Mechanisms of Beta Cell Coordination

项目摘要 II型糖尿病影响着超过三千万的美国人，其每年的经济成本估计超过300美元。 十亿美元，是NIH年度预算的十倍。外周胰岛素抵抗导致进行性功能障碍 分泌胰岛素的胰岛。由于胰岛素抵抗的补偿不足， 由于胰岛素是糖尿病进展的重要因素，因此迫切需要了解胰岛素分泌失败是如何发生的。 单个细胞内胰岛素分泌的原型途径是明确的。葡萄糖代谢导致 ATP生成、ATP敏感性K+通道关闭、膜去极化、钙内流和胰岛素 分泌物葡萄糖激酶，胰腺β细胞中的葡萄糖敏感酶，是这一过程中的限速酶。 通过改变酶活性来控制胰岛素分泌的途径。即便如此，贝塔细胞的组织 对胰岛内细胞间的分泌反应知之甚少。缝隙连接促进胰岛内β 细胞通过耦合细胞之间的电信号和代谢信号进行通信。最近的研究提供了 胰岛内的“中枢”β细胞不成比例地影响甚至控制胰岛电和 钙活性这些细胞具有升高的葡萄糖激酶水平，这表明代谢增加的细胞 活性控制胰岛活性。我们希望了解葡萄糖激酶的活性和表达如何影响 代谢、电和钙动力学的协调。我们假设， 葡萄糖激酶活性和表达指导胰岛活性且协调依赖于代谢物 通过间隙连接的扩散。为了验证我们的假设，我们将首先确定是否增加葡萄糖激酶 单细胞中的活性增强了它们对胰岛葡萄糖代谢的影响（具体目标1）。接下来，我们将测试 代谢物通过缝隙连接的扩散如何影响胰岛活性（具体目标2）。调查这些 问题，我们的实验室已经产生了两种新的转基因小鼠模型，表达遗传编码的工具， 尤其是胰腺β细胞。我们将使用葡萄糖激酶生物传感器来量化其在活细胞中的活性， 和内质网定位的通道视紫红质2（ER-ChR）来操纵单个细胞中的钙水平。 细胞光遗传ER-ChR允许通过细胞内钙的变化精确激活葡萄糖激酶。 此外，我们将使用微流控移液管来选择性地影响胰岛小区域中的葡萄糖激酶活性， 和数学建模来探测胰岛协调。这项研究的结果将提供深入了解如何 II型糖尿病可能出现分泌衰竭，并确定了潜在的治疗途径。

Project Summary Type II diabetes affects over thirty million Americans, and its yearly economic cost is estimated to be over 300 billion dollars, ten times the annual NIH budget. Peripheral insulin resistance leads to progressive dysfunction of the insulin-secreting pancreatic islet. Since inadequate compensation for insulin resistance is a prime contributor to diabetes progression, there is an urgent need to understand how insulin secretory failure occurs. The prototypical pathway for insulin secretion within individual cells is well defined. Glucose metabolism leads to ATP generation, closure of ATP-sensitive K+ channels, membrane depolarization, calcium influx, and insulin secretion. Glucokinase, the glucose-sensing enzyme in pancreatic beta cells, is the rate-limiting enzyme in this pathway allowing changes in enzyme activity to control insulin secretion. Even so, the organization of beta-cell secretory responses across cells within an islet is poorly understood. Gap junctions facilitate intra-islet beta cells communication by coupling electrical and metabolic signals between cells. Recent studies have provided evidence that ‘hub’ beta cells within an islet disproportionately affect and even control islet electrical and calcium activity. These cells have elevated glucokinase levels, suggesting that cells with increased metabolic activity control islet activity. We wish to understand how glucokinase activity and expression affect the coordination of metabolic, electrical, and calcium dynamics. We hypothesize that cells with enhanced glucokinase activity and expression guide islet activity and that coordination is dependent on metabolite diffusion across gap junctions. To test our hypothesis, we will first determine whether increased glucokinase activity in single cells enhances their influence over islet glucose metabolism (Specific Aim 1). Next, we will test how metabolite diffusion across gap junctions affects islet activity (Specific Aim 2). To investigate these questions, our lab has generated two novel transgenic mouse models that express genetically-encoded tools specifically in pancreatic beta cells. We will use a glucokinase biosensor to quantify its activity in living cells, and an endoplasmic reticulum-localized channelrhodopsin2 (ER-ChR) to manipulate calcium levels in single cells. Optogenetic ER-ChR allows for precise activation of glucokinase via changes in intracellular calcium. Further, we will use a microfluidic pipette to influence glucokinase activity in small areas of the islet selectively, and mathematical modeling to probe islet coordination. Findings from this study will provide insight into how secretory failure may arise in type II diabetes and identify potential avenues for treatment.