Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

Silke Morris; Niall D Geoghegan; Jessica B A Sadler; Anna M Koester; Hannah L Black; Marco Laub; Lucy Miller; Linda Heffernan; Jeremy C Simpson; Cynthia C Mastick; Jon Cooper; Nikolaj Gadegaard; Nia J Bryant; Gwyn W Gould

doi:10.7717/peerj.8751

Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

PeerJ. 2020 Mar 5:8:e8751. doi: 10.7717/peerj.8751. eCollection 2020.

Authors

Affiliations

¹ Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow, UK.
² School of Engineering, University of Glasgow, Glasgow, UK.
³ Department of Biology, University of York, York, UK.
⁴ School of Biology & Environmental Science, University College Dublin, Dublin, Ireland.
⁵ Molecular Biosciences, University of Nevada - Reno, Reno, NV, USA.
⁶ Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK.

^# Contributed equally.

Abstract

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.

Keywords: Diabetes; Endosome; GLUT4; Insulin; Membrane; Transport.

Grants and funding

This work was supported by a Lord Kelvin/Adam Smith PhD studentship from the University of Glasgow to Silke Morris, the Arthur and Sadie Pethybridge studentship from Diabetes UK to Jessica Sadler, BHF studentship number FS/16/55/32731 to Anna Koester and grant 13/0004725 and 17/0005605 from Diabetes UK to Nia Bryant and Gwyn Gould. Linda Heffernan was supported by a postgraduate scholarship from the Irish Research Council. Nikolaj Gadegaard acknowledges support from the European Research Council through the Consolidator Award “FAKIR”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.