Half of US adults have diabetes or pre-diabetes, illustrating a critical need for novel treatments. There is a fundamental gap in the understanding of how paracrine feedback in the islet controls insulin and glucagon. The long-term goal is to elucidate the (patho)physiological crosstalk within pancreatic islets in order to identify novel therapeutic targets. The overall objective in this application is to understand how endogenous SST inhibits Ca2+ and cAMP pathways in alpha and beta cells concurrently to balance inhibition of insulin and glucagon exocytosis. How a single inhibitory hormone can balance the inhibition of beta and alpha cells is a major and poorly understood aspect of islet physiology. The central hypothesis is that the pancreatic delta cell coordinates with beta cells to attenuate insulin and glucagon secretion by inhibiting Ca2+ and cAMP while concurrently remodeling the F-actin cytoskeleton to limit exocytosis. The rationale for the proposed research is that elucidating the mechanisms and circumstances of delta inhibition of alpha and beta cells would establish a better understanding of the physiological role of delta cells. This hypothesis will be tested in 3 specific aims. Aim 1 tests the hypothesis that beta and delta cells coordinate responses largely independently of gap junctions via the exchange of paracrine factors. Beta and delta cell calcium behavior will be quantified across hundreds of beta and delta cells in the same intact islets by GCaMP6. Dye injection experiments will determine if gap junctions can account for the coordinated delta and beta cell behavior. Aim 2 tests the hypothesis that endogenous delta cell feedback inhibition leads to robust intra-islet elevations of SST that coordinate alpha and beta cell behaviors via the combined inhibition of cAMP and Ca2+. Beta cells primarily respond to glucose with Ca2+. Insulin release is potentiated by cAMP. Ca2+ and cAMP will be quantified across hundreds of alpha and beta cells expressing biosensors for Ca2+ and cAMP in islets with or without SST. We will quantify local SST release via unique Sstr2 and Sstr3 (ant)agonists to determine the relative importance of cilia SSTR3 and cell-surface Sstr2. Aim 3 tests the hypothesis that activation of SSTR2 and SSTR3 on alpha and beta cells differentially inhibits exocytosis by remodeling the cortical F-actin network by activating RhoA GTPase. F-actin remodeling will be tested directly using FRET biosensors for RhoA expressed by stable MIN6 cells and primary islets in response to SST, by measuring dynamic remodeling of cortical F-actin via LifeAct, and by quantifying transcriptional changes to stimulation of primary alpha and beta cells with SSTR2 and SSTR3-selective agonists. The research is conceptually and technically innovative, in the applicants' opinion, as it evaluates the important physiological role of delta cells within intact islets in attenuating alpha and beta cell activity. It attains this by applying new techno...