Aldose reductase (AR), the first enzyme of the polyol pathway of glucose metabolism, has been implicated in the pathophysiology of such diabetic complications as cataractogenesis, retinopathy, nephropathy, neuropathy and microangiopathy. Since AR inhibitors (ARIs) delay or prevent some of these complications, the therapeutic use of ARIs in diabetes has been advocated, and is currently in U.S. clinical trials. The main physiological substrate of this enzyme is thought to be glucose. However, our recent results suggest that lipid-derived aldehydes (LDAs), such as 4-hydroxynonenal (HNE), and their glutathione conjugates are more likely the physiological substrates of AR since the Km HNE and Km GS-HNE are 10 to 30 __M, vs, a Km glucose in the millimolar range. Hyperglycemia is known to cause oxidative stress, leading to LDA-formation via lipid peroxidation. HNE is the most abundant and toxic LDA which can readily form adducts with GSH and proteins. It has been proposed that under hyperglycemia AR is activated and becomes resistant to inhibition by sorbinil-type ARIs, due to oxidation of Cys-298. The PI has now demonstrated that LDAs and nitrosothiols bind at Cys-298 and can modify AR, and either activate of inactivate the enzyme, depending on the ligand. The PI studies suggest that AR may have an antioxidative role. We thus plan to investigate: 1) the kinetic mechanisms by which AR reduces LDAs; 2) AR's role in the metabolism of LDA and mercapturic acid pathway intermediates during hyperglycemia; 3) the mechanisms of AR's regulation by LDAs and nitrosothiols: and 4) post-translational modifications induced in AR of cultured hyperglycemic vascular endothelial cells and tissues from streptozotocin-induced diabetic rats. All the procedures to be used in achieving these aims are in place, including electrospray ionization-and gas chromatography-mass spectometry, synthesis of radiolabeled LDA, labeling of AR with such ligands as 3H-Hne for tryptic digestion, separation of peptides, and amino acid sequence analysis and HPLC separation of LDA-metabolites. The investigations of the kinetic mechanisms of AR catalysis and regulation, plus X-ray crystallography and modeling, will be helpful in making specific ARIs that, unlike present ARIs, will not inhibit other aldo-keto reductases. Moreover, understanding the anti-oxidative role of AR under normoglycemic and hyperglycemic conditions will provide the information necessary to evaluate long-term ARI therapy. The PI results should show whether inclusions of antioxidants as part of ARI therapy in diabetes would be useful to counteract and decrease in cellular antioxidant defense due to inhibition of AR.
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