Multi-cellular Interactions and Dynamics underlying Insulin Secretion

描述(由申请人提供)： 项目总结。朗格汉斯胰岛是位于胰腺内的多细胞微器官。它是通过分泌胰岛素和胰升糖素等激素来维持血糖平衡的核心。胰岛细胞之间的相互作用对葡萄糖刺激的胰岛素分泌(GSIS)的调节至关重要。胰岛内的β细胞分泌胰岛素，与孤立的β细胞相比，GSIS反应增加了许多倍。此外，胰岛中的β细胞表现出同步振荡，这导致整个胰腺出现搏动性胰岛素。这些协调的胰岛素脉冲被认为在降低血糖和维持胰岛素敏感性方面更有效。通过移植完整的胰岛(但不能通过分离的β细胞)，1型糖尿病可以有效地逆转，这突显了胰岛内结构和信号的重要性。以往的研究和初步数据表明，缝隙连接偶联在胰岛功能中具有生理作用。然而，其他研究也表明旁分泌和旁分泌机制在细胞间通信中可能发挥作用。我们推测，主要是电活动的缝隙连接耦合起到协调和增强GSIS的作用，但cAMP介导的胰岛内α和β细胞之间的信号耦合可以进一步调节GSIS。为了验证这一假设，有必要引入精确的实验扰动，并使用定量技术来监测由此对GSIS潜在的信号通路的影响。在这项建议的指导阶段，我们将建立必要的生理和生化分析，以及完善目前使用的定量数学模型。然后提出了独立研究阶段的两个具体目标：1)量化缝隙连接偶联和KATP通道调节的膜极化在调节GSIS中的相对作用；2)确定胰岛内β细胞和α细胞之间cAMP信号偶联的机制和作用。这两个目标可以独立进行，尽管每个目标的结果将是对整个假设检验的补充。实验将在一些缝隙连接基因敲除和KATP通道转基因小鼠模型以及人类胰岛上进行，并将利用最先进的定量成像方法、荧光蛋白生物传感器和数学模型以及更成熟的生化和生理分析。这些实验将更全面地了解胰岛内的信号机制，这对于开发1型糖尿病的移植和干细胞疗法以及确定2型糖尿病的新治疗药物靶点将是重要的。 公共卫生相关性： 关联性。胰岛在血糖动态平衡中起着核心作用，胰岛功能不全会导致糖尿病的发生。细胞之间的通讯对胰岛的功能至关重要，但对其中涉及的机制的了解尚不清楚。准确了解胰岛内不同细胞之间的信号传递将促进移植和干细胞疗法的发展，并确定有效治疗糖尿病患者的药物靶点。

DESCRIPTION (provided by applicant): Project summary. The islet of Langerhans is a multi-cellular micro-organ located in the pancreas. It is central to maintaining blood glucose homeostasis through secretion of hormones such as insulin and glucagon. Interactions between cells in the islet are crucial to the regulation of glucose stimulated insulin secretion (GSIS). Beta cells within the islet secrete insulin with a many-fold increase in GSIS response compared to isolated beta cells. Furthermore beta cells in the islet exhibit synchronized oscillations which lead to pulsatile insulin from the whole pancreas. These coordinated insulin pulses are thought to be more effective in lowering blood glucose and maintaining insulin sensitivity. The importance of the structure and signaling within the islet is highlighted by the fact that type 1 diabetes can be effectively reversed by the transplantation of intact islets (but not by isolated beta cells). Previous research and preliminary data shows that gap junction coupling has a physiological role in islet function. However other research has also shown possible roles for paracrine and juxtacrine mechanisms in cell-cell communication. We hypothesize that it is primarily gap junction coupling of electrical activity which serves to coordinate and enhance GSIS, but coupling of cAMP mediated signaling between alpha and beta cells within the islet can further modulate GSIS. To test this hypothesis it will be necessary to introduce precise experimental perturbations, and use quantitative techniques to monitor the resulting impact on signaling pathways underlying GSIS. During the mentored phase of this proposal we will establish the necessary physiological and biochemical assays, as well as to refine the quantitative mathematical models currently in use. Two specific aims are then proposed for the independent research phase: 1) quantify the relative role of gap junction coupling and KATP channel regulated membrane polarization in regulating GSIS, 2) determine the mechanism and role for coupling of cAMP signaling between beta cells and alpha cells within the islet. These two aims can proceed independently, although the results from each aim will be complimentary to testing the overall hypothesis. Experiments will be performed on a number of gap junction knockout and KATP channel transgenic mouse models as well as human islets, and will utilize state-of- the art quantitative imaging approaches, fluorescent protein biosensors and mathematical models along with more established biochemical and physiological assays. These experiments will yield a more complete understanding of signaling mechanisms within the islet which will be important for the development of transplantation and stem cell therapies for type1 diabetes as well as identifying novel therapeutic drug targets for type2 diabetes. PUBLIC HEALTH RELEVANCE: Relevance. The pancreatic islet plays a central role in glucose homeostasis and the failure of the islet leads to the development of diabetes. Communication between cells is crucial to the function of the islet, but knowledge of the mechanisms involved is unclear. A precise understanding of the signaling between different cells within the islet will improve the development of transplantation and stem cell therapies and identify drug targets to effectively treat individuals with diabetes.