INSULIN ACTION ON THE FAT CELL SURFACE MEMBRANE

胰岛素对脂肪细胞表面膜的作用

• 批准号: 3229722
• 负责人: MICHAEL P CZECH
• 金额: $ 30.45万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-09-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3229722
• 关键词: adipocytes; affinity chromatography; affinity labeling; antireceptor antibody; cell membrane; chimeric proteins; fibroblasts; genetic library; glucose transport; high performance liquid chromatography; hormone regulation /control mechanism; immunoprecipitation; insulin; insulin receptor; intracellular transport; laboratory rabbit; laboratory rat; membrane channels; membrane proteins; membrane reconstitution /synthesis; membrane structure; molecular cloning; radiotracer; receptor mediated endocytosis; tissue /cell culture; transfection; transport proteins; western blottings

The overall objective of these proposed studies is to provide insight into the detailed molecular mechanisms underlying glucose transport regulation by insulin. Insulin-sensitive cells such as muscle and fat express one predominant glucose transporter isoform, GLUT4, but also contain a HepG2/brain-type (GLUT1) and perhaps others. It is established that a major action of insulin is to increase the levels of glucose transporters as well as certain receptors in the cell surface membrane at the expense of those in intracellular membranes. During the previous grant period, we have obtained substantial data indicating that the intrinsic activity of GLUT1, and perhaps GLUT4 can also be markedly regulated. In this proposal, we seek to investigate these two potential modes by which transporters are regulated in 3T3-L1 adipocytes which contain both GLUT4 and GLUT1. Our experiments shall address three key questions related to these mechanisms: 1. Does GLUT4 translocation to the cell surface solely account for the effect of insulin on glucose uptake? We have shown that expression of the human GLUT1 protein in transfected mouse 3T3-L1 cells leads to increased basal but no changes in insulin-stimulated glucose transport. We shall perform similar transfections with rat GLUT4 cDNA and a number of chimeric GLUT1/GLUT4 constructs we have engineered to directly determine the contribution of GLUT4 (insulin) using [2-3H]BMPA and a novel anti-exofacial peptide antibody we have produced that recognizes GLUT4 on the surface of intact cells. 2. Do the GLUT1 and GLUT4 transporters continuously recycle between intracellular and cell surface membranes? During the previous grant period we demonstrated that both the endocytic rate and exocytotic rate of transferrin receptor recycling are regulated processes. We propose to identify the step or steps in the trafficking pathway of the transporter proteins that are regulated by insulin. We shall study the cell surface residency times, coated vesicle localization, endocytic rates, and exocytotic rates for these transporters. These experiments will be conducted using the anti-exofacial GLUT4 antibody, [2-3H]BMPA, and metabolic labeling of transporters in pulse-chase studies, in conjunction with specific immunoprecipitations of GLUT1 and GLUT4 using protocols we have established. 3. How is the intrinsic activity of GLUT1 (and GLUT4?) regulated in intact rate fat and 3T3-L1 adipocytes? We have established that plasma membranes from 3T3-L1 adipocytes treated with protein synthesis inhibitors exhibit 7 fold elevations in glucose transport activity without a change in GLUT1 or GLUT4 levels. Reconstitution experiments will be performed to determine whether transporter inhibitory activity can be observed in extracts of control membranes. Affinity-columns will be prepared using large quantities of GLUT1 and GLUT4 obtained from baculovirus infected Sf9 cells that we have established. In these studies, we shall attempt to absorb and isolate transporter regulator proteins that suppress transport activity in control adipocytes.

这些拟议的研究的总体目的是提供对 葡萄糖转运调节的详细分子机制 由胰岛素。 胰岛素敏感细胞（例如肌肉和脂肪）表达 主要的葡萄糖转运蛋白同工型GLUT4，但也包含一个 HEPG2/脑型（GLUT1）以及其他人。 已经确定 胰岛素的主要作用是增加葡萄糖转运蛋白的水平 以及细胞表面膜中的某些受体，以代价 那些在细胞内膜中。 在上一个赠款期间，我们 已经获得了大量数据，表明 GLUT1，也许GLUT4也可以显着调节。 在此提案中， 我们试图调查运输者的这两种潜在模式 在包含GLUT4和GLUT1的3T3-L1脂肪细胞中调节。 我们的 实验应解决与这些机制有关的三个关键问题： 1。glut4易位到细胞表面仅考虑 胰岛素对葡萄糖摄取的影响？ 我们已经表明了 转染的小鼠3T3-L1细胞中的人Glut1蛋白导致增加 基础但胰岛素刺激的葡萄糖转运没有变化。我们将 用大鼠Glut4 cDNA和许多嵌合进行类似的转染 GLUT1/GLUT4构建体我们已经设计了直接确定 使用[2-3H] BMPA和一种新型的抗副异构的GLUT4（胰岛素）的贡献 我们已经生产的肽抗体在表面上识别glut4 完整的细胞。 2。执行GLUT1和GLUT4转运器连续回收 在细胞内和细胞表面膜之间？ 在上一个 赠款期我们证明了内吞率和胞吞 转铁蛋白受体回收率是调节过程。 我们建议 确定运输者贩运途径的步骤或步骤 受胰岛素调节的蛋白质。 我们将研究细胞表面 居住时间，涂层囊泡定位，内吞率和 这些转运蛋白的胞吐率。 这些实验将是 使用抗副glut4抗体[2-3H] BMPA和 转运蛋白在脉搏学习研究中的代谢标记，结合 使用方案对GLUT1和GLUT4进行特定的免疫沉淀，我们 已经建立了。 3。glut1（和glut4？）的固有活性如何？ 由完整速率脂肪和3T3-L1脂肪细胞调节？ 我们已经建立了 来自用蛋白质合成处理的3T3-L1脂肪细胞的质膜 抑制剂在无葡萄糖转运活性中表现出7倍的海拔 GLUT1或GLUT4级别的变化。 重建实验将是 执行以确定转运蛋白抑制活性是否可以是 在对照膜提取物中观察到。 亲和力列将是 使用大量的GLUT1和GLUT4从 杆状病毒感染了我们已经建立的SF9细胞。 在这些研究中， 我们将尝试吸收并分离转运蛋白调节蛋白 抑制控制脂肪细胞中的运输活性。