PROJECT SUMMARY: We propose to use state-of-the-art mouse modeling, genetically modified human islets and a unique single cell RNA-seq technique to generate new insights into the regulation of pancreatic islet function that will serve as the basis for developing new therapeutics for type 2 diabetes mellitus (T2DM). The proglucagon-derived peptides, glucagon and glucagon-like peptide-1 (GLP-1), potentiate glucose-stimulated insulin secretion (GSIS). However, unlike GLP-1, glucagon promotes hepatic glucose production and is a weaker inducer of GSIS. We identified a pathway that shifts the alpha cell from producing glucagon to producing GLP-1. We propose to define this pathway to establish a new model describing GLP-1 function and to identify drug targets that increase alpha cell GLP-1 production. Proglucagon is expressed in gut L cells and islet alpha cells and is differentially cleaved depending on the prohormone convertase (PC) present. Canonically, PC1/3 (Pcsk1) is expressed in L cells to produce GLP-1 and PC2 (Pcsk2) is expressed in alpha cells to produce glucagon. However, our data reveal that beta cell GLP-1 receptor (GLP-1R) signaling increases alpha cell GLP-1 and Pcsk1 expression. We hypothesize that beta cell GLP-1R signaling increases alpha cell GLP-1 production to augment GSIS in a paracrine positive feedback loop. We will pursue three aims to define the alpha cell to beta cell (aims 1 and 2) and the beta cell to alpha cell (aim 3) cross-talk involved in our model of beta cell GLP-1R function to identify novel targets that raise alpha cell GLP-1:glucagon levels for T2DM treatment. In aim 1, we will define the contribution of alpha cells to beta cell GLP-1R function in response to endogenous GLP-1 in high fat diet-fed mice. To this end, we will assess glucose regulation and GSIS in low fat diet-fed or high fat diet-fed beta cell GLP-1R wild-type and knockout mice, with or without alpha cell ablation, with or without L cell-derived GLP-1 stimulation. In aim 2, we will define the contribution of alpha cells to beta cell GLP-1R function in response to exogenous GLP-1. To this end, we will assess glucose regulation and GSIS in beta cell GLP-1R wild-type and knockout mice, with or without alpha cell ablation, with or without treatment with a GLP-1R agonist. In aim 3, we will determine the impact of beta cell GLP-1R signaling on alpha cell fate. We show that beta cell GLP-1R signaling increases the expression of Pcsk1 and other beta cell-specific genes in human alpha cells and extend upon previous work showing that alpha cells that express GLP-1 are immature. Thus, we hypothesize that beta cell GLP-1R signaling regulates alpha cell transcriptional programs to promote conversion of alpha cells into beta-like cells. To test this hypothesis and identify putative mediators we will perform IHC, lineage tracing and conditioned media studies in mice. In parallel, we will use a highly sensitive single cell RNA-seq platform that we have developed to as...