Mechanism of insulin-like effect of chromium(III) ions on glucose uptake in C2C12 mouse myotubes involves ROS formation

Chromium is considered a trace element which improves glucose tolerance, but mechanism accounting for this insulin-like action is not recognized. The main purpose of this study was to examine the role of reactive oxygen species (ROS) in chromium and insulin stimulated glucose transport using antioxidants. Effect of chromium ions on phosphatases, enzymes involved in inhibition of insulin signaling was also investigated. Experiments were performed in vitro on C2C12 mouse myotubes. ROS level was measured with the use of confocal microscope and 2',7' dichlorodihydrofluorescein diacetate (DCFH-DA). Glucose metabolism was assayed by the measurement of 2-[H-3]-deoxyglucose uptake. Cr3+ ions and insulin treatment caused significant increase of ROS formation and also stimulated glucose uptake in C2C12 cells in concentration dependent manner. Antioxidants (L-ascorbic acid and N-acetyl cysteine 100 mu M) and DPI (diphenyleneiodonium-NADPH oxidase inhibitor, 10 mu M) abolished insulin- and Cr-inducted glucose transport. Our results confirm the hypothesis that the ROS are integral part of insulin signaling pathway and that the insulin mimetic effect of Cr3+ ions depends on the antioxidant status of the cells. Surprisingly, chromium treatment resulted in increased activity of membrane phosphatases.