Identification of three distinct functional sites of insulin-mediated GLUT4 trafficking in adipocytes using quantitative single molecule imaging.
Insulin stimulation of glucose uptake is achieved by redistribution of insulin-responsive glucose transporters, GLUT4, from intracellular storage compartment(s) to the plasma membrane in adipocytes and muscle cells. Although GLUT4 translocation has been investigated using various approaches, GLUT4 trafficking properties within the cell are largely unknown. Our novel method allows direct analysis of intracellular GLUT4 dynamics at the single molecule level by using Quantum dot technology, quantitatively establishing the behavioral nature of GLUT4. Our data demonstrate the predominant mechanism for intracellular GLUT4 sequestration in the basal state to be "static retention" in fully differentiated 3T3L1 adipocytes. We also directly defined three distinct insulin-stimulated GLUT4 trafficking processes: 1) release from the putative GLUT4 anchoring system in storage compartment(s), 2) the speed at which transport GLUT4-containing vesicles move, and 3) the tethering/docking steps at the plasma membrane. Intriguingly, insulin-induced GLUT4 liberation from its static state appeared to be abolished by either pretreatment with an inhibitor of phosphatidylinositol 3-kinase or overexpression of a dominant-interfering AS160 mutant (AS160/T642A). In addition, our novel approach revealed the possibility that, in certain insulin-resistant states, derangements in GLUT4 behavior can impair insulin-responsive GLUT4 translocation.
Tohoku University Biomedical Engineering Research Organization, Sendai, Miyagi, 980-8575, Japan.
Hideaki
H
Fujita
Hiroyasu
H
Hatakeyama
Tomonobu M
TM
Watanabe
Masaaki
M
Sato
Hideo
H
Higuchi
Makoto
M
Kanzaki
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D021381:Protein Transport / Q000187:drug effects; D045663:Quantum Dots; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism
D019332:Endothelin-1; D051275:Glucose Transporter Type 4; D055767:Immobilized Proteins; D007328:Insulin