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 ...