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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.

项目摘要在II型糖尿病中，胰岛分泌胰岛素的β细胞不能分泌足够的胰岛素。维持正常血糖水平所需的数量。由此产生的高血糖可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预防II型糖尿病发病和并发症的治疗。L型钙的两个亚类通道、Cav1.2和Cav1.3在胰岛β细胞中表达。我们开发了一种“敲进去”的方法来引入对L类二氢吡啶不敏感的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。在这个系统中，内生的L式的经络可以用DHP药物“关闭”，从而从药理上隔离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。荷尔蒙GLP-1通过与GLP-1受体结合来增强胰岛素的分泌。我们有鉴定出一种来源于GLP-1受体初级氨基酸序列的短肽，它可以模仿一些，但并不是GLP-1的所有作用。我们假设，在受体的上下文中，该肽包括一个配体结合时未被屏蔽的自动激活结构域。在本项目的目标1中，我们将对两者进行描述该多肽作为小分子激动剂的活性及其在GLP-1受体激活中的作用 GLP-1受体结构的背景。在目标2中，我们将进一步研究L类型的耦合机制钙通道对胰岛素分泌和β细胞增殖的影响及其受GLP-1受体的调节激活。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导入大鼠原代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、胰岛素分泌试验、免疫沉淀试验和蛋白质印迹化验。这一建议与PI了解L型钙通道的长期目标是一致的调制和细胞功能。

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。