A Novel Tool to Understand Insulin Production and Failure in Pancreatic beta-Cell

一种了解胰腺β细胞胰岛素产生和衰竭的新工具

• 批准号: 7455821
• 负责人: ISRAEL HODISH
• 金额: $ 9万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7455821
• 关键词: Affect; Age; Amino Acids; Anabolism; Animals; Antibodies; Applications Grants; Behavior; Beta Cell; Biochemical; Biological Assay; Blood Glucose; Body Weight; Breeding; C-Peptide; C57BL/6 Mouse; Calnexin; Cell model; Cells; Chimera organism; Chimeric Proteins; Complementary DNA; Confocal Microscopy; Coupled; Development; Diabetes Mellitus; Disease Progression; Dominant-Negative Mutation; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Exhibits; Exocytosis; Failure; Fasting; Female; Fluorescence; Fluorescence Microscopy; Freezing; Functional disorder; Genetic; Glass; Glucose; Green Fluorescent Proteins; Heterozygote; Human; Individual; Insulin; Insulin Antibodies; Islets of Langerhans; Israel; Knock-out; Label; Lead; Life; Measures; Methodology; Modeling; Molecular; Monitor; Mus; Mutation; Neonatal; Operative Surgical Procedures; Pancreas; Pathway interactions; Pattern; Phase; Physiologic pulse; Point Mutation; Prevalence; Process; Production; Proinsulin; Pulse taking; Radioimmunoassay; Relative (related person); Research Personnel; Rodent; Secretory Vesicles; Slice; Stimulus; Stress; Structure of beta Cell of islet; Techniques; Testing; Therapeutic Intervention; Time; Tissues; Toxic effect; Transgenes; Transgenic Mice; Ursidae Family; Wild Type Mouse; enhanced green fluorescent protein; fluorescence imaging; in vivo; insulin secretion; interest; islet; male; mutant; novel; novel strategies; programs; promoter; response; secretion process; secretory protein; selective expression; tool; trafficking

DESCRIPTION (provided by applicant): As the prevalence of Diabetes Mellitus increases worldwide, the need for recognizing the major cellular and molecular processes that underlie progression of the disease becomes more urgent. It is now clear that at the time or shortly after onset of "beta cell dysfunction" in diabetes, beta cells develop "secretory pathway stress" initiated within a compartment in the cell called the endoplasmic reticulum (ER). ER stress includes misfolding of proinsulin, the beta cell's major secretory protein product - the precursor in insulin biosynthesis. I propose that proinsulin misfolding causes further beta cell dysfunction and demise, ultimately decreasing pancreatic insulin mass. Indeed in the Akita mouse, a point mutation in just one genetic copy of proinsulin-ll (with two normal copies of proinsulin-l and one normal proinsulin-ll) causes sufficient misfolded proinsulin to produce diabetes in all animals with the mutation (so-called "dominant negative" behavior). I have been interested to know whether misfolded proinsulin can attack other proinsulin molecules in the ER and confer upon them the mutant behavior, thereby leading to cell toxicity and loss of beta cell (and insulin) mass. However, conventional methodologies cannot distinguish normal proinsulin from misfolded proinsulin. In this application, I have developed a novel approach to model the folding of normal proinsulin and to examine possible interactions with misfolded proinsulin by generating a transgenic mouse expressing (selectively in (-cells) a human proinsulin fusion protein containing enhanced green fluorescent protein within the C-peptide (midregion) of the proinsulin molecule (called hProins-CpepGFP). Preliminary studies are presented, indicating that this fusion protein exhibits the typical features of normal proinsulin folding, trafficking, processing, and secretion. In this grant application I intend to breed this transgenic mouse with Akita mice or, alternatively, to introduce the Akita mutation into the hProins-CpepGFP construct, in order to directly examine interactions of hProins-CpepGFP with misfolded proinsulin. Using these models, (-cell (tagged) insulin mass can be followed. A combination of biochemical and morphological approaches are proposed to understand the relationship of proinsulin misfolding to progression of diabetes. The model presented opens new avenues to the development and testing of potential therapeutic interventions that may ultimately help preserve insulin secretory function in humans with diabetes mellitus.

描述（由申请人提供）： 随着糖尿病在世界范围内的流行率的增加，认识到疾病进展的主要细胞和分子过程的需要变得更加迫切。现在清楚的是，在糖尿病中的“β细胞功能障碍”发作时或发作后不久，β细胞在称为内质网（ER）的细胞隔室内产生“分泌途径应激”。ER应激包括胰岛素原的错误折叠，胰岛素原是β细胞的主要分泌蛋白产物-胰岛素生物合成的前体。我认为胰岛素原错误折叠会导致β细胞功能障碍和死亡，最终降低胰腺胰岛素质量。事实上，在秋田小鼠中，仅胰岛素原-II的一个基因拷贝中的点突变（具有胰岛素原-I的两个正常拷贝和胰岛素原-II的一个正常拷贝）导致足够的错误折叠的胰岛素原在具有突变的所有动物中产生糖尿病（所谓的“显性负性”行为）。我一直有兴趣知道错误折叠的胰岛素原是否可以攻击ER中的其他胰岛素原分子，并赋予它们突变行为，从而导致细胞毒性和β细胞（和胰岛素）质量的损失。然而，常规方法不能区分正常胰岛素原和错误折叠的胰岛素原。在本申请中，我开发了一种新的方法来模拟正常胰岛素原的折叠，并通过产生转基因小鼠来研究与错误折叠的胰岛素原的可能相互作用，所述转基因小鼠表达（选择性地在（-细胞）中）人胰岛素原融合蛋白，所述人胰岛素原融合蛋白在胰岛素原分子的C肽（中间区）内含有增强的绿色荧光蛋白（称为hProins-CpepGFP）。初步研究表明，该融合蛋白具有正常胰岛素原折叠、运输、加工和分泌的典型特征。在本授权申请中，我打算用秋田小鼠繁殖这种转基因小鼠，或者，将秋田突变引入hProins-CpepGFP构建体，以便直接检查hProins-CpepGFP与错误折叠的胰岛素原的相互作用。使用这些模型，可以跟踪（-细胞（标记的）胰岛素质量。 生物化学和形态学的方法相结合，提出了解胰岛素原错误折叠的糖尿病进展的关系。该模型为开发和测试潜在的治疗干预措施开辟了新的途径，这些干预措施可能最终有助于保护糖尿病患者的胰岛素分泌功能。