Defining alpha-cell proglucagon processing for type 2 diabetes treatment

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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 assess markers of islet cell identity and transcriptome on mouse and human islets. These data will enable the development of therapies that increase alpha cell GLP-1 production for T2DM treatment.