6-Fluoro-6-deoxy-D-glucose as a tracer of glucose transport.

Glucose transport rates are estimated noninvasively in physiological and pathological states by kinetic imaging using PET. The glucose analog most often used is (18)F-labeled 2FDG. Compared with glucose, 2FDG is poorly transported by intestine and kidney. We examined the possible use of 6FDG as a tracer of glucose transport. Lacking a hydroxyl at its 6th position, 6FDG cannot be phosphorylated as 2FDG is. Prior studies have shown that 6FDG competes with glucose for transport in yeast and is actively transported by intestine. Its uptake by muscle has been reported to be unresponsive to insulin, but that study is suspect. We found that insulin stimulated 6FDG uptake 1.6-fold in 3T3-L1 adipocytes and azide stimulated the uptake 3.7-fold in Clone 9 cells. Stimulations of the uptake of 3OMG, commonly used in transport assays, were similar, and the uptakes were inhibited by cyclochalasin B. Glucose transport is by GLUT1 and GLUT4 transporters in 3T3-L1 adipocyte and by the GLUT1 transporter in Clone 9 cells. Cytochalasin B inhibits those transporters. Rats were also imaged in vivo by PET using 6(18)FDG. There was no excretion of (18)F into the urinary bladder unless phlorizin, an inhibitor of active renal transport, was also injected. (18)F activity in brain, liver, and heart over the time of scanning reached a constant level, in keeping with the 6FDG being distributed in body water. In contrast, (18)F from 2(18)FDG was excreted in relatively large amounts into the bladder, and (18)F activity rose with time in heart and brain in accord with accumulation of 2(18)FDG-6-P in those organs. We conclude that 6FDG is actively transported by kidney as well as intestine and is insulin responsive. In trace quantity, it appears to be distributed in body water unchanged. These results provide support for its use as a valid tracer of glucose transport.

Distribution of Glut1 in detergent-resistant membranes (DRMs) and non-DRM domains: effect of treatment with azide.

We have previously shown that the acute stimulation of glucose transport in Clone 9 cells in response to azide is mediated by activation of Glut1 and that stomatin, a Glut1-binding protein, appears to inhibit Glut1 function. In Clone 9 cells under basal conditions, approximately 38% of Glut1, approximately 70% of stomatin, and the bulk of caveolin-1 was localized in the detergent-resistant membrane (DRM) fraction; a significant fraction of Glut1 is also present in DRMs of 3T3-L1 fibroblasts and human red blood cells (RBCs). Acute exposure to azide resulted in 40 and 50% decreases in the content of Glut1 in DRMs of Clone 9 cells and 3T3-L1 fibroblasts, respectively, whereas the distribution of stomatin and caveolin-1 in Clone 9 cells remained unchanged. In addition, treatment of Clone 9 cells with azide resulted in a approximately 50% decrease in the content of Glut1 in the DRM fraction of plasma membranes. We conclude that 1) a significant fraction of Glut1 is localized in DRMs, and 2) treatment of cells with azide results in a partial redistribution of Glut1 out of the DRM fraction.

Differential accumulation of Glut1 in the non-DRM domain of the plasma membrane in response to the inhibition of oxidative phosphorylation.

Approximately 50% of Glut1 in the plasma membrane of Clone 9 cells is localized to the detergent-resistant membrane (DRM) fraction. Acute exposure (90 min) to 5mM azide stimulated glucose transport by approximately 4.7-fold and increased the abundance of Glut1 in the non-DRM fraction of the plasma membrane by approximately 2.9-fold while the abundance of Glut1 in the DRMs was not changed. In parallel experiments, approximately 17 h exposure to azide further increased the rate of glucose transport over that observed at 90 min by approximately 33% and increased plasma membrane Glut1 content by approximately 3.5-fold over control. The increase in total plasma membrane Glut1 reflected a approximately 4.7-fold increase of Glut1 content in the non-DRM fraction and a approximately 2.6-fold increase in the DRMs. We conclude that acute exposure to azide increases Glut1 content in the non-DRM fractions, while prolonged exposure to azide increases the Glut1 content in both non-DRM and DRM fractions. These changes may play an important role in the stimulation of glucose transport in response to the inhibition of oxidative phosphorylation.