title: 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.
abstract: 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.
  
  
  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 microm) 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 microm) 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 microm) or more distant (20 microm) 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 microm) takes place between perinuclear depots and sarcolemma but not at t-tubules.
authors.affiliation: Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, MA 02215, USA. hans.lauritzen@joslin.harvard.edu
authors.forename: Hans P M M
authors.initials: HP
authors.lastname: Lauritzen
authors.forename: Henrik
authors.initials: H
authors.lastname: Galbo
authors.forename: Josef
authors.initials: J
authors.lastname: Brandauer
authors.forename: Laurie J
authors.initials: LJ
authors.lastname: Goodyear
authors.forename: Thorkil
authors.initials: T
authors.lastname: Ploug
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000502:physiology; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D007328:Insulin / Q000097:blood / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012508:Sarcolemma / Q000502:physiology; D014162:Transfection
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D011993:Recombinant Fusion Proteins; C495693:Slc2a4 protein, mouse; D049452:Green Fluorescent Proteins; D005947:Glucose