The depletion of cellular mitochondrial DNA causes insulin resistance through the alteration of insulin receptor substrate-1 in rat myocytes

Since the bioenergetic capacity of skeletal muscle mitochondria is decreased in type 2 diabetes and obesity, the reduction of mitochondrial DNA (mtDNA) content may be involved in the development of insulin resistance in skeletal muscle. 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 (EtBr). L6 GLUT4myc cells cultured with 0.2 μg/ml EtBr (termed depleted cells) revealed a marked decrease in cellular mtDNA, 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 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. Interestingly, the expression of IRS-1 associated with insulin signaling was decreased by 90% in the depleted cells, and the insulin-stimulated phosphorylation of IRS-1 and Akt2/PKB 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 reduction in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/PKB are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.