Diabetes mellitus is a debilitating disease affecting millions of people worldwide, and is the cause of significant morbidity and mortality due to progressive impairment of the visual, renal, nervous, and vascular systems. Damage to these tissues results from biochemical and metabolic alterations occurring in response chronic hyperglycemia. Recent evidence suggests that the metabolic products of aldose reductase, the first and rate limiting enzyme of the polyol pathway of glucose metabolism, may represent a biochemical link between hyperglycemia and diabetic complications in the eye and other major organ systems. Increased levels of aldose reductase in the human retina and lens appear to correlate with diabetic retinopathy and cataract. Pharmacological inhibition of aldose reductase provides a therapeutically rational means to delay the onset and diminish the severity of diabetic retinopathy and cataract. However, development and use of potent and selective enzyme inhibitors will depend on a thorough understanding of the molecular mechanisms leading to aldose reductase biosynthesis, the mechanism of its catalytic activity, and structural features of its active site domain. In this project, we will isolate and characterize the gene encoding human aldose reductase. Our studies will include determination of initiation site of gene transcription, identification and characterization of DNA sequences involved in transcription regulation of the aldose reductase gene, and examination of mechanisms involved in modulation of gene transcription in cells cultured under hyperglycemic and hyperosmotic conditions.The level of aldose reductase gene transcripts will be assessed in diabetic and nondiabetic peripheral leucocytes, lens, and retina. Correlations between tissue levels of aldose reductase messenger RNA and presence and extent of diabetic complications will be explored. The functional domains of aldose reductase will be studied by expressing the human enzyme in vitro. Site-directed mutagenesis will be used to introduce defined amino acid substitutions in the enzyme structure. The functional consequences of amino acid replacements in mutant aldose reductases will be assessed by kinetic and biophysical analysis.
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