Control of beta cell function and survival by RYR2-mediated calcium signals

项目总结/摘要 尽管β细胞衰竭在糖尿病进展中的重要性（1型、2型和多单基因糖尿病）， 尽管β细胞功能障碍的潜在分子机制尚未完全了解。 胰岛素从胰腺β细胞的释放响应于细胞溶质钙（Ca 2+）的动态变化而发生。 程度.迄今为止，大多数β细胞生理学的研究都集中在调节Ca 2+内流，通过 质膜离子通道，传统上更适合通过电生理学研究 接近。这些研究支持KATP通道依赖性质膜的主导作用， 在调节胰岛素释放，特别是在胰岛素抵抗中， 对高浓度葡萄糖的反应。然而，最近在高时间分辨率方面的改进 我们小组开创的成像技术能够检测到β细胞中强有力的Ca 2+信号， 生理相关的葡萄糖浓度，即使在质膜电位没有变化的情况下， 指出了从细胞内储存的Ca 2+的调节释放的关键作用。为了支持这一点，我们的出版物 初步数据表明，对细胞内兰尼碱受体活性的药理学操作 (RYR)独特地调节这些超快Ca 2+释放事件，即使在电压钳条件下， 影响葡萄糖刺激的胰岛素分泌。与其他小组一起，我们发现RYR 2亚型 在胰腺β细胞中占主导地位，并且重要的是使用qRT-PCR和基于质谱的 RYR 2 mRNA和蛋白质可以在人和小鼠胰岛和纯化的β细胞中鉴定。 此外，我们的初步数据表明，β细胞选择性Ryr 2基因敲除小鼠的胰岛素水平降低， 而离体研究表明ER Ca 2+通过RYR活性失调而泄漏 导致β细胞Ca 2+信号传导改变和加速β细胞死亡。在此背景下，我们 假设RYR 2通道作为β细胞健康和功能的关键变阻器， 在糖尿病进展中的作用。为了验证这一假设，我们组建了一个多学科的MPI团队， 在β细胞生物学、糖尿病生理学、定量成像和疾病建模方面的专业知识，以及三个具体的 提出了目标。在具体目标1中，我们将定量β细胞RYR 2活性和胰腺切片中的作用， 使用活体成像的活体。特异性目的2将确定β细胞RYR 2在胰岛素分泌中的体内作用， 细胞存活和葡萄糖稳态。第3章将 利用质谱来确定ER应激诱导的RYR 2功能障碍的潜在机制， 和体内小鼠模型，以确定ER应激诱导的RYR 2功能障碍如何影响β细胞Ca 2+信号传导， 生存

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.