In vivo mechanisms of amyloid-induced pancreatic islet dysfunction in type 2 diabetes

项目总结/摘要 2型糖尿病（T2 D）影响20%的退伍军人，每年花费VA近15亿美元。除了 胰岛素抵抗，定义T2 D的高血糖症是由胰岛素分泌不足引起的， 胰岛β和α细胞的胰高血糖素分泌失调。T2 D胰岛也被表征为 血管系统的变化、炎症增加和不溶性淀粉样蛋白的沉积，主要由 胰岛淀粉样多肽（IAPP）。可溶性IAPP寡聚体，而不是淀粉样蛋白本身，对β细胞有毒， 通过各种假定的机制，包括ER应激、氧化应激、膜透化， 受体介导的信号传导。晚期糖基化终末产物受体（AGEs），可结合多种 细胞外配体和激活细胞内炎症信号通路，最近被证明可以结合 IAPP寡聚体和介导IAPP寡聚体诱导的β细胞毒性，使用细胞和胰岛培养模型， 转基因小鼠模型。然而，目前尚不清楚人类胰岛中是否存在IAPP-RAGE信号传导，如果存在的话， 信号发生在非β胰岛细胞，包括α细胞，什么影响特定细胞类型的IAPP-β信号 对人类胰岛功能的影响，以及在人类胰岛细胞中激活了哪些特定的信号通路。我 假设IAPP寡聚体诱导的β和β 2细胞上的β 2受体的活化调节 人体胰岛功能和健康研究。为了验证我的假设并填补这些知识空白，我 将利用四种新的实验技术来研究原代人类胰岛细胞：1）最近开发的 假胰岛方法，其允许对人类胰岛进行有效的细胞特异性遗传操作; 2） 将人假胰岛移植到小鼠中，以能够纵向分析胰岛的结构和功能， 体内; 3）改良的活体成像技术，以纵向观察淀粉样蛋白形成和细胞死亡; 4） 单核分离和测序技术，以检测转导细胞中基因表达的变化， 假胰岛在目的1中，我将检验以下假设，即β-淀粉样蛋白介导IAPP寡聚体诱导的β细胞 在体外和体内的人胰岛功能障碍。在目标2中，我将测试IAPP-β信号转导在 胰岛细胞在体外和体内引起人胰岛中胰高血糖素分泌失调。这些实验将 阐明T2 D发病机制的基本过程，并帮助确定治疗和预防T2 D的新靶点。我 将完成这些目标，作为强化监督的职业发展计划的一部分， 多学科专家指导委员会的指导。我将接受正式和非正式的培训， 五个基本领域：1）新兴实验技术; 2）科学教育; 3）演示 和沟通技巧; 4）专业发展;和5）实验室管理。这些技能和 我提出的实验结果将为我的独立研究计划奠定坚实的基础，因为我 努力提高对糖尿病的认识和治疗。 项目摘要-第1页

PROJECT SUMMARY / ABSTRACT Type 2 diabetes (T2D) affects 20% of veterans and costs the VA almost $1.5 billion annually. In addition to insulin resistance, the hyperglycemia that defines T2D is caused by insufficient insulin secretion and dysregulated glucagon secretion from the β and α cells of pancreatic islets. T2D islets are also characterized by changes in vasculature, increased inflammation, and deposition of insoluble amyloid, composed primarily of islet amyloid polypeptide (IAPP). Soluble IAPP oligomers, rather than the amyloid itself, are toxic to β cells, through various postulated mechanisms including ER stress, oxidative stress, membrane permeabilization, and receptor-mediated signaling. The receptor for advanced glycation endproducts (RAGE), which binds several extracellular ligands and activates intracellular inflammatory signaling pathways, was recently shown to bind IAPP oligomers and mediate IAPP oligomer-induced toxicity in β cells using cell and islet culture models and transgenic mouse models. However, it is unknown if IAPP-RAGE signaling occurs in human islets, if such signaling occurs in non-β islet cells including α cells, what effect IAPP-RAGE signaling in specific cell types has on human islet function, and what specific RAGE signaling pathways are activated in human islet cells. I hypothesize that IAPP oligomer-induced activation of RAGE receptors on β and ⍺ cells modulates human islet function and health in vitro and in vivo. To test my hypothesis and fill these knowledge gaps, I will leverage four new experimental techniques to study primary human islet cells: 1) recently developed pseudoislet methodology that allows efficient cell-specific genetic manipulation of human islets; 2) transplantation of human pseudoislets into mice to enable longitudinal analysis of structure and function in vivo; 3) modified intravital imaging techniques to visualize amyloid formation and cell death longitudinally; 4) single nuclear isolation and sequencing technologies to detect changes in gene expression in transduced 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. These experiments will clarify fundamental processes in T2D pathogenesis and help identify novel targets to treat and prevent T2D. I will complete these aims as part of an intensive supervised career development plan with oversight and guidance from an expert multi-disciplinary mentoring committee. I will receive formal and informal training in five fundamental areas: 1) new and emerging experimental techniques; 2) scientific education; 3) presentation and communication skills; 4) professional development; and 5) laboratory management. These skills and the results of my proposed experiments will form a strong foundation for my independent research program as I work to improve the understanding and treatment of diabetes mellitus. Project Summary-Page 1