ALDOSE REDUCTASE AND DIABETIC COMPLICATIONS

醛糖还原酶和糖尿病并发症

• 批准号: 6380528
• 负责人: SATISH K SRIVASTAVA
• 金额: $ 20.87万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6380528
• 关键词: X ray crystallography; aldehyde reductase; animal tissue; antioxidants; chemical binding; diabetes mellitus; electrospray ionization mass spectrometry; enzyme induction /repression; enzyme inhibitors; enzyme substrate; enzyme therapy; free radicals; gas chromatography mass spectrometry; high performance liquid chromatography; human tissue; hyperglycemia; laboratory rat; medical complication; molecular pathology; molecular site; oxidative stress; oxidoreductase inhibitor; posttranslational modifications; protein sequence; sorbinil

DESCRIPTION: 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.

描述：醛糖还原酶（AR），多元醇途径的第一种酶 葡萄糖代谢，已牵连在这种病理生理学 糖尿病并发症，如白内障、视网膜病、肾病， 神经病和微血管病。 由于AR抑制剂（阿里斯）延迟或阻止 对于这些并发症中的一些，阿里斯在糖尿病中的治疗用途已经 目前正在美国进行临床试验。 主要 这种酶的生理底物被认为是葡萄糖。 然而，在这方面， 我们最近的研究结果表明，脂质衍生的醛（LDAs），如 4-羟基壬烯醛（HNE）及其谷胱甘肽结合物更可能是 由于Km HNE和Km GS-HNE为10至30， __M，vs，在毫摩尔范围内的Km葡萄糖。 众所周知，hyperplasia 引起氧化应激，导致通过脂质过氧化形成LDA。 HNE是最丰富和毒性最大的LDA，其可以容易地与 GSH和蛋白质。 已经提出，在高血糖症下， 激活并对山梨醇型阿里斯抑制产生抗性， Cys-298的氧化。 PI现已证明，LDAs和 亚硝基硫醇在Cys-298处结合，并且可以修饰AR，并且激活 根据配体的不同，使酶发生变性。 PI研究表明， AR可能具有抗氧化作用。 因此，我们计划调查：1） AR减少LDAs的动力学机制; 2）AR在代谢中的作用 高血糖期间LDA和巯基尿酸途径中间体的浓度; 3） LDAs和亚硝基硫醇调节AR的机制; 4） 培养的高血糖大鼠AR中诱导的翻译后修饰 链脲佐菌素诱导的糖尿病血管内皮细胞和组织 大鼠 实现这些目标所采用的所有程序都已到位， 包括电喷雾电离-和气相色谱-质谱法， 放射性标记的LDA的合成，用诸如3 H-Hne的配体标记AR 用于胰蛋白酶消化、肽分离和氨基酸序列测定 LDA-代谢物的分析和HPLC分离。 的调查工作 AR催化和调节的动力学机制，加上X射线 晶体学和建模，将有助于制作特定的阿里斯， 与现有的阿里斯不同，它不会抑制其它醛酮还原酶。 此外，委员会认为， 了解AR在正常血糖和 高血糖状况将提供必要的信息， 长期ARI治疗。 PI结果应显示是否包含 抗氧化剂作为ARI治疗糖尿病的一部分， 抵消和减少细胞的抗氧化防御，由于抑制 AR.