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

• 批准号: 8291618
• 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
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8291618
• 关键词: 2,4-thiazolidinedione; 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; 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; cell type; diabetic; genetic regulatory protein; glucose tolerance; humoral immunity deficiency; impaired glucose tolerance; insulin granule; insulin secretion; insulinoma; islet; prospective; protein function; public health relevance; response; restoration; translocase; two-photon; uptake

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）：该提案涉及糖尿病相关研究的一个新领域，即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（包括构成该提议基础的研究）表明，胰腺细胞中 ATP 结合盒转运蛋白 ABCA1 和 ABCG1 的缺陷（这些缺陷与大多数细胞类型促进胆固醇向血浆脂蛋白的细胞输出有关）通过抑制胰岛素分泌而损害全身葡萄糖耐量。此外，这些转运蛋白的表达在 2 型糖尿病模型中减少，并且还影响抗糖尿病噻唑烷二酮类药物的葡萄糖敏感性。对胰岛细胞和胰岛衍生细胞系中 ABCG1 的详细分析出人意料地表明，这种转运蛋白主要存在于胰岛素颗粒的膜中；它的缺乏会导致颗粒增大、颗粒胆固醇含量降低以及颗粒响应葡萄糖或 K+ 刺激而进行胞吐作用的能力降低。看起来好像颗粒的形成和/或成熟受到干扰。初步研究结果表明，ABCA1 也定位于颗粒，并且其缺乏会导致与 ABCG1 减少引起的类似细胞内效应。引人注目的是，ABCG1 缺乏引起的所有变化都可以通过添加外源胆固醇来逆转，这表明胆固醇可能是共同点。总而言之，这些观察结果得出了这样的工作假设：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节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/生物物理方法用于胰岛衍生细胞和胰岛素瘤细胞系，以实现四个特定目标。首先，ABCA1 与 ABCG1 在胰岛素分泌途径中的功能将使用已成功应用于 ABCG1 的程序进行测试。其次，将检查分泌途径内的脂质组成和蛋白质分选活性，以评估两种 ABC 在胆固醇依赖性颗粒形成/成熟中的作用。第三，使用分离的胰岛素颗粒部分，荧光脂质类似物将用于测定ABC转运蛋白和相关的脂质易位机制，这些机制被认为有助于胰岛素颗粒的形成。第四，实时显微成像将用于确定 ABC 缺陷影响胰岛素颗粒胞吐作用的水平。拟议的研究强调胰岛素颗粒是主要的胆固醇调节区室；它们代表了对 ABC A1 和 G1 与细胞胆固醇流出无关的细胞内作用的首次探索；它们补充了其他人正在进行的研究，旨在了解胰岛素颗粒胞吐作用和可能与 2 型糖尿病细胞衰竭相关的扰动机制。