游离脂肪酸对大鼠肝细胞瘦素受体基因、蛋白表达及酪氨酸磷酸化的影响

目的 :探讨游离脂肪酸 (FFA)是否会对大鼠肝细胞瘦素 (leptin)受体的基因、蛋白表达及酪氨酸磷酸化产生一定的影响。　方法 :分离、培养新生Sprague Dawley大鼠肝细胞 ,分别与软脂酸 (0 .2 5mmol/L)或油酸 (0 .12 5mmol/L)孵育 12、2 4和 36h ,提取蛋白后用Western印迹法检测瘦素受体的蛋白水平。用RT PCR检测肝细胞内瘦素受体RNA含量的变化。应用免疫沉淀法检测瘦素受体的酪氨酸磷酸化程度。　结果 :软脂酸和油酸孵育后大鼠肝细胞瘦素受体的蛋白表达水平在孵育 12和 2 4h后无显著变化 ,在孵育 36h后显著下调 (P <0 .0 5 ) ;瘦素受体的RNA水平在孵育 12h后无显著变化 ,孵育 2 4和 36h后显著下调 (P <0 .0 5 ) ;瘦素受体的酪氨酸磷酸化程度在孵育 12h后无显著变化 ,孵育 2 4和 36h后显著下调 (P <0 .0 5 )。　结论 :FFA可对大鼠肝细胞瘦素受体的基因、蛋白表达水平及磷酸化程度产生一定的抑制作用 ,这可能是FFA导致胰岛素抵抗的机制之一。

Effects of free fatty acids on gene and protein expression and tyrosine phosphorylation of leptin-receptor in rat hepatic cells

Objectives: The effects of free fatty acids on gene and protein expression and tyrosine phosphorylation of leptin receptor in rat hepatic cells were investigated. Methods: Hepatic cells were isolated from new born Sprague Dawley rats. After the rat hepatic cells were incubated with palmitate (0.25 mmol/L) or oleate (0.125 mmol/L) for 12, 24 and 36 hours, Western bolt was used to assess the protein abundance of leptin receptor in rat hepatic cells. The RNA expression level of leptin receptor was revealed by RT PCR .The tyrosine phosphorylation of leptin receptor was revealed by immuno precipitation. Results: After incubated with free fatty acids(palmitate and oleate) for 12 and 24 hours, there was no difference of the protein content of leptin receptor among rat hepatic cells treated by palmitate and oleate and the control cells( P >0.05). The protein abundance of leptin receptor in rat hepatic cells was significantly decreased compared with the control after incubated with palmitate and oleate for 36 hours( P <0.05). The RNA content of leptin receptor in rat hepatic cells was similar to the control after incubated with palmitate and oleate for 12 hours( P >0.05),but significantly decreased after 24 or 36 hours( P <0.05).The tyrosine phosphorylation of Leptin receptor was remarkably reduced after incubated with palmitate and oleate for 24 or 36 hours( P <0.05). Conclusions: Free fatty acids(palmitate and oleate) may down regulate leptin receptor gene and protein expression and suppress its tyrosine phosphorylation in rat hepatic cells and may contribute to the occurrence of insulin resistance through inhibiting glucose metabolism and insulin action.