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

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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 defined 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 fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent 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.

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Abbreviations

CHO:

Chinese hamster ovary

eGFP:

enhanced green fluorescent protein

FSE:

fenugreek seeds extract

GLUT4:

glucose transporter 4

HA:

hemagglutinin

HIRc:

human insulin receptor

IR:

insulin receptor

PM:

plasma membrane

TIRF:

total internal reflection fluorescence

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Authors and Affiliations

  1. National Centre for Cell Science, NCCS Complex, Ganeshkhind, Pune, 411 007, India

    Maleppillil Vavachan Vijayakumar, Amrendra Kumar Ajay & Manoj Kumar Bhat

Authors
  1. Maleppillil Vavachan Vijayakumar
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  2. Amrendra Kumar Ajay
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  3. Manoj Kumar Bhat
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Correspondence to Manoj Kumar Bhat.

Additional information

Supplementary information, figures and movies pertaining to this article are available on the Journal of Biosciences Website at http://www.ias.ac.in/jbiosci/Dec2010/pp525-531/suppl.pdf

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Vijayakumar, M.V., Ajay, A.K. & Bhat, M.K. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time. J Biosci 35, 525–531 (2010). https://doi.org/10.1007/s12038-010-0060-0

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  • DOI: https://doi.org/10.1007/s12038-010-0060-0

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