Glycation-induced inactivation of NADP+-dependent isocitrate dehydrogenase: implications for diabetes and aging

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH), because it supplies NADPH for antioxidant systems. When exposed to reducing sugars such as glucose, glucose 6-phosphate, and fructose, ICDH was susceptible to oxidative modification and damage, which was indicated by a loss of activity and fragmentation of the peptide as well as by the formation of carbonyl groups. The glycated ICDH was isolated and identified by boronate-affinity chromatography and immunoblotting with anti-hexitol-lysine antibody. The active site lysine residue, Lys212, was identified as one of the major sites of nonenzymatic glycation of ICDH. The structural alterations of modified enzymes were indicated by changes in thermal stability, intrinsic tryptophan fluorescence, and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid. When we examined the antioxidant role of mitochondrial ICDH against glycation-induced cytotoxicity with HEK293 cells transfected with the cDNA for mouse mitochondrial ICDH in sense and antisense orientations, a clear inverse relationship was observed between the amount of mitochondrial ICDH expressed in target cells and their susceptibility to glycation-mediated cytotoxicity. Mitochondrial ICDH was purified by immunoprecipitation and probed with anti-hexitol-lysine antibody, which revealed increased levels of glycated ICDH in the kidneys of diabetic rats and in the lenses of diabetic patients suffering from cataracts. A decrease in ICDH activity was observed in those tissues. We also found that levels of glycated ICDH increased in IMR-90 cells and rat kidney during normal aging. The glycation-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition and may contribute to various pathologies associated with the general aging process and long-term complications of diabetes.