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

通过 RYR2 介导的钙信号控制 β 细胞功能和存活

基本信息

批准号：
10375087
负责人：
Carmella Evans-Molina
金额：
$ 47.96万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-20 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10375087
关键词：
Address Age Alleles Apoptosis Beta Cell Biophysics Bolus Infusion Ca(2+)-Transporting ATPase Calcium Calcium Signaling Cell Death Cell Survival Cell membrane Cell physiology Cells Cellular biology Closure by clamp Complex Data Data Science Detection Diabetes Mellitus Disease model Electrophysiology (science)Endoplasmic Reticulum Equilibrium Event Failure Functional disorder Genes Genetic Glucose Glucose Intolerance Health Human ITPR1 gene Image In Vitro Individual Inflammatory Inositol Insulin Insulin-Dependent Diabetes Mellitus Ion Channel Knock-out Knockout Mice Knowledge Link Maintenance Mass Spectrum Analysis Measures Mediating Membrane Membrane Potentials Messenger RNA Metabolic Metabolic stress Modeling Molecular Monitor Morphology Mus Non-Insulin-Dependent Diabetes Mellitus Organelles Pancreas Pattern Pharmacology Physics Physiological Physiology Play Process Production Protein Isoforms Proteins Proteomics Publishing Pump Quantitative Reverse Transcriptase PCR Rattus Reagent Regulation Research Role RyR2 Ryanodine Receptor Calcium Release Channel Ryanodine Receptors Shapes Signal Transduction Slice Stress Structure of beta Cell of islet Testing Tissues Work base blood glucose regulation diet-induced obesity endoplasmic reticulum stress experimental study glucose tolerance humoral immunity deficiency imaging approach imaging modality impaired glucose tolerance in vivo insulin regulation insulin secretion intravital imaging intravital microscopy islet light microscopy mitochondrial metabolism mouse model multidisciplinary novel quantitative imaging receptor response temporal measurement tool tripolyphosphate two-photon uptake voltage voltage clamp

项目摘要

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.

项目摘要/摘要尽管β细胞衰竭在糖尿病进展（类型1，2型和多个单基因的进展中都是重要的形式），导致β细胞功能障碍的基本分子机制尚不完全理解。从胰腺β细胞中释放胰岛素是响应胞质钙的动态变化而发生的（Ca2+）水平。迄今为止，大多数β细胞生理研究的研究都集中在通过质膜离子通道传统上可以通过电生理学研究方法。这些研究支持KATP通道依赖性质膜的主要作用在调节胰岛素释放中，通过电压门控Ca2+通道的沉积和Ca2+影响，尤其是在对非常高的葡萄糖的反应。但是，高临时分辨率的最新改进我们组率先进行的成像使得在刺激更多生理相关的葡萄糖浓度，即使在质膜电位变化的情况下，指出了从细胞内存储中释放Ca2+的关键作用。为此，我们出版了和初步数据表明，细胞内ryanodine受体活性的药物操纵（RYR）独特地调节这些超快CA2+释放事件，即使在电压钳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RYR2同工型在胰腺β细胞中占主导地位，并使用QRT-PCR和基于质量镜头的β细胞表示 RyR2 mRNA和蛋白质的蛋白质组学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RYR2敲除的小鼠减少了胰岛素分泌和葡萄糖intlerance，而离体研究表明ER Ca2+通过RYR活性失调而泄漏导致β细胞Ca2+信号传导和加速β细胞死亡的改变。在这个背景下，我们假设RYR2通道是β细胞健康和功能的关键风湿病，并且它们发挥了关键在糖尿病进展中的作用。为了检验这一假设，我们已经组装了一个多学科的MPI团队 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，三个特定提出了目标。在特定的目标1中，我们将量化β细胞RyR2活性和在胰腺切片中的作用，并在使用插入成像的体内。特定的目标2将确定β细胞RyR2在胰岛素分泌中的体内作用β 使用高度β细胞特异性敲除小鼠模型的细胞存活和葡萄糖稳态。具体的目标3将利用质谱法来确定ER应力诱导的RYR2功能障碍和体外的机制和体内小鼠模型，以定义ER应力诱导的RyR2功能障碍如何影响β细胞Ca2+信号传导和生存。