ALDOSE REDUCTASE AND DIABETIC COMPLICATIONS

描述：醛糖还原酶(AR)，多元醇途径的第一种酶 葡萄糖代谢，已被牵涉到这种病理生理学 糖尿病并发症，如白内障、视网膜病变、肾病、 神经病和微血管病。因为AR抑制剂(ARI)延迟或阻止 其中一些并发症，ARIS在糖尿病中的治疗应用 一直在提倡，目前正在美国进行临床试验。主 这种酶的生理底物被认为是葡萄糖。然而， 我们最近的研究结果表明，脂质衍生的醛(LDA)，如 4-羟基壬烯醛(HNE)及其谷胱甘肽结合物更有可能是 由于KMHNE和KMGS-HNE的AR的生理底物是10到30 __M，vs，毫摩尔范围内的Km葡萄糖。已知的高血糖症 导致氧化应激，通过脂质过氧化导致LDA的形成。 HNE是含量最多、毒性最强的LDA，它很容易与 谷胱甘肽和蛋白质。已有研究提出，在高血糖状态下，AR 激活并对山梨醇型ARIs的抑制产生抵抗力，原因是 半胱氨酸-298的氧化。PI现在已经证明了LDAS和 亚硝硫醇结合在Cys-298上，可修饰AR，并激活 根据配基的不同，使酶失活。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降低LDAs的动力学机制；2)AR在代谢中的作用 高血糖时LDA和硫代尿酸途径中间产物的变化；3) 乳酸脱氢酶和亚硝硫醇对AR的调节机制 培养高血糖大鼠AR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 老鼠。实现这些目标所需的所有程序都已到位， 包括电喷雾电离和气相色谱-质谱仪， 放射性标记LDA的合成及~3H-HNE等配体标记AR 用于胰酶消化、多肽分离和氨基酸序列 LDA代谢物的分析及高效液相色谱分离在调查中 AR催化和调节的动力学机制，加上X射线 结晶学和建模，将有助于制造具体的ARI， 与目前的ARIs不同，它不会抑制其他醛酮还原酶。此外， 了解AR在正常血糖和正常血糖条件下的抗氧化作用 高血糖状态将提供评估所需的信息 长期急性呼吸窘迫综合征治疗。PI结果应显示是否包含 抗氧化剂作为糖尿病ARI治疗的一部分将有助于 拮抗和降低细胞抗氧化防御能力 阿。

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.