Cholinergic signaling in the human pancreatic islet

神经递质乙酰胆碱在调节胰岛素分泌方面起着重要作用。在几个物种中 副交感神经支配为胰岛β细胞提供胆碱能输入，但最近的研究表明，分泌胰高血糖素的α细胞是人类胰岛中乙酰胆碱的主要来源。胰岛中这种新的乙酰胆碱来源的存在意味着胰岛素的分泌和葡萄糖代谢可以通过尚不清楚的局部胆碱能机制来调节。这项研究计划的长期目标是了解胆碱能信号对人类胰岛生物学在健康和疾病中的贡献。这项应用的目的是结合体外和体内的创新方法，确定乙酰胆碱是如何分泌的，它是如何降解的，以及它是如何影响β细胞生物学的。中心假设是，从阿尔法细胞输入的旁分泌胆碱能影响人类的β细胞功能。在我们的模型中，乙酰胆碱的释放不依赖于胰高血糖素，并激活β细胞M受体，刺激信号级联反应，促进胰岛素分泌和葡萄糖稳态。由β细胞产生的胆碱酯酶决定胆碱能信号的持续时间和大小。提出这项研究的理由是，研究结果将为基本知识贡献一个缺失的基本元素，如果没有这些元素，就无法理解小岛生物学。因此，这项拟议的研究与美国国立卫生研究院的使命有关，该使命与追求关于生命系统的性质和行为的基本知识有关。在强大的初步数据的指导下，我们的中心假设将通过追求三个具体目标来检验：1)确定人类α细胞释放乙酰胆碱的机制；2)确定乙酰胆碱酯酶在α-β细胞通讯中的位置和作用；以及3)确定胆碱能信号对人类β细胞功能的影响。在第一个目标下，我们将使用胞吐作用的光学指示剂(PHluorins)可视化乙酰胆碱和高血糖素的分泌，并使用生物传感器细胞实时测量人类胰岛的乙酰胆碱和高血糖素释放。在第二个目标下，我们将使用生化分析、RT-PCR和免疫组织化学方法研究胆碱酯酶在人胰岛中的表达，并测试FDA批准的胆碱酯酶抑制剂增加胰岛素分泌的能力。在第三个目标下，我们将用新的信号分子探针刺激阿尔法细胞和测量贝塔细胞的反应。为了研究胰岛胆碱能信号在体内的长期影响，我们将使用人源化的小鼠模型，在这个模型中，人的胰岛被移植到小鼠的眼睛里。我们将抑制人眼内胰岛移植物的乙酰胆碱分泌、乙酰胆碱分解和M受体，并测量其对人胰岛素血浆水平和受体小鼠血糖的影响。这项拟议的研究意义重大，因为它有望对我们理解胆碱能信号在人类胰岛生物学中的作用产生强大而持久的影响。归根结底，这些知识有可能影响糖尿病的治疗方式。

The neurotransmitter acetylcholine plays a major role in regulating insulin secretion. In several species parasympathetic innervation provides cholinergic input to beta cells, but recent studies show that the glucagon secreting alpha cell is a major source of acetylcholine in human islets. The existence of this novel source of acetylcholine in the islet implies that insulin secretion and glucose metabolism can be regulated by local cholinergic mechanisms that remain unknown. The long-term goal of this research program is to understand the contribution of cholinergic signaling to human islet biology in health and disease. The objective of this application is to determine how acetylcholine is secreted, how it is degraded, and how it impacts beta cell biology, using a combination of innovative in vitro and in vivo approaches. The central hypothesis is that paracrine cholinergic input from the alpha cell influences human beta cell function. In our model, acetylcholine is released independently of glucagon and activates beta cell muscarinic receptors, stimulating signaling cascades that promote insulin secretion and glucose homeostasis. Cholinesterases produced by beta cells shape the duration and magnitude of cholinergic signaling. The rationale for the proposed research is that the results will contribute a missing, fundamental element to basic knowledge, without which islet biology cannot be understood. The proposed research is therefore relevant to the mission of the NIH that pertains to the pursuit of fundamental knowledge about the nature and behavior of living systems. Guided by strong preliminary data, our central hypothesis will be tested by pursuing three specific aims: 1) Identify the mechanisms of acetylcholine release from human alpha cells; 2) Determine the location and role of acetylcholinesterase in alpha-beta cell communication; and 3) Determine the impact of cholinergic signaling on human beta cell function. Under the first aim, we will visualize secretion of acetylcholine and glucagon using optical indicators of exocytosis (pHluorins) and measure acetylcholine and glucagon release from human islets in real time using biosensor cells. Under the second aim, we will study the expression of cholinesterases in human islets using biochemical assays, RT-PCR, and immunohistochemistry, and test FDA-approved cholinesterase inhibitors for their ability to increase insulin secretion. Under the third aim, we will stimulate alpha cells and measure beta cell responses with novel probes for signaling molecules. To investigate long-term effects of islet cholinergic signaling in vivo we will use a humanized mouse model in which human islets are transplanted into the mouse eye. We will inhibit acetylcholine secretion, acetylcholine breakdown, and muscarinic receptors in intraocular human islet grafts and measure the effects on human insulin plasma levels and glycemia in the recipient mouse. The proposed research is significant because it is expected to make a strong and lasting impact on our understanding of the role of cholinergic signaling in human islet biology. Ultimately, such knowledge has the potential to impact the way diabetes is treated.