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目的:探讨游离脂肪酸是否通过改变胰岛素信号传导蛋白的表达和功能状态影响骨骼肌细胞的葡萄糖代谢。方法:分离培养Wistar大鼠骨骼肌细胞。软脂酸(0.25mmol·L-1)或油酸(0.125mmol·L-1)与骨骼肌细胞共同孵育6、12、24h,提取蛋白后用Western杂交方法检测胰岛素受体和胰岛素受体底物1(IRS1)的蛋白水平,同时应用免疫沉淀和免疫杂交技术检测胰岛素刺激后胰岛素受体β亚单位和IRS1的酪氨酸磷酸化程度。结果:(1)软脂酸或油酸作用后骨骼肌细胞的胰岛素受体蛋白质表达量无改变。(2)骨骼肌细胞与软脂酸或油酸共同孵育12和24h后胰岛素受体β亚单位的酪氨酸磷酸化显著降低。(3)骨骼肌细胞在软脂酸或油酸作用后各个时间点的IRS1蛋白质量均明显降低。(4)骨骼肌细胞经软脂酸或油酸作用后IRS1的酪氨酸磷酸化在各个时间点均显著降低。结论:游离脂肪酸可能通过抑制骨骼肌细胞的IRS1蛋白质表达和胰岛素受体的酪氨酸磷酸化以及IRS1的酪氨酸磷酸化阻滞胰岛素信号传导,引起骨骼肌胰岛素抵抗
AIM: To investigate whether free fatty acids affect glucose metabolism in skeletal muscle cells by altering the expression and functional status of insulin signaling proteins. Methods: Skeletal muscle cells of Wistar rats were isolated and cultured. Palmitic acid (0.25mmol·L-1) or oleic acid (0.125mmol·L-1) were incubated with skeletal muscle cells for 6, 12 and 24 hours respectively. After the protein was extracted, the insulin receptor and insulin receptor Body substrate 1 (IRS 1) protein levels, immunoprecipitation and immune hybridization techniques used to detect insulin-stimulated insulin receptor β subunit and IRS 1 tyrosine phosphorylation. Results: (1) There was no change of insulin receptor protein expression in skeletal muscle cells after palmitate or oleic acid. (2) Tyrosine phosphorylation of insulin receptor β subunit was significantly reduced after co-incubation of skeletal muscle cells with palmitate or oleic acid for 12 and 24 h. (3) skeletal muscle cells in palmitic acid or oleic acid at various time points after the IRS 1 protein levels were significantly lower. (4) tyrosine phosphorylation of skeletal muscle cells after palmitate or oleic acid IRS 1 were significantly reduced at all time points. Conclusion: Free fatty acids may cause skeletal muscle insulin resistance by inhibiting IRS-1 protein expression in skeletal muscle cells and tyrosine phosphorylation of insulin receptor and tyrosine phosphorylation of IRS-1 in insulin signaling