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

项目摘要/摘要 尽管β细胞衰竭在糖尿病进展中的重要性(1型、2型和多发性单基因 尽管如此，导致β细胞功能障碍的潜在分子机制仍不完全清楚。 胰腺β细胞胰岛素的释放是对细胞内钙离子动态变化的反应 级别。到目前为止，β细胞生理学的研究主要集中在钙离子内流的调节上。 质膜离子通道，传统上更易于通过电生理进行研究 接近了。这些研究支持依赖KATP通道的质膜起主导作用 电压门控性钙通道在胰岛素释放调节中的去极化和钙内流 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 我们团队开创的成像技术使我们能够在更多的刺激下检测到β细胞中强大的钙信号 生理上相关的葡萄糖浓度，即使在质膜电位没有变化的情况下， 这表明，细胞内钙离子的调节释放起着关键作用。为了支持这一点，我们出版的 初步数据显示，药物调控细胞内兰尼定受体的活性 (RyR)独一无二地调节这些超快的钙释放事件，即使在电压钳制条件下也是如此 影响葡萄糖刺激的胰岛素分泌。与其他组一起，我们发现了RYR2亚型 在胰腺β细胞中占主导地位，重要的是用定量RT-PCR法和基于质谱学的方法显示 蛋白质组学表明，在人和小鼠胰岛和纯化的β细胞中都可以鉴定出RYR2基因和蛋白。 此外，我们的初步数据表明，β细胞选择性Ryr2基因敲除的小鼠胰岛素减少。 分泌和糖耐量异常，而体外研究表明，ER钙离子通过调节失调的RyR活性泄漏 导致β细胞钙信号的改变，加速β细胞死亡。在此背景下，我们 假设RYR2通道是β细胞健康和功能的关键变阻器，并发挥关键作用 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，以及三个特定的 提出了目标。在特定的目标1中，我们将量化β细胞ryr2的活性和在胰腺切片和 活体使用活体成像。特异性目标2将确定β细胞RYR2在体内对胰岛素分泌的作用，β 使用高度β细胞特异性基因敲除小鼠模型的细胞存活和葡萄糖动态平衡。具体目标3将 利用质谱法确定内质网应激诱导的RYR2功能障碍的机制及体外实验 和活体小鼠模型，以确定内质网应激诱导的RYR2功能障碍如何影响β细胞钙信号和 生死存亡。

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.