磷脂酰肌醇3激酶抑制剂诱导猪卵巢颗粒细胞胰岛素抵抗的研究

Induction of insulin resistance induced by PI-3K inhibitor in porcine granulosa cells

目的观察磷脂酰肌醇3激酶（PI-3K）抑制剂——沃曼青霉素（wortmannin）诱导猪卵巢颗粒细胞胰岛素抵抗（IR）后，其信号传导通路中葡萄糖转运蛋白4（GLUT4）、丝裂原激活蛋白激酶（MAPK）基因表达的变化。方法采用猪卵巢颗粒细胞体外培养，分别以浓度为0、1．0、1．5、3．0、5．0μmol／L的wortmannin作用48h后，以氚标记葡萄糖法及葡萄糖氧化酶法检测细胞的葡萄糖消耗量，以免疫荧光法分析GLUT4、MAPK蛋白定位表达情况，以RT-PCR方法对GLUT4、MAPK的mRNA进行半定量分析。结果浓度为1．5μmol／L的wortmannin降低细胞对葡萄糖的摄取量最高达40％（P〈0.05），残余葡萄糖含量增多约13．76％，出现明显的IR。免疫荧光法结果显示GLUT4、MAPK蛋白主要在细胞质表达。RT-PCR结果提示，GLUT4基因表达水平降低21．06％，MAPK表达水平升高56．43％。结论wortmannin可干扰细胞内的糖代谢信号传导通路，诱导IR的发生；同时可放大有丝分裂信号传导通路。

Objective To investigate the biological effects of insulin resistance （IR） on the porcine granulosa cells which is induced by wortmannin, the PI-3K inhibitor and mediated by key molecules including GLUT4 and MAPK during insulin signaling. Methods The model of IR porcine granulosa cell was established in in vitro culture by treatment of wortmannin, and was assessed the amount of ^3H glucose uptake as well as medium glucose levels by glucose oxidase method. The protein and mRNA expression of GLUT4 and MAPK were evaluated by immunofluorescence and RT-PCR respectively. Results The glucose intake was decreased by 40% with treatment of wortmannin at 1.5 μmol/L（P 〈0. 05）. GLUT4 and MAPK were localized mainly to cytoplasm of granulose cells. When granulosa cells were insulin resistant, the expression of GLUT4 was down-regulated whereas MAPK was up-regulated as compared with the controls. Conclusions Wortmannin treatment can lead to decreased expression of GLUT4 and increase of IR granulose cells. This metabolic phenotype could induce increased expression of MAPK and mitogenic potential, indicating the cross-talk between two pathways of insulin signaling within ovarian cells.