L-type Ca2+ channel modulation of beta cell function

L 型 Ca2 通道调节 β 细胞功能

基本信息

批准号：
8098808
负责人：
GREGORY Howard HOCKERMAN
金额：
$ 28.02万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-04-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8098808
关键词：
Adenylate Cyclase Agonist Amino Acid Sequence Beta Cell Binding Biological Assay Blood Glucose Cell Line Cell Proliferation Cell model Cell physiology Cells Chemosensitization Complex Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Data Development Dihydropyridines Electrophysiology (science)Environment Event Exocytosis Extracellular Domain Fluorescence Fluorescence Microscopy GTP-Binding Proteins Glucose Goals Health Hormones Hyperglycemia Image Immunoprecipitation Insulin Islets of Langerhans Knock-in Mouse L-Type Calcium Channels Lead Ligand Binding Link Mediating Methods Molecular Non-Insulin-Dependent Diabetes Mellitus Output Pathway interactions Pattern Peptide Receptor Peptides Pharmaceutical Preparations Phosphorylation Physiological Proinsulin Property Proteins Rattus Receptor Activation Role Signal Pathway Signal Transduction Stimulus Structure Structure of beta Cell of islet System Techniques Viral Vector Western Blotting Work channel blockers dihydropyridine glucagon-like peptide 1 insulin granule insulin secretion insulinoma knock-down mutant novel patch clamp peptide B preference prevent protein protein interaction prototype receptor response sensor small molecule

项目摘要

DESCRIPTION (provided by applicant): In type II diabetes, the insulin secreting beta cells of pancreatic islets fail to secrete insulin in sufficient quantities to maintain normal blood glucose levels. The resulting hyperglycemia can lead to many serious complications. Therefore, understanding the mechanisms that mediate insulin secretion could lead to new therapies to prevent the onset and complications of Type II diabetes. Two sub-classes of L-type calcium channels, Cav1.2 and Cav1.3 are expressed in pancreatic beta cells. We have developed a "knock in" method to introduce Cav1.2 and Cav1.3 mutant channels that are insensitive to the dihydropyridine (DHP) class of L-type channel blockers into the insulinoma cell line INS-1. In this system, the endogenous L-type channels can be "shut off" with DHP drugs, thus pharmacologically isolating either Cav1.2 of Cav1.3 channels. Using this system, we have shown that Cav1.3 but not Cav1.2 channels can mediate glucose-stimulated insulin secretion. Insulin secretion is potentiated by the hormone GLP-1, by its binding to the GLP-1 receptor. We have identified a short peptide, derived from the GLP-1 receptor primary amino acid sequence, that can mimic some, but not all of the actions of GLP-1. We hypothesize that in the context of the receptor this peptide comprises an autoactivation domain that is unmasked upon ligand binding. In Aim 1 of this project, we will characterize both the activity of this peptide as a small molecule agonist, and its contribution to GLP-1 receptor activation in the context of the GLP-1 receptor structure. In Aim 2, we will further examine the mechanisms that couple L-type calcium channels to insulin secretion and beta cell proliferation, and how they are modulated by GLP-1 receptor activation. Although most of this work will be done in the INS-1 cell model, viral vectors have been developed to introduce mutant channels and channel fragments into rat primary beta cells. This proposal will utilize techniques such as patch clamp whole-cell electrophysiology, Fluorescence Lifetime Imaging, Total Internal Reflection Fluorescence Microscopy, insulin secretion assays, immunoprecipitation assays, and western blot assays. This proposal is consistent with the PI's long term goal of understanding L-type calcium channel modulation and cellular function. PUBLIC HEALTH RELEVANCE: This project will explore a set of novel molecules that stimulate beta cells of the pancreas to secret insulin in the presence of glucose, and may also promote the proliferation of beta cells. It will also examine the mechanisms by which the influx of Ca2+ into beta cells through specific types of Ca2+ channels regulates insulin secretion. The results of these studies may provide information for the development of new drugs to treat type 2 diabetes.

描述（由申请人提供）：在II型糖尿病中，胰岛的胰岛素分泌β细胞无法分泌足够量的胰岛素来维持正常的血糖水平。由此产生的高血糖会导致许多严重的并发症。因此，了解介导胰岛素分泌的机制可能会带来预防 II 型糖尿病发病和并发症的新疗法。 L 型钙通道的两个亚类 Cav1.2 和 Cav1.3 在胰腺 β 细胞中表达。我们开发了一种“敲入”方法，将对二氢吡啶 (DHP) 类 L 型通道阻滞剂不敏感的 Cav1.2 和 Cav1.3 突变通道引入胰岛素瘤细胞系 INS-1 中。在该系统中，内源性L型通道可以用DHP药物“关闭”，从而在药理学上隔离Cav1.2或Cav1.3通道。使用该系统，我们已经证明 Cav1.3 而不是 Cav1.2 通道可以介导葡萄糖刺激的胰岛素分泌。激素 GLP-1 通过与 GLP-1 受体结合来增强胰岛素分泌。我们已经鉴定出一种源自 GLP-1 受体一级氨基酸序列的短肽，它可以模拟 GLP-1 的部分但不是全部作用。我们假设在受体的背景下，该肽包含一个在配体结合后暴露的自激活结构域。在该项目的目标 1 中，我们将表征该肽作为小分子激动剂的活性，及其在 GLP-1 受体结构背景下对 GLP-1 受体激活的贡献。在目标 2，我们将进一步研究 L 型钙通道与胰岛素分泌和 β 细胞增殖的耦合机制，以及它们如何通过 GLP-1 受体激活进行调节。尽管大部分工作将在 INS-1 细胞模型中完成，但已开发出病毒载体以将突变通道和通道片段引入大鼠原代 β 细胞中。该提案将利用膜片钳全细胞电生理学、荧光寿命成像、全内反射荧光显微镜、胰岛素分泌测定、免疫沉淀测定和蛋白质印迹测定等技术。该提案与 PI 了解 L 型钙通道调节和细胞功能的长期目标一致。公共健康相关性：该项目将探索一组新颖的分子，这些分子可以在葡萄糖存在的情况下刺激胰腺β细胞分泌胰岛素，并且还可能促进β细胞的增殖。它还将研究 Ca2+ 通过特定类型的 Ca2+ 通道流入 β 细胞调节胰岛素分泌的机制。这些研究结果可能为开发治疗2型糖尿病的新药提供信息。