Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time.

ABSTRACT

Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defi ned target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fl uorescence by employing a cooled charge-coupled device camera attached to a fl uorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.