Oxidative Stress and Pteridine Metabolism in Diabetes

描述(申请人提供)：糖尿病的一个标志是内皮功能障碍。我们的长期目标是了解糖尿病内皮细胞功能障碍的细胞和分子机制，以便确定新的干预靶点，以预防和/或减少这种疾病的血管并发症。该提案的目的是确定高血糖如何减少II型糖尿病患者四氢生物蝶呤(BH4)的可获得性，四氢生物蝶呤是一种抗氧化剂，也是内皮型一氧化氮合酶(ENOS)催化NO生成的重要辅助因子。这一应用的假设是，高血糖降低了GTP环水解酶I(GTPCH)的表达，GTPCH是合成BH4的第一个和速度控制酶，并增加了BH4的氧化，从而减少了BH4对NO生成的可获得性。这反过来会促进氧化损伤，降低抗氧化能力，并损害内皮依赖的血管功能。这一假说是基于我们实验室的初步数据提出的，即抑制BH4合成会导致培养的EC氧化损伤。提出了两个特定的目标：(1)阐明BH4在内皮细胞和2型糖尿病大鼠血管中的可获得性降低的机制；(2)寻找能够增加BH4在EC中的获得性和抗氧化能力的药物，从而改善NO介导的血管反应性。利用新鲜分离的Zucker糖尿病脂肪(ZDF)大鼠的EC[作为I型！[糖尿病]，我们将在疾病进展的不同时间点检测GTPCH、BH4、二氢生物蝶呤(加生物蝶呤)和eNOS的细胞含量，以及GTPCH活性和NO合成，并与年龄匹配的瘦身对照组大鼠的EC进行比较。我们还将分析BH4水平与二氢蝶呤还原酶活性(BH4再生所必需的)和氧化应激之间的相关性。此外，饮食中补充精氨酸、他汀类药物、谷胱甘肽治疗和抑制NAD(P)H氧化酶活性将测试它们提高EC中BH4水平和恢复NO介导的血管反应性的能力。预计这项拟议的研究将产生关于BH4在细胞防御氧化应激中的作用的新知识。确定内皮细胞BH4缺乏的机制将导致有效的治疗干预措施，以减少糖尿病患者的氧化应激，这反过来将减少血管并发症的发生率，血管并发症是这种疾病的发病率和死亡率的主要原因。

DESCRIPTION (provided by applicant): A hallmark of diabetes is endothelial dysfunction. Our long-range goal is to understand the cellular and molecular mechanisms of endothelial cell dysfunction in diabetes in order to identify new targets for intervention that will prevent and/or minimize vascular complications of this disease. The objective of this proposal is to determine how hyperglycemia reduces availability of tetrahydrobiopterin (BH4), an antioxidant and essential cofactor for endothelial NO synthase- (eNOS) catalyzed NO generation, in type II diabetes. The hypothesis of this application is that hyperglycemia reduces expression of GTP cyclohydrolase I (GTPCH), the first and rate-controlling enzyme for BH4 synthesis, and increases oxidation of BH4, thereby reducing BH4 availability for NO generation. This, in turn, promotes oxidative damage, decreases antioxidant capacity, and impairs endothelium-dependent vascular function. This hypothesis has been formulated on the basis of preliminary data from our laboratory that inhibition of BH4 synthesis resulted in oxidative damage in cultured EC. Two specific aims are proposed: (1) To elucidate mechanisms for reduced BH4 availability in EC and vessels of type II diabetic rats and (2) To identify agents that increase BH4 availability and antioxidant capacity in EC, thereby improving NO-mediated vascular reactivity. Utilizing freshly isolated EC from the Zucker diabetic fatty (ZDF) rat [as a model of type I! diabetes], we will measure the cellular content of GTPCH, BH4, dihydrobiopterin (plus biopterin), and eNOS, as well as GTPCH activity and NO synthesis at different time points during disease progression and compare with EC from age-matched lean control rats. We will also analyze the correlation between BH4 levels and dihydropteridine reductase activity (required for regeneration of BH4) and oxidative stress. Further, dietary arginine supplementation, statin therapy, glutathione treatment and inhibition of NAD(P)H oxidase activity will be tested for their ability to increase BH4 levels in EC and restore NO-mediated vascular reactivity. It is expected that the proposed research will generate new knowledge about the role of BH4 in cellular defense against oxidative stress. Identification of the mechanisms responsible for endothelial BH4 deficiency will lead to effective therapeutic interventions for reducing oxidative stress in diabetic patients, which in turn will decrease the incidence of vascular complications, the leading cause of morbidity and mortality in this disease.