Determination of Glucose Utilization Rates in Cultured Astrocytes and Neurons with [14C]deoxyglucose: Progress, Pitfalls, and Discovery of Intracellular Glucose Compartmentation

Abstract

2-Deoxy-D-[¹⁴C]glucose ([¹⁴C]DG) is commonly used to determine local glucose utilization rates (CMR_glc) in living brain and to estimate CMR_glc in cultured brain cells as rates of [¹⁴C]DG phosphorylation. Phosphorylation rates of [¹⁴C]DG and its metabolizable fluorescent analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), however, do not take into account differences in the kinetics of transport and metabolism of [¹⁴C]DG or 2-NBDG and glucose in neuronal and astrocytic cells in cultures or in single cells in brain tissue, and conclusions drawn from these data may, therefore, not be correct. As a first step toward the goal of quantitative determination of CMR_glc in astrocytes and neurons in cultures, the steady-state intracellular-to-extracellular concentration ratios (distribution spaces) for glucose and [¹⁴C]DG were determined in cultured striatal neurons and astrocytes as functions of extracellular glucose concentration. Unexpectedly, the glucose distribution spaces rose during extreme hypoglycemia, exceeding 1.0 in astrocytes, whereas the [¹⁴C]DG distribution space fell at the lowest glucose levels. Calculated CMR_glc was greatly overestimated in hypoglycemic and normoglycemic cells because the intracellular glucose concentrations were too high. Determination of the distribution space for [¹⁴C]glucose revealed compartmentation of intracellular glucose in astrocytes, and probably, also in neurons. A smaller metabolic pool is readily accessible to hexokinase and communicates with extracellular glucose, whereas the larger pool is sequestered from hexokinase activity. A new experimental approach using double-labeled assays with DG and glucose is suggested to avoid the limitations imposed by glucose compartmentation on metabolic assays.

Keywords: Astrocyte; Compartmentation; Deoxyglucose; Glucose; Lumped constant; Neuron.