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Effects and Molecular Mechanism of Sirt2 during Porcine Preadipocytes Differentiation

Author: LiuBingZuo
Tutor: YangGongShe
School: Northwest University of Science and Technology
Course: Zoology
Keywords: Sirt2 Adipocyte differentiation Overexpression Adenovirus vectors PGC-1β
CLC: S828
Type: Master's thesis
Year: 2010
Downloads: 171
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Abstract

Adipose tissue is an important metabolic organ, insulin sensitivity and the overall energy balance is essential. When the energy consumption is less than energy intake, excess triglycerides stored in the white adipose tissue, adipose tissue which causes or obese. Adipose tissue is also endocrine organs, regulates many physiological and pathological processes. Adipogenic factors such as secretion (adipokine), which regulate the body's physiological processes, including glucose metabolism, blood pressure, and reproductive functions. Therefore, adipose tissue and human health is closely related to research fat cell proliferation, differentiation, and apoptosis is very meaningful. In addition, pig and human physiological and biochemical characteristics closest human genes with the highest sequence conservation, but it's growth and fat deposition patterns of genetic regulation is similar to the study of human obesity and diabetes as an ideal model animal. Therefore, the study porcine preadipocytes differentiation is particularly important. Sirtuin family is a mammalian silent information regulator, is a dependent on nicotinamide adenine dinucleotide (NAD) of the deacetylase. Which, Sirt1 has been very in-depth research, but few studies Sirt2-7. And Sirt2 is the highest content of fat cells Sirtuin member. Has been demonstrated in 3T3-L1 cell line can Sirt2 interaction with FoxO1 to regulate adipocyte differentiation, but are there any other methods of control is unclear, and its porcine preadipocytes differentiation and its mechanism The studies have not been reported. 1 to 3-day-old Landrace pigs as experimental animals by collagenase digestion of pig preadipocytes. While building Sirt2 super expression vector, the adenovirus shuttle vector into Sirt2 pAdTrack-CMV, and the backbone vector pAdEasy-1 homologous recombination in E. coli BJ5183, with Lipofectamine2000 packaging recombinant HEK293 cells transfected, recombinant adenovirus vAd -Sirt2. With vAd-Sirt2 swine preadipocytes. Then using oil red O staining fat differentiation; Realtime PCR and Western Blot detection Sirt2 and differentiation marker genes PPARγ, aP2, C / EBPαmRNA and protein expression; while taking advantage of co-immunoprecipitation studies Sirt2 interaction with PGC-1β, to clear Sirt2 in the pig preadipocytes differentiation in the initial regulation to clarify Sirt2 pig preadipocytes differentiation molecular mechanisms for the control of pig fat deposition as well as provide the basis for human obesity and related diseases and prevention foundation. The main results obtained are as follows: 1. Cloned pigs Sirt2 length CDS, get two full-length isoforms, Sirt2 and splice mutant Sirt2T, respectively Login Genbank (FJ903384 and FJ903385). 2 splice mutant pig Sirt2 Sirt2T bioinformatic analysis, we found Sirt2T length of 1059bp, encoding 352 amino acids, the N-terminal deletion Sirt2 than 39 amino acids, belonging to the 5 'end of the cut form. 3 on the biological characteristics of Sirt2T found Sirt2T mRNA in the pig widely expressed in various tissues, especially highly expressed in the brain, fat, secondly; their porcine preadipocytes sustained expression during differentiation, differentiation of the first four days the highest expression ; and in both the nucleus and cytoplasm localization. Therefore, Sirt2T with Sirt2 have similar expression and localization, suggesting that it may have a similar function with Sirt2, inhibits adipocyte differentiation. 4 constructed Sirt2 adenovirus expression vector super vAd-Sirt2. 5. VAd-Sirt2 swine preadipocytes, compared with the control oil red O lipid droplets reduced morphology, Realtime PCR and Western blot adipocyte differentiation key genes PPARγ, aP2, C / EBPαmRNA and protein levels decreased, suggesting that overexpression inhibited Sirt2 pig preadipocytes differentiation. 6 overexpression Sirt2 not directly affect PGC-1βmRNA and protein levels, but through interaction with PGC-1β and deacetylation of PGC-1β to regulate. In summary, this study is the first discovered the existence of splice mutant pig Sirt2 Sirt2T, belonging to the 5 'end splicing forms; Sirt2T with Sirt2 have similar expression and localization, suggesting Sirt2T with Sirt2 have similar inhibitory role in adipocyte differentiation . Successfully constructed Sirt2 adenovirus vectors infected pigs overexpressing preadipocytes, morphology and molecular biology detected Sirt2 inhibiting differentiation of porcine preadipocytes. In addition, Sirt2 interaction with PGC-1β and deacetylation PGC-1β, thereby regulating pig preadipocytes differentiation.

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