Preferential utilization of acetate by astrocytes is attributable to transport

Abstract

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.

Fig. 1.

Astrocytes produce ¹⁴CO₂from 1-[¹⁴C]acetate much more rapidly than do synaptosomes. Both preparations were incubated with 0.2 mmsodium acetate under identical conditions in an oxygenated, glucose-containing, HEPES-buffered HBSS at 37°C for the indicated times. Data points represent the mean ± SD of three determinations. Synaptosomal production of¹⁴CO₂ was significantly less than astrocytic production at all time points after 0 min; p < 0.01.

Fig. 2.

Acetyl-CoA synthetase activity is greater in synaptosomes than astrocytes. Data points represent the mean ± SD. Three preparations of cortex and synaptosomes were used. Three plates of astrocytes were pooled and a single preparation was used. This experiment was repeated once for astrocytes and cortex, and the same results were obtained.

Fig. 3.

Preincubation conditions strongly affect [¹⁴C]acetate uptake by astrocytes. Astrocytes that were not preincubated (No preinc.) were rinsed on transfer from the culture incubator, and [¹⁴C]acetate was immediately added to the wells. The cells in the other treatment groups were preincubated for 15 min at 4°C with glucose-free buffer alone or buffer containing 10 mml-lactate, pyruvate, or glyoxylate. After preincubation, cells were rinsed three times with fresh buffer alone before adding medium containing [¹⁴C]acetate. The initial rate of uptake was measured after a 15 sec incubation at 4°C with 0.2 mm [¹⁴C]acetate.Bars represent mean ± SD for triplicate determinations. Uptake was significantly reduced by preincubating cells in buffer alone compared with not preincubating cells in buffer. *Significant differences from cells preincubated in buffer alone;p < 0.05.

Fig. 4.

Uptake by astrocytes of [¹⁴C]acetate at concentrations ranging from 0.2 to 20 mm at 4°C. Data points are mean ± SD of triplicate determinations. The line represents a fit of the data to the Michaelis–Menten equation. This fit provided a calculatedK_m of 9.3 ± 3.0 mm and aV_max of 648 ± 102 nmol/min per milligram of protein. An Eadie–Hofstee plot of the average uptake at each concentration (inset) was linear, indicating a single kinetic component. This experiment was repeated three times, and similar results were obtained.

Fig. 5.

Effect of increasing concentrations ofl-lactate, d-lactate, and CHC on [¹⁴C]acetate uptake by astrocytes at 4°C. Individual compounds were added to the cells simultaneously with 0.2 mm [¹⁴C]acetate after a 15 min preincubation with 10 mml-lactate. Data points represent the mean ± SD (n = 4) for each concentration. Separate controls were included for each compound. This experiment was repeated and similar results were obtained. *Significant difference from control uptake;p < 0.05.

Fig. 6.

Effect of pyruvate, fluoroacetate, and CHC on [¹⁴C]acetate uptake by astrocytes at 4°C. This experiment was performed and is presented as described in the legend to Figure 5. CHC was only tested at 10 mm to allow comparisons between experiments.

Fig. 7.

Synaptosomes incubated at 4 or 37°C do not increase their uptake of [¹⁴C]acetate over time. The observed association of [¹⁴C] label at 37°C is not altered by addition of 10 mm CHC. Synaptosomal suspensions were incubated with 0.2 mm[¹⁴C]acetate alone at 4 or 37°C in the presence or absence of CHC. At the indicated times, aliquots of the suspension were diluted into cold buffer containing 10 mm CHC and immediately centrifuged through a layer of silicone oil at room temperature. Synaptosomes from the same preparation were kept on ice in 0.32 m sucrose until the acetate-uptake experiments were completed and then were incubated with 0.01 mm[¹⁴C]glutamate at 37°C. Data points represent the mean ± SD of five determinations. The 4°C experiment was repeated two additional times and the same results were obtained. There were no significant differences in [¹⁴C]acetate uptake between individual time points or between synaptosomes incubated with or without CHC.

Fig. 8.

Effect of CHC on ¹⁴CO₂production from 1-[¹⁴C]acetate in cortical astrocytes and synaptosomes. Both preparations were incubated with 0.2 mm sodium acetate under identical conditions in an oxygenated, glucose-containing, HEPES-buffered HBSS at 37°C for the indicated times. CHC (10 mm) was added to the tissues at the same time as the 1-[¹⁴C]acetate. Data points represent the mean ± SD of four determinations. *Significant differences between CHC and control-treated astrocytes or synaptosomes at the times indicated; p < 0.05. This experiment was repeated two additional times, and the same results were obtained.