Mechanisms Driving the Kinetics of Incretin-Mediated Beta Cell Responses

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are therapeutic Type 2 Diabetes Mellitus (T2DM) targets. Incretins bind to their respective receptors on beta () cells to activate adenylyl cyclases (ACs) and generate cAMP, the second messenger necessary to potentiate glucose-stimulated insulin secretion. Although GIPR and GLP-1R are Gs- coupled GPCRs that share the same downstream signaling cascades, I discovered that they elicit profoundly different kinetics of cAMP generation in primary  cells. The mechanisms underlying the difference between GIPR and GLP-1R signaling are unknown. Furthermore, a direct comparison of the signaling and trafficking between GIPR and GLP-1R in primary  cells has not been performed. This knowledge gap prompts the need to improve our understanding of incretin signaling towards more effective T2D treatments. Details of the kinetics of incretin-induced cAMP responses and how they are affected by GPCR trafficking and the nutrient stimulated Ca2+ responses, are not well established. By imaging genetically encoded cAMP sensors expressed in  cells, I have identified differences in the cAMP kinetics of  cells to GIP and GLP-1 stimulation. I propose that these stark differences connect to differences in receptor trafficking and may explain in part the known differences in effectiveness between both incretins. Furthermore, I also discovered that incretin-mediated cAMP production is paradoxically inhibited by Ca2+ induced by glucose and other stimuli, suggesting a dynamic interaction between Ca2+ and Ca2+-regulated ACs that shapes the kinetics of cAMP formation and determines the  cell insulin secretory response to nutrient and incretin co-stimulation. My overarching hypothesis is that receptor trafficking, -Arrestin preferences, and the interplay between Ca2+ and ACs underlie dynamic cAMP kinetics of  cells in response to nutrient and incretin co-stimulation. I will test this hypothesis in two separate aims that converge on the functional imaging of primary  cells. In Aim 1, I will quantify trafficking of SNAP-tag incretin receptors co- expressed with a genetically encoded cAMP sensor in HEK293 cells and primary mouse  cells to determine how incretin receptor trafficking influences cAMP responses. I will also assess changes in incretin-mediated cAMP responses in the absence of -Arrestins. In Aim 2 I will multiplex genetically encoded cAMP and Ca2+ sensors to determine the interplay between cAMP and Ca2+ across hundreds of  cells in islets that lack key ACs. These approaches are innovative as they leverage novel transgenic mouse that expresses endogenous SNAP-tag GLP-1R in every  cell in islets. Separately, I can quantify cAMP and Ca2+ dynamics in the same cells using genetically encoded spectrally compatible fluorescent sensors. These proposed aims are significant as they will provide a comprehensive understanding of the mechanisms and kinetics that dictate how different incretins achieve insulin release under nutrient stimulation. This understanding carries significant weight in the development of improved incretin dual agonists to treat T2DM and improve patient outcomes.