Effect Of Sirt1 On Apoptosis Of Porcine Preadipocyte

Obesity is characterized by an increase in adipocyte number and size. Adipose tissue cellularity is linked to the dynamic role played by preadipocytes. Preadipocytes, which reside in the adipose tissue stromal-vascular compartment, are specialized fibroblast-like progenitor cells which proceed through a complex program of gene expression to differentiate into terminal mature adipocytes when appropriately cued.Mechanisms to reduce adipose mass include decreased proliferation and differentiation of preadipocytes, and also involves the loss of preadipocytes through apoptosis. So, to decrease fat deposition through preadipocyte apoptosis is effective.Apoptosis is a form of programmed cell death that results in removal of undesired cells without inflammation, and it plays a key role in maintenance of homeostasis in organisms. Evidence for apoptosis in preadipocytes has been obtained through the use of in vitro cell culture as well as in vivo studies in rodents or humans. Compared with other animal models, model of pig is more similar to human's in the physiological characteristics. So it is a more ideal animal model to investigate human obesity.Sirt1, the mammalian homologue of Sir2 (silent information regulator 2), is a NAD-dependent class III deacetylase. Growing evidences have indicated that Sirt1 plays an important role in mediating survival of several types of cells by deacetylating other substrates, such as p53, FoxOs, Ku70. However, the effect of Sirt1 on porcine preadipocyte apoptosis is unknown. Therefore, in the current study, we treated cells with Sirt1 shRNA or camptothecin, which has been widely used to induce apoptosis under experimental conditions, and apoptosis was assessed to determine whether Sirt1 had role in the normal condition and whether it exerted a protective action against camptothecin-induced apoptosis in porcine preadipocytes. The results are as follows:1. This study successfully constructed the recombinant lentivirus interfering vector target on porcine Sirt1 gene, Western blotting showed that Sirt1 protein expression was remarkably decreased in porcine preadipocytes infected with recombinant lentivirus, of which, Sirt1 protein expression were suppressed by approximate 55% by Sirt1 shRNA-2055, it may lay solid foundation for the further research on the function of Sirt1.2. After Sirt1 expression was inhibited by lentiviral Sirt1 shRNA infection, preadipocytes showed distinguished morphological features of apoptosis under fluorescence microscope. Apoptosis rate in preadipocytes infected with Sirt1 shRNA was significantly increased compared with that of non-transfected or empty vector group, assessed with flow cytometric analysis. Western blotting analysis showed that inhibition of Sirt1 also upregulated the cleaved Caspase-3 expression. These results showed that lentiviral Sirt1 shRNA inhibit Sirt1 expression could induce cell apopotosis in porcine preadipocytes.3. In this study, we found that knockdown of Sirt1 enhanced camptothecin-induced apoptosis in porcine preadipocytes, which may due to the upregulation of p53, acetylated p53, Bax and the downregulation of Bcl-2.