Both cell growth (e.g. proliferative retinopathy and renal hypertrophy) and cell death are seen in diabetic complications. Of the proposed mechanisms responsible for diabetic complications, oxidative stress is considered to be of central importance. Redox regulation in most cells relies on glucose 6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, as G6PD produces the main intracellular reductant, NADPH. The critical importance of G6PD to cell survival has been recently shown as follows: 1) The investigator's lab has shown that G6PD activity per se plays a critical role in supporting cell growth and preventing cell death. 2) The investigator's lab has shown that inadequate activation of G6PD predisposes cells to oxidative damage and cell death. 3) Other labs have shown that G6PD activity is required for cellular antioxidant defense. Thus if G6PD activity is impaired in diabetic patients, this impairment could play a critical role in the development of diabetic complications. Preliminary work by us and work by others has shown that, indeed, endothelial cells exposed to high glucose have impaired activation of G6PD. High glucose inhibits G6PD activity and impairs G6PD response to oxidant stress. Preliminary work from our lab suggests that increases in CAMP levels and protein kinase C (PKC) activity (signals known to be increased by high glucose) mediate the high glucose-induced changes in G6PD activity. Previous work by the investigator has shown that G6PD is regulated both in its activity and intracellular location. Based on our preliminary work and published data, we hypothesize that high glucose-stimulated cell death is associated with increased cAMP and increases in a specific isoform of PKC(delta), whose combined effect is to cause changes in G6PD phosphorylation and/or intracellular location thus inhibiting G6PD activity. In cells in which high glucose increases cell proliferation or hypertrophy, decreased CAMP and increased PKC(beta) leads to increased G6PD activity. Thus we propose to: 1) Determine whether CAMP mediates the high glucose-induced changes in G6PD. 2) Determine whether specific PKC isoforms mediate the high glucose-induced changes in G6PD. 3) Determine the importance of G6PD activity in high glucose-induced cell death. And 4) Determine whether cells from rats with diabetes also have altered G6PD activity. Endothelial, mesangial, and smooth muscle cells will be studied utilizing overexpression, phosphopeptide mapping, site-directed mutagenesis, and immunofluorescence. This work will provide new insights into the effects of high glucose on cells and likely provide a new approach for preventing diabetic complications.
NIH ExPorter
NIH ExPorter