Insulin-regulated trafficking of dual-labeled glucose transporter 4 in primary rat adipose cells.
In isolated rat adipose cells, physiologically relevant insulin target cells, glucose transporter 4 (GLUT4) subcellular trafficking can be assessed by transfection of exofacially HA-tagged GLUT4. To simultaneously visualize the transfected GLUT4, we fused GFP with HA-GLUT4. With the resulting chimeras, GFP-HA-GLUT4 and HA-GLUT4-GFP, we were able to visualize for the first time the cell-surface localization, total expression, and intracellular distribution of GLUT4 in a single cell. Confocal microscopy reveals that the intracellular proportions of both GFP-HA-GLUT4 and HA-GLUT4-GFP are properly targeted to the insulin-responsive aminopeptidase-positive vesicles. Dynamic studies demonstrate close similarities in the trafficking kinetics between the two constructs and with native GLUT4. However, while the basal subcellular distribution of HA-GLUT4-GFP and the response to insulin are indistinguishable from those of HA-GLUT4 and endogenous GLUT4, most of the GFP-HA-GLUT4 is targeted to the plasma membrane with little further insulin response. Thus, HA-GLUT4-GFP will be useful to study GLUT4 trafficking in vivo while GFP on the N-terminus interferes with intracellular retention.
Dawson
K
K
Experimental Diabetes, Metabolism, and Nutrition Section, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, Maryland 20892-0842, USA.
Aviles-Hernandez
A
A
Cushman
S W
SW
Malide
D
D
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D015870:Gene Expression; D051275:Glucose Transporter Type 4; D049452:Green Fluorescent Proteins; D006388:Hemagglutinins / Q000235:genetics; D007328:Insulin / Q000502:physiology*; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D009124:Muscle Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013347:Subcellular Fractions; D014162:Transfection; D014178:Translocation, Genetic
D051275:Glucose Transporter Type 4; D006388:Hemagglutinins; D007328:Insulin; D008164:Luminescent Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C495694:Slc2a4 protein, rat; D049452:Green Fluorescent Proteins