Kinetic validation of 6-NBDG as a probe for the glucose transporter GLUT1 in astrocytes.
In recent years, the use of fluorescent glucose analogs has allowed the study of rapid transport modulation in heterogeneous cell cultures and complex tissues. However, the kinetic behavior of these tracers is not conventional. For instance, the fluorescent glucose analog 6-NBDG permeates the cell 50-100 times slower than glucose but the uptake of 6-NBDG is almost insensitive to glucose, an observation that casts doubts as to the specificity of the uptake pathway. To investigate this apparent anomaly in cultured astrocytes, which are rich in the glucose transporter GLUT1, we first estimated the kinetic parameters of 6-NBDG uptake, which were then incorporated into the kinetic model of GLUT1. The main outcome of the analysis was that 6-NBDG binds to GLUT1 with 300 times higher affinity than glucose, which explains why its uptake is not efficiently displaced by glucose. The high binding affinity of 6-NBDG also explains why cytochalasin B is less effective at inhibiting 6-NBDG uptake than at inhibiting glucose uptake. We conclude that 6-NBDG, used at low concentrations, permeates into astrocytes chiefly through GLUT1, and advise that the exofacial GLUT1 inhibitor 4,6-ethylidine-D-glucose be used, instead of glucose, as the tool of choice to confirm the specificity of 6-NBDG uptake.
Barros
Luis Felipe
LF
Centro de Estudios Científicos (CECS), Valdivia, Chile. fbarros@cecs.cl
Bittner
Carla X
CX
Loaiza
Anitsi
A
Ruminot
Iván
I
Larenas
Valeria
V
Moldenhauer
Hans
H
Oyarzún
Carolina
C
Alvarez
Mauro
M
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000494:pharmacology; D004058:Diffusion; D005944:Glucosamine / Q000031:analogs & derivatives*; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D015233:Models, Statistical; D015203:Reproducibility of Results; D013379:Substrate Specificity
D051272:Glucose Transporter Type 1; C045400:6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoglucose; D003571:Cytochalasin B; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; D005944:Glucosamine