二甲双胍通过抑制核糖体40S小亚基S6K蛋白激酶(P70S6)调节体外人颗粒细胞胰岛素受体底物-1(IRS-1)的表达  

Effect of metformin-induced stimulation on the expression of insulin receptor substrate 1(IRS-1) through negative regulation of P70S6k

作　　者：张永芳[1] 王蒙蒙[1] 李昕[1] 王永峰[1] 裴秀英[1] 王燕蓉[1] 马会明[1] 徐仙[1] 

机构地区：[1]宁夏医科大学,生育力保持教育部重点实验室/宁夏回族自治区生殖与遗传重点实验室,宁夏医科大学总医院生殖医学中心,人体解剖与组织胚胎学系,银川753600

出　　处：《生殖与避孕》2015年第3期146-153,共8页Reproduction and Contraception

基　　金：国家自然科学基金(31200882);宁夏医科大学校级(XZ2012006)资助项目

摘　　要：目的:探讨二甲双胍对核糖体40S小亚基S6K蛋白激酶(P70S6k)及胰岛素受体底物-1(IRS-1)蛋白及其ser307位点磷酸化表达的影响。方法:利用0.1 mmol/L二甲双胍体外连续作用多囊卵巢综合症(PCOS)患者黄素化颗粒细胞24 h为实验组,设未加二甲双胍培养的颗粒细胞为对照组。通过RT-PCR和Real-time PCR检测P70S6k和IRS-1的表达,细胞免疫荧光化学和Western blotting的方法检测P70S6k、p-thr389-P70S6k、IRS-1及p-ser307-IRS-1蛋白的表达。结果:Real-time PCR结果显示实验组P70S6k和IRS-1m RNA水平显著低于对照组,差异有统计学意义(P<0.05)。细胞免疫荧光化学检测IRS-1和p-ser307-IRS-1蛋白在颗粒细胞胞质表达,P70S6k和p-thr389-P70S6k蛋白在细胞核表达。Western blotting法结果显示二甲双胍作用24 h前、后卵巢颗粒细胞P70S6k、p-thr389-P70S6k、IRS-1及p-ser307-IRS-1蛋白表达有统计学差异(P<0.05),灰度值分析显示添加二甲双胍组P70S6k、p-thr389-P70S6k蛋白表达显著升高,IRS-1及p-ser307-IRS-1蛋白表达显著下降(P<0.05)。结论:二甲双胍可抑制人颗粒细胞P70S6k的表达,并通过Akt/P70S6k/IRS途径调节IRS-1的表达,从而增加颗粒细胞胰岛素的敏感性。Objective： To study the effects of metformin treatment on the expression of 70 kDa ribosomal protein S6 kinase （P70S6k）, insulin receptor substrate 1 （IRS-1） and IRS-1 Ser307 phosphorylation in granulosa cells. Methods： Granulosa cells were cultured in DMEM media supplemented with 0.1 mmol/L metformin for 24 h, and only cultured in DMEM media in the control. The expressions ofP70S6k, 1RS-1 mRNA were detected by RT-PCR and Real-time PCR. P70S6k, IRS-1, p-ser307-IRS-1 and p-thr389-P70S6k protein expressions were detected by immunohistochemistry and Western blotting. Results： P70S6k mRNA level showed an increasing tendency, whereas a decrease on the IRS-1 mRNA level was observed by Real-time PCR （P〈0.05）.IRS-1, p-ser307-IRS-1, P70S6k and p-thr389-P70S6k protein level were significantly different after metformin treatment for 24 h, as compared with the control （P〈0.05）. Compared the mean intensity of bands of Western blotting, in the metformin treatment group P70S6k and p-thr389-P70S6k protein level was significantly higher, and IRS-1, p-ser307-IRS-1 level was significantly lower （P〈0.05）. Conclusion： Metformin treatment inhibited the expression of P70S6k mRNA and protein levels in granulosa cells, and improved insulin sensitivity with altered IRS-1 expression via an Akt/P70S6k/IRS-1-dependent pathway.

关 键 词：二甲双胍 核糖体40S小亚基S6K蛋白激酶(P70S6k) 胰岛素受体底物-1(IRS-1) 人颗粒细胞 

分 类 号：R965[医药卫生—药理学]