Effect And Molecular Mechanism Of The Sirt1 Gene Expression On Porcine Lipolysis

Sirtl and ATGL were recently identified and described as important factors in fat metabolism. In this study, the full-length cDNA sequences of porcine Sirtl (pSirtl) and porcine ATGL (pATGL) were cloned. The tissue-specific, developmental expression pattern and the breed differences of pSirtl and the key functional gene pATGL of porcine fat metabolism, the effects of pSirtl on the expression of fat metabolism related genes and the regulation mechanism of Sirtl on the fat metabolism were studied by using real-time PCR, RNA interference (RNAi) and immunohistochemical techniques. Furthermore, the regulations of pSirtl by TNF-a and Insulin were also studied. Results are as bellow:1. The full-length cDNA sequence of pSirtl and pATGL cloning and analysisThe full-length cDNA sequence of pSirtl and pATGL were obtained by RACE technology. The full-length cDNA sequences of pSirtl was 4024 bp (accession no: EU030283), which contains 15 bp of 5'-untranslated region (5'-UTR),2226 bp of coding region that encodes a 742-AA protein (accession no. ABS29571), and 1780 bp of 3'-UTR. The calculated molecular mass of this protein is 80.9 kDa, contained a Sirtl familial conserved domain. The BLAST analysis of the deduced AA sequences of pSirtl with those of the other species indicated that pSirtl sharing 87% similarity with human,92% with dog,91% with cattle and 82% with mouse. The full-length cDNA sequences of pATGL was 1958 bp (accession no:EF583921), which contains 33 bp of 5'-untranslated region (5'-UTR),1461 bp of coding region that encodes a 486-AA protein (accession no. ABS58651), and 464 bp of 3'-UTR. The calculated molecular mass of this protein is 53.2 kDa, contained a patatin-like domain. The BLAST analysis of the deduced AA sequences of pATGL with those of the other species indicated that pATGL sharing 87% similarity with cattle,84% with mouse, 83% with rat,81% with dog, and 80% with human. 2. The study on the tissue-specific, developmental expression pattern and breed differences of pSirtl and pATGLTissue-specific study results show that the pSirtl mRNA levels was highly expressed in brain, to a lesser degree in subcutaneous adipose tissue (SAT); pATGL mRNA levels was highly expressed in SAT. The developmental expression pattern of different growth stage study results show that in SAT, pSirtl and pATGL genes expression increased with body weight (BW). In peritoneal adipose tissue and in omental adipose tissue, the expression pattern of pSirtl and pATGL was not obviously. The breed differences study results showed that in the adipose tissue (SAT) the mRNA levels of pSirtl and pATGL in Jinhua pigs were lower than that of Landrace, and inversely proportional to the rate of fat (The fat rate of Jinhua pigs was significantly higher than the Landrace). In muscle (longissimus dorsi muscle), the mRNA levels pSirtl and pATGL in Jinhua pigs were higher than that of Landrace, and the mRNA levels of pSirtl and pATGL in longissimus dorsi muscle were positively correlated with the IMF (The IMF of Jinhua pigs was significantly higher than Landrace). These results indicated that pSirtl and pATGL play an important role in adipose metabolism and IMF deposition.3. Study on the effect and the regulation mechanism of Sirtl on the expression of porcine fat metabolism related genesIn vitro culture system of porcine adipocytes was successfully established. Using the pSirtl inhibitors (NAM) and agonist (RES) to explore the effect and the molecular mechanism of Sirtl on the fat metabolism. The effects of pSirtl on the expression of fat metabolism related genes were further studied by using RNA interference (RNAi) technology. The results showed that the expression of pSirtl and pATGL gene expression were increased with gradually, reaching a maximal value at 10 d and 8 d, respectively. pSirtl inhibitor (NAM) significantly decreased pSirtl, pATGL and HSL gene mRNA levels, promoted the differentiation and lipid accumulation of porcine adipocytes, and reduced the medium glycerol content. pSirtl agonist (RES) not only increased pSirtl gene expression, but also improve pATGL and HSL gene expression, inhibited the differentiation and lipid accumulation of porcine adipocytes, and increased the medium glycerol content. The effective siRNA expression vector of pSirtl (RS3) and pATGL (RA1) were screened out. The conditions were optimized and the interference effects reached 70% and 60%. Using pSirtl-siRNA interfered the pSirtl gene expression, the gene expression of pATGL and HSL was significantly reduced. Treated with RES50 and pSirtl-siRNA further proved that pSirtl can positive regulate pATGL and HSL gene expression. At the same time, we also found that inhibited the gene expression of pSirtl by RNAi or NAM significantly enhanced the PPARγgene expression; activated the pSirtl gene expression by RES significantly inhibitd the gene expression of PPARy. These results revealed that the reduced fat deposition and promoted lipolysis by pSirtl may be raised through the achieved expression of the fat metabolism key functional genes pATGL and HSL by pSirtl. PPARy signal pathway may play an important role in this process.4. Regulation of TNF-a and Insulin on the gene expression of pSirtlThe results showed that treated with different concentrations of TNF-α(1,12.5, 25,50,100 ng/ml) for 24 h or treated with 25 ng/ml TNF-αfor 12,24,36,48 h significantly decreased pSirtl gene expression and medium glycerin content. Treated with different concentrations of Insulin (10,50,100,1000 nM) for 24 h or treated with 50 nmol/L insulin for 24,36,48 h significantly reduced the gene expression of pSirtl and the medium glycerol content. These results indicated that treated with a certain concentration of TNF-αand Insulin for a certain period of time can inhibit pSirtl gene expression and lipid droplets lipolysis in adipocytes. At the same time, The study also found that TNF-αand Insulin treatment can also significantly reduce pATGL and HSL gene expression. This results further confirmed that pSirtl control fat lipolysis may be through the achieved gene expression of pATGL and HSL by pSirtl. Through RNAi technology and real-time PCR technology to further study the Insulin and pSirtl-siRNA synergies on pSirtl gene expression. The results showed that Insulin+pSirtl-siRNA-treated group has additive inhibitory effect on the pSirtl, at the same time significantly down-regulated the pATGL and HSL gene expression. These results revealed that Insulin and pSirtl-siRNA has synergetic inhibition on the expression of fat mobilization key genes pSirtl, pATGL and HSL.