ALDOSE REDUCTASE ACTIVATION IN DIABETIC COMPLICATIONS

糖尿病并发症中醛糖还原酶的激活

• 批准号: 3244960
• 负责人: CHARLES EDWARD GRIMSHAW
• 金额: $ 20.07万
• 依托单位: WHITTIER INSTITUTE FOR DIABETES & ENDOC
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-25 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3244960
• 关键词: aldehyde reductase; chemical binding; computer assisted sequence analysis; diabetes mellitus; enzyme inhibitors; enzyme mechanism; human tissue; kidney disorder; laboratory rabbit; medical complication; oxidation; oxidoreductase inhibitor

There is a growing appreciation of aldose reductase "activation" as a potential risk factor in diabetic complications. Activation may be central to why some diabetic patients experience severe tissue degeneration while others with comparable hyperglycemia survive 40 years of disease with little or not complications. This new perspective stems, in part, from our studies of purified bovine kidney aldose reductase showing that the enzyme can exist in either and unactivated or an activated state, with a 17-fold difference in turnover number for the two enzyme forms. Evidence for activation of aldose reductase has been reported in several mammalian species, including human. The hyperglycemia-induced increase in reaction flux through the polyol pathway (aldose reductase-catalyzed NADPH-dependent reduction of D-glucose to D- sorbitol followed by NDA+-dependent reoxidation to D-fructose) has been directly linked to the subsequent development of diabetic complications in general, and of diabetic kidney disease in particular. Activation of aldose reductase can magnify this effect leading to greater tissue damage at a fixed level of hyperglycemia. The design of specific aldose reductase inhibitors (ARI) has thus become a major area of research effort. Yet, results from clinical trials in humans have not been as promising as expected based on the success in animal models. We believe that activation in vivo may be at fault. Activation of bovine kidney aldose reductase in vitro leads to a pronounced change in the potency of some, but not other ARI. Combined with evidence for a difference in ARI binding stoichiometry, these results suggest that ARI can bind to aldose reductase in different modes and/or site(s), and have lead to a proposal for a new ARI classification system based on the sensitivity to the activation state of the enzyme. In this application, we will directly test the hypothesis that aldose reductase activation is a predictor for increased risk of developing diabetic kidney complications in humans. We have developed methods that allow direct quantitation of the amount of aldose reductase protein, the total activity, and the activation state of the enzyme in tissue samples. This method will be used to correlate the extent of enzyme activation with the severity of diabetic kidney disease using tissue from normal and diabetic patients. We ill use conventional and tight-binding kinetic methods and pH studies to compare and contrast the two ARI classes. Computer-aided QSAR studies will then be used to identify and analyze the important structural factors (electrostatic, steric, hydrophobic) that determine the sensitivity of ARI potency to the activation state of the enzyme. Using the protein chemistry knowledge base developed during the determination of the primary sequence for bovine lens aldose reductase, we will investigate the molecular basis for enzyme activation, focusing on the role of sulfhydryl oxidation.

越来越多的人认识到醛糖还原酶的“活化”是一种重要的酶。 糖尿病并发症的潜在危险因素。 激活可以 为什么一些糖尿病患者会出现严重的组织损伤 而其他患有类似高血糖症的人存活了40年 几乎没有并发症的疾病。 这种新的观点源于， 部分来自我们对纯化的牛肾醛糖还原酶的研究， 表明酶可以存在于未活化的或未活化的细胞中， 激活状态，两者的营业额相差17倍 酶的形式。 已发现激活醛糖还原酶的证据， 在包括人类在内的几种哺乳动物中均有报道。 的 高血糖诱导的通过多元醇途径的反应通量增加 （醛糖还原酶催化的NADPH依赖性还原D-葡萄糖为D-葡萄糖） 山梨糖醇，然后NDA+依赖性再氧化为D-果糖）已经被 与糖尿病并发症的后续发展直接相关， 一般来说，尤其是糖尿病肾病。 激活 醛糖还原酶可以放大这种效应，导致更大的组织损伤 在一个固定的高血糖水平。 特定醛糖的设计 还原酶抑制剂（ARI）因此成为研究的一个主要领域 努力 然而，人类临床试验的结果并不像 基于动物模型的成功，如预期的那样有希望。 我们认为 体内的激活可能是错误的。 牛肾激活 醛糖还原酶在体外导致 一些，但不是其他ARI。 结合ARI差异的证据 结合化学计量，这些结果表明ARI可以结合醛糖 还原酶在不同的模式和/或网站（S），并导致一个建议 一个新的ARI分类系统的基础上的敏感性， 酶的活化状态。 在这个应用程序中，我们将直接 验证醛糖还原酶激活是 增加人类患糖尿病肾脏并发症的风险。 我们 已经开发出允许直接定量的方法， 醛糖还原酶蛋白，总活性，和激活状态， 组织样本中的酶 此方法将用于关联 酶激活程度与糖尿病肾病的严重程度 使用正常和糖尿病患者的组织。 我们将使用传统的 紧结合动力学方法和pH研究进行比较和对比 两个班级。 计算机辅助QSAR研究将用于 识别和分析重要的结构因素（静电， 空间的、疏水的），其决定ARI效力对 酶的活化状态。 利用蛋白质化学知识 在确定主要序列期间开发的基础， 牛透镜醛糖还原酶，我们将研究的分子基础， 酶的激活，重点是巯基氧化的作用。