Relationships Between SIRT1 Genes And Insulin Receptors In 3T3-L1 Adipocytes

Background and objectiveWith the economic and social development, our eating habits have been changed in recent years. The incidence of obesity and type 2 diabetes mellitus (T2DM) is increasing year by year. So the research on diabetes and obesity has become a hot point in biological and medical fields. Current understanding of pathogenesis of T2DM has not stayed in the level of insulin deficiency, while a growing number of studies have shown that obesity and insulin resistance play a more and more important role in the development of T2DM.Insulin is the principal hormone that regulates uptake of glucose from the blood into most cells. Therefore deficiency of insulin or insensitivity of its receptors plays a central role in all forms of diabetes mellitus. It is known that insulin plays its role by following these steps: first, the insulin binds to the insulin receptor'sαsubunit, and then its conformation changes which leads to an activation of the insulin receptor'sβsubunit. The auto-phosphorylation ofβsubunit can lead to a tyrosine phosphorylation of IRS-1 which can activate PI3K. The activated PI3K can promote the glyco-transportation and play a role in other functions.Silent information regulator 2 (Sir2), firstly discovered in yeast cell in the eighties, is an important regulation gene which can extend life of a considerable number of species. It participates in gene silencing and maintains the stability of rDNA recombination. It has histone deacetylation activity. In mammals, there are seven Sir2 homologues, named SIRT1-SIRT7. They play a significant part in cell cycle regulation, DNA damage repair, inhibition of apoptosis and longevity. In recent years, it has been found that SIRT1 also plays an important role in the development of lipid metabolism, glycometabolism, cardiovascular diseases and tumor.It has been proved that SIRT1 can act on many factors in Insulin/IGF-1 signaling pathway, such as PPARγ, FOXO, IRS-1, etc, to improve the insulin signaling transportation, so as to regulate the lipid metabolism and glyco-metabolism. Can SIRT1 act on the most upstream gene INSR of Insulin/IGF-1 signaling pathway?Can SIRT1 regulate the number and the affinity of insulin receptors, to control the whole pathway of glycometabolism? We conclude that, there must be some relationships between SIRT1 and insulin receptors in adipocytes. SIRT1 probably act on the most upstream gene INSR of Insulin/IGF-1 signaling pathway to regulate the expression of INSR, thus participate in the glycometabolism and lipid metabolism.In this study, we will research the effect of SIRT1 gene upon INSR gene and protein in adipocytes by transfecting 3T3-L1 cells. Our research helps studying on the molecular mechanisms of diabetes, as well as exploring the mechanisms and treatment of insulin resistence.Materials and methodsWe use the internationally recognized 3T3-L1 preadipocytes as the study objects. 3T3-L1 cells were cultured and differentiated into mature adipocytes under certain conditions, then used red oil O straining to identify. Transfected the adipocytes with Ad-mSirt1-IRES-eGFP in order to make a high expression of SIRT1 gene, and then detected the expression of INSR gene and protein. Samples were divided into 2 groups: control group and Ad-mSirt1-IRES-eGFP transfection group. We extracted total RNA and detected SIRT1 gene and INSR gene by real time PCR (Q-PCR). We detected SIRT1 protein and INSR protein by Immunocytochemical assay and Western blot.Results1. Immunocytochemical assay showed that, both SIRT1 protein and INSR protein were highly expressed contrast to the control group.2. Western blot showed that, both SIRT1 protein and INSR protein were high expressed contrast to the control group.3. Q-PCR showed that, contrast to the control group, SIRT1 gene was up-regulated while the INSR was down-regulated in the transfection group (it was probably due to the inaccuracy in the process of the Q-PCR).ConclusionsThe up-regulation of SIRT1 gene in mature adipocytes differentiated from 3T3-L1 preadipocytes can affect the expression of INSR. In our experiment, it showed that, compared with the control group, SIRT1 gene was down-regulated, while the expression of INSR protein was up-regulated in the transfection group. There is a contradiction here between the expression of gene and protein. It was probably due to the inaccuracy in the process of the Q-PCR. Generally speaking, The WB is more authoritative and reliable,and it can show the final expression of the protein, which was also coincidence with the results of the immunocytochemical assay and our expectation, so the WB result shall prevail. From the above, we can see that SIRT1 can act on the most upstream gene INSR of Insulin/IGF-1 signaling pathway and regulate the expression of INSR, thus elevate the insulin sensitivity, relieve the insulin resistance and delay the development of T2DM.