胰岛素抵抗骨骼肌细胞中P-Ser473PKB及糖原合成酶-3表达

摘要

Objective To study on the expression of P-Ser473PKB and GSK-3 with insulin resistance (IR)in skeletal muscle cells induced by palmitinic acid(PA) and the role of P-Ser473PKB and GSK-3 in IR of skeletal muscle cells.Methods Rat skeletal muscle cells were cultivated in Dulbecco's modified eagle's medium(DMEM) ,and the cells were identified by the technology of immunofluorescence.Control group and PA group were set,and the glucose concentrations were measured by GOD-POD method at 6 h,12 h and 24 h before the cells were suscitated with insulin for 15 minutes.Phosphorylated forms of protein kinase B (P-Ser473 PKB) and glycogen synthase kinase-3 alpha/beta ( P-Ser21/9 GSK-3α/β) were determined by Western blot.Results Immunofluorescence showed that over 90 ％ of cells were positive reflection for α-sarcometric actin monoclonal antibody staining,which proved the cells to be the skeletal muscle cells.After 0.6 mmol/L PA treatment for 24 h,the glucose concentrations in DMEM were found to be higher than the control group.Protein level of P-Ser473 PKB in PA group significantly decreased while P-Ser21/9 GSK-3α/β significantly increased compared to control group.Conclusion In high fat condition,the ability of skeletal muscle cells treatment to glucose was decreased in insulin suscitation condition,and this showed high fat could induce insulin resistance in skeletal muscle cells,the mechamsm possibly associated with the depression in protein level of P-Ser473 PKB and increase of P-Ser21/9 GSK-3α/β.%目的 探讨棕榈酸(PA)诱导的胰岛素抵抗(IR)骨骼肌细胞中P-Ser473PKB及糖原合成酶(GSK)-3的表达.方法 培养大鼠骨骼肌细胞,免疫荧光鉴定原代大鼠骨骼肌细胞;设立对照组、PA组(0.6 mmol/L PA),在细胞培养6、12、24 h后,胰岛素刺激15 min,葡萄糖氧化酶-过氧化物酶偶联(GOD-POD)法测定培养液中葡萄糖的浓度;Western blot检测P-Ser473PKB及P-Ser21/9GSK-3α/β的蛋白表达.结果 90%以上的肌细胞α-sarcometric actin单克隆抗体染色呈阳性,证明培养的为骨骼肌细胞;培养24 h后,PA组培养液中葡萄糖浓度高于对照组,P-Ser473PKB 蛋白水平低于对照组,而P-Ser21/9GSK-3α/β高于对照组(P值均＜0.05).结论 PA作用下,胰岛素刺激的骨骼肌细胞对葡萄糖的处理能力下降,即PA可诱导骨骼肌细胞产生胰岛素抵抗,其机制可能与P-Ser473PKB蛋白表达下调,P-Ser21/9GSK-3α/β表达增加有关.