Study Of Expression And Regulation Of Resistin Gene

Adipose tissue (AT) is a major metabolic organ. It can synthesize and storetriglyceride, and release it when organ needs to maintain energy balance. AT can secrete anumber of adipokines, such as TNFα, leptin, 1L-6, adiponectin and resistin etc. Theseadipokines express greatly in AT and are involve in regulating insulin action and glucosemetabolism linking to obesity and diabetes. The insulin resistance is namely theattenuation of the insulin physiological functions. Current studies indicated that manyadipokines were important in lipid metabolism, energy balance and insulin resistance.Resistin was a newly found adipokine. Researchers found it when use rosiglitazone totreat 3T3-L1 cells. Current studies indicated that resistin played key roles in inflammation,glucose tolerance and insulin resistance. However, resistin function in normal status,resistin's target, resistin's acceptor and signaling regulation need been clarified further.In this study, by choosing mouse and human resistin as subjects and usingsemi-quantitative RT-PCR, western blotting and transfection, we studied thetranscriptional regulation, expressional regulation and function of resistin and got thefollowing results:1. By the comparative analysis and semi-quantitative RT-PCR, according rat resistinsplicing mechanism, mouse resistin splicing form was cloned and the expressionpattern of resistin splicing form in nine tissue samples was determined. By analyzingmouse resistin splicing form sequence and comparing with rat and human resistinsplicing form, mouse resistin splicing form was found neither secretes, nor codesprotein.2. By RT-PCR, CDS of SREBP, CREB, C/EBPαand resistin were cloned; by PCR, thepromoter of resistin was cloned. Subsequently, their expression vectors wereconstructed, respectively. By transfection or co-transfection, their effects ontranscription of resistin were investigated. The results indicated that C/EBPαcouldenhance resistin transcription, but SREBP and CREB could not.3. By using insulin, rosiglitazone, LPS, dexamethasone and clenbuteroi, the effects on resisitn expression in macrophages were explored. Our data showed that lowerconcentration insulin inhibited expression of resistin and TNFα, stimulated PPARγexpression; high concentration insulin increased expression of resistin and TNFα,suppressed PPARγexpression. And rosiglitazone decreased resistin expression. ButLPS, dexamethasone and clenbuterol increased expression of resistin and TNFα.These data were the same as that obtained from adipocytes. It suggests resistin has thesame regulated mechanism between adipocytes and macrophages.4. By transfection, resistin was overexpressed in hepatic cells. Resistin overexpressionrepressed glucose uptake dramatically. By analyzing the expression of relative genes,the data indicated G6Pase expression was enhanced which controls hepatic glucoseproduction; however GLUT2 expression had no change which transfers glucose fromextracellular to intracellular. And the results demonstrated resistin overexpression alsoimpaired insulin sensitivity in hepatic cells.5. By western blotting, the effect of adipocyte-conditioned medium (CM) on hepaticinsulin resistance was monitored. The data showed CM induced a marked hepaticinsulin resistance. Subsequently, to investigate the role of recombinant resistin in thisprocess, its effect on insulin signal were measured. The results demonstratedrecombinant resistin suppressed insulin action and induced insulin resistance inhepatocyte. And its effect was dose-dependent. Therefore, recombinant resistin canuse to build insulin resistance model in hepatocyte.6. With different time and different dose treatment, by western blotting, the effects ofresistin on other signal pathways in hepatocytes were determined. It showed resistinactivated Erk, enhanced p65 expression and impaired phosphorylation of AMPK andACC. These might be the reason to explain why resistin could impair glucosetolerance and insulin sensitivity.