乳酸不依赖于丙酮酸参与调节C2C12细胞线粒体功能维持的研究

摘要

Objective:Long time exercise training significantly increase mitochondrial function in skeletal muscle cells,but the mechanism is unclear.The aim of this study is to investigate whether lactate is involved in the regulation of mitochondrial function maintenance in C2C12 cell,and to investigate whether its role is dependent on the pyruvate.Methods:The C2C12 myotubes were treated with lactate,pyruvate and Galloflavin.The concentration of pyruvate and the ac-tivity of pyruvate dehydrogenase were measured by colorimetric method.The protein contents of MCAT,lipoylation,PDHE2 and β-actin were detected by West-ern blot.Results:1.Compared with the control,16mmol/L lactate very significantly increase the expression of MCAT protein in myotube in time course manner (P<0.01).And 2-64mmol/L lactate very significantly increased the protein expression of MCAT in myotubes in dose-dependent fashion(P<0.01).2.Com-pared with the control,16mmol/L lactate very significantly increase lipoylation level of PDH in myotubes in time course manner(P<0.01),but the protein con-tent of PDHE2 did not change.3.Compared with the control,the protein activity of PDH in myotubes was increased by 16mmol/L lactate(P<0.05).4.Compared with the control,16mmol/L pyruvate did not significantly change the protein level of MCAT in time course manner,even the protein level of MCAT was de?creased after 8h treatment(P<0.05).5.Compared with the control,16mmol/L pyruvate did not significantly change the lipoylation level of PDH in time course manner,even the lipoylation level of PDH was decreased after 8h treatment(P<0.05).6.Compared with the control,intracellular pyruvate content significantly increased after the lactate treatment(P<0.05).The content of pyruvate increase by lactate was significantly attenuated by pre-treatment of C2C12 cells with Galloflavin(P<0.05).However,50 μmol/L Galloflavin treatment did not affect the effect of lactate on the protein expression of MCAT(P>0.05).Conclusions:Lactate is involved in the regulation of cell mitochondrial function maintenance in C2C12 cells independent pyruvate.%目的:长时间运动训练能够显著增加骨骼肌细胞线粒体功能,但是其机制还不清楚.本研究旨在观察乳酸是否参与调节C2C12肌管细胞线粒体功能维持以及是否依赖于丙酮酸发挥作用.方法:用乳酸、丙酮酸和Galloflavin等化学药物处理C2C12肌管细胞,比色法检测细胞中丙酮酸含量和PDH活性,Western blot方法检测MCAT、lipoylation、PDHE2和β-actin等蛋白含量.结果:1.与对照组相比,16 mmol/L乳酸在时间效应上能够非常显著增加C2C12肌管细胞中MCAT蛋白表达量(P<0.01);2~64 mmol/L乳酸在剂量效应上非常显著增加肌管细胞中MCAT蛋白表达量(P<0.01).2.与对照组相比,16 mmol/L乳酸在时间效应上显著增加PDH脂化信号水平(P<0.01),但未影响PDHE2蛋白含量(P>0.05).3.与对照组相比,16 mmol/L乳酸诱发C2C12肌管细胞中PDH活性增加(P<0.05).4.与对照组相比,16 mmol/L丙酮酸在时间效应上不能影响C2C12肌管细胞中MCAT蛋白表达量,甚至在处理8 h后降低MCAT蛋白表达量(P<0.05).5.与对照组相比,16 mmol/L丙酮酸在时间效应上不能影响细胞中PDH脂化信号水平,甚至在处理8 h后降低PDH脂化水平(P<0.05).6.与对照组相比,乳酸处理后显著增加细胞内丙酮酸含量(P<0.05);Galloflavin能够减弱乳酸增加C2C12肌管细胞中丙酮酸含量的作用(P<0.05);50 μmol/L Galloflavin处理后,却并未影响乳酸增加MCAT蛋白表达量的作用(P>0.05).结论:乳酸参与调节C2C12肌管细胞线粒体功能维持而不依赖于丙酮酸.