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

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, analyzed the steady-state recycling and subsequent re-internalization of GLUT4 on an insulin bolus. RESEARCH DESIGN AND METHODS-A confocal imaging technique was used in GLUT4-enhanced green fluorescent protein-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 (> 1 mu m) 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. Photo-bleaching experiments revealed that during initial translocation and steady-state recycling, GLUT4 inicrovesicles (< 1 mu m) 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 (1 mu m) or more distant (20 mu m) t-tubule areas. During waning of insulin effect, GLUT4 was re-internalized 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 (10-20 mu m) takes place between perinuclear depots and sarcolemma but not at t-tubules.