Diabetic complications have been increasing rapidly due to the prevalence of diabetes worldwide. The mechanisms responsible for the complications are incompletely understood. Many laboratories have shown that oxidative stress plays a central mechanistic role. Oxidative stress can occur as a result of increased oxidant production and/or decreased antioxidant function. The investigator's lab has focused on the role of decreased antioxidants in diabetes. The antioxidant system relies on NADPH, the main intracellular reductant, which is principally produced by glucose-6-phosphate dehydrogenase (G6PD). Work from the investigator showed that G6PD plays a critical role in supporting cell growth and preventing cell damage/death. Work from the lab also showed that high glucose causes a decrease in G6PD activity mediated, at least in part, by cAMP-dependent protein kinase A (PKA) in cultured cells. Preliminary work by the investigator's lab in animals is consistent with the cell culture findings and, in addition, has shown that G6PD protein expression is decreased in diabetic animals. Specifically, research showed that diabetic animals have decreased G6PD activity and lower NADPH in such tissues as kidney, aorta, and heart as compared to non-diabetic animals. In addition to affecting the antioxidant system, lower NADPH could decrease any NADPH-requiring process. Since endothelial dysfunction is a hallmark of diabetes, the lab has focused on a critical endothelial cell function, nitric oxide (NO) production. Work by the investigator's lab and collaborators showed that decreased NADPH due to G6PD inhibition leads to lower NO and increased superoxide production by endothelial nitric oxide synthase (eNOS). Thus, we hypothesize that diabetes mellitus causes a decrease in G6PD activity that leads to lower levels of NADPH thus increasing oxidant stress. We propose to: 1) Determine whether diabetic animals have decreased G6PD activity, whether G6PD deficiency per se predisposes to diabetic complications, and whether overexpression of G6PD ameliorates the deleterious effects of diabetes. 2) Determine how PKA-dependent phosphorylation regulates G6PD. 3) Determine if the high glucose-induced decrease in G6PD activity leads to decreased NO and increased production of reactive oxygen species by eNOS. Taken together, these studies will provide critical insights into the mechanisms responsible for diabetic complications and will provide new therapeutic targets. ? ?
