TY - JOUR
T1 - Kinetic validation of 6-NBDG as a probe for the glucose transporter GLUT1 in astrocytes
AU - Barros, Luis Felipe
AU - Bittner, Carla X.
AU - Loaiza, Anitsi
AU - Ruminot, Iván
AU - Larenas, Valeria
AU - Moldenhauer, Hans
AU - Oyarzún, Carolina
AU - Alvarez, Mauro
PY - 2009/5
Y1 - 2009/5
N2 - 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.
AB - 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.
KW - Cytochalasin B
KW - ETDG
KW - Kinetic analysis
KW - Mathematical modeling
KW - Numerical simulation
UR - http://www.scopus.com/inward/record.url?scp=64349098703&partnerID=8YFLogxK
U2 - 10.1111/j.1471-4159.2009.05885.x
DO - 10.1111/j.1471-4159.2009.05885.x
M3 - Article
C2 - 19393014
AN - SCOPUS:64349098703
SN - 0022-3042
VL - 109
SP - 94
EP - 100
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
IS - SUPPL. 1
ER -