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

描述(由申请人提供)： 随着糖尿病在世界范围内的流行，认识疾病进展的主要细胞和分子过程的必要性变得更加迫切。现在很清楚的是，在糖尿病患者出现“β细胞功能障碍”的时候或之后不久，β细胞就会产生“分泌途径应激”，起始于细胞内质网(ER)。内质网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，是胰岛素生物合成的前体。我认为，胰岛素原的错误折叠会导致进一步的胰岛细胞功能障碍和死亡，最终减少胰腺胰岛素质量。事实上，在秋田小鼠中，仅一个胰岛素原基因拷贝的点突变(带有两个正常的胰岛素原拷贝-L和一个正常的胰岛素原-11)就会导致足够多的错误折叠的胰岛素原，从而在所有具有该突变的动物中产生糖尿病(所谓的“显性阴性”行为)。我一直想知道错误折叠的胰岛素原是否会攻击内质网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)质量的丧失。然而，传统的方法不能区分正常的胰岛素原和错误折叠的胰岛素原。在这项应用中，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(在细胞中选择性地)表达人胰岛素原融合蛋白的转基因小鼠(在胰岛素原分子的C肽(中间区域)中含有增强的绿色荧光蛋白)(称为hProins-CPepGFP)，来研究与错误折叠的胰岛素原可能的相互作用。初步研究表明，该融合蛋白具有正常的胰岛素原折叠、运输、加工和分泌的典型特征。在这次拨款申请中，我打算用Akita小鼠培育这只转基因小鼠，或者，将Akita突变引入hProins-CPepGFP结构中，以便直接检测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(-cell(标记))胰岛素质量。 我们建议结合生化和形态学的方法来了解胰岛素原错误折叠与糖尿病进展的关系。提出的模型为开发和测试潜在的治疗干预措施开辟了新的途径，这些干预措施最终可能有助于保护糖尿病患者的胰岛素分泌功能。

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.