visfatin对体外脂肪细胞胰岛素受体底物和PI3K表达的影响

目的：从胰岛素经典信号传导通路磷脂酰肌醇-3-激酶（PI3K）角度探讨内脏脂肪素（visfatin）与2型糖尿病（T2DM）胰岛素抵抗（IR）的关系。方法：复苏、传代和诱导分化人源T2DM前脂肪细胞，构建 visfatin过表达载体，进行载体转化、培养和提取；以4个不同表达梯度（0.0、1.0、2.5和5.0 μg）转染传代脂肪细胞，以0.0 μg组为对照组，其余3组为观察组；Q-PCR法检测visfatin 、胰岛素受体底物1（IRS-1）、胰岛素受体底物2（IRS-2）和 PI3K（P85α） mRNA表达水平，Western blotting法检测visfatin、IRS-1、IRS-2和PI3K（P85α） 蛋白表达水平及IRS-1和IRS-2酪氨酸磷酸化水平，[³H]-2-脱氧-D-葡萄糖摄取法测定细胞葡萄糖摄取率的变化。结果：各组 visfatin mRNA及蛋白表达水平随转染浓度梯度升高而升高（P<0.01），所构建visfatin过表达载体有效。随visfatin表达增加，各组IRS-1和PI3K（P85α） mRNA和蛋白表达水平以及IRS-1磷酸化程度均明显升高（P< 0.01），但IRS-2 mRNA和蛋白表达水平未出现明显变化（P>0.05）。脂肪细胞的葡萄糖摄取率随visfatin表达增加而升高（P<0.05）。结论：体外脂肪细胞visfatin过表达可增加IRS-1和PI3K的表达水平。

Influence of visfatin in expressions of insulin receptor substrate and PI3K in fat cells in vitro

Objective:To investigate the relationship between visfatin and insulin resistance (IR)of type 2 diabetes mellitus(T2DM) through the classic insulin signaling pathway phosphatidy inositol-3 kinase (PI3K).Methods:The human T2DM preadipocyte cells were recoveried, extended and differentiated.The visfatin overexpression vectors were built, transformated, cultivated and extracted.The fat cells were transfected with different overexpression levels (0.0, 1.0, 2.5 and 5.0 μg);0.0 μg group was used as control group,and the remaining three groups as observation groups.The mRNA expression levels of visfatin, insulin receptor substrate-1(IRS-1), insulin receptor substrate-2(IRS-2) and PI3K (P85α) were detected by Q-PCR.The protein expression levels of visfatin, IRS-1, IRS-2, PI3K (P85α), IRS-1 and IRS-2 phosphorylation levels were measured by Western blotting method.The glucose uptake rates of fat cells were determined by [3H]-2-deoxidation-D-glucose uptake assay.Results:The expression levels of visfatin mRNA and protein in various groups were increased with the increase of transfection concentration gradient (P<0.01);and the constructed overexpression vector of visfatin was effective.With the increase of visfatin expression, the mRNA and protein expression levels of IRS-1 and PI3K (P85α) and IRS-1 phosphorylation degrees in various groups were increased (P<0.01), but the IRS-2 mRNA and protein expression levels had no obvious changes(P>0.05).The glucose uptake rates of fat cells were elevated with the increasing of visfatin expression (P<0.05).Conclusion:The visfatin overexpression of fat cells in vitro can increase the expression levels of IRS-1 and PI3K.