The role of Ndufb6 subunit of the electron transport system complex I in the regulation of mitochondrial energy metabolism and insulin sensitivity in C2C12 myotubes

Introduction:

Impaired mitochondrial function associates with insulin resistance in skeletal muscle, yet the causal relationships and mechanisms are unclear. We have shown that a polymorphism in the Ndufb6 subunit of the mitochondrial complex I relates to impaired mitochondrial plasticity after exercise and age-related susceptibility to insulin resistance in humans. We hypothesize that decreased Ndufb6 activity reduces oxidative capacity and in turn insulin signaling in myotubes.

Methods:

Differentiated C2C12 myotubes treated with Ndufb6 siRNA to induce its knockdown (siNdufb6) and with negative control siRNA (NT) were studied under basal, palmitate-treated, and/or electrical pulse-stimulated (EPS) conditions (n = 4 – 6). Mitochondrial oxidative capacity was measured by high-resolution respirometry and reactive oxygen species (ROS) by DCF fluorimetry. Insuling signaling was assessed in insulin-treated cells by Western blots.

Results:

Ndufb6 mRNA and protein levels were silenced by 70% and by 40%, respectively, after 24h of siRNA treatment. Complex I-linked state u respiration was 36% lower in siNdufb6 than in NT (p < 0.05). While there were no differences in respiration with octanoyl-carnitine, EPS-stimulated respiration was abolished in siNdufb6 myotubes. ROS production was 18% higher in siNdufb6 (p < 0.01) and not further stimulated by palmitate. In contrast to NT, EPS did not rescue the palmitate-induced decrease in pAkt(Ser473), which was decreased by 48% in siNdufb6 under these conditions (p < 0.001).

Conclusions:

Reduced Ndufb6 activity redirects electrons from oxidative phosphorylation towards electron leakage. Lower oxidative capacity and higher ROS production could contribute to the impairment of both insulin sensitivity and lower exercise responsiveness in humans with G/G-single nucleotide polymorphism in Ndufb6.