Insulin action on GLUT4 traffic visualized in single 3T3-l1 adipocytes by using ultra-fast microscopy.
A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20-30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5-7 min at 37 degrees C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.
Patki
V
V
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Buxton
J
J
Chawla
A
A
Lifshitz
L
L
Fogarty
K
K
Carrington
W
W
Tuft
R
R
Corvera
S
S
D016475:3T3 Cells / Q000166:cytology*; D017667:Adipocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D003599:Cytoskeleton; D003952:Diagnostic Imaging / Q000295:instrumentation / Q000379:methods; D005089:Exocytosis / Q000187:drug effects; D051275:Glucose Transporter Type 4; D049452:Green Fluorescent Proteins; D006207:Half-Life; D007328:Insulin / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D022161:Transport Vesicles
D051275:Glucose Transporter Type 4; D007328:Insulin; D008164:Luminescent Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D049452:Green Fluorescent Proteins