PROJECT SUMMARY/ABSTRACT Despite the importance of β cell failure in the progression of diabetes (type 1, type 2, and multiple monogenic forms), the underlying molecular mechanisms responsible for β cell dysfunction remain incompletely understood. Release of insulin from the pancreatic β cell occurs in response to dynamic changes in cytosolic calcium (Ca2+) levels. To date, the majority of studies of β cell physiology have focused on the regulation of Ca2+ influx via plasma membrane ion channels, which have traditionally been more amenable to study via electrophysiological approaches. These studies have supported a dominant role for KATP channel-dependent plasma membrane depolarization and Ca2+ influx via voltage-gated Ca2+ channels in the regulation of insulin release, especially in response to very high concentrations of glucose. However, recent improvements in high temporal resolution imaging pioneered by our group have enabled the detection of robust Ca2+ signals in β cells stimulated with more physiologically relevant glucose concentrations, even in the absence of changes in plasma membrane potential, pointing to a critical role for the regulated release of Ca2+ from intracellular stores. In support of this, our published and preliminary data show that pharmacological manipulation of the activity of intracellular ryanodine receptors (RYR) uniquely modulates these ultrafast Ca2+ release events, even in voltage-clamp conditions, while also impacting glucose-stimulated insulin secretion. Together with other groups, we have found that the RYR2 isoform predominates in pancreatic β cells, and importantly showed using qRT-PCR and mass spectometry-based proteomics that RYR2 mRNA and protein can be identified in human and mouse islets and purified β cells. Furthermore, our preliminary data indicate that mice with β cell selective Ryr2 knockout have reduced insulin secretion and glucose intolerance, while ex vivo studies suggest that ER Ca2+ leak via dysregulated RYR activity leads to alterations in β cell Ca2+ signalling and accelerated β cell death. Against this background, we hypothesize that RYR2 channels serve as critical rheostats of β cell health and function and that they play key roles in diabetes progression. To test this hypothesis, we have assembled a multidisciplinary MPI team with expertise in β cell biology, diabetes physiology, quantitative imaging, and disease modelling, and three specific aims are proposed. In Specific Aim 1, we will quantify β cell RYR2 activity and roles in pancreatic slices and in vivo using intravital imaging. Specific Aim 2 will determine the in vivo role of β cell RYR2 in insulin secretion, β cell survival, and glucose homeostasis using a highly β cell-specific knockout mouse model. Specific Aim 3 will utilize mass spectrometry to determine mechanisms underlying ER stress-induced RYR2 dysfunction and in vitro and in vivo mouse models to define how ER stress-induced RYR2 dysfunction impacts β cell Ca2+ signaling and survival.

PROJECT NARRATIVE Calcium signaling plays a central role in pancreatic β cell function, and alterations in β cell calcium signaling are observed in both major forms of diabetes. Ryanodine receptors are large-conductance calcium release channels in the endoplasmic reticulum membrane. The proposed research will define how ryanodine receptor dysfunction in the β cell contributes to diabetes pathophysiology and will test the hypothesis that the ryanodine receptor serves as a critical hub in the pancreatic β cell, regulating calcium dynamics, insulin secretion, and cell survival.