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.

Grant Number: 5F31DK136313-03
NIH Institute/Center: NIH
Principal Investigator: Michelle Chan