Synthesis of Deoxyglucose-1-Phosphate, Deoxyglucose-1,6-Bisphosphate, and Other Metabolites of 2-Deoxy-D-[14C]Glucose in Rat Brain In Vivo: Influence of Time and Tissue Glucose Level

Abstract: Abstract: When the kinetics of interconversion of deoxy¹⁴C]glucose (¹⁴C]DG) and ¹⁴C]DG-6-phosphate (¹⁴C]DG-6-P) in brain in vivo are estimated by direct chemical measurement of precursor and products in acid extracts of brain, the predicted rate of product formation exceeds the experimentally measured rate. This discrepancy is due, in part, to the fact that acid extraction regenerates ¹⁴C]DG from unidentified labeled metabolites in vitro. In the present study, we have attempted to identify the ¹⁴C-labeled compounds in ethanol extracts of brains of rats given ¹⁴C]DG. Six ¹⁴C-labeled metabolites, in addition to ¹⁴C]DG-6-P, were detected and separated. The major acid-labile derivatives, DG-1-phosphate (DG-1-P) and DG-1,6-bisphosphate (DG-1,6-P₂), comprised ?5 and ?10–15%, respectively, of the total ¹⁴C in the brain 45 min after a pulse or square-wave infusion of ¹⁴C]DG, and their levels were influenced by tissue glucose concentration. Both of these acid-labile compounds could be synthesized from DG-6-P by phosphoglucomutase in vitro. DG-6-P, DG-1-P, DG-1,6-P₂, and ethanol-insoluble compounds were rapidly labeled after a pulse of ¹⁴C]DG, whereas there was a 10–30-min lag before there was significant labeling of minor labeled derivatives. During the time when there was net loss of ¹⁴C]DG-6-P from the brain (i.e., between 60 and 180 min after the pulse), there was also further metabolism of ¹⁴C]DG-6-P into other ethanol-soluble and ethanol-insoluble ¹⁴C-labeled compounds. These results demonstrate that DG is more extensively metabolized in rat brain than commonly recognized and that hydrolysis of ¹⁴C]DG-1-P can explain the overestimation of the ¹⁴C]DG content and underestimation of the metabolite pools of acid extracts of brain. Further metabolism of DG does not interfere with the autoradiographic DG method.