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229-LB: Quantification of GLP-1R Internalization and Recycling in Live ß Cells

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor that is predominantly expressed on pancreatic β-cells in mice. Endogenous and synthetic ligands for GLP-1R exhibit distinct intracellular signaling cascades and promote insulin secretion to various degrees. Ligand binding typically induces receptor internalization followed by recycling back to the membrane or degradation within the lysosome. We hypothesized that endogenous and synthetic GLP-1R ligands would have distinct rates of internalization and recycling in β-cells. To test this hypothesis, we combined a recently-validated, highly-specific GLP-1R antibody (Glp1r0017) with flow cytometry and applied ligands (GLP-1, exendin-4 [Ex4], glucagon, and tirzepatide [TZP]) to characterize GLP-1R trafficking in freshly-isolated mouse islet cells. Under basal conditions, ~90% of β-cells were GLP1R+, with very low or absent binding in α- and δ-cells. Increasing ligand concentrations (0.01-100 nM) demonstrated distinct rates of internalization between ligands. Most potent was Ex4, followed by the native ligand GLP-1. TZP, a dual-receptor ligand for GLP-1R and GIPR, was more modest in inducing internalization. While showing different potencies, Ex4, GLP-1, and TZP were able to induce maximal internalization. In contrast, maximal treatment with glucagon only resulted in ~70% of β-cells being GLP-1R+. To test the rate of recycling back to the plasma membrane, we treated islet cells with Ex4 or GLP-1 with a dose that induced ~50% internalization, removed the ligand, and then monitored GLP1R+ β-cells over time. Within the first 15 minutes, GLP1R+ β-cells increased linearly and rapidly. This plateaued from 30-60 minutes, resulting in 60-70% of β-cells being GLP1R+ at the end of the experiment. In summary, our assay allows for investigation of GLP-1R trafficking in live β-cells. Understanding into the factors that regulate GLP-1R internalization and recycling may shed light on the insulinotropic properties of these ligands. Disclosure S. M. Gray: None. K. Sloop: Employee; Self; Eli Lilly and Company. P. Ravn: None. J. Campbell: None. D. A. D’alessio: Advisory Panel; Self; Eli Lilly and Company, Sun Pharmaceutical Industries Ltd., Research Support; Self; Eli Lilly and Company, Merck & Co., Inc. Funding National Institutes of Health (F32DK121420, R01DK123075, R01DK101991); Lilly Research Award Program