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（由适用提供）：该提案介绍了与糖尿病相关研究中有关胆固醇分布及其在胰岛素秘书途径中的调节作用的新领域。最近的研究（包括构成该提案基础的研究）表明，ATP结合盒磁带转运蛋白ABCA1和ABCG1中胰腺细胞的缺乏，这些细胞已暗示，这些细胞被暗示地通过大多数细胞类型促进胆固醇向血浆脂蛋白促进血浆脂蛋白的细胞导出，从而损害了系统的系统性葡萄糖耐受性，从而损害了胰岛素的胰岛素。此外，在2型糖尿病模型中，这些转运蛋白的表达降低了，以及葡萄糖敏感性的数字，响应于抗糖尿病性噻唑烷二酮。胰岛细胞和胰岛衍生细胞系中ABCG1的详细分析表明，该转运蛋白主要驻留在胰岛素颗粒的膜中。它的缺乏会导致颗粒的扩张，颗粒胆固醇含量降低以及颗粒能够响应葡萄糖或K+刺激的颗粒能力降低。看起来好像颗粒形成和/或成熟是干扰的。初步发现表明，ABCA1还定位于颗粒，其缺乏会导致与ABCG1减少引起的相似细胞内作用。令人惊讶的是，ABCG1缺乏症引起的所有变化都通过添加外源胆固醇逆转，从而将胆固醇鉴定为可能性的共同点。综上所述，这些观察结果导致了这样的假设：ABCG1与ABCA1和其他脂质调节蛋白合作，通过促进胆固醇 - 烯烃限制膜的组装来形成胰岛素颗粒的形成，能够支持调节的特定秘密路径的分类和出口活动。为了探讨这一假设，将使用细胞生物学和生化/生物物理方法的结合，用于胰岛衍生的细胞和胰岛素瘤细胞系，以追求四个特定的目标。首先，将使用已经成功应用于ABCG1的程序对ABCA1的功能与胰岛素秘密途径中的ABCG1一起进行测试。其次，将检查秘密途径内的脂质组成和蛋白质分选活性，以评估两个ABC在胆固醇依赖性颗粒形成/成熟中的作用。第三，使用孤立的胰岛素颗粒级分，将雇用荧光脂质类似物来进行ASSAY ABC转运蛋白和相关的脂质翻译机制，这些机制被认为有助于胰岛素颗粒的形成。第四，实时微观成像将用于确定ABC缺乏影响胰岛素颗粒胞吐作用的水平。拟议的研究强调了胰岛素颗粒作为主要的胆固醇调节室。它们代表了与细胞胆固醇外排无关的ABCS A1和G1细胞内作用的首次探索。他们补充了其他人寻求了解胰岛素颗粒外胞菌病和扰动机制的持续调查，这些机制可能与2型糖尿病中的细胞衰竭有关。