游离脂肪酸对大鼠骨骼肌细胞胰岛素受体与胰岛素受体底物1的表达和酪氨酸磷酸化的影响

EFFECTS OF FREE FATTY ACIDS ON INSULIN RECEPTOR SUBSTRATE 1 AND INSULIN RECEPTOR EXPRESSION AND TYROSINE PHOSPHORYLATION IN RAT SKELETAL MUSCLE CELLS

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（ＩＲＳ１）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ＩＲＳ１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ＩＲＳ１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ＩＲＳ１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ＩＲＳ１蛋白质表达和胰岛素受体的酪氨酸磷酸化以及ＩＲＳ１的酪氨酸磷酸化阻滞胰岛素信号传导。

Objective: To elucidate cellular mechanisms of insulin resistance induced by high free fatty acids, and study the protein abundance of insulin receptor, insulin receptor substrate 1(IRS 1) and their tyrosine phosphorylation in palmitate or oleate treated rats skeletal muscle cells in vitro . Methods: The skeletal muscle cells were isolated from Wistar rat. When the cells differentiated to confluent and auto contraction, the media were replaced by 199 with palmitate (0.25 mmol·L -1 ) or oleate (0.125 mmol·L -1 ) then cells were incubated for 6, 12 and 24 hours. The abundance of insulin receptor and IRS 1 was assessed by Western blot. The tyrosine phosphorylation of insulin receptor β subunit and IRS 1 was measured using immunoprecipitation and immunobloting with anti phosphor tyrosine antibodies. Results: (1)There was no change of insulin receptor levels in both fatty acids treated muscle cells. (2)The protein abundance of IRS 1 in palmitate and oleate treated muscle cells was decreased compared with control groups. (3)The insulin was less effective in stimulating tyrosine phospholation of insulin receptor and IRS 1 in fatty acids treated muscle cells. Conclusion: Decrease of IRS 1 protein abundance and tyrosine phosphorylation of insulin receptor β subunit and IRS 1 contributed significantly to the muscle insulin resistance induced by fatty acids.