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ABCs of Cholesterol Regulation in the Insulin Secretory Pathway

胰岛素分泌途径中胆固醇调节的基础知识

基本信息

批准号：
8856221
负责人：
John David Castle
金额：
$ 34.37万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8856221
关键词：
ATP binding cassette transporter 1 ATP phosphohydrolase ATP-Binding Cassette Transporters Address Affect Anabolism Area B-Lymphocytes Biochemical Biological Biological Assay Cell Line Cells Cholesterol Cholesterol Homeostasis Collaborations Complement Cytoplasmic Granules Data Defect Diabetes Mellitus Docking Endocrine Equilibrium Etiology Exocytosis Failure Figs - dietary Fluorescence Microscopy Genetic Glucose Goals Health Homeostasis Image Insulin Investigation Islet Cell Islets of Langerhans Life Lipids Lipoproteins Membrane Microscopic Modeling Non-Insulin-Dependent Diabetes Mellitus Pancreas Pathway interactions Plasma Procedures Process Proteins Regulation Research Role Secretory Vesicles Series Sorting - Cell Movement Testing Thiazolidinediones Time Work aminophospholipid transporter analog base biophysical techniques cell type diabetic genetic regulatory protein glucose tolerance humoral immunity deficiency impaired glucose tolerance insulin granule insulin secretion insulinoma islet prospective protein function response restoration translocase two-photon uptake

项目摘要

DESCRIPTION (provided by applicant): This proposal addresses a new area in diabetes-related research regarding cholesterol distribution and its regulatory roles in the insulin secretory pathway. Recent studies including those forming the basis of this proposal have shown that deficiencies in pancreatic � cells in the ATP-binding cassette transporters ABCA1 and ABCG1, which have been implicated to promote cellular export of cholesterol to plasma lipoproteins by most cell types, impair systemic glucose tolerance through inhibition of insulin secretion. Moreover, expression of these transporters is reduced in models of type 2 diabetes and also figures in glucose sensitivity in response to anti-diabetic thiazolidinediones. Detailed analysis of ABCG1 in islet cells and islet-derived cell lines has shown unexpectedly that this transporter mostly resides in the membranes of insulin granules; its deficiency leads to enlargement of granules, reduced granule cholesterol content and reduced ability of granules to undergo exocytosis in response to glucose or K+ stimulation. It appears as if granule formation and/or maturation are perturbed. Preliminary findings show that ABCA1 also localizes to granules and that its deficiency causes similar intracellular effects as caused by reduced ABCG1. Strikingly, all changes elicited by ABCG1 deficiency are reversed by addition of exogenous cholesterol, identifying cholesterol as the likely common denominator. Taken together, these observations have led to the working hypothesis that ABCG1, in collaboration with ABCA1 and other lipid regulatory proteins, functions in the formation of insulin granules by promoting assembly of a cholesterol-enriched limiting membrane that is able to support the sorting and export activities of the regulated secretory pathway. To explore this hypothesis, a combination of cell biological and biochemical/biophysical approaches will be used on pancreatic islet derived cells and insulinoma cell lines to pursue four specific aims. First, ABCA1's functions alongside ABCG1 in insulin's secretory pathway will be tested using procedures already successfully applied to ABCG1. Second, lipid composition and protein sorting activities within the secretory pathway will be examined to evaluate the roles of the two ABCs in cholesterol dependent granule formation/maturation. Third, using isolated insulin granule fractions, fluorescent lipid analogs will be employed to assay ABC transporter and related lipid translocation mechanisms that are thought to contribute to insulin granule formation. Fourth, real-time microscopic imaging will be used to identify the level(s) at which ABC deficiency affects insulin granule exocytosis. The proposed studies highlight the insulin granule as a major cholesterol regulatory compartment; they represent the first exploration of intracellular roles of ABCs A1 and G1 that are unrelated to cellular cholesterol efflux; and they complement ongoing investigations by others seeking to understand mechanisms of insulin granule exocytosis and perturbations that might relate to � cell failure in type 2 diabetes.

描述(由申请人提供)：这项建议涉及糖尿病相关研究中的一个新领域，即关于胆固醇分布及其在胰岛素分泌途径中的调节作用。最近的研究，包括那些构成这一建议的基础的研究表明，胰腺�细胞三磷酸腺苷结合盒转运体ABCA1和ABCC1的缺陷通过抑制胰岛素的分泌而损害全身葡萄糖耐量，这两种转运体被认为是大多数细胞类型促进细胞向血浆脂蛋白输出胆固醇。此外，在2型糖尿病模型中，这些转运体的表达减少，而且抗糖尿病药物噻唑烷二酮类药物对葡萄糖的敏感性也有所降低。对胰岛细胞和胰岛来源细胞系中Abcg1的详细分析表明，该转运蛋白主要存在于胰岛素颗粒的膜上；它的缺失导致胰岛素颗粒增大，颗粒胆固醇含量降低，颗粒对葡萄糖或K+刺激进行胞吐的能力降低。似乎颗粒的形成和/或成熟受到了干扰。初步研究结果表明，ABCA1也定位于颗粒，其缺陷引起的细胞内效应与Abcg1减少引起的相似。引人注目的是，由Abcg1缺乏引起的所有变化都被外源性胆固醇的添加逆转，确定胆固醇可能是共同的分母。综上所述，这些观察结果导致了一个工作假说，即Abcg1与ABCA1和其他脂质调节蛋白合作，通过促进富含胆固醇的限制膜的组装而在胰岛素颗粒的形成中发挥作用，该限制膜能够支持受调控的分泌途径的分选和输出活动。为了探索这一假设，将结合细胞生物学和生化/生物物理方法对胰岛来源的细胞和胰岛素瘤细胞系进行研究，以追求四个特定的目标。首先，ABCA1的S在胰岛素分泌途径中与Abcg1的功能将使用已经成功应用于Abcg1的程序进行测试。其次，将检测分泌途径中的脂质组成和蛋白质分选活动，以评估这两种ABC在胆固醇依赖颗粒形成/成熟中的作用。第三，使用分离的胰岛素颗粒组分，荧光脂质类似物将被用于分析ABC转运蛋白和相关的脂转移机制，这些机制被认为有助于胰岛素颗粒的形成。第四，实时显微成像将被用来确定ABC缺乏影响胰岛素颗粒胞吐的水平(S)。拟议中的研究强调胰岛素颗粒是主要的胆固醇调节间隔；它们代表了对与细胞胆固醇外流无关的ABC A1和G1在细胞内的作用的首次探索；它们补充了其他人正在进行的研究，这些研究试图了解胰岛素颗粒吐出的机制和可能与2型糖尿病�细胞衰竭有关的扰动。