Morris Silke, Geoghegan Niall D, Sadler Jessica B A, Koester Anna M, Black Hannah L, Laub Marco, Miller Lucy, Heffernan Linda, Simpson Jeremy C, Mastick Cynthia C, Cooper Jon, Gadegaard Nikolaj, Bryant Nia J, Gould Gwyn W
Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow, UK.
School of Engineering, University of Glasgow, Glasgow, UK.
PeerJ. 2020 Mar 5;8:e8751. doi: 10.7717/peerj.8751. eCollection 2020.
Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA-GLUT4-GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA-GLUT4-GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells.
胰岛素刺激的葡萄糖转运是脂肪细胞和肌肉细胞的一个特征性特性，涉及将含有葡萄糖转运蛋白4（GLUT4）的囊泡从细胞内储存部位有调控地转运至细胞表面。这些囊泡的融合导致细胞表面GLUT4分子数量增加。为了克服与原代脂肪细胞和培养脂肪细胞相关的一些局限性，我们在HeLa细胞中表达了一种带有表位和绿色荧光蛋白标签的GLUT4（HA-GLUT4-GFP）。在此我们报告与3T3-L1脂肪细胞相比该系统的特性。我们表明，胰岛素利用直系同源的转运机制以相似的动力学促进HA-GLUT4-GFP向这两种细胞类型的表面转运。虽然胰岛素刺激的GLUT4转位幅度小于小鼠3T3-L1脂肪细胞，但HeLa细胞提供了一个有用的、实验上易于处理的人类模型系统。在此，我们通过小规模的siRNA筛选举例说明了它们的实用性，以鉴定GOSR1和YKT6作为人类细胞中GLUT4转运的潜在新型调节因子。
