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 ind...