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Imaging of Insulin Signaling in Skeletal Muscle of Living Mice Shows Major Role of T-Tubules

¹ Copenhagen Muscle Research Centre, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
² Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, U.K

Address correspondence and reprint requests to H.P.M. Mortensen Lauritzen, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark. E-mail: h.p.lauritz{at}mfi.ku.dk

Abbreviations: ARNO, ADP ribosylation factor 1 nucleotide binding site opener; GFP, green fluorescent protein; PI3K, phosphatidylinositol 3 kinase; PIP₃, phosphatidylinositol-3,4,5 triphosphate; ROI, region of interest

Insulin stimulates glucose transport in skeletal muscle by glucose transporter GLUT4 translocation to sarcolemma and membrane invaginations, the t-tubules. Although muscle glucose uptake plays a key role in insulin resistance and type 2 diabetes, the dynamics of GLUT4 translocation and the signaling involved are not well described. We have now developed a confocal imaging technique to follow trafficking of green fluorescent protein–labeled proteins in living muscle fibers in situ in anesthetized mice. Using this technique, by imaging the dynamics of GLUT4 translocation and phosphatidylinositol 3,4,5 P₃ (PIP₃) production in response to insulin, here, for the first time, we delineate the temporal and spatial distribution of these processes in a living animal. We find a 10-min delay of maximal GLUT4 recruitment and translocation to t-tubules compared with sarcolemma. Time-lapse imaging of a fluorescent dye after intravenous injection shows that this delay is similar to the time needed for insulin diffusion into the t-tubule system. Correspondingly, immunostaining of muscle fibers shows that insulin receptors are present throughout the t-tubule system. Finally, PIP₃ production, an early event in insulin signaling, progresses slowly along the t-tubules with a 10-min delay between maximal PIP₃ production at sarcolemma compared with deep t-tubules following the appearance of dye-labeled insulin. Our findings in living mice indicate a major role of the t-tubules in insulin signaling in skeletal muscle and show a diffusion-associated delay in insulin action between sarcolemma and inner t-tubules.

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