Insulin regulation of cell nutrition

细胞营养的胰岛素调节

• 批准号: 6609593
• 负责人: Konstantin V Kandror
• 金额: $ 32.3万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-28 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6609593
• 关键词: 3T3 cells; SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; adipocytes; aminopeptidase; chemical kinetics; confocal scanning microscopy; endocytosis; enzyme activity; glucose metabolism; glucose transport; glucose transporter; hormone regulation /control mechanism; immunoelectron microscopy; insulin; insulin receptor; laboratory rat; mass spectrometry; matrix assisted laser desorption ionization; membrane transport proteins; nuclear matrix; protein structure function; proteomics

DESCRIPTION (provided by applicant): Insulin action on glucose uptake in fat and skeletal muscle tissues is mediated by translocation of glucose transporter Glut4 from an "intracellular storage pool" to the plasma membrane. The intracellular pool of Glut4 is not homogenous and consists of several distinct membrane compartments only one of which, insulin-responsive vesicles, or IRVs, is believed to be translocated to the plasma membrane upon insulin stimulation. The rest of intracellular Glut4 is localized in early and recycling endosomes as well as in intermediate transport vesicles. In the previous funding period, we developed a procedure for the biochemical separation of Glut4-containing endosomes from post-endosomal membrane vesicles. In addition, we identified a specific marker for one type of Glut4-containing vesicles, cellugyrin, and prepared antibodies to this protein. Thus, we can now separate individual intracellular Glut4-containing compartments and, for the first time, isolate pure IRVs. Based on these findings, we propose to determine their protein composition and to identify specific sequence(s) in the Glut4 molecule which target this protein into IRVs. We have also developed an in vitro budding assay for Glut4-vesicles. We propose to use this approach in order to reconstitute major trafficking steps of the Glut4 pathway in vitro and to study molecular mechanisms of these events. Membrane budding from endosomes usually requires a scaffold or a sorting receptor that gathers cargo proteins together and recruits protein coats to this complex. In the previous funding period, we have identified a novel component protein, sortilin, in the total pool of intracellular Glut4-vesicles. We suggest that this protein may play an important role in the organization of the insulin-sensitive glucose transporting machinery and, in particular, in formation of IRVs. Thus, we propose to study the biological role of sortilin in adipocytes using the antisense technique.

描述（由申请人提供）：胰岛素对脂肪和骨骼肌组织中葡萄糖摄取的作用是通过葡萄糖转运蛋白Glut4从“细胞内储存池”易位至质膜来介导的。 Glut4 的细胞内池不是同质的，由几个不同的膜区室组成，其中只有其中之一，胰岛素反应性囊泡 (IRV)，被认为在胰岛素刺激后易位到质膜。细胞内 Glut4 的其余部分位于早期和再循环内体以及中间转运囊泡中。在之前的资助期间，我们开发了一种从内体后膜囊泡中生化分离含有 Glut4 的内体的程序。此外，我们还鉴定了一种含有 Glut4 的囊泡（cellugyrin）的特异性标记物，并制备了针对该蛋白的抗体。因此，我们现在可以分离单个细胞内含有 Glut4 的区室，并首次分离出纯 IRV。基于这些发现，我们建议确定它们的蛋白质组成，并鉴定 Glut4 分子中将该蛋白质靶向 IRV 的特定序列。我们还开发了 Glut4 囊泡的体外出芽测定。我们建议使用这种方法在体外重建 Glut4 途径的主要运输步骤并研究这些事件的分子机制。内体的膜出芽通常需要支架或分选受体，将货物蛋白聚集在一起并将蛋白外壳招募到该复合物中。在之前的资助期间，我们在细胞内 Glut4 囊泡的总池中发现了一种新的成分蛋白，即分拣蛋白。我们认为这种蛋白质可能在胰岛素敏感的葡萄糖转运机制的组织中发挥重要作用，特别是在 IRV 的形成中。因此，我们建议使用反义技术研究分选蛋白在脂肪细胞中的生物学作用。