Identification of a distal GLUT4 trafficking event controlled by actin polymerization

Mol Biol Cell. 2009 Sep;20(17):3918-29. doi: 10.1091/mbc.e09-03-0187. Epub 2009 Jul 15.

Abstract

The insulin-stimulated trafficking of GLUT4 to the plasma membrane in muscle and fat tissue constitutes a central process in blood glucose homeostasis. The tethering, docking, and fusion of GLUT4 vesicles with the plasma membrane (PM) represent the most distal steps in this pathway and have been recently shown to be key targets of insulin action. However, it remains unclear how insulin influences these processes to promote the insertion of the glucose transporter into the PM. In this study we have identified a previously uncharacterized role for cortical actin in the distal trafficking of GLUT4. Using high-frequency total internal reflection fluorescence microscopy (TIRFM) imaging, we show that insulin increases actin polymerization near the PM and that disruption of this process inhibited GLUT4 exocytosis. Using TIRFM in combination with probes that could distinguish between vesicle transport and fusion, we found that defective actin remodeling was accompanied by normal insulin-regulated accumulation of GLUT4 vesicles close to the PM, but the final exocytotic fusion step was impaired. These data clearly resolve multiple steps of the final stages of GLUT4 trafficking, demonstrating a crucial role for actin in the final stage of this process.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3-L1 Cells / cytology
  • 3T3-L1 Cells / metabolism
  • Actins / chemistry
  • Actins / metabolism*
  • Adipocytes / cytology
  • Adipocytes / metabolism
  • Animals
  • Cell Membrane / metabolism*
  • Cystinyl Aminopeptidase / genetics
  • Cystinyl Aminopeptidase / metabolism
  • Cytoskeleton / metabolism*
  • Exocytosis / physiology
  • Glucose Transporter Type 4 / genetics
  • Glucose Transporter Type 4 / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Insulin / metabolism
  • Membrane Fusion / physiology
  • Mice
  • Microscopy, Fluorescence / methods
  • Proto-Oncogene Proteins c-akt / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / physiology
  • Transport Vesicles / metabolism*

Substances

  • Actins
  • Glucose Transporter Type 4
  • Insulin
  • PHluorin
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Proto-Oncogene Proteins c-akt
  • Cystinyl Aminopeptidase
  • leucyl-cystinyl aminopeptidase