CAJ | 학술논문

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;葡萄糖氧化酶法检测二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测二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FOXO1、CCAAT增强子结合蛋白(C/EBP)α、C/EBPβ的mRNA水平.2 mmol/L二甲双胍作用24 h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.2 mmol/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Compound C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.
목적 연구이갑쌍고억제간장당이생적분자궤제.방법 채용개량적효원매관류법분리소서간장원대세포;포도당양화매법검측이갑쌍고대간장원대세포포도당산생적영향;실시정량PCR검측당이생상관기인mRNA수평적변화;쌍형광소매보고기인법검측이갑쌍고대당이생관건기인계동자활성적조절;Western인적법검측상관기인적단백표체급린산화수평.결과 이갑쌍고정시간화제량의뢰성지강저간장원대세포이유산화병동산위저물적당이생,강저간당이생관건매린산희순형병동산최격매(PEPCK)、포도당-6-린산매(G6pase)화상관전록인자FOXO1、CCAAT증강자결합단백(C/EBP)α、C/EBPβ적mRNA수평.2 mmol/L이갑쌍고작용24 h균사PEPCK、G6pase계동자활성강저34％.이갑쌍고야명현강저PEPCK적단백표체.2 mmol/L이갑쌍고작용1h자격AMP활화적단백격매(AMPK)화을선보매A최화매(ACC)적린산화,해작용가피AMPK억제제Compound C대항.이갑쌍고대cAMP화지새미송자격적cAMP반응원건결합단백(CREB)린산화무명현작용.결론 이갑쌍고통과격활AMPK하조당이생관건기인적표체,억제소서간원대세포적당이생,기작용불의뢰우CREB린산화적억제.
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/enhancer-binding protein (C/EBP) α,and C/EBPβ.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.