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：
8292152
负责人：
GREGORY Howard HOCKERMAN
金额：
$ 27.94万
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 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8292152
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 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

Project Summary 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.

项目概要在 II 型糖尿病中，胰岛分泌胰岛素的 β 细胞无法分泌足够的胰岛素维持正常血糖水平的量。由此产生的高血糖可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导致新的发现预防 II 型糖尿病发病和并发症的疗法。 L型钙的两个亚类通道、Cav1.2 和 Cav1.3 在胰腺 β 细胞中表达。我们开发了一种“敲入”方法引入对 L 型二氢吡啶 (DHP) 类不敏感的 Cav1.2 和 Cav1.3 突变通道通道阻滞剂进入胰岛素瘤细胞系 INS-1。在该系统中，内源性 L 型通道可以是用 DHP 药物“关闭”，从而在药理学上隔离 Cav1.2 或 Cav1.3 通道。使用这个系统中，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。我们有鉴定出一种源自 GLP-1 受体一级氨基酸序列的短肽，它可以模仿一些，但不是 GLP-1 的所有作用。我们假设在受体的背景下，该肽包含配体结合后暴露的自激活结构域。在该项目的目标 1 中，我们将描述该肽作为小分子激动剂的活性及其对 GLP-1 受体激活的贡献 GLP-1 受体结构的背景。在目标2中，我们将进一步研究L型偶联的机制胰岛素分泌和 β 细胞增殖的钙通道，以及它们如何受 GLP-1 受体调节激活。尽管大部分工作将在 INS-1 细胞模型中完成，但病毒载体已经开发出来将突变通道和通道片段引入大鼠原代β细胞中。本提案将利用膜片钳全细胞电生理学、荧光寿命成像、Total Internal 等技术反射荧光显微镜、胰岛素分泌测定、免疫沉淀测定和蛋白质印迹化验。该提案与 PI 了解 L 型钙通道的长期目标一致调节和细胞功能。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Uncoupling of Cav1.2 from Ca(2+)-induced Ca(2+) release and SK channel regulation in pancreatic β-cells.

Cav1.2 与 Ca(2 ) 诱导的 Ca(2 ) 释放和胰腺 β 细胞中 SK 通道调节的解偶联。

DOI：
10.1210/me.2013-1094
发表时间：
2014
期刊：
Molecular endocrinology (Baltimore, Md.)
影响因子：
0
作者：
Wang,Yuchen;Jarrard,RachelE;Pratt,EvanPS;Guerra,MarcyL;Salyer,AmyE;Lange,AllisonM;Soderling,IanM;Hockerman,GregoryH
通讯作者：
Hockerman,GregoryH

Potentiation of sulfonylurea action by an EPAC-selective cAMP analog in INS-1 cells: comparison of tolbutamide and gliclazide and a potential role for EPAC activation of a 2-APB-sensitive Ca2+ influx.

EPAC 选择性 cAMP 类似物在 INS-1 细胞中增强磺酰脲作用：甲苯磺丁脲和格列齐特的比较以及 EPAC 激活 2-APB 敏感 Ca2 流入的潜在作用。

DOI：
10.1124/mol.112.081943
发表时间：
2013
期刊：
Molecular pharmacology
影响因子：
3.6
作者：
Jarrard,RachelE;Wang,Yuchen;Salyer,AmyE;Pratt,EvanPS;Soderling,IanM;Guerra,MarcyL;Lange,AllisonM;Broderick,HilaryJ;Hockerman,GregoryH
通讯作者：
Hockerman,GregoryH

Antibody inhibition of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 reduces rapid exocytosis in insulin-secreting cells.

25 kDa 突触体蛋白 (SNAP-25) 和突触蛋白 1 的抗体抑制可减少胰岛素分泌细胞中的快速胞吐作用。