Robert A. Waniewski

Exogenous acetate is preferentially metabolized by astrocytes in the CNS, but the biochemical basis for this selectivity is unknown. We observed that rat cortical astrocytes produce 14CO2 from 0.2 mM [14C]acetate at a rate of 0.43 nmol/min per milligram of protein, 18 times faster than cortical synaptosomes. Subsequent studies examined whether this was attributable to cellular differences in the transport or metabolism of acetate. The activity of acetyl-CoA synthetase, the first enzymatic step in acetate utilization, was greater in synaptosomes than in astrocytes (5.0 and 2.9 nmol/min per milligram of protein), indicating that slower metabolism in synaptosomes cannot be attributed to lack of enzymatic activity. [14C]Acetate uptake in astrocytes is rapid and time-dependent and follows saturation kinetics (Vmax, 498 nmol/min per milligram of protein; Km, 9.3 mM). Uptake is inhibited stereospecifically by L-lactate as well as by pyruvate, fluoroacetate, propionate, and alpha-cyano-4-hydroxycinnamate (CHC). Preloading astrocytes with L-lactate or acetate, but not D-lactate, pyruvate, or glyoxylate, transaccelerates [14C]acetate uptake. Acetate uptake by astrocytes appears to be mediated by a carrier with properties similar to that of monocarboxylate transport. In contrast, studies with synaptosomes provided no evidence for time-dependent, saturable, transaccelerated, or CHC-inhibitable uptake of [14C]acetate. The high rate of transport in astrocytes compared with synaptosomes explains the rapid incorporation of [14C]acetate into brain glutamine over glutamate. These findings provide support for the use of acetate as a marker for glial metabolism and suggest that extracellular acetate in the brain generated from acetylcholine and ethanol metabolism is accumulated first by astrocytes.

Rat cortical astrocytes in primary culture were examined for their capacity to transport and metabolize exogenous L-[U-14C]glutamate. After incubation for time periods up to 120 min, cells and incubation media were analyzed for labelled and endogenous glutamate and its metabolic products by HPLC coupled with fluorescence detection and liquid scintillation counting. Glutamine was the major labelled metabolite after 120 min, accounted for 38% of the original glutamate label, and was found primarily in the incubation medium. A further 13.5% of the label was recovered in deaminated metabolites of glutamate, 1.2% was associated with aspartate, 23% remained in glutamate, and 10.2% was found in an acid-precipitated cell fraction. More than 84% of the label was recovered in these fraction. suggesting that the maximum possible formation and loss of 14CO2 was 16%. The rate of total glutamine synthesis was 1.1 nmol X mg protein-1 X min-1 when 9 microM exogenous glutamate was present. The total amount of glutamine synthesized greatly exceeded the consumption of glutamate, indicating that a substantial proportion of glutamine was synthesized from other carbon sources. Almost all of the newly formed glutamine was exported into the medium. These results indicate that astrocytes in primary culture, by accumulating glutamate, producing glutamine, and exporting it, are capable of carrying out the glial component of the glutamine cycle.

The effect of ammonia on glutamate accumulation and metabolism was examined in astrocyte cultures prepared from neonatal rat cortices. Intact astrocytes were incubated with 70 microM L-[14C(U)]glutamate and varying amounts of ammonium chloride. The media and cells were analyzed separately by HPLC for amino acids and labelled metabolites. Extracellular glutamate was reduced to 8 microM by 60 min. Removal of glutamate from the extracellular space was not altered by addition of ammonia. The rate of glutamine synthesis was increased from 3.6 to 9.3 nmol/mg of protein/min by addition of 100 microM ammonia, and intracellular glutamate was reduced from 262 to 86 nmol/mg of protein after 30 min. The metabolism of accumulated glutamate was matched nearly perfectly by the synthesis of glutamine, and both processes were proportional to the amount of added ammonia. The transamination and deamination products of glutamate were minor metabolites that either decreased or remained unchanged with increasing ammonia. Thus, ammonia addition stimulates the conversion of glutamate to glutamine in intact astrocyte cultures. At physiological concentrations of ammonia, glutamine synthesis appears to be limited by the rate of glutamate accumulation and the activity of competing reactions and not by the activity of glutamine synthetase.