Role of Ncb5or in Insulin Production

Ncb5or 在胰岛素生产中的作用

• 批准号: 6985070
• 负责人: H. Franklin Bunn
• 金额: $ 29.49万
• 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/6985070
• 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）：开发针对1型和2型糖尿病的确定性治疗取决于对胰岛中胰岛素产生所涉及的分子事件的透彻理解。我们的实验室已克隆并表征了高度保守的氧化还原酶NCB5OR，该氧化还原酶位于内质网（ER）中。该基因在小鼠中的靶向失活导致糖尿病表型。到7周龄的NCB5OR - / - 小鼠患有严重的高血糖，血清胰岛素水平明显降低。胰岛在β细胞中显示出明显的缺乏，但α，三角洲和PP细胞的正常数字。我们的研究计划是基于以下假设：NCB5OR保护胰腺β细胞免受ER中氧化剂诱导的损伤。在第一个特定目的中，我们计划的研究进一步表征了NCB5OR缺乏对完整小鼠的影响。我们将通过用正常的β细胞移植敲除小鼠来解决NCB5OR是否在胰腺β细胞之外起生物学上重要的作用的重要问题。我们还将研究表达转基因的NCB5OR - / - 和 +/ +小鼠，该转基因报告了不同器官和组织中的ER应力。最后，我们将为NCB5OR和CHOP的缺陷纯合子准备小鼠，这是ER应力诱导的凋亡所需的转录因子。具体目标2的重点是NCB5OR对ER反应和氧化应激的影响。胰腺β细胞特别容易出现ER应力。因此，我们设计了实验来确定NCB5或缺陷型胰岛，胰岛素瘤细胞系和小鼠胚胎成纤维细胞（MEFS）是否证明了基因表达的变化和ER应激反应的信号转导特征。我们还将评估活性氧的产生，以及在NCB5OR - / - 组织和MEF的ER以及NCB5或耗尽的胰岛素瘤细胞中还原与氧化的谷胱甘肽的比率。最后的特定目的需要对NCB5OR的生化功能进行全面评估。将采用无细胞和无细胞下拉实验来测试和确认潜在的伴侣蛋白。我们还将开发一个无细胞的系统，以识别NCB5OR的生物底物和产物。我们将通过分析NCB5OR - / - 和 +/ +小鼠的ER制剂中的脂质谱分析ER膜中介导脂肪酸的脂肪酸介导的脂肪酸去饱和度的假设。在这三个特定目标中计划的实验密切相关，并且总体上应该促进我们对Beta-Cell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理和治疗提供新的见解。