The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor that is insulinotropic in β-cells. Multiple endogenous and synthetic ligands exist for the GLP-1R and each ligand interacts with the receptor differently to produce specific activation of intracellular signaling pathways. Ligand binding typically induces receptor internalization, leading to either i) receptor degradation or ii) recycling to the cell membrane. We hypothesized that each specific GLP-1R ligand would produce a unique trafficking profile in primary β-cells. To test this hypothesis, we developed flow cytometry-based assay that can be utilized in primary dispersed mouse islets. First, we validated a GLP-1R antibody (GLP-1R-APC) for flow cytometry application. The GLP-1R-APC stained β-cells, but not α-cells, aligning with the reported expression pattern in islets. Post-sort analysis confirmed GLP-1R expression only in the GLP-1R-APC+ population. Finally, GLP-1R-APC did not stain any cell populations in GLP1R-/- islets. Next, we used this assay to quantify GLP-1R internalization in wild type β-cells. To induce internalization, we treated dispersed islets with either vehicle or a GLP-1R ligand for 30 minutes prior to antibody staining. After vehicle treatment, 90% of the β-cells stained GLP-1R-APC+. Treatment with either GLP-1 or exendin-4 reduced the number of GLP-1R-APC+ β-cells by 97 and 89%, respectively, suggesting near complete internalization of all GLP-1Rs. In contrast, treatment with the GLP-1R antagonist exendin-9 produced staining similar to PBS controls. Finally, we used image-stream technology, a combination of brightfield microscopy and flow cytometry, to visually confirm GLP-1R internalization. Here, vehicle-treated cells produced GLP-1R staining only on the cell membrane, which was nearly ablated by the treatment with a ligand.
In conclusion, we have developed an assay that allows for quantification of GLP-1R trafficking in primary mouse islets.
S.M. Gray: None. P. Ravn: Employee; Self; AstraZeneca. K. Sloop: None. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S.