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Identification of Three Distinct Functional Sites of Insulin-mediated GLUT4 Trafficking in Adipocytes Using Quantitative Single Molecule Imaging

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      Abstract

      We developed a novel approach allowing intracellular GLUT4 dynamics to be analyzed directly at the single molecule level using Quantum dot to quantitatively establish the behavioral nature of GLUT4. With this approach, we defined the actual steps at which insulin signals directly converge and impact the process of dynamic GLUT4 trafficking events.

      Abstract

      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.

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      Most cited references 51

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            Author and article information

            Affiliations
            *Tohoku University Biomedical Engineering Research Organization, Sendai, Miyagi, 980-8575, Japan;
            Graduate School of Biomedical Engineering, Tohoku University, Sendai, Miyagi, 980-8575, Japan;
            §World Premier International Research Center, Immunology Frontier Research Center, Osaka University, Suita, Osaka, 565-0871, Japan;
            Department of Physics, Graduate School of Science, University of Tokyo, Tokyo, 113-0033, Japan; and
            Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
            Author notes
            Address correspondence to: Makoto Kanzaki ( kanzaki@ 123456bme.tohoku.ac.jp ).

            These authors contributed equally to this work.

            Contributors
            Role: Monitoring Editor
            Journal
            Mol Biol Cell
            mbc
            mbc
            Mol. Bio. Cell
            Molecular Biology of the Cell
            The American Society for Cell Biology
            1059-1524
            1939-4586
            1 August 2010
            : 21
            : 15
            : 2721-2731
            20519436 2912357 3610334 10.1091/mbc.E10-01-0029
            © 2010 by The American Society for Cell Biology
            Product
            Categories
            Articles
            Membrane Trafficking

            Molecular biology

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