Pathogenesis and Impact of Islet Amyloid

描述（由申请人提供）： 项目总结-摘要人类2型糖尿病的胰岛病变包括ss细胞功能和质量的降低，这两者共同导致高血糖症随时间的发展和进展。胰岛淀粉样蛋白沉积是导致ss细胞丢失的一个因素，在大多数2型糖尿病患者中观察到。这些淀粉样蛋白沉积物含有作为其独特肽组分的胰岛淀粉样蛋白多肽（IAPP），也称为胰淀素，其是β细胞的正常β细胞分泌产物。 在胰岛淀粉样蛋白形成过程中，IAPP聚集并具有细胞毒性。这种聚集过程导致原纤维的形成，并最终导致通常位于细胞外的淀粉样蛋白沉积。这些沉积物在ss细胞外的发展和积累表明，在淀粉样蛋白形成的条件下，控制IAPP正常清除的机制可能受损。 虽然我们对胰岛淀粉样蛋白及其毒性了解很多，但对IAPP减少原纤维形成和/或增强原纤维清除的正常机制知之甚少。我们最近开发的数据表明，两种蛋白酶，脑啡肽酶和基质金属蛋白酶-9（MMP-9），通常由胰岛产生，可以限制原纤维形成的IAPP。其他研究表明，另一种胰岛蛋白酶，胰岛素降解酶（IDE），作用类似。基于这些发现，我们现在建议在体外确定这三种蛋白酶减少胰岛淀粉样蛋白形成的机制，以及在体内确定脑啡肽酶和MMP-9是否有效限制淀粉样蛋白沉积和β细胞毒性。 为了实现这一目标，我们确定了以下三大具体目标：1.确定脑啡肽酶减少胰岛淀粉样蛋白形成的机制; 2.确定MMP-9减少胰岛淀粉样蛋白形成的机制;和3.确定IDE减少胰岛淀粉样蛋白形成的机制。 使用体外方法，我们将确定是否neprilysin可以解离或降解IAPP纤维和/或胰岛淀粉样蛋白沉积物，以及是否由MMP-9和IDE的IAPP裂解产物是淀粉样蛋白。将使用化学抑制剂、siRNA和慢病毒构建体在培养的人和人IAPP（hIAPP）转基因小鼠胰岛中进行额外的体外研究。在这些研究中，我们将检测蛋白酶活性降低是否会增加淀粉样蛋白沉积和ss细胞凋亡，以及蛋白酶活性增加是否会减少淀粉样蛋白沉积和ss细胞凋亡。体内研究将采用我们的hIAPP转基因小鼠胰岛移植模型的淀粉样蛋白的形成，以确定是否增加脑啡肽酶和MMP-9的活性产生的移植慢病毒转导的胰岛与减少淀粉样蛋白的形成和减少β-细胞损失。 总之，这些研究的目的是增强我们对脑啡肽酶、MMP-9和IDE的生理学和病理生理学的了解，因为它们与hIAPP及其聚集形式有关。通过检测它们在调节胰岛淀粉样蛋白形成中的作用，我们希望找到治疗靶点，用于减少胰岛淀粉样蛋白，从而保护2型糖尿病患者的ss细胞群。 公共卫生相关性： 2型糖尿病正在达到流行病的程度，在退伍军人中很常见。导致糖尿病特征的葡萄糖水平升高的部分过程是负责产生和释放胰岛素的胰岛ss细胞的损失。ss细胞的这种破坏部分是由于淀粉样蛋白沉积物的形成而发生的，淀粉样蛋白沉积物由一种称为胰岛淀粉样多肽的蛋白质组成。这些沉积物形成的原因尚不清楚。我们现在建议使用来自人类和动物模型的胰岛来研究这些破坏性淀粉样蛋白沉积物形成的机制。其目标是开发一种方法，可以用来防止胰岛淀粉样蛋白的发展，并最终可以应用于治疗2型糖尿病患者，以保护他们的ss细胞。

DESCRIPTION (provided by applicant): PROJECT SUMMARY - ABSTRACT The islet lesion of human type 2 diabetes includes decreases in both ss-cell function and mass, which together contribute to the development and progression of hyperglycemia over time. Islet amyloid deposition is one factor that contributes to this loss of ss-cells and is observed in most patients with type 2 diabetes. These amyloid deposits contain as their unique peptide component islet amyloid polypeptide (IAPP), also known as amylin, which is a normal ss-cell secretory product of the ss-cell. During the process of islet amyloid formation, IAPP aggregates and is cytotoxic. This aggregation process results in the formation of fibrils and ultimately amyloid deposits that are typically located extracellularly. The development and accumulation of these deposits outside the ss-cell suggests that mechanisms controlling the normal clearance of IAPP may be impaired under conditions of amyloid formation. While we know much about islet amyloid and its toxicity, a great deal less is known about the normal mechanism(s) that decrease fibril formation by IAPP and/or enhance fibril clearance. Our recently developed data demonstrate that two proteases, neprilysin and matrix metalloproteinase-9 (MMP-9), normally produced by the islet, can limit fibril formation by IAPP. Others have shown that another islet protease, insulin-degrading enzyme (IDE), acts similarly. Based on these findings, we now propose to determine in vitro the mechanisms by which these three proteases decrease islet amyloid formation and in vivo whether neprilysin and MMP-9 are effective in limiting amyloid deposition and ss-cell toxicity. In order to achieve this, we have identified the following three major specific objectives: 1. To determine the mechanism(s) by which neprilysin decreases islet amyloid formation; 2. To determine the mechanism(s) by which MMP-9 decreases islet amyloid formation; and 3. To determine the mechanism(s) by which IDE decreases islet amyloid formation. Using in vitro approaches, we will determine whether neprilysin can dissociate or degrade IAPP fibrils and/or islet amyloid deposits and whether the products of IAPP cleavage by MMP-9 and IDE are amyloidogenic. Additional in vitro studies will be done in cultured human and human IAPP (hIAPP) transgenic mouse islets using chemical inhibitors, siRNA and lentiviral constructs. In these studies we will examine whether decreasing protease activity increases amyloid deposition and ss-cell apoptosis, and whether increasing protease activity decreases amyloid deposition and ss-cell apoptosis. The in vivo studies will employ our hIAPP transgenic mouse islet transplant model of amyloid formation to determine whether increased neprilysin and MMP-9 activity produced by transplanting lentivirus-transduced islets is associated with reduced amyloid formation and decreased ss-cell loss. In summary, the goal of these studies is to enhance our knowledge of the physiology and pathophysiology of neprilysin, MMP-9 and IDE as they relate to hIAPP and its aggregated forms. By examining their role in modifying islet amyloid formation, we hope to find therapeutic targets that could be used to reduce islet amyloid and thereby preserve ss-cell mass in type 2 diabetes. PUBLIC HEALTH RELEVANCE: NARRATIVE Type 2 diabetes is reaching epidemic proportions and is common amongst Veterans. Part of the process that results in the increase in glucose levels that characterizes diabetes is the loss of pancreatic islet ss-cells that are responsible for producing and releasing insulin. This destruction of ss-cells occurs in part by the formation of amyloid deposits, which are comprised of a protein called islet amyloid polypeptide. The reasons why these deposits form are unclear. We now propose to use islets from humans and animal models to study the mechanisms responsible for the formation of these destructive amyloid deposits. The goal is to develop approaches that can be used to prevent the development of islet amyloid and can ultimately be applied to treat patients with type 2 diabetes in order to preserve their ss-cells.