Accumulation of 2-deoxyglucose against its concentration gradient in rat adipocytes

Rat adipocytes were incubated at 37 degrees C with 2-deoxy-D-[1-14C]glucose ([14C]2dGlc) at various concentrations and the intracellular concentrations of [14C]2dGlc and deoxy[14C]glucose phosphate ([14C]2dGlcP) were measured. Using 7 microM extracellular [14C]2dGlc, the intracellular [14C]2dGlc concentration approached the extra-cellular by 5 min insulin-stimulated cells and by 60 min it exceeded the extracellular concentration by 50-fold. A maximum accumulation ratio of 3.5 was reached by 7 min using 1 mM and a ratio of 1.6 was reached by 1 to 3 min using 10 mM extracellular 2dGlc. The time at which the concentration of intracellular 2dGlc exceeded the extracellular was inversely related to the accumulation of 2dGlcP. The rate of accumulation of total radioactivity ([14C]2dGlc plus [14C]2dGlcP) decreased after 20 min using 7 microM extracellular [14C]2dGlc. This change occurred later at 22 degrees C or in the absence of insulin and sooner at higher concentrations of 2dGlc. Experiments where uptake was stopped by dilution indicated that radioactivity appearing in the medium was [14C]2dGlc, but radio-activity disappearing from the cells was largely [14C]2dGlcP. Addition of 10 mM unlabelled 2dGlc or glucose to cells preincubated with 7 microM [14C]2dGlc resulted in a more rapid loss of accumulated label from the cells, while addition of 10 mM 3-O-methylglucose, a non-metabolizeable sugar analogue with about the same affinity for the transport system as 2dGlc, was without effect. The results show that 2dGlc is accumulated against its concentration gradient. It is suggested that the mechanism involves first, dephosphorylation of 2dGlcP and second, the presence of a diffusion barrier between the site of dephosphorylation and the transport site.