Molecular Regulatory Mechanisms of Insulin Secretion

描述(申请人提供)：胰岛素是由胰岛β细胞分泌的，以应对血糖水平升高和控制全身新陈代谢和生长的功能。葡萄糖刺激的胰岛素分泌功能障碍(GSIS)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都是至关重要的。我们最近的工作提供了证据支持分泌型胰岛素对GSIS的积极调节作用。对培养的β细胞进行胰岛素处理会导致葡萄糖敏感酶葡萄糖激活酶的激活。这一过程是通过一氧化氮合酶在分泌颗粒上产生一氧化氮来实现的。然而，导致该酶激活的信号通路尚不清楚。此外，目前还不清楚胰岛素是否像在胰岛中一样，在一群β细胞和其他类型的细胞中刺激了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赋予了类似的调节潜力。根据我们的初步研究，我们假设胰岛素通过刺激细胞内钙离子的释放来激活一氧化氮合酶，这一过程发生在胰岛。因此，本研究的具体目的是：1)确定从激活的胰岛素受体到分泌颗粒上的一氧化氮合酶激活的分子信号事件，以及 2)确定胰岛素治疗是否激活活体胰岛一氧化氮合酶和葡糖激活酶。为了实现这些目标，我们开发了针对特定细胞隔间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的贝塔细胞中单个颗粒上一氧化氮的产生。此外，还将开发慢病毒载体系统，以便将生物传感器构建物特异性地运送到分离的胰岛中的β细胞。这些研究将进一步了解GSIS的生理调节，并可能为I型和II型糖尿病的药物干预寻找新的靶点。

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.