Role of Ncb5or in Insulin Production

描述（由申请人提供）：1型和2型糖尿病的确定性治疗的开发取决于对胰岛内胰岛素产生所涉及的分子事件的透彻理解。我们的实验室已经克隆和表征了一个高度保守的氧化还原酶，NCB 5 OR，它位于内质网（ER）。小鼠中该基因的靶向失活导致糖尿病表型。到7周龄时，Ncb 5或-/-小鼠发展为严重的高血糖症，血清胰岛素水平显著降低。胰岛显示β细胞明显缺乏，但α、δ和PP细胞数量正常。我们的研究计划是基于NCB 5 OR保护胰腺β细胞免受ER中氧化剂诱导的损伤的假设。在第一个具体目标中，我们计划进一步表征NCB 5 OR缺陷对完整小鼠的影响的研究。我们将通过移植正常β细胞的敲除小鼠来解决NCB 5 OR是否在胰腺β细胞外发挥生物学重要作用的重要问题。我们还将研究表达转基因的Ncb 5或-/-和+/+小鼠，该转基因报告不同器官和组织中的ER应激。最后，我们将制备NCB 5 OR和CHOP（ER应激诱导细胞凋亡所需的转录因子）缺陷的纯合子小鼠。具体目标2关注NCB 5 OR对ER和氧化应激反应的影响。胰腺β细胞特别容易受到ER应激。因此，我们设计了实验来确定NCB 5 OR缺陷的胰岛、胰岛素瘤细胞系和小鼠胚胎成纤维细胞（MEFs）是否表现出ER应激反应的基因表达和信号转导特征的变化。我们还将评估NCB 5 OR-/-组织和MEFs的ER以及NCB 5 OR耗竭的胰岛素瘤细胞中活性氧的产生和还原型谷胱甘肽与氧化型谷胱甘肽的比率。最后一个特定目的需要全面评估NCB 5 OR的生化功能。将采用细胞和无细胞下拉实验来测试和确认潜在的伴侣蛋白。我们还将开发一种无细胞系统来鉴定NCB 5 OR的生物底物和产物。我们将通过分析来自Ncb 5 or-/-和+/+小鼠肝脏的ER制剂中的脂质谱来检验NCB 5 OR介导ER膜中脂肪酸去饱和的假设。在这三个特定目标中计划的实验是密切相关的，并且共同地，应该促进我们对β细胞对氧化应激的防御的理解。这些研究可能为糖尿病的发病机制和治疗提供新的见解。

DESCRIPTION (provided by applicant): Development of definitive therapy for both type 1 and type 2 diabetes depends on a thorough understanding of the molecular events involved in the production of insulin within pancreatic islets. Our lab has cloned and characterized a highly conserved oxidoreductase, NCB5OR, that is localized in the endoplasmic reticulum (ER). The targeted inactivation of this gene in mice results in a diabetic phenotype. By 7 weeks of age Ncb5or-/- mice develop severe hyperglycemia with markedly decreased serum insulin levels. Pancreatic islets show marked deficiency in beta-cells but normal numbers of alpha, delta and PP cells. Our Research Plan is predicated on the hypothesis that NCB5OR protects the pancreatic beta-cell against oxidant-induced damage in the ER. In the first Specific Aim we plan studies that further characterize the impact of NCB5OR deficiency in the intact mouse. We will address the important question of whether NCB5OR plays a biologically important role outside the pancreatic beta-cell by transplanting the knockout mouse with normal beta-cells. We will also study Ncb5or -/- and +/+ mice expressing a transgene that reports ER stress in different organs and tissues. Finally, we will prepare mice homozygous for deficiencies in both NCB5OR and CHOP, a transcription factor required for ER stress induced apoptosis. Specific Aim 2 focuses on the impact of NCB5OR on responses to ER and oxidative stress. Pancreatic beta-cells are particularly prone to ER stress. Therefore we have designed experiments to determine whether NCB5OR-deficient pancreatic islets, insulinoma cell lines and mouse embryonic fibroblasts (MEFs) evince changes in gene expression and signal transduction characteristic of the ER stress response. We will also assess the production of reactive oxygen species and the ratio of reduced to oxidized glutathione in the ER of Ncb5or-/- tissues and MEFs as well as in NCB5OR depleted insulinoma cells. The last Specific Aim entails a comprehensive assessment of the biochemical function of NCB5OR. Cellular and cell-free pull-down experiments will be employed to test and confirm potential partner proteins. We will also develop a cell-free system to identify the biologic substrate(s) and product(s) of NCB5OR. We will test the hypothesis that NCB5OR mediates fatty acid desaturation in the ER membrane by analyzing lipid profiles in ER preparations from livers of Ncb5or-/- and +/+ mice. The experiments planned in these three Specific Aims are closely inter-related and, collectively, should advance our understanding of beta-cell's defense against oxidant stress. These studies may provide new insights into the pathogenesis and treatment of diabetes.