Regulation Of Sirt1 On Pig Adipocytes Proliferation And Differentiation

The biology of adipose tissue has identified the adipocyte as an important mediator in many physiologic and pathologic processes regarding energy metabolism. Excess of fat deposition leads to human obesity and related metabolism disease, also causes the decrease in quality of domestic product. The decrease of fat deposition lies in controlling the proliferation and differentiation of adipocytes. The proliferation and differentiation of adipocytes is a dynamic and plastic process under the influence of various hormones, genes and signaling pathway. Sirt1, a novel regulatory factor of adipocyte and myocyte, interacts with target FoxO1 to modulate cell proliferation, differentiation, ageing, apoptosis and metabolism. It was recently hypothesized that Sirt1 might be a key candidate for the reduced adiposity. Thus, pig, which is the highest natural obese animal, was used as the experimental animal. Sirt1 cDNA sequence of pig was cloned and the pattern of tissue expression was investigated. Then, the regulatory role of Sirt1 in adipocytes proliferation and differentiation of pig was explored in this study. The main results were as follows:1. The length of Sirt1 cDNA sequence cloned by PCR technology was 530 bp and GenBank accession number was DQ868430. Homologous alignment analysis using Clustal W software showed that pig Sirt1 gene had 94%, 90% and 88% homology to that of human, rat and mouse respectively. Further CDD analysis indicated that pig Sirt1 protein translated from the cloned cDNA sequence contained the conserved core domain of Sirt family. This identified that pig Sirt1 was evolutionarily conserved in history.2. The expression of Sirt1 mRNA in various pig tissues from different developmental stages was investigated by Semi-quatitive RT-PCR. The results showed that Sirt1 was widely expressed in heart, liver, spleen, lung, kidney, skeletal muscle, subcutaneous adipose and visceral adipose from 3-day-old piglets and 180-day-old adult pigs, hinting that Sirt1 has extensively biological role in growth and development of pig. For piglets, Sirt1 mRNA was abundant in lung, kidney and spleen tissue and deficient in skeletal muscle and subcutaneous adipose. For adult pigs, the expression of Sirt1 was highest in kidney, higher in spleen and lowest in heart. Furthermore, the expression of Sirt1 mRNA in visceral adipose was significantly higher than subcutaneous adipose (P<0.05), showing that Sirt1 may lead to the differential fat deposition between subcutaneous adipose and visceral adipose tissue from adult pigs. Additionally, Sirt1 mRNA expression in liver (P<0.05), kidney, subcutaneous adipose and visceral adipose (P<0.01) from adult pigs was remarkably higher than piglets. However, Sirt1 mRNA expression in skeletal muscle from adult pigs was significantly lower than piglets (P<0.01). These results suggested that Sirt1 may exert different modulatory role in fat deposition and muscle development of pig from different developmental stages.3. The time-spatial expression of Sirt1 during primary pig adipocyte differentiation was detected by SQ RT-PCR. The results showed that Sirt1 was expressed throughout the entire differentiation process of pig preadipocytes. According to these results, it presumed that Sirt1 may be involved in regulating the process during pig preadipocytes differentiation. Moreover, the expression of Sirt1 mRNA in cells treatment with resveratrol was significantly higher than control (P<0.01). This confirmed that resveratrol is the potent activator for Sirt1.4. The effects of resveratrol, a potent activator of Sirt1, and nicotinamide, a strong inhibitor of Sirt1, on proliferation and differentiation of pig preadipocyte were tested by methylthiazolyldiphenyl-tetrazolium bromide (MTT), and Oil red O staining and extraction, and SQ RT-PCR. It showed that 100μmol/L resveratrol had the remarkable inhibitory role on proliferation and differentiation of pig preadipocyte, and up-regulated Sirt1 mRNA expression but down-regulated the expression of FoxO1 and PPARγ2. Conversely, 300μmol/L nicotinamide significantly promote the proliferation and differentiation of pig preadipocyte, and down-regulated Sirt1 mRNA expression but up-regulated the expression of FoxO1, PPARγ2, C/EBPα, aP2 and LPL. These findings indicated that activation of Sirt1 positively regulated the proliferation and differentiation of pig preadipocytes, and inhibition of Sirt1 negatively modulated the proliferation and differentiation of pig preadipocytes. In addition, it suggested that Sirt1 influenced the proliferation and differentiation of pig preadipocytes by regulating FoxO1 and adipocyte marker genes.