Beta Cell Intracellular Calcium and Diabetes

传统观点认为胰腺β细胞的胰岛素释放机制主要依赖于细胞外介质中钙离子的内流，最终导致胰岛素的分泌。在后生动物细胞中，以内质网(ER)为代表的主要细胞内储存室的钙动员机制尚不清楚。细胞内钙释放的主要通道是肌醇-1，4，5-三磷酸受体(IP3Rs)和兰尼定受体(RyRs)。RyR在2型糖尿病(T2 DM)病理生理学中的作用最近被阐明，表明RyR在葡萄糖刺激的胰岛素分泌中起着至关重要的作用。相反，β细胞IP3R在T2 DM中的确切作用还知之甚少，在代谢领域仍然是一个明显的知识空白。已在哺乳动物中发现了三种IP3R亚型(IP3R1-3)；它们的表达模式是重叠的，功能上是冗余的。胰腺β细胞表达所有的IP3R亚型，其水平在慢性葡萄糖刺激下上调。我们推测IP3Rs在T2 DM的病理生理过程中起关键作用。在目前的提案中，我们将使用包括遗传学和药理学工具在内的最先进的模型，在体内、体外和体外检验这一假说。科学前提和理论基础：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紊乱和饮食诱导的糖尿病的易感性增加有关；类似地，基因图谱确定IP3Rs是T2 DM的危险因素；然而，这些联系尚未从功能上解释。此外，在试图研究IP3Rs在β细胞中的实际作用时，也有报道称有争议的发现。我们有强有力的初步数据表明，与非糖尿病患者相比，T2 DM患者的胰岛中IP3R显著上调；类似地，我们检测到，与饲喂标准食物的非糖尿病小鼠相比，高脂饮食(HFD)小鼠和db/db小鼠的胰岛中IP3Rs显著上调。在此基础上，我们将探索以下具体目标：目标1将在体内定义β细胞IP3Rs在T2 DM发病机制中的功能作用，描述一种新的β细胞特异性动物模型的代谢表型，而Aim 2将识别将IP3Rs与T2 DM的胰腺β细胞(Dys)功能联系起来的分子机制，重点是线粒体适合性和自噬。重要的是，尽管已知内质网-线粒体界面是自噬小体形成的主要部位，但IP3Rs在自噬和有丝分裂吞噬中的确切作用仍存在极大争议。我们将在小鼠、胰岛和β细胞中进行分析；包括人类胰岛研究，以研究小鼠和人类β细胞之间的潜在相似性和差异。我们还设计了救援性研究，以验证所提出的机制是否必要且足以在β细胞中介导IP3R的影响。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；实际上，我们的研究将对β细胞IP3Rs的功能角色提供公正的评估，并确定在T2 DM的病理生理学中将IP3Rs、线粒体功能障碍和自噬/有丝分裂吞噬联系起来的先前未知的途径。

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.