An improved understanding of the pathophysiologic mechanisms contributing to insulin deficiency in type 1 diabetes (T1D) is crucial to optimize approaches to disease prevention and treatment. Multiple groups have described increases in beta cell prohormones in individuals at different stages of T1D development, both at the level of the islet and in circulation. These findings have highlighted the concept that dysfunctional beta cell prohormone processing may contribute to insulin deficiency and T1D progression. Because of historical limitations of access to human pancreatic tissue, prior human analyses have been unable to combine analysis of prohormone secretory dynamics with tissue–level phenotyping of prohormone expression and processing. The introduction of nPOD and other like programs, alongside innovative technologies like the tissue slice effort (the subject of this RFA), is markedly changing this situation. For this proposal, our overarching hypothesis is that dysfunctional prohormone processing at the level of the islet will be correlated with altered islet prohormone release. Specifically, we predict that, relative to non–diabetic control donors, islets from donors with T1D will exhibit: 1) increased basal release of islet proinsulin and pro–islet amyloid polypeptide (proIAPP) relative to mature insulin and IAPP, 2) an abrogated increase in prohormone release under stimulated conditions, and that 3) these functional secretory abnormalities will correlate with islet prohormone accumulation. To test this, we propose analysis of islet prohormone secretion relative to mature hormone secretion from slices from nondiabetic donors and donors with T1D. Histologic analyses of proinsulin, proIAPP, and insulin and amyloid in slice sections will be performed. These results will determine if islet prohormone expression and processing is linked to altered hormone secretion, yielding critical insight into association of altered prohormone processing and release with insulin deficiency in T1D. Additional exploratory mechanistic studies will test EV content using the remaining perifusate samples already at our possession at IU, using the Exoview platform, with quantification of PD–L1+ and LC3+ positive EVs (absolute and as a percentage of total EVs) as well as bead–based pulldown and permeablization to identify proinsulin+ EVs. These results will determine if heterogeneity islet proinsulin secretion is linked to altered EV composition, yielding critical insight into association of altered prohormone processing and release with insulin deficiency in T1D.
