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内皮细胞功能障碍的细胞和分子机制，以确定新的干预目标，预防和/或减少这种疾病的血管并发症。本提案的目的是确定高血糖如何降低II型糖尿病患者四氢生物蝶呤（BH 4）的可用性，BH 4是一种抗氧化剂和内皮NO合酶（eNOS）催化NO生成的必需辅助因子。本申请的假设是，高血糖症降低了GTP环化水解酶I（GTPCH）（BH 4合成的第一和速率控制酶）的表达，并增加了BH 4的氧化，从而降低了用于NO生成的BH 4的可用性。这反过来又会促进氧化损伤，降低抗氧化能力，并损害内皮依赖性血管功能。这一假设已制定的基础上，从我们的实验室的初步数据，抑制BH 4合成导致氧化损伤培养EC。提出了两个具体的目标：（1）阐明降低BH 4在EC和II型糖尿病大鼠的血管中的可用性的机制和（2）确定增加BH 4在EC中的可用性和抗氧化能力的试剂，从而改善NO介导的血管反应性。利用新鲜分离的EC从Zucker糖尿病脂肪（ZDF）大鼠[作为模型的I型！糖尿病]，我们将在疾病进展期间的不同时间点测量GTPCH、BH 4、二氢生物蝶呤（加上生物蝶呤）和eNOS的细胞含量，以及GTPCH活性和NO合成，并与来自年龄匹配的瘦对照大鼠的EC进行比较。我们还将分析BH 4水平和二氢蝶啶还原酶活性（BH 4再生所需）和氧化应激之间的相关性。此外，将测试饮食精氨酸补充剂、他汀类药物治疗、谷胱甘肽治疗和NAD（P）H氧化酶活性抑制剂增加EC中BH 4水平和恢复NO介导的血管反应性的能力。预计这项研究将产生关于BH 4在细胞防御氧化应激中作用的新知识。确定负责内皮BH 4缺乏的机制将导致有效的治疗干预，以减少糖尿病患者的氧化应激，这反过来又会降低血管并发症的发病率，发病率和死亡率在这种疾病的主要原因。

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.