Role of Ncb5or in Insulin Production

Ncb5or 在胰岛素生产中的作用

• 批准号: 7116993
• 负责人: H. Franklin Bunn
• 金额: $ 28.81万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7116993
• 关键词: apoptosis; cell line; cytoprotection; diabetes mellitus; disease /disorder model; endoplasmic reticulum; enzyme deficiency; enzyme mechanism; enzyme substrate; genetically modified animals; hormone regulation /control mechanism; insulin; laboratory mouse; molecular pathology; oxidative stress; oxidoreductase; pancreatic islet function; pancreatic islets; protein protein interaction; transcription factor

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.

描述（由申请人提供）：针对 1 型和 2 型糖尿病的明确疗法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。该基因在小鼠体内的定向失活会导致糖尿病表型。到 7 周龄时，Ncb5or-/- 小鼠出现严重高血糖，血清胰岛素水平显着下降。胰岛的 β 细胞明显缺乏，但 α、δ 和 PP 细胞数量正常。我们的研究计划基于这样的假设：NCB5OR 可以保护胰腺 β 细胞免受内质网氧化诱导的损伤。在第一个具体目标中，我们计划进一步研究 NCB5OR 缺陷对完整小鼠的影响。我们将通过向敲除小鼠移植正常 β 细胞来解决 NCB5OR 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 Ncb5or -/- 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 NCB5OR 和 CHOP 缺陷的纯合小鼠，CHOP 是 ER 应激诱导细胞凋亡所需的转录因子。具体目标 2 重点关注 NCB5OR 对 ER 和氧化应激反应的影响。胰腺β细胞特别容易受到内质网应激。因此，我们设计了实验来确定 NCB5OR 缺陷的胰岛、胰岛素瘤细胞系和小鼠胚胎成纤维细胞 (MEF) 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 Ncb5or-/- 组织和 MEF 以及 NCB5OR 耗尽的胰岛素瘤细胞的 ER 中活性氧的产生以及还原型谷胱甘肽与氧化型谷胱甘肽的比率。最后一个具体目标需要对 NCB5OR 的生化功能进行全面评估。将采用细胞和无细胞下拉实验来测试和确认潜在的伙伴蛋白。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 ER 制剂中的脂质谱来检验 NCB5OR 介导 ER 膜中脂肪酸去饱和的假设。这三个具体目标中计划的实验是密切相关的，总的来说，应该增进我们对 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。