Role of RAGE in amyloid-induced pancreatic islet dysfunction in diabetes

项目摘要/摘要 在2型糖尿病(T2D)中，由胰岛淀粉样多肽(IAPP)组成的淀粉样沉积在 胰岛。T2D胰岛还损害了β细胞的胰岛素分泌，胰高血糖素分泌失调 来自α细胞，炎症增加，血管系统改变。在多种潜在机制中 将淀粉样蛋白沉积与胰岛功能障碍联系起来，晚期糖基化终产物受体(RAGE)是 最近发现在体外可以结合IAPP寡聚体并介导β细胞毒性，这一结果也得到了支持 使用转基因啮齿动物模型。但是，体外细胞培养模型虽然有价值，但并不能完全复制 生物体内复杂的环境变化、细胞间变化或时间变化。此外，人类和啮齿动物 胰岛在功能、结构、细胞组成和基因表达方面存在差异。因此，要充分理解 人类疾病的发病机制，必须研究在人类细胞和组织中的这些过程 VIVO上下文。这类研究一直受到无法获得和操纵这些相对 由于无法接触到人体组织，以及缺乏对其进行纵向研究的活体模型。因此，它 仍不清楚内源性分泌的IAPP寡聚体是否作用于原代人β的RAGE受体 细胞，如果这种信号发生在α细胞中，以及特定细胞类型中的IAPP-RAGE信号对胰岛有什么影响 功能。我推测IAPP寡聚体诱导β和α细胞RAGE受体的激活会损害 体外和体内人类胰岛功能与健康。为了用人类的胰岛来检验我的假设，我将使用 四种新的技术和试剂。1)我们最近报道的伪胰岛方法将使高效的遗传 在重新聚集成功能细胞团之前操纵特定的胰岛细胞类型。2)新的生命中 成像技术将允许纵向监测移植的人类假性胰岛中淀粉样蛋白的形成 进入老鼠的眼睛前房。3)将假性胰岛移植到新近开发的 高血糖素基因敲除小鼠将可以在体内准确测量人的高血糖素分泌。4) 应用单一的核RNA测序方法将能够评估转录对 移植的假性胰岛中的特定细胞类型。在目标1中，我将检验愤怒调节IAPP的假设 低聚物在体外和体内诱导人胰岛β细胞功能障碍。在目标2中，我将测试假设 ⍺细胞中的IAPP-RAGE信号在体外和体内引起人胰岛胰高血糖素分泌失调。 这些目标的完成将阐明T2D、OPEN发病的关键机制 研究新的预防和治疗方法的途径。我将受益于杰出的 范德比尔特糖尿病研究和培训中心的环境、协作和指导 作为一名内科科学家过渡到独立。

PROJECT SUMMARY / ABSTRACT In type 2 diabetes (T2D), amyloid deposits composed of islet amyloid polypeptide (IAPP) are found within pancreatic islets. T2D islets also have impaired insulin secretion from β cells, dysregulated glucagon secretion from α cells, increased inflammation, and alterations in vasculature. Among multiple potential mechanisms linking amyloid deposition and islet dysfunction, the receptor for advanced glycation endproducts (RAGE) was recently shown to bind IAPP oligomers and mediate β cell toxicity in vitro, which results were also supported using a transgenic rodent model. But in vitro cell culture models, while valuable, do not fully replicate the complex environmental, intercellular, or temporal changes in living organisms. Furthermore, human and rodent islets differ in function, structure, cellular composition, and gene expression. Thus, to fully understand the pathogenesis of human disease, one must study these processes in human cells and tissues in the in vivo context. Such studies have been limited by the inability to obtain and manipulate these relatively inaccessible human tissues and by the lack of in vivo models in which to study them longitudinally. It therefore remains unknown if endogenously secreted IAPP oligomers act on the RAGE receptor in primary human β cells, if such signaling occurs in α cells, and what effect IAPP-RAGE signaling in specific cell types has on islet function. I hypothesize that IAPP oligomer-induced activation of RAGE receptors on β and α cells impairs human islet function and health in vitro and in vivo. To test my hypothesis using human islets, I will employ four novel techniques and reagents. 1) Our recently reported pseudoislet method will enable efficient genetic manipulation of specific islet cell types prior to reaggregation into functional cell clusters. 2) New intravital imaging techniques will allow longitudinal monitoring of amyloid formation in human pseudoislets transplanted into the mouse anterior chamber of the eye. 3) Transplantation of pseudoislets into a recently developed glucagon knockout mouse will permit accurate measurement of human glucagon secretion in vivo. 4) Application of single nuclear RNA sequencing approaches will permit assessment of transcriptional effects on specific cell types in transplanted pseudoislets. In Aim 1, I will test the hypothesis that RAGE mediates IAPP oligomer-induced β cell dysfunction in human islets in vitro and in vivo. In Aim 2, I will test the hypothesis that IAPP-RAGE signaling in ⍺ cells causes dysregulated glucagon secretion in human islets in vitro and in vivo. Completion of these aims will elucidate key mechanisms responsible for pathogenesis of T2D, opening avenues for study into new preventive and therapeutic approaches. I will benefit from the outstanding environment, collaboration, and mentorship at the Vanderbilt Diabetes Research and Training Center as I transition to independence as a physician-scientist.