ALDOSE REDUCTASE ACTIVATION IN DIABETIC COMPLICATIONS

越来越多的人认识到，醛糖还原酶“激活”是一种 糖尿病并发症的潜在危险因素。激活可能是 一些糖尿病患者经历严重组织的核心原因 退化，而其他有类似高血糖的人则存活40年 很少或没有并发症的疾病。这种新的视角源于， 部分来自我们对纯化的牛肾醛糖还原酶的研究 表明该酶可以存在于和未激活的或 激活状态，两者的成交额相差17倍 酶的形式。醛糖还原酶激活的证据已经被证明 在包括人类在内的几种哺乳动物中都有报道。这个 高血糖通过多元醇途径引起的反应通量增加 醛糖还原酶催化NADPH依赖的D-葡萄糖还原为D-葡萄糖 山梨醇，然后依赖于NDA再氧化成D-果糖)已经被 与糖尿病并发症的后续发展直接相关 一般，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。 处于固定的高血糖水平。特定醛糖的设计 因此，还原酶抑制剂(ARI)已成为一个重要的研究领域 努力。然而，在人类身上进行的临床试验的结果还没有达到 基于动物模型的成功，正如预期的那样，前景看好。我们相信 体内的激活可能是错误的。激活牛肾 醛糖还原酶在体外可引起细胞活性的显著变化 有一些，但不是其他ARI。结合ARI差异的证据 结合化学计量学，这些结果表明ARI可以与醛糖结合 不同方式和/或地点的还原酶(S)，并导致了一项建议 用于新的ARI分类系统 酶的激活状态。在此应用程序中，我们将直接 检验醛糖还原酶激活是预测 增加人类患糖尿病肾病并发症的风险。我们 已经开发出方法，可以直接量化 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 酶活化程度与糖尿病肾病严重程度的关系 使用来自正常和糖尿病患者的组织。我们将使用传统的 与紧束缚动力学方法和pH研究进行比较和对比 两个ARI班级。然后，计算机辅助的QSAR研究将用于 确定和分析重要的结构因素(静电、 立体的，疏水的)，决定了ARI效力对 酶的激活状态。利用蛋白质化学知识 在确定初级序列过程中形成的碱基 牛晶状体醛糖还原酶，我们将对其分子基础进行研究 酶的活化，侧重于巯基氧化的作用。

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.