Effects And Molecular Mechanism Of Sirt2 During Porcine Preadipocytes Differentiation

Adipose tissue is an essential metabolism organ which plays a key role in insulin sensitivity and energy balance. When energy expenditure is lower than energy intake, excess triglyceride stores in white adipose tissue, which leads to fat hyperplasia and obesity. Adipose tissue also acts as an endocrine organ regulating multiple physiological and pathological processes, such as, secreting adipokine which modulates wholebody physiological processes, including glucose metabolism, blood pressure and reproduction. Therefore, according to adipose tissue which is closely related to human health, it is extraordinary useful to study adipocyte proliferation, differention and apoptosis processes. In addition, physiological and biochemical characteristics of pig are close to that of human, gene sequences of pig have the highest conservation with that of human, and growth and fat deposition genetic regulating modes of pig are similar with that of human, thus pig is the ideal animal of studying human obesity and diabetes. In a word, to study pig preadipocyte differentiation is especially important .In mammals, the homologs of Sir2 have been named Sirtuins (Sirt) which are NAD+-dependent deacetylase, with seven members in a family termed Sirt1 through Sirt7. Sirt1 is the best-charactered number of Sirtuins. It is known that Sirt2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. There are studies revealed that Sirt2 could interact with and deacetylate FoxO1 to suppress preadipocyte differentiation in 3T3-L1 cells. It is unclear that there are other regulating modes, and no reports show the role and the mechanism of Sirt2 in porcine preadipocyte differentiation.In this study, Sirt2 were inserted into pAdTrack-CMV. Then the linearized shuttle vector were introduced into a strain of BJ5183 bacterial cells that harbored the supercoiled backbone vector pAdEasy-1. Finally, PacⅠ-digested recombinant adenoviral DNA were transfected into HEK293 cells by Lipofectamine2000. Consequently we successfully gained recombinant adenoviruse vAd-Sirt2. Next, porcine preadipocytes were infected with vAd-Sirt2, after 48 h cells were observed by Oil Red O staining and adipocyte differentiation marker genes (like PPARγ, aP2 and C/EBPα) were examined by Realtime PCR and Western Blot. We found that overexpression of Sirt2 decreases lipid droplets, meanwhile the mRNA and protein level of marker genes are significantly reduced. In addition, IP revealed that Sirt2 could interact with and deacetylate PGC-1β. In a word, evidences suggested that Sirt2 could suppress porcine preadipocytes differentiation, which lay foundation for controlling pig body fat deposition and treating and preventing human obesity and related diseases.The main results were as follows:1. Pig Sirt2 gene was successfully cloned the complete CDS. Sirt2 and an alternative splice variant of Sirt2 were gained and submitted to Genbank (FJ903384 and FJ903385).2. According to bioinformatics analysis, alternative splicing variant of Sirt2 (Sirt2T) CDS is 1059bp, encoded 352 amino acids that is lack 39aa than Sirt2. It belongs to 5'splice sites.3. Sirt2T mRNA express in nearly all tissues of porcine, and continuously express at 0-6 day during porcine preadipocytes differentiation and reach the peak at the 4th day. In addition, Sirt2T are in nuclear and in cytoclasm. Sirt2T and Sirt2 have the same expression and distribution which reveals that Sirt2T may also suppress preadipocytes differentiation.4. Recombinant Adenovirus Vector vAd-Sirt2 was successfully constructed.5. Overexpression of Sirt2 gene could suppress porcine preadipocytes differentiation. It was shown that the number of adipocytes, fat content in adipocytes, and the abundance of PPARγ,aP2 and C/EBPαmRNA and protein decreased.6. Overexpression of Sirt2 didn't direactly affect the mRNA and protein level of PGC-1β, but could interact with and deacetylate PGC-1β.Taken together, an alternative splicing variant of porcine Sirt2 (Sirt2T) was found at the first time, which belongs to 5'splice sites. Sirt2T and Sirt2 have the same expression and distribution which reveals that Sirt2T may also suppress preadipocytes differentiation. Overexpression of Sirt2 gene could suppress porcine preadipocytes differentiation. The effects of Sirt2 on the porcine preadipocyte differentiation closely relate to its influence on the expression of PPARγ, aP2 and C/EBPαmRNA and protein. In addition, Sirt2 could interact with and deacetylate PGC-1β. Therefore, Sirt2 may suppress adipocyte differentiation by regulating PGC-1βacetylation.