawal Sirt1 protein cells with increased or decreased levels of Sirt1 (Fig. 1c), suggesting binds to and represses genes controlled by the fat regulator that Sirt1 affects regulators that act downstream of insulin/IGF-1 PPARg(peroxisome proliferatoractivated receptorg), includ signalling. ing genes mediating fat storage. Sirt1 represses PPARgby Differentiation of 3T3-L1 fibroblasts increases levels of the docking with its cofactors NCoR (nuclear receptor corepressor) transcription factor C/EBP-d, which stimulates the expression of and SMRT (silencing mediator of retinoid and thyroid hormone 10 PPAR-gand C/EBP-a. PPAR-ginduces expression of target genes, receptors). Mobilization of fatty acids from white adipocytes 1/2 such as the fatty-acid-binding protein aP2 (also known as adipose upon fasting is compromised inSirt1mice. Repression of 13 tissue-specific FABP). Moreover, PPAR-gcan maintain expression PPARgby Sirt1 is also evident in 3T3L1 adipocytes, where of itself, perhaps by binding to PPAR-gsites in the promoter of the overexpression of Sirt1 attenuates adipogenesis, and RNA inter 9,14 PPAR-ggene (PpargTo gain an insight into the mechanisms by) . ference of Sirt1 enhances it. In differentiated fat cells, upregula which Sirt1 represses fat accretion, we measured protein and tion of Sirt1 triggers lipolysis and loss of fat. As a reduction in fat messenger RNA expression of key factors in the transcriptional 3 is sufficient to extend murine lifespan , our results provide a programme in the different virus-infected 3T3-L1 adipocytes. We possible molecular pathway connecting calorie restriction to life observed a reduction in C/EBP-a, C/EBP-dand aP2 mRNA, but extension in mammals. not C/EBP-b, upon Sirt1 overexpression (Fig. 1e). A reduction in Calorie restriction is the first regimen shown to extend mamma- PPAR-gand C/EBP-awas also observed by western blotting lian lifespan. Calorie restriction induces a shedding of body fat from (Fig. 1f). In contrast, cells in which Sirt1 had been downregulated white adipose tissue (WAT), a decrease in body temperature and an showed higher levels of PPAR-g, C/EBP-d, C/EBP-aand aP2 4 increase in insulin sensitivity . It has been suggested that calorie (Fig. 1e, f). These findings are consistent with the model that restriction functions in a passive way by lowering oxidative and Sirt1 functions as a repressor of genes that drive white adipocyte other damage, thereby resulting in a longer lifespan. However, differentiation and fat storage. studies in yeast suggest that calorie restriction is a highly regulated The above experiments do not address whether Sirt1 regulates fat response that requires a sensing step followed by the execution of accumulation in fully differentiated adipocytes. Thus, we fully 5 a programme to extend lifespan. The regulatory gene that differentiated 3T3-L1 cells and subsequently (12 days after induction) 15 mediates this programme isSIR2, which promotes survival in applied the Sirt1 activator resveratrolover a wide range of concen- 6 7 yeast ,Caenorhabditis elegansand, perhaps, higher eukaryotes in trations (Fig. 2a). After staining the cells for fat content, a strong response to food scarcity.SIR2is well suited for this function reduction in fat was observed at 50 and 100mM resveratrol. The loss of 1 because it encodes an NAD-dependent protein deacetylase, fat was due to activation of Sirt1, because there was no drug-mediated enabling it to monitor cellular metabolism and exert corresponding fat reduction in cells in which Sirt1 levels were knocked down (Fig. effects on gene expression. 2b). To validate further these visual results, we measured triglyceride WAT seems to be a primary factor in longevity, as mice engineered content and free fatty acid (FFA) release in these cells. Triglyceride 3 to have reduced levels of it live longer . This effect on lifespan may content was reduced and release of FFA was stimulated by resveratrol result from changes in hormones that are normally secreted from in the control cells, but not in the Sirt1 knockdown cells (Fig. 2c). WAT in proportion to fat mass. Thus, we evaluated whether the These findings strongly suggest that upregulation of Sirt1 stimulates mammalian Sir2 orthologue, Sirt1, senses nutrient availability in fat mobilization in fully differentiated 3T3-L1 adipocytes. WAT and mediates corresponding effects on fat accumulation. To To determine whether Sirt1 stimulates fat mobilization in bona probe whether Sirt1 may actually modulate adipogenesis, we used fide adipocytes, we cultured primary rat white adipocytes and 8 mouse 3T3-L1 fibroblasts as anin vitro activated fat mobilization with the known model . Adipogenesis in b-adrenergic inducer 9 these cells is promoted by the nuclear receptor PPAR-g. Upon adrenalin. Addition of resveratrol greatly stimulated the release of induction of adipogenesis by insulin, dexamethasone and isobutyl-FFA triggered by adrenalin (Fig. 2d), consistent with the findings in NATURE | VOL 429 | 17 JUNE 2004 | www.nature.com/nature 771 ©2004 Nature Publishing Group