Cholinergic signaling in the human pancreatic islet

人胰岛中的胆碱能信号传导

• 批准号: 9540886
• 负责人: Alejandro Caicedo
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-19 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9540886
• 关键词: Acetylcholine; Acetylcholinesterase; Alpha Cell; Animals; Axon; Behavior; Beta Cell; Biochemical; Biological Assay; Biology; Biosensor; C-Peptide; Cell Communication; Cell Shape; Cell physiology; Cells; Cellular biology; Cholinesterase Inhibitors; Cholinesterases; Clinical Trials; Cytoplasmic Granules; Data; Development; Diabetes Mellitus; Disease; Electron Microscopy; Elements; Endocrine; Exocytosis; Eye; FDA approved; Future; Glucagon; Glucose; Goals; Health; Human; Image; Immunohistochemistry; In Vitro; Insulin; Islets of Langerhans; Knowledge; Laboratories; Location; Long-Term Effects; Measures; Mind; Mission; Modeling; Molecular; Mus; Muscarinic Acetylcholine Receptor; Nature; Neurotransmitters; Optics; Organism; Outcomes Research; PHluorin; Pancreas; Paracrine Communication; Pharmacology; Plasma; Play; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Shapes; Signal Transduction; Signaling Molecule; Source; Synapses; Technical Expertise; Testing; Transplantation; United States National Institutes of Health; Vesicle; Work; acetylcholine transporter; base; blood glucose regulation; cholinergic; glucose metabolism; humanized mouse; in vivo; innovation; insulin secretion; islet; mouse model; nerve supply; nonhuman primate; novel; novel therapeutic intervention; paracrine; polypeptide C; programs; response; time use; tool

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.

神经递质乙酰胆碱在调节胰岛素分泌中起主要作用。在好几个物种 副交感神经支配向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。胰岛中存在这种新的乙酰胆碱来源意味着胰岛素分泌和葡萄糖代谢可以通过局部胆碱能机制进行调节，这一机制尚不清楚。这项研究计划的长期目标是了解胆碱能信号对健康和疾病中人类胰岛生物学的贡献。该应用的目的是使用创新的体外和体内方法相结合，确定乙酰胆碱如何分泌、如何降解以及如何影响β细胞生物学。中心假设是来自α细胞的旁分泌胆碱能输入影响人类β细胞功能。在我们的模型中，乙酰胆碱独立于胰高血糖素释放，并激活β细胞毒蕈碱受体，刺激促进胰岛素分泌和葡萄糖稳态的信号级联。由β细胞产生的胆碱酯酶形成胆碱能信号传导的持续时间和幅度。这项研究的基本原理是，研究结果将为基础知识提供一个缺失的基本要素，没有这些要素，就无法理解胰岛生物学。因此，拟议的研究与NIH的使命有关，该使命涉及对生命系统的性质和行为的基础知识的追求。在强有力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进行测试：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; 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）可视化乙酰胆碱和胰高血糖素的分泌，并使用生物传感器细胞在真实的时间内测量从人类胰岛释放的乙酰胆碱和胰高血糖素。在第二个目标下，我们将使用生化测定、RT-PCR和免疫组织化学研究人类胰岛中胆碱酯酶的表达，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，我们将刺激α细胞，并使用信号分子的新型探针测量β细胞反应。为了研究胰岛胆碱能信号在体内的长期作用，我们将使用人源化小鼠模型，其中将人胰岛移植到小鼠眼中。我们将抑制眼内人胰岛移植物中的乙酰胆碱分泌、乙酰胆碱分解和毒蕈碱受体，并测量对受体小鼠中人胰岛素血浆水平和胰岛素水平的影响。这项研究意义重大，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烈而持久的影响。最终，这些知识有可能影响糖尿病的治疗方式。