游离脂肪酸抑制大鼠肝细胞胰岛素受体和胰岛素受体底物-1酪氨酸磷酸化

Free fatty acids down regulate tyrosine phosphorylation of insulin receptor and insulin receptor substrate 1 in rat hepatic 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胰岛素在肝细胞内的信号传导。方法 分离培养Ｗｉｓｔａｒ 大鼠肝细胞，以软脂酸（０ ．２５ ｍ ｍｏｌ／Ｌ） 或油酸（０．１２５ ｍ ｍｏｌ／Ｌ） 与肝细胞共同孵育６ 、１２ 、２４ 小时，提取蛋白后用Ｗｅｓｔｅｒｎ 印迹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） 软脂酸和油酸实验组肝细胞的胰岛素受体蛋白表达量在各个时间点均无改变。（２） 肝细胞与软脂酸和油酸共同孵育１２ 和２４ 小时后胰岛素受体β亚单位的酪氨酸磷酸化显著降低（ Ｐ＜ ０ ．０５） 。（３） 肝细胞与软脂酸或油酸共同孵育６ 小时，ＩＲＳ１ 蛋白量无明显变化，而１２ 和２４ 小时后则显著降低。（４）软脂酸和油酸实验组的肝细胞在胰岛素刺激后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ＩＲＳ１蛋白表达、胰岛素受体和ＩＲＳ１ 的酪氨酸磷酸化阻滞胰岛素信号传导，引起胰岛素抵抗。

Objective To examine whether the insulin resistance induced by free fatty acids is associated with altered insulin receptor signal system. Methods Hepatic cells were isolated from Wistar rats. The hepatic cells were incubated with palmitate (0.25 mmol/L) or oleate (0.125 mmol/L) for 6, 12 and 24 hours respectively. One group of cells served as control and the cells were not incubated with any free fatty acid. The abundance of insulin receptor and IRS 1 was assessed by Western blot. The tyrosine phosphorylation of insulin receptor subunit and IRS 1 was measured by immunobloting with anti phosphotyrosine antibodies. Results (1) There was no difference of insulin receptor levels among hepatic cells treated by palmitate and oleate and contorl cells. (2) The protein abundance of IRS 1 in palmitate and oleate treated hepatic cells decreased at 12 and 24 time points. (P<0.05, vs control). (3) In palmitate or oleate treated hepatic cells, insulin was less effective in stimulating tyrosine phosphorylation of insulin receptor at 12 and 24 hour time points (P<0.05, vs control). (4) The insulin stimulated tyrosine phosphorylation of IRS 1 decreased at all time points in palmitate or oleate treated hepatic cells (P<0.05, vs control). Conclusion Decrease of IRS 1 protein abundance and tyrosine phosphorylation of insulin receptor β subunit and IRS 1 might play a role in free fatty acid induced hepatic insulin resistance.