ABCs of Cholesterol Regulation in the Insulin Secretory Pathway

描述（由申请人提供）：本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，包括构成这一建议基础的研究表明，胰腺细胞中atp结合盒转运体ABCA1和ABCG1的缺陷，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损害全身葡萄糖耐量。此外，在2型糖尿病模型中，这些转运蛋白的表达减少，并且在抗糖尿病噻唑烷二酮类药物的葡萄糖敏感性中也有所体现。对胰岛细胞和胰岛衍生细胞系中ABCG1的详细分析显示，这种转运蛋白主要存在于胰岛素颗粒的膜中；其缺乏导致颗粒增大，颗粒胆固醇含量降低，颗粒在葡萄糖或K+刺激下发生胞吐的能力降低。看起来好像颗粒的形成和/或成熟受到干扰。初步发现ABCA1也定位于颗粒，其缺乏引起的细胞内效应与ABCG1减少引起的细胞内效应相似。引人注目的是，所有由ABCG1缺乏引起的变化都可以通过添加外源性胆固醇而逆转，这表明胆固醇可能是共同因素。综上所述，这些观察结果导致了一个有效的假设，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，在胰岛素颗粒的形成中起作用，该限制膜能够支持受调节的分泌途径的分类和输出活动。为了探索这一假设，我们将结合细胞生物学和生物化学/生物物理学的方法，对胰岛来源的细胞和胰岛素瘤细胞系进行研究，以实现四个特定的目标。首先，ABCA1与ABCG1在胰岛素分泌通路中的功能将使用已经成功应用于ABCG1的方法进行测试。其次，将检查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在胆固醇依赖性颗粒形成/成熟中的作用。第三，使用分离的胰岛素颗粒，荧光脂质类似物将被用来检测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缺乏影响胰岛素颗粒胞吐的水平。拟议的研究强调胰岛素颗粒是一个主要的胆固醇调节室；它们首次探索了与细胞胆固醇外排无关的abc A1和G1在细胞内的作用；他们补充了其他人正在进行的研究，试图了解胰岛素颗粒胞吐和扰动的机制，这可能与2型糖尿病的细胞衰竭有关。

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.