Abstract
Chronic oxidative stress results in decreased responsiveness to insulin, eventually leading to diabetes and cardiovascular disease. Activation of the JNK signaling pathway can mediate many of the effects of stress on insulin resistance through inhibitory phosphorylation of insulin receptor substrate 1. By contrast, exercise, which acutely increases oxidative stress in the muscle, improves insulin sensitivity and glucose tolerance in patients with Type 2 diabetes. To elucidate the mechanism underlying the contrasting effects of acute versus chronic oxidative stress on insulin sensitivity, we used a cellular model of insulin-resistant muscle to induce either chronic or acute oxidative stress and investigate their effects on insulin and JNK signaling. Chronic oxidative stress resulted in increased levels of phosphorylated (activated) JNK in the cytoplasm, whereas acute oxidative stress led to redistribution of JNK-specific phosphatase MKP7 from the nucleus into the cytoplasm, reduction in cytoplasmic phospho-JNK, and a concurrent accumulation of phospho-JNK in the nucleus. Acute oxidative stress restored normal insulin sensitivity and glucose uptake in insulin-resistant muscle cells, and this effect was dependent on MKP7. We propose that the contrasting effects of acute and chronic stress on insulin sensitivity are driven by changes in subcellular distribution of MKP7 and activated JNK.
Highlights
The mechanistic link between increased reactive oxygen species (ROS) levels and insulin resistance is activation of several signaling pathways, primarily mitogen-activated protein kinases (MAPK) pathways
It is unclear what mechanistic differences lead to opposite effects of acute and chronic stress on muscle insulin sensitivity and glucose uptake, or why activation of MAPK/JNK signaling causes insulin resistance in the case of chronic, but not acute, oxidative stress
Our findings suggest that differential subcellular distributions of JNK-specific phosphatase MAP kinase phosphatase 7 (MKP7) and activated JNK determine the opposite effects of acute and chronic oxidative stress on insulin sensitivity
Summary
C2C12 cells were cultured in DMEM containing 15 g/liter of glucose, 10% fetal bovine serum, sodium pyruvate, 1 g/ml of insulin, 100 nM dexamethasone, and 1% penicillin/ streptomycin for 1 week. This medium was used in normal passaging procedures. At the end of the treatments, cells were fixed in 4% paraformaldehyde for 10 min, washed, and treated with primary antibodies overnight at 4 °C, followed by a 1-h incubation with Alexa Fluor 488- or 573-conjugated goat anti-rabbit, mouse, or donkey anti-goat IgG (1:500 dilution). We found that chronic peroxide stress resulted in a decrease in insulin-induced AKT activation (Fig. 1A), consistent with the development of insulin resistance in individuals with hyperglycemia and hyperlipidemia that have chronically elevated plasma ROS levels
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