Impact of mitochondrial reactive oxygen species and apoptosis signal-regulating kinase 1 on insulin signaling.

Tumor necrosis factor (TNF)-alpha inhibits insulin action; however, the precise mechanisms are unknown. It was reported that TNF-alpha could increase mitochondrial reactive oxygen species (ROS) production, and apoptosis signal-regulating kinase 1 (ASK1) was reported to be required for TNF-alpha-induced apoptosis. Here, we examined roles of mitochondrial ROS and ASK1 in TNF-alpha-induced impaired insulin signaling in cultured human hepatoma (Huh7) cells. Using reduced MitoTracker Red probe, we confirmed that TNF-alpha increased mitochondrial ROS production, which was suppressed by overexpression of either uncoupling protein-1 (UCP)-1 or manganese superoxide dismutase (MnSOD). TNF-alpha significantly activated ASK1, increased serine phosphorylation of insulin receptor substrate (IRS)-1, and decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, and all of these effects were inhibited by overexpression of either UCP-1 or MnSOD. Similar to TNF-alpha, overexpression of wild-type ASK1 increased serine phosphorylation of IRS-1 and decreased insulin-stimulated tyrosine phosphorylation of IRS-1, whereas overexpression of dominant-negative ASK1 ameliorated these TNF-alpha-induced events. In addition, TNF-alpha activated c-jun NH(2)-terminal kinases (JNKs), and this observation was partially inhibited by overexpression of UCP-1, MnSOD, or dominant-negative ASK1. These results suggest that TNF-alpha increases mitochondrial ROS and activates ASK1 in Huh7 cells and that these TNF-alpha-induced phenomena contribute, at least in part, to impaired insulin signaling.

Activation of AMP-activated protein kinase reduces hyperglycemia-induced mitochondrial reactive oxygen species production and promotes mitochondrial biogenesis in human umbilical vein endothelial cells.

We previously proposed that the production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetes complications. The association between the pathogenesis of diabetes and its complications and mitochondrial biogenesis has been recently reported. Because metformin has been reported to exert a possible additional benefit in preventing diabetes complications, we investigated the effect of metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on mtROS production and mitochondrial biogenesis in cultured human umbilical vein endothelial cells. Treatment with metformin and AICAR inhibited hyperglycemia-induced intracellular and mtROS production, stimulated AMP-activated protein kinase (AMPK) activity, and increased the expression of peroxisome proliferator-activated response-gamma coactivator-1alpha (PGC-1alpha) and manganese superoxide dismutase (MnSOD) mRNAs. The dominant negative form of AMPKalpha1 diminished the effects of metformin and AICAR on these events, and an overexpression of PGC-1alpha completely blocked the hyperglycemia-induced mtROS production. In addition, metformin and AICAR increased the mRNA expression of nuclear respiratory factor-1 and mitochondrial DNA transcription factor A (mtTFA) and stimulated the mitochondrial proliferation. Dominant negative-AMPK also reduced the effects of metformin and AICAR on these observations. These results suggest that metformin normalizes hyperglycemia-induced mtROS production by induction of MnSOD and promotion of mitochondrial biogenesis through the activation of AMPK-PGC-1alpha pathway.