title: Real time qualitative and quantitative GLUT4 translocation assay.
abstract: Insulin-stimulated glucose transporter 4 (GLUT4) translocation promoting glucose uptake is vital to glucose homeostasis and is a defined target of antidiabetic drug research. Existing functional assays to detect the process of GLUT4 translocation are hampered due to assay variability and low sensitivity, thus slowing down the progress towards the development of preferred alternative to insulin. This chapter describes a real time, visual, cell-based qualitative GLUT4 translocation assay suitable for screening insulin mimetics. The basic strategy consists of establishment of insulin-sensitive CHO-HIRc-myc-GLUT4eGFP cells those stably express myc and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by tracking the movement of GLUT4 associated GFP fluorescence from perinuclear space to the plasma membrane by employing cooled charge-coupled device (CCD) camera attached to a simple fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provides rapid and fool-proof visual evidence suitable for screening GLUT4 translocation modulators. This assay is further validated by complementary assays.
authors.affiliation: National Centre for Cell Science, NCCS Complex, Pune University Campus, Ganeshkhind, Pune, India.
authors.forename: Maleppillil Vavachan
authors.initials: MV
authors.lastname: Vijayakumar
authors.forename: Manoj Kumar
authors.initials: MK
authors.lastname: Bhat
mesh_terms: D000818:Animals; D032701:Biomimetics; D016466:CHO Cells; D006224:Cricetinae; D055808:Drug Discovery; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4 / Q000032:analysis* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video / Q000379:methods*; D021381:Protein Transport
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; C495692:SLC2A4 protein, human; D049452:Green Fluorescent Proteins; D005947:Glucose