Beta Cell Intracellular Calcium and Diabetes

传统观点认为胰腺β细胞中的胰岛素释放机制主要依赖于细胞外介质中Ca2+的流入，最终导致胰岛素分泌。Ca2+从主要的细胞内储存室（在后生动物细胞中由内质网（ER）代表）中动员的机制仍然知之甚少。细胞内Ca2+释放通道主要是肌醇1,4,5-三磷酸受体（IP3Rs）和红嘌呤受体（RyRs）。RyR在2型糖尿病（T2DM）病理生理中的作用最近得到澄清，表明RyR在葡萄糖刺激的胰岛素分泌中起关键作用。相反，β细胞IP3R在T2DM中的确切作用知之甚少，并且在代谢领域仍然是一个明显的知识缺口。在哺乳动物中已经鉴定出IP3R的三种亚型（IP3R1-3）；它们的表达模式重叠且功能冗余。胰腺β细胞表达所有IP3R亚型，其水平在慢性葡萄糖刺激下上调。我们假设IP3Rs在T2DM的病理生理中起关键作用。在目前的建议中，我们将使用最先进的模型，包括遗传和药理学工具，在体内，离体和体外测试这一假设。科学前提和基本原理：基于人类基因组的研究已经证明，编码IP3Rs的基因的功能获得突变与葡萄糖稳态的扰动和饮食诱导糖尿病的易感性增加有关；类似地，遗传图谱确定IP3Rs为2型糖尿病的危险因素；然而，这些关联还没有得到功能上的解释。此外，当试图检查IP3Rs在β细胞中的实际作用时，有争议的发现已被报道。我们有可靠的初步数据显示，与非糖尿病患者相比，T2DM患者胰岛中的IP3Rs显著上调；同样，我们发现，与喂食标准食物的非糖尿病幼鼠相比，喂食高脂肪饮食（HFD）的小鼠和db/db小鼠的胰岛IP3Rs显著上调。基于这些原因，我们将探讨以下具体目标：目的1将确定β细胞IP3Rs在T2DM发病机制中的体内功能作用，表征一种新的β细胞特异性动物模型的代谢表型，而目的2将确定IP3Rs与胰腺β细胞（dys）功能在T2DM中的分子机制，重点关注线粒体适应性和自噬。重要的是，尽管已知er -线粒体界面是自噬体形成的主要位点，但IP3Rs在自噬和有丝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细胞中进行实验；人类胰岛研究包括研究小鼠和人类β细胞之间潜在的异同。我们还设计了补救研究来验证所提出的机制是否必要和充分地介导IP3Rs在β细胞中的作用。计划的实验具有高度相关性，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IP3Rs的功能作用进行公正的评估，并确定在T2DM病理生理学中连接IP3Rs、线粒体功能障碍和自噬/线粒体自噬的先前未被认识的途径。

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.