Park Seung Y, Choi Guem H, Choi Hyo I, Ryu Jiwon, Jung Chan Y, Lee Wan
Department of Biochemistry, College of Medicine, Dongguk University, Kyungju 780-714, Korea.
J Biol Chem. 2005 Mar 18;280(11):9855-64. doi: 10.1074/jbc.M409399200. Epub 2004 Dec 17.
Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 mug/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.
线粒体功能障碍与许多人类疾病有关，如高脂血症、肥胖症和糖尿病。线粒体DNA（mtDNA）的突变和减少被认为是糖尿病发病机制中的因素。为了阐明细胞mtDNA含量与胰岛素抵抗之间的关联，我们通过用溴化乙锭长期处理产生了mtDNA缺失的L6 GLUT4myc肌细胞。用0.2μg/ml溴化乙锭培养的L6 GLUT4myc细胞（称为缺失细胞）显示细胞mtDNA和ATP含量显著降低，同时缺乏由mtDNA编码的mRNA。有趣的是，mtDNA缺失的细胞在基础和胰岛素刺激的葡萄糖摄取方面显著降低，表明L6 GLUT4myc细胞出现葡萄糖利用受损和胰岛素抵抗。mtDNA的补充使基础和胰岛素刺激的葡萄糖摄取恢复正常。与胰岛素信号相关的胰岛素受体底物（IRS）-1的mRNA水平和表达在缺失细胞中分别降低了76%和90%。基础状态下质膜（PM）上的GLUT4减少，mtDNA缺失使胰岛素刺激的GLUT4向PM的转位显著减少。此外，在缺失细胞中，胰岛素刺激的IRS-1和Akt2/蛋白激酶B的磷酸化显著降低。mtDNA补充后，这些变化恢复到对照水平。综上所述，我们的数据表明，PM GLUT4含量和胰岛素信号通路中间体受到细胞mtDNA含量改变的调节，IRS-1表达的降低以及胰岛素刺激的IRS-1和Akt2/蛋白激酶B的磷酸化与mtDNA缺失的L6 GLUT4myc肌细胞中的胰岛素抵抗有关。
