IMAGING BETA CELL FUNCTION WITH BIOSENSORS

本提案的总体目标是了解CaZ* 依赖性刺激-分泌偶联 从生物物理学、生理学和免疫学的角度， 分子生物学这一目标将通过研究细胞内Ca 2+浓度、线粒体 功能，葡萄糖代谢和胰岛素分泌在小鼠和人类胰岛中，其中13-细胞具有 是专门为功能成像设计的钙依赖性信号转导机制 在体外使用完整的胰岛、13- 细胞和胰岛素瘤细胞。尽管体外方法做出了重要贡献， 体内完整胰岛中13-细胞功能的潜在机制的知识仍然不完全。 目前成像方法的一些技术限制不允许研究13-细胞 在胰腺原位复杂的多细胞环境中发挥作用。的核心重点 本提案中描述的实验是开发和表征一种新型成像方法，该方法 将促进原位胰岛细胞功能的研究。在具体目标1中，病毒基因转移载体将是 用于用遗传靶向生物合成荧光Ca 2+传感器刺激小鼠细胞。他们的 研究促分泌素后Q细胞生物物理和生理反应的有效性 将对刺激进行评估。在特定目标2中，这些传感器作为功能成像的实用性 将评估完整小鼠胰岛中的指标。在特定目标3中，完整人胰岛内的13-细胞将 被设计成表达Ca 2+生物传感器和体外研究的人类胰岛功能， 鼠岛在具体目标4中，转基因小鼠模型，其中13-细胞已被遗传修饰。 增强表达Ca 2+的生物传感器将在体外和体内开发和表征， 共聚焦显微荧光测定法和测量胰岛素分泌。这些研究将提供新的 了解胰岛细胞生物学，这将有利于临床策略，以保持和维持功能 13-细胞团

The overall goal of this proposal is to understand the CaZ*-dependent stimulus-secretion coupling mechanisms that regulate 13-cell function in vivo from the perspectives of biophysics, physiology and molecular biology. This goal will be achieved by studying intracellular Ca 2¿concentration, mitochondrial function, glucose metabolism, and insulin secretion in mouse and human islets in which the 13-cells have been engineered specifically for functional imaging. Ca2¿-dependent signal transduction mechanisms that regulate insulin secretion have been well-defined in vitro using intact islets, primary cultures of 13- cells, and insulinoma cells. Notwithstanding the important contributions of the in vitro approaches, knowledge of the mechanisms underlying 13-cell function in intact islets in vivo remains incomplete. Several technical limitations of the current imaging methodologies do not permit the study of 13-cell function within the complex multicellular environment of the pancreas in situ. The central focus of the experiments described in this proposal is to develop and characterize a novel imaging approach, which will facilitate the study of islet cell function in situ. In Specific Aim 1, viral gene transfer vectors will be used to transduce mouse _-cells with genetically-targeted biosynthetic fluorescent Ca 2¿ sensors. Their effectiveness in studying Q-cell biophysical and physiological responses following secretagogue stimulation will be evaluated. In Specific Aim 2, the utility of these sensors as functional imaging indicators in intact mouse islets will be assessed. In Specific Aim 3, 13-cells within intact human islets will be engineered to express Ca 2¿ biosensors and human islet function studied in vitro with comparison to mouse islets. In Specific Aim 4, transgenic mouse models in which the 13-cells have been genetically enhanced to express Ca 2+ biosensors will be developed and characterized in vitro and in vivo by confocal microfluorometry and by measurements of insulin secretion. The studies will provide new understanding of islet cell biology that will benefit clinical strategies to preserve and maintain functional 13-cell mass.