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Title: Sirt1 attenuates camptothecin-induced apoptosis through caspase-3 pathway in porcine preadipocytes

Author
item Pang, Wei-jun - Northwest Agricultural & Forestry University
item Xiong, Yan - Northwest Agricultural & Forestry University
item Wang, Yu - Northwest Agricultural & Forestry University
item Tong, Qiang - Children'S Nutrition Research Center (CNRC)
item Yang, Gong-she - Northwest Agricultural & Forestry University

Submitted to: Experimental Cell Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/31/2012
Publication Date: 1/9/2013
Citation: Pang, W., Xiong, Y., Wang, Y., Tong, Q., Yang, G. 2013. Sirt1 attenuates camptothecin-induced apoptosis through caspase-3 pathway in porcine preadipocytes. Experimental Cell Research. 319(5):670-683.

Interpretive Summary: The SIRT1 protein belongs to a family of enzymes called sirtuins that can bind and modify (deacetylate) other proteins. Sirtuin proteins regulate many aspects of cellular functions, including cell growth, cell death, stress response, metabolism and aging. This article reports the finding that suppressing SIRT1 expression in swine fat cells renders these cells susceptible to cell death. Conversely, increasing SIRT1 level in these cells make them more resistant to cell death. The underlying mechanism is that SIRT1 modifies (deacetylates) and inhibits the p53 protein. SIRT1 protein also binds to the caspase-3 protein. Both p53 and caspase-3 are key promoters for cell death. In summary, this study uncovers a role of SIRT1 protein in maintaining the survival of fat cells and this may contribute to the development of obesity.

Technical Abstract: Adipose tissue is an important energy reservoir, and its over-development results in obesity in humans or body fat over-deposition in livestock. Loss of preadipocytes through apoptosis has been proposed as an alternative way to reduce adipose tissue mass. At present, the effect and regulatory mechanism of Sirt1 and camptothecin on porcine preadipocyte apoptosis are still largely unknown. Here, we evaluated whether Sirt1 had any role in the basal cellular and camptothecin-induced conditions in porcine preadipocytes. Flow cytometric analysis shows that viable cells decrease as well as early apoptotic and late apoptotic cells increase after knockdown of Sirt1 in porcine preadipocytes. Camptothecin induces porcine preadipocyte apoptosis in a dose-dependent manner, assessed with the Hoechst staining and western blot analysis. Interestingly, silencing of Sirt1 significantly enhances sensitivity of porcine preadipocytes to camptothecin, which may be due to upregulation of p53, acetylated p53, Bax, cleaved caspase-3 and downregulation of Bcl-2. On the contrary, under the Sirt1 overexpression condition viable cells' number significantly increases when challenging with camptothecin, and the protein levels of cleaved caspase-3, p53, acetylated p53 and Bax are downregulated. We also find that hyperacetylated p53 is the major effect of Sirt1 knockdown by overexpression of a mutated p53, whereas Sirt1 overexpression prevents camptothecin-induced apoptosis through p53 deacetylation in preadipocytes. Furthermore, repressing preadipocyte apoptosis of Sirt1 is mediated by direct interaction with cleaved caspase-3 using immunoprecipitation and inhibition of caspase-3 transcriptional activity using luciferase reporter assays.