The role of innervation for hormone secretion in human islets

描述(申请人提供)：无论是在胰腺的自然环境中，还是在移植治疗1型糖尿病后，在了解朗格汉斯胰岛如何在体内发挥作用方面，都存在着根本的空白。大多数关于胰岛功能的研究都是在体外进行的，因此对神经支配对胰岛激素分泌的作用知之甚少。我们研究的长期目标是了解活体中朗格汉斯胰岛的细胞生物学。这一特殊应用的目的是利用一种新的技术平台来确定神经在胰岛激素分泌中所起的作用，该技术平台允许在移植后对血运和再神经支配的胰岛进行活体成像。在这项新技术中，胰岛被移植到眼睛的前房，在操纵眼睛的神经输入后，它们的功能被局部和系统地记录下来。中心假设是，自主神经系统通过调节胰岛内的血液流动来调节人类胰岛的功能。与小鼠胰岛不同，人的内分泌细胞不受自主神经支配的直接影响。在所提出的机制中，自主神经纤维强烈地神经支配作为括约肌的收缩周细胞，以改变局部血流，从而改变葡萄糖感知和激素释放到循环的效率。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，就无法理解人类胰岛的生物学。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，这一假说将通过追求三个具体目标来验证：1)体外胰岛神经支配模式的物种差异；2)眼内胰岛移植物决定其自身的神经支配模式；3)神经输入在体内对血糖稳态的作用。在第一个目标下，将利用免疫组织化学和细胞内[Ca~(2+)]成像技术，系统地研究小鼠和人类胰岛的神经支配模式和神经递质受体类型的分布。在第二个目标下，将在免疫组织化学染色模式和自主神经纤维的活体追踪方面比较人和小鼠眼内胰岛移植物。在第三个目标下，通过激活瞳孔反射的副交感和交感成分、药物阻断和选择性地消除神经输入，将挑战局部胰岛细胞的反应和通过小鼠和人类胰岛移植物对葡萄糖稳态的调节。这项拟议的工作具有创新性，因为它利用了一种新的技术平台，首次允许在体内成像受神经支配的人和鼠的胰岛功能。这项拟议的研究具有重要意义，因为它有望推进和扩展目前胰岛调节葡萄糖稳态的模型。 公共卫生相关性：拟议的工作将展示神经对人类胰岛细胞功能的作用。它利用了我们团队开发的新技术平台，首次允许对神经支配的人和小鼠胰岛进行活体成像。作为一个主要的预期结果，我们将获得胰岛神经支配的全面图景，这将对目前胰岛葡萄糖稳态调节模型产生强烈影响。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how the islets of Langerhans function in vivo, either in the native environment in the pancreas or after transplantation to treat type 1 diabetes. Most studies on islet function have been performed in vitro, and as a consequence little is known about the role of innervation on islet hormone secretion. The long-term goal of our research is to understand the cell biology of islets of Langerhans in the living organism. The objective of this particular application is to determine the role innervation plays in the secretion of islet hormones using a new technological platform allowing in vivo imaging of vascularized and reinnervated islets after transplantation. In this new technology, islets are transplanted into the anterior chamber of the eye, and their function is recorded locally and systemically after manipulation of the eye's neural input. The central hypothesis is that the autonomic nervous system modulates human islet function by regulating blood flow within the islet. In contrast to mouse islets, human endocrine cells are not affected directly by the autonomic innervation. In the proposed mechanism, autonomic nerve fibers strongly innervate contractile pericytes that work as sphincters to change blood flow locally, thus changing the efficiency of glucose sensing and of hormone release into the circulation. The rationale for the proposed research is that the results will contribute a missing, fundamental element to basic knowledge, without which the biology of human islets 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, this hypothesis will be tested by pursuing three specific aims: 1) Species differences in the innervation patterns of pancreatic islets in vitro; 2) Intraocular islet grafts determine their own innervation pattern; and 3) The role of neural input on glucose homeostasis in vivo. Under the first aim, the innervation patterns of islets and the neurotransmitter receptor profiles of the innervated endocrine cell types will be systematically examined in mouse and human islets using immunohistochemistry and imaging of intracellular [Ca2+]. Under the second aim, intraocular human and mouse islet grafts will be compared in terms of immunohistochemical staining patterns and in vivo tracing of autonomic nervous fibers. Under the third aim, local islet cell responses and regulation of glucose homeostasis by mouse and human islet grafts will be challenged by activating the parasympathetic and sympathetic components of the pupillary reflex, by pharmacological blockade, and by selective elimination of the neural input. The proposed work is innovative because it capitalizes on a new technological platform that allows for the first time in vivo imaging the function of innervated human and mouse islet. The proposed research is significant because it is expected to advance and expand current models of the regulation of glucose homeostasis by pancreatic islets. PUBLIC HEALTH RELEVANCE: The proposed work will demonstrate the role of innervation for human islet cell function. It capitalizes on a new technological platform developed by our group allowing for the first time in vivo imaging of innervated human and mouse islets. As a major expected outcome we will obtain a comprehensive picture of islet innervation, which will have a strong impact on current models of islet regulation of glucose homeostasis.