L-type Ca2+ channel modulation of beta cell function

L 型 Ca2 通道调节 β 细胞功能

• 批准号: 7925825
• 负责人: GREGORY Howard HOCKERMAN
• 金额: $ 31.32万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 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/7925825
• 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; GLP-I receptor; GTP-Binding Proteins; Glucose; Goals; 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; public health relevance; response; sensor; small molecule

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(申请人提供)：在II型糖尿病中，胰岛分泌胰岛素的β细胞不能分泌足够数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，了解调节胰岛素分泌的机制可能会导致新的治疗方法来预防II型糖尿病的发生和并发症。L类钙通道的两个亚类Cav1.2和Cav1.3在胰岛β细胞中表达。我们开发了一种“敲入”方法，将对二氢吡啶类L类通道阻断剂不敏感的Cav1.2和Cav1.3突变通道导入胰岛素瘤细胞株INS-1。在这个系统中，内源性的L类通道可以被DHP药物“关闭”，从而从药理上隔离Cav1.2或Cav1.3的任一个通道。使用这个系统，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。荷尔蒙GLP-1通过与GLP-1受体结合来增强胰岛素的分泌。我们已经鉴定出一种来源于GLP-1受体初级氨基酸序列的短肽，它可以模拟GLP-1的部分但不是所有的作用。我们假设，在受体的背景下，该肽包含一个在配体结合时未被屏蔽的自激活化域。在本项目的目标1中，我们将在GLP-1受体结构的背景下，表征该肽作为小分子激动剂的活性，以及它对GLP-1受体激活的贡献。在……里面 目的2，我们将进一步研究L钙通道与胰岛素分泌和β细胞增殖的偶联机制，以及GLP-1受体激活是如何调节这些机制的。虽然大部分工作将在INS-1细胞模型中完成，但已开发出病毒载体将突变通道和通道片段引入大鼠原代β细胞。这项建议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、胰岛素分泌分析、免疫沉淀分析和蛋白质印迹分析等技术。这一建议与PI了解L型钙通道调节和细胞功能的长期目标是一致的。与公共健康相关：该项目将探索一组新的分子，在葡萄糖存在的情况下刺激胰腺的β细胞分泌胰岛素，并可能促进β细胞的增殖。它还将研究通过特定类型的钙通道进入β细胞的钙离子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。