Beta Cell Intracellular Calcium and Diabetes

胰腺β细胞中胰岛素释放机制的传统观点基本上依赖于细胞外介质中Ca 2+的流入，最终导致胰岛素分泌。从主要的细胞内储存室，这在后生动物细胞中是由内质网（ER）的Ca 2+动员所涉及的机制，仍然不太清楚。细胞内主要的钙释放通道是肌醇1，4，5-三磷酸受体（IP 3Rs）和兰尼碱受体（RyRs）。RyR在2型糖尿病（T2 DM）的病理生理学中的作用最近已被阐明，表明RyR在葡萄糖刺激的胰岛素分泌中至关重要。相反，β细胞IP 3R在T2 DM中的确切作用知之甚少，仍然是代谢领域的一个明显的知识空白。已在哺乳动物中鉴定出三种IP 3R亚型（IP 3R 1 -3）;它们的表达模式是重叠的且功能冗余。胰腺β细胞表达所有IP 3R亚型，并且其水平通过慢性葡萄糖刺激上调。我们推测IP 3R在T2 DM的病理生理学中起关键作用。在本提案中，我们将使用最先进的模型（包括遗传学和药理学工具）在体内、离体和体外测试这一假设。科学前提和原理：基于人类基因组的研究表明，编码IP 3R的基因中的功能获得性突变与葡萄糖稳态的扰动和对饮食诱导的糖尿病的易感性增强有关;类似地，遗传图谱将IP 3R确定为T2 DM的风险因素;然而，这些关联尚未得到功能性解释。此外，当试图检查IP 3R在β细胞中的实际作用时，已经报道了有争议的发现。我们有稳健的初步数据显示，与非糖尿病个体相比，来自T2 DM患者的胰岛中的IP 3R显著上调;类似地，与喂食标准食物的非糖尿病同窝小鼠相比，我们检测到喂食高脂饮食（HFD）的小鼠和db/db小鼠的胰岛中的IP 3R显著上调。基于这些理由，我们将探索以下具体目标：目标1将在体内定义β细胞IP 3R在T2 DM发病机制中的功能作用，表征新型β细胞特异性动物模型的代谢表型，而目标2将确定将IP 3R与T2 DM中胰腺β细胞（dys）功能联系起来的分子机制，重点关注线粒体适应性和自噬。重要的是，虽然ER-线粒体界面已知是自噬体形成的主要位点，但IP 3Rs在自噬和线粒体自噬中的确切作用仍然存在极大的争议。我们将在小鼠、胰岛和β细胞中进行试验;包括人胰岛研究，以研究鼠和人β细胞之间的潜在相似性和差异。我们还设计了补救研究，以验证所提出的机制是否是必要的，并足以介导β细胞中IP 3R的作用。计划中的实验具有高度的相关性，具有高度的概念和技术创新;事实上，我们的研究将对β细胞IP 3R的功能作用提供无偏见的评估，并定义先前未被识别的T2 DM病理生理学中连接IP 3R、线粒体功能障碍和自噬/线粒体自噬的途径。

The traditional view of insulin release machinery in pancreatic β cells relies essentially on the influx of Ca2+ from the extracellular medium, eventually leading to insulin secretion. The mechanisms involved in Ca2+ mobilization from the major intracellular storage compartment, which in metazoan cells is represented by the endoplasmic reticulum (ER), remain less understood. The main intracellular Ca2+ release channels are Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). The role of RyR in the pathophysiology of type 2 diabetes mellitus (T2DM) has been recently clarified, demonstrating that RyR is crucial in glucose-stimulated insulin secretion. Instead, the exact role of β cell IP3R in T2DM is poorly understood and remains a glaring knowledge gap in the metabolic field. Three isoforms of IP3R have been identified in mammals (IP3R1-3); their expression pattern is overlapping and functionally redundant. Pancreatic β cells express all IP3R isoforms, and their levels are upregulated by chronic glucose stimulation. We hypothesize that IP3Rs play a key role in the pathophysiology of T2DM. In the present proposal, we will test this hypothesis in vivo, ex vivo, and in vitro using state-of-the-art models including both genetic and pharmacologic tools. Scientific premise and rationale: Genome-based studies in humans have demonstrated that gain-of-function mutations in genes encoding for IP3Rs are linked to perturbations in glucose homeostasis and enhanced susceptibility to diet-induced diabetes; similarly, genetic mapping identified IP3Rs as a risk factor for T2DM; however, these associations have not been functionally explained. Moreover, controversial findings have been reported when attempting to examine the actual role of IP3Rs in β cells. We have robust preliminary data showing that IP3Rs are significantly upregulated in islets from T2DM patients compared with non-diabetic individuals; similarly, we detected a marked upregulation of IP3Rs in islets from mice fed high-fat diet (HFD) and db/db mice compared with non-diabetic littermates fed standard chow. On these grounds, we will explore the following specific aims: Aim 1 will define in vivo the functional role of β cell IP3Rs in the pathogenesis of T2DM, characterizing the metabolic phenotype of a novel, β cell-specific, animal model, whereas Aim 2 will identify the molecular mechanisms linking IP3Rs to pancreatic β cell (dys)function in T2DM, focusing on mitochondrial fitness and autophagy. Importantly, although the ER-mitochondrial interface is known to be a primary site for autophagosome formation, the exact role of IP3Rs in autophagy and mitophagy remain extremely debated. We will conduct assays in mice, in islets, and in β cells; human islet studies are included to investigate potential similarities and differences between murine and human β cells. We also designed rescue studies to verify if the proposed mechanisms are necessary and sufficient to mediate the effects of IP3Rs in β cells. The planned experiments are highly relevant, with a high degree of conceptual and technical innovation; indeed, our studies will provide an unbiased assessment of the functional role of β cell IP3Rs and define previously unrecognized pathways linking IP3Rs, mitochondrial dysfunction, and autophagy/mitophagy in the pathophysiology of T2DM.