Generation of reactive oxygen species by endothelial and smooth muscle cells: influence of hyperglycemia and metformin.

There is evidence that reactive oxygen intermediates (ROI) play an important role in the pathogenesis of vascular complications in diabetes. On the other hand, metformin, one of the most often used antidiabetic compounds has not only been shown to reduce the risk for vascular complications, but in addition these protective effects are largely independent of its well-known antihyperglycemic action. Therefore, to explain the vasculoprotective effects of metformin, a direct antioxidative action of this compound has been suggested. We show here that human endothelial cells (HUVEC) generate ROI not only in response to high glucose (30 mmol/l glucose), but also in response to palmitic acid, and advanced glycation end-products (carboxymethyllysine and S100 proteins). Metformin inhibited the production of ROI in response to all these stimuli. By double staining-dichlorofluorescein as marker of ROI and Mitotracker CMH-Ros for mitochondria-the mechanism of ROI generation was analyzed in more detail in smooth muscle cells. Our data suggest that ROI are generated by uncoupling of the mitochondrial respiratory chain as well as by activation of the cytosolic NADPH-oxidase. A complete inhibition of ROI generation is only achieved by simultaneous inhibition of the mitochondrial electron flux (theonyltrifluoroacetone) and NADPH-oxidase (apocynin). Our data suggest that the various processes contributing to generation of ROI are closely linked. Activation of AMP kinase may represent an important mechanism to understand the antioxidative effects of metformin on the mitochondrial and cytosolic generation of ROI.