Molecular Regulatory Mechanisms of Insulin Secretion

描述（由申请方提供）：胰岛素由胰岛β细胞分泌，以响应血糖水平升高，并具有控制全身代谢和生长的功能。葡萄糖刺激的胰岛素分泌（GSIS）功能障碍是I型和II型糖尿病的标志。因此，了解参与调节GSIS的分子机制对于了解代谢稳态的系统控制以及糖尿病疾病状态的起源和治疗至关重要。我们最近的工作提供了证据支持分泌的胰岛素对GSIS的积极调节作用。培养的β细胞的胰岛素处理导致葡萄糖敏感酶葡萄糖激酶的活化。这一过程是由一氧化氮合酶在分泌颗粒上产生一氧化氮介导的。然而，导致这种酶活化的信号通路尚不清楚。此外，目前还不清楚胰岛素是否在β细胞群和其他细胞类型中刺激与胰岛中发生的类似的自动反馈途径，并通过激活一氧化氮合酶和葡萄糖激酶对GSIS赋予类似的调节潜力。基于我们的初步研究，我们假设胰岛素通过刺激细胞内钙离子库的释放激活一氧化氮合酶，并且该过程发生在胰岛中。因此，本研究的具体目的是：1）确定从激活的胰岛素受体到激活分泌颗粒上的NOS的分子信号传导事件， 2)确定胰岛素治疗是否激活活胰岛中的一氧化氮合酶和葡萄糖激酶。为了实现这些目标，我们开发了针对特定细胞区室的遗传编码生物传感器，以测量活细胞中的胰岛素信号转导。这将允许通过荧光成像技术直接检查活β细胞中单个颗粒上的一氧化氮产生。此外，将开发慢病毒载体系统，以便将生物传感器构建体特异性递送至分离的胰岛中的β细胞。这些研究将进一步了解GSIS的生理调节，并可能确定I型和II型糖尿病药物干预的新靶点。

DESCRIPTION (provided by applicant): Insulin is secreted from the pancreatic islet beta cells in response to elevated blood glucose levels and functions to control systemic metabolism and growth. Dysfunction of glucose-stimulated insulin secretion (GSIS) is a hallmark of both Type I and Type II diabetes. Thus, an understanding of the molecular mechanisms that participate in regulating GSIS is essential to understanding both systemic control of metabolic homeostasis as well as the origin and treatment of diabetic disease states. Our recent work has provided evidence supporting a positive regulatory effect of secreted insulin on GSIS. Insulin treatment of cultured beta cells results in activation of the glucose-sensing enzyme, glucokinase. This process is mediated by production of nitric oxide on secretory granules by nitric oxide synthase. However, the signaling pathway leading to the activation of this enzyme is not understood. In addition, it is unclear whether insulin stimulates a similar auto feedback pathway in the crowd of beta cells and other cell types as occurs in an islet and confers a similar regulatory potential on GSIS through the activation of nitric oxide synthase and glucokinase. Based on our preliminary studies, we hypothesize that insulin activates nitric oxide synthase by stimulating the release of calcium ions from intracellular stores and that this process occurs in pancreatic islets. Therefore, the Specific Aims of this study are to 1) determine the molecular signaling events leading from activated insulin receptors to activation of NOS on secretory granules and 2) determine whether insulin treatment activates nitric oxide synthase and glucokinase in living pancreatic islets. To accomplish these goals, we have developed genetically-encoded biosensors targeted to specific cellular compartments in order to measure insulin signal transduction in living cells. This will allow direct examination of nitric oxide production on individual granules in living beta cells by fluorescence imaging techniques. In addition, a lentiviral vector system will be developed in order to specifically deliver the biosensor constructs to beta cells in isolated pancreatic islets. These studies will further understanding of the physiological regulation of GSIS and may identify novel targets for pharmacological intervention of Type I and Type II diabetes.