Role of Ncb5or in Insulin Production

描述（由申请人提供）：1型和2型糖尿病的决定性治疗的发展取决于对胰岛内胰岛素产生的分子事件的透彻理解。我们的实验室已经克隆并鉴定了一种高度保守的氧化还原酶NCB5OR，它位于内质网（ER）中。该基因在小鼠中的靶向失活导致糖尿病表型。7周龄时，ncb5或-/-小鼠出现严重高血糖，血清胰岛素水平显著降低。胰岛细胞明显缺乏，但α、δ和PP细胞数量正常。我们的研究计划是基于NCB5OR保护胰腺β细胞免受内质网氧化引起的损伤的假设。在第一个特异性目标中，我们计划进一步研究NCB5OR缺乏对完整小鼠的影响。我们将通过将敲除小鼠与正常β细胞移植，来解决NCB5OR是否在胰腺β细胞外发挥重要的生物学作用这一重要问题。我们还将研究表达不同器官和组织内质网应激的转基因ncb5或-/-和+/+小鼠。最后，我们将制备NCB5OR和CHOP缺失的小鼠纯合子，CHOP是内质网应激诱导细胞凋亡所需的转录因子。具体目标2侧重于NCB5OR对内质网和氧化应激反应的影响。胰腺细胞特别容易受到内质网应激的影响。因此，我们设计了实验来确定缺乏ncb5or的胰岛、胰岛素瘤细胞系和小鼠胚胎成纤维细胞（MEFs）是否会改变内质网应激反应的基因表达和信号转导特征。我们还将评估ncb5或-/-组织和mef以及ncb5或耗尽的胰岛素瘤细胞内质膜中活性氧的产生和还原性谷胱甘肽与氧化性谷胱甘肽的比例。最后一个特定目标需要对NCB5OR的生化功能进行全面评估。细胞和无细胞下拉实验将用于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NCB5OR的生物底物和产物。我们将通过分析NCB5OR -/-和+/+小鼠肝脏内质网制剂的脂质谱来验证NCB5OR介导内质网膜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，总的来说，应该推进我们对β细胞防御氧化应激的理解。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.