Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers

Shuhei Ishikura1, Costin N. Antonescu1, Amira Klip1

1 Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.15
DOI:  10.1002/0471143030.cb1515s46
Online Posting Date:  March, 2010
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The elevated blood glucose following a meal is cleared by insulin‐stimulated glucose entry into muscle and fat cells. The hormone increases the amount of the glucose transporter GLUT4 at the plasma membrane in these tissues at the expense of preformed intracellular pools. In addition, muscle contraction also increases glucose uptake via a gain in GLUT4 at the plasma membrane. Regulation of GLUT4 levels at the cell surface could arise from alterations in the rate of its exocytosis, endocytosis, or both. Hence, methods that can independently measure these traffic parameters for GLUT4 are essential to understanding the mechanism of regulation of membrane traffic of the transporter. Here, we describe cell population–based assays to measure the steady‐state levels of GLUT4 at the cell surface, as well as to separately measure the rates of GLUT4 endocytosis and endocytosis. Curr. Protoc. Cell Biol. 46:15.15.1‐15.15.19. © 2010 by John Wiley & Sons, Inc.

Keywords: glucose; internalization; insulin; muscle contraction; regulated membrane traffic

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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring Steady‐State Cell Surface GLUT4myc
  • Basic Protocol 2: Measuring GLUT4myc Endocytosis
  • Basic Protocol 3: Measuring GLUT4myc Exocytosis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Measuring Steady‐State Cell Surface GLUT4myc

  • GLUT4myc cells [L6‐GLUT4myc; Wang et al., ; L6 cell lines can be obtained from ATCC (, cat. no. CRL‐1458); these cells can be made to stably express epitope‐tagged GLUT4 as desired; for further inquiries regarding currently existing L6‐GLUT4myc stable cell lines, please contact Dr. Amira Klip (]
  • L6 growth medium (see recipe) or L6 differentiation medium (see recipe)
  • α‐modification of Eagle's medium (α‐MEM; Wisent Bioproducts;
  • Stimulatory agents: e.g., human insulin (Humulin R, Lilly), sucrose (Sigma‐Aldrich), or platelet‐derived growth factor (PDGF; Sigma‐Aldrich)
  • PBS+: phosphate‐buffered saline (PBS; see recipe) supplemented with 1 mM CaCl 2 and 1 mM MgCl 2
  • Blocking solution: PBS+ (see above) 5% (v/v) goat serum (Sigma‐Aldrich)
  • Anti‐myc rabbit polyclonal antibody (Sigma‐Aldrich)
  • 4% (w/v) paraformaldehyde in PBS+, ice cold
  • PBS+ (see above) containing 100 mM glycine
  • Horseradish peroxidase (HRP)–conjugated goat anti‐rabbit antibody (Jackson Immunoresearch)
  • OPD assay solution (see recipe)
  • 3 N HCl or H 2SO 4
  • 24‐well culture plates (BD Biosciences)
  • 96‐well microtiter plate
  • Spectrophotometer with microtiter plate reader
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Literature Cited

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