title: 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 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.
authors.affiliation: National Centre for Cell Science, NCCS Complex, Ganeshkhind, Pune 411 007, India.
authors.forename: Maleppillil Vavachan
authors.initials: MV
authors.lastname: Vijayakumar
authors.forename: Amrendra Kumar
authors.initials: AK
authors.lastname: Ajay
authors.forename: Manoj Kumar
authors.initials: MK
authors.lastname: Bhat
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003571:Cytochalasin B / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D019833:Genistein / Q000494:pharmacology; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007329:Insulin Antagonists / Q000494:pharmacology; D018613:Microscopy, Confocal / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D010936:Plant Extracts / Q000494:pharmacology; D021381:Protein Transport; D011972:Receptor, Insulin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods*; D029910:Trigonella; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D015703:Antigens, CD; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D007329:Insulin Antagonists; D010936:Plant Extracts; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003571:Cytochalasin B; C056164:fenugreek seed meal; D019833:Genistein; C435941:INSR protein, human; D011972:Receptor, Insulin; D000077191:Wortmannin