%0 Journal Article %T 支链氨基酸和胰岛素对大鼠骨骼肌细胞蛋白质代谢的分子调控机制研究 %A 罗钧秋 %A 陈代文 %A 余冰 %A 张峻豪 %A 刘杰 %J 南京农业大学学报 %D 2016 %R 10.7685/jnau.201512004 %X [目的]研究支链氨基酸（BCAA）对大鼠骨骼肌细胞蛋白质代谢的影响，探讨氨基酸转运载体在BCAA调控细胞蛋白质代谢途径中的作用机制。[方法]用缺失支链氨基酸（Leu-，Ile-，Val-）的无血清高糖型DMEM培养液培养L6肌管细胞，先后在培养基质中添加100 nmol？L-1雷帕霉素、支链氨基酸（Leu+，Ile+，Val+）和胎牛胰岛素，RT-PCR法检测细胞表面氨基酸转运载体LAT1、CD98和SNAT2以及氨基酸调控因子GCN2和ATF4 mRNA表达，并结合Western-blot法考察蛋白合成关键因子磷酸化S6K1蛋白表达量及蛋白降解关键因子MuRF1和MAFbx mRNA表达水平。[结果]BCAA依赖于mTOR途径显著促进磷酸化S6K1（Thr389）蛋白表达（P<0.05），此过程与提高LAT1和CD98的mRNA表达水平（P<0.05）有关。SNAT2在胰岛素参与下，其表达量显著增加（P<0.05），并依赖于mTOR途径；BCAA对GCN2和ATF4 mRNA水平没有产生显著影响，但能够依赖mTOR途径显著提高ATF4蛋白表达量（P<0.05）；在没有胰岛素参与下，BCAA未能够引起MuRF1和MAFbx表达发生变化，但在胰岛素协同下，BCAA能够使MAFbx表达显著下降（P<0.05），但该抑制效应并不能被雷帕霉素所阻断；无胰岛素下MuRF1和MAFbx表达量显著高于胰岛素参与下的表达量（P<0.05）。[结论]BCAA不依赖胰岛素调控蛋白质合成代谢，通过细胞膜氨基酸转运受体（LAT1、CD98和SNAT2）介导，作用于mTOR途径发挥促进蛋白合成作用；胰岛素使BCAA的促蛋白合成作用更加明显；BCAA调控蛋白质分解代谢过程依赖胰岛素，与胰岛素协同抑制蛋白降解。</br>[Objectives] This experiment was to study the effects of BCAA on molecular mechanisms of protein metabolism in L6 myotubes. [Methods] L6 myotubes were treated with rapamycin,branched amino acids((Leu,Ile,Val) and insulin. The mRNA expressions of LAT1,CD98,SNAT2,GCN2,ATF4,MuRF1 and MAFbx were determined by RT-PCR. Phosphorylated levels of S6K1(Thr389) and ATF4 were determined by Western-blot analysis. [Results] Transcriptional levels of LAT1 and CD98 were induced by BCAA dependent of mTOR pathway(P<0.05). mRNA level of SNAT2 was up-regulated by BCAA dependent of insulin involved,which was mTOR-dependent(P<0.05). BCAA did not affect mRNA levels of GCN2 and ATF4,but translational level of ATF4 was up-regulated by BCAA,dependent on mTOR signaling pathway(P<0.05). Insulin may promote protein synthesis mediated by BCAA. mRNA level of MAFbx was reduced by BCAA in insulin involved(P<0.05),which could not be completely blocked by rapamycin. And expressions of MuRF1 and MAFbx were higher without insulin involved than those in insulin involved(P<0.05). [Conclusions] Protein synthesis may be induced by amino acids(BCAA) through LAT1-, CD98- and SNAT2-mediated mTOR signaling pathway. Meanwhile proteolysis may be regulated by Ubiquitin(Ub)-proteasome signal transduction pathways,which is dependent of insulin %K 支链氨基酸 %K 氨基酸转运载体 %K 蛋白合成 %K 骨骼肌< %K /br> %K branched chain amino acid %K amino acid transporter %K protein synthesis %K skeletal muscle %U http://nauxb.njau.edu.cn/oa/darticle.aspx?type=view&id=201606017