title: 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 (CMRglc) in living brain and to estimate CMRglcin 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 CMRglcin 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 CMRglcwas 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.
authors.affiliation: Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, MD, USA. gadienel@uams.edu.|Department of Neurology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Mail Slot 500, Little Rock, AR, 72205, USA. gadienel@uams.edu.
authors.forename: Gerald A
authors.initials: GA
authors.lastname: Dienel
authors.affiliation: Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, MD, USA.
authors.forename: Nancy F
authors.initials: NF
authors.lastname: Cruz
authors.affiliation: Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, MD, USA.
authors.forename: Louis
authors.initials: L
authors.lastname: Sokoloff
authors.affiliation: Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, MD, USA.
authors.forename: Bernard F
authors.initials: BF
authors.lastname: Driscoll
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D005947:Glucose / Q000378:metabolism*; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002250:Carbon Radioisotopes; D003847:Deoxyglucose; D005947:Glucose
keywords: Astrocyte; Compartmentation; Deoxyglucose; Glucose; Lumped constant; Neuron