Insulin regulation of cell nutrition

细胞营养的胰岛素调节

• 批准号: 7034588
• 负责人: Konstantin V Kandror
• 金额: $ 31.54万
• 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/7034588
• 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将葡萄糖转运蛋白Glut4转移到“细胞内存储池”到质膜的介导的。 GLUT4的细胞内池不是同质的，由几个不同的膜室组成，其中仅一个胰岛素反应性囊泡或IRV被认为是在胰岛素刺激后转移到质膜的。其余的细胞内GLUT4位于早期和回收的内体以及中间运输囊泡中。在上一个资金期间，我们开发了一种从粘液后膜囊泡中含有GLUT4的内体生化分离的程序。此外，我们确定了一种含有GLUT4的囊泡，Cellugyrin和该蛋白质制备的抗体的特异性标记。因此，我们现在可以分离单个含有细胞内GLUT4的隔室，并首次分离出纯IRV。基于这些发现，我们建议确定其蛋白质组成，并确定GLUT4分子中的特定序列，将该蛋白靶向IRV。我们还开发了用于Glut4-vesicles的体外出芽测定法。我们建议使用这种方法来重建体外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类受体，将货物蛋白聚集在一起，并将蛋白质涂层募集到该复合物中。在上一个资金期间，我们已经在细胞内Glut4-sepicles的总库中鉴定了一种新型的成分蛋白Tortilin。我们建议该蛋白可能在胰岛素敏感的葡萄糖转运机械的组织中起重要作用，尤其是在IRV的形成中。因此，我们建议使用反义技术研究Torsilin在脂肪细胞中的生物学作用。