Depletion of Mitochondrial DNA Causes Impaired Glucose Utilization and Insulin Resistance in L6 GLUT4myc Myocytes

Abstract

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 g/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.

Insulin resistance is a cellular antioxidant defense mechanism

“…First, how does O 2 •Ϫ produced in the mitochondria regulate the translocation of GLUT4 to the plasma membrane? Intriguingly, cells that lack mitochondria are not insulin responsive (31), suggesting that the insulin signal must traverse this organelle as a checkpoint before signaling to GLUT4. Second, is mitochondrial antioxidant therapy likely to be of benefit in the treatment of metabolic disease?…”

Mitochondrial dysfunction induces aberrant insulin signalling and glucose utilisation in murine C2C12 myotube cells

“…Recently, Park et al [43] showed that reductions in Irs1 expression and insulin-stimulated phosphorylation of IRS1 and Akt2/protein kinase B are associated with insulin resistance in L6 Glut4myc myocytes, where 95% of mtDNA was depleted by EtBr treatment. They showed no discernible changes in total SLC2A4, whereas we observed reduced SLC2A4 production in oligomycin-or EtBr-treated C2C12 cells.…”

C1q Tumor Necrosis Factor α-related Protein Isoform 5 Is Increased in Mitochondrial DNA-depleted Myocytes and Activates AMP-activated Protein Kinase

“…The control parental L6 GLUT4myc myocytes were maintained for the same time period in normal culture medium. The mtDNA content of L6 GLUT4myc myocytes cultured with or without EtBr was monitored routinely by amplifying genomic DNA as described previously (2). HEK293 and SK-Hep1 cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS.…”

“…After amplification, melting curve analysis and agarose-gel electrophoresis verified the authenticity of the PCR products. The qRT-PCR results were analyzed using Rotor-Gene analysis software version 6.0 (Corbett Research) as described previously (2).…”

“…Previously, we demonstrated that the depletion of mtDNA in myocytes reduces the expression of insulin receptor substrate-1 (IRS-1), 2 which results in insulin resistance and impaired glucose utilization (2). The signals from mitochondrial stress cause a variety of changes in nuclear gene expressions (3).…”