Oxidative Stress and Pteridine Metabolism in Diabetes

描述（由申请人提供）：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中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。本研究的目的是确定高血糖如何降低四氢生物蝶呤（BH4）的可用性，BH4是内皮NO合成酶（eNOS）催化NO生成的抗氧化剂和必需的辅助因子。本应用的假设是高血糖降低了GTP环水解酶I （GTPCH）的表达，GTPCH是BH4合成的第一个和速率控制酶，并增加了BH4的氧化，从而降低了BH4生成NO的可用性。这反过来又会促进氧化损伤，降低抗氧化能力，损害内皮依赖性血管功能。这一假设是基于我们实验室的初步数据，即抑制BH4合成导致培养EC的氧化损伤。本研究提出了两个具体目标：(1)阐明II型糖尿病大鼠EC和血管中BH4可用性降低的机制；(2)确定增加EC中BH4可用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Zucker糖尿病脂肪（ZDF）大鼠新鲜分离的EC作为I型模型。我们将在疾病进展的不同时间点测量GTPCH、BH4、双氢生物蝶呤（加生物蝶呤）和eNOS的细胞含量，以及GTPCH活性和NO合成，并与年龄匹配的瘦对照大鼠的EC进行比较。我们还将分析BH4水平与二氢蝶啶还原酶活性（BH4再生所需）和氧化应激之间的相关性。此外，将测试膳食精氨酸补充、他汀类药物治疗、谷胱甘肽治疗和抑制NAD(P)H氧化酶活性的能力，以提高EC中BH4水平并恢复no介导的血管反应性。预计本研究将对BH4在细胞防御氧化应激中的作用产生新的认识。确定内皮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，从而减少血管并发症的发生率，血管并发症是糖尿病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.