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Large GLUT4 vesicles are stationary while locally and reversibly depleted during transient insulin stimulation of skeletal muscle of living mice. Imaging analysis of GLUT4-EGFP vesicle dynamics

¹ Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, MA 02215, USA
² Department of Rheumatology, Bispebjerg Hospital, DK-2400, Copenhagen NV, Denmark
³Copenhagen Muscle Research Centre, Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark

Objective: Insulin stimulates glucose transport in skeletal muscle by GLUT4 translocation from intracellular compartments to sarcolemma and t-tubules. We studied in living animals the recruitment of GLUT4-vesicles in more detail than previously done and, for the first time, analysed the steady state recycling and subsequent re-internalisation of GLUT4 upon an insulin bolus.

Research Design and Methods: A confocal imaging technique was used in GLUT4-EGFP transfected superficial muscle fibers in living mice.

Results: During the first 30 min of insulin stimulation very few superficially or deeply located GLUT4 storage vesicles (>1µm) moved in toto. Rather, big vesicles were stationary in their original position at sarcolemma or t-tubules and were locally depleted of GLUT4 by budding off of smaller vesicles. Photobleaching experiments revealed that during initial translocation and steady state recycling, GLUT4 microvesicles (<1 µm) move from perinuclear GLUT4 depots out along the plasma membrane. Furthermore, after photobleaching of t-tubule areas recovery of GLUT4 was slow or absent indicating no recycling of GLUT4 from perinuclear or adjacent (1µm) or more distant (20µm) t-tubule areas. During waning of insulin effect GLUT4 was re-internalised to basal stores with a delay in t-tubules compared with sarcolemma, probably reflecting delayed disappearance of insulin from t-tubules.

Conclusions: In skeletal muscle insulin reversibly stimulates local depletion of GLUT4 storage vesicles at sarcolemma and t-tubules rather than inducing movement of intact storage vesicles. During steady state stimulation, recycling of GLUT4 containing microvesicles over longer distances (10-20µm) takes place between perinuclear depots and sarcolemma but not at t-tubules.

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