二甲双胍抑制肝脏糖异生的机制研究  

On the mechanism of metformin-inhibited hepatic gluconeogenesis

作　　者：唐红菊[1] 张玉青[1] 王晓[1] 张娟[1] 邓儒元[1] 刘赟[1] 李凤英[1] 周丽斌[1] 

机构地区：[1]上海市内分泌代谢病研究所,上海交通大学医学院附属瑞金医院内分泌代谢病科,上海市内分泌代谢病临床医学中心,200025

出　　处：《中华内分泌代谢杂志》2013年第11期971-976,共6页Chinese Journal of Endocrinology and Metabolism

基　　金：国家自然科学基金项目（81070652,81070617,81170720,81270910）;上海市卫生局课题（2010-093,2011-217）

摘　　要：目的研究二甲双胍抑制肝脏糖异生的分子机制。方法采用改良的胶原酶灌流法分离小鼠肝脏原代细胞；葡萄糖氧化酶法检N--甲双胍对肝脏原代细胞葡萄糖产生的影响；实时定量PCR检测糖异生相关基因mRNA水平的变化；双荧光素酶报告基因法检N--甲双胍对糖异生关键基因启动子活性的调节；Western印迹法检测相关基因的蛋白表达及磷酸化水平。结果二甲双胍呈时间和剂量依赖性地降低肝脏原代细胞以乳酸和丙酮酸为底物的糖异生，降低肝糖异生关键酶磷酸烯醇型丙酮酸羧激酶（PEPCK）、葡萄糖-6-磷酸酶（G6pase）和相关转录因子FOX01、CCAAT增强子结合蛋白（C／EBP）a、C／EBPβ的mRNA水平。2mmol／L二甲双胍作用24h均使PEPCK、G6pase启动子活性降低34％。二甲双胍也明显降低PEPCK的蛋白表达。2mmol／L二甲双胍作用1h刺激AMP活化的蛋白激酶（AMPK）和乙酰辅酶A羧化酶（ACC）的磷酸化，该作用可被AMPK抑制剂CompoundC对抗。二甲双胍对cAMP和地塞米松刺激的cAMP反应元件结合蛋白（CREB）磷酸化无明显作用。结论二甲双胍通过激活AMPK下调糖异生关键基因的表达，抑制小鼠肝原代细胞的糖异生，其作用不依赖于CREB磷酸化的抑制。Objective To investigate the underlying molecular mechanisms of metformin in inhibiting hepatic gluconeogenesis. Methods Primary hepatocytes of mice were isolated by a modified version of the collagenase method. The effect of metformin on glucose production in primary hepatocytes was detected by glucose oxidation method. The expression of mRNA of hepatic gluconeogenesis genes was tested by real time PCR. The effects of metformin on promoter activity of hepatic gluconeonesis key genes were measured by Dual-luciferase Reporter Assay. Western blot was used to detect the protein expression and phosphorylation of related genes. Results Metformin inhibited hepatic gluconeogenesis from the substrate pyruvate/lactate in a dose- and time-dependent manner, along with decreased mRNA levels of hepatic gluconeogenesis genes such as phosphoenolpyruvate carboxykinase （PEPCK） , glucose-6-phosphatase （G6pase） , FOXO1, CCAAT/enhaneer-binding protein （C/EBP） a, and C/EBP. The promoter activities of both PEPCK and G6pase were decreased by 34% after 24h exposure to 2 mmol/L merformin. The protein level of PEPCK was significantly reduced in the presence of 2 mmol/L metformin. The phosphorylations of AMP-activated protein kinase （AMPK） and acetyl-CoA carboxylase （ ACC ） were elevated after treatment with 2 mmol/L metformin for 1 h, and were blunted by pretreatment with compound C. Metformin had no effect on cAMP/dexamethasone-stimulated cAMP response element-binding protein （ CREB ） phosphorylation. Conclusions Metformin suppresses hepatic gluconeogenesis via activating AMPK and decreasing the expressions of the key genes of hepatic gluconeogenesis without decreasing the phosphorylation of CREB.

关 键 词：二甲双胍 肝糖异生 磷酸烯醇型丙酮酸羧激酶 AMP活化的蛋白激酶 cAMP反应元件 结合蛋白 

分 类 号：R587.1[医药卫生—内分泌]