title: Imaging of insulin signaling in skeletal muscle of living mice shows major role of T-tubules.
abstract: 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 P(3) (PIP(3)) 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, PIP(3) production, an early event in insulin signaling, progresses slowly along the t-tubules with a 10-min delay between maximal PIP(3) 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.
authors.affiliation: Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark. h.p.lauritz@mfi.ku.dk
authors.forename: Hans P M M
authors.initials: HP
authors.lastname: Lauritzen
authors.forename: Thorkil
authors.initials: T
authors.lastname: Ploug
authors.forename: Clara
authors.initials: C
authors.lastname: Prats
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Tavaré
authors.forename: Henrik
authors.initials: H
authors.lastname: Galbo
mesh_terms: D000818:Animals; D001692:Biological Transport; D005456:Fluorescent Dyes; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism; D018482:Muscle, Skeletal / Q000502:physiology*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D012508:Sarcolemma / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D051275:Glucose Transporter Type 4; D007328:Insulin; D018129:Phosphatidylinositol Phosphates; C495693:Slc2a4 protein, mouse; C060974:phosphatidylinositol 3,4,5-triphosphate; D011972:Receptor, Insulin