The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U‐13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 mM [U‐13C]glutamate 85% of the 13C metabolized was converted to glutamine. The formation of [1,2,3‐13C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 mM glutamate, 13C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [13C]lactate was essentially unchanged within the range of 0.2–0.5 mM glutamate, whereas the amount of [13C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 mM, suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 mM and astrocytes from both rat and mouse brain are consistent with these findings.

Several recent studies have demonstrated that the metabolism of energy substrates takes place in multiple compartments in both astrocytes and synaptic terminals from brain. There are a number of differences in the metabolism of astrocytes and synaptic terminals primarily due to the localization of key enzymes such as pyruvate carboxylase and glutamine synthetase in astrocytes. The present study determined the rates of ¹⁴CO₂ production from several energy substrates by primary cultures of astrocytes and cortical synaptic terminals from rat brain. The rates of ¹⁴CO₂ production from labelled substrates by astrocytes were 0.96 ± 0.13, 11.13 ± 0.67, 10.51 ± 0.35, 24.92 ± 1.66 and 4.80 ± 0.50 for D-[6-¹⁴C]gIucose, L-[U-¹⁴C]lactate, D-3-hydroxy[3-¹⁴C]butyrate, L-[U-¹⁴C]gIutamine and L-[U-¹⁴C]ma-late, respectively. The rates of ¹⁴CO₂ production were also measured in the presence of 5 mM aminooxyacetate (AOAA) to determine the effect of inhibiting the malate-aspartate shuttle and other transaminase reactions on the oxidation of energy substrates. In astrocytes the addition of AOAA decreased the rate of glutamine oxidation 5-fold, consistent with other studies showing that glutamine enters the TCA cycle via transamination. AOAA increased the rate of ¹⁴CO₂ production from labelled glucose 4-fold, suggesting that inhibition of alanine biosynthesis profoundly alters the utilization of glucose by astrocytes. AOAA also increased the oxidation of lactate and 3-hydroxybutyrate 36 and 58%, respectively. The rates of ¹⁴CO₂ production from labelled substrates by synaptic terminals were 13.12 ± 1.05, 35.29 ± 3.58, 17.66 ± 1.95, 30.18 ± 1.10 and 9.95 ± 1.29, respectively, for glucose, lactate, 3-hydroxybutyrate, glutamine and malate, demonstrating that all substrates were oxidized at a higher rate by synaptic terminals than by astrocytes. The addition of AOAA decreased the rate of ¹⁴CO₂ production from labelled lactate by 57% suggesting that the use of lactate for energy in synaptic terminals is tightly coupled to the activity of the malate-aspartate shuttle. AOAA had no effect on the rate of ¹⁴CO₂ production from labelled glutamine, demonstrating that exogenous glutamine enters the TCA cycle in synaptic terminals via glutamate dehydrogenase, not via transamination as is the case with astrocytes. AOAA had no significant effect on the rates of oxidation of glucose, 3-hydroxybutyrate-and malate by synaptic terminals. These findings demonstrate that inhibiting transamination with AOAA had very different effects on the oxidation of energy substrates in the two preparations, suggesting that the regulation of metabolism is quite different in astrocytes and synaptic terminals. These studies also underscore the importance of utilizing multiple energy substrates since the presence of AOAA altered energy metabolism in some, but not all, co...

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Several reports indicate that lactate can serve as an energy substrate for the brain. The rate of oxidation of this substrate by cultured rat brain astrocytes was 3-fold higher than the rate with glucose, suggesting that lactate can serve as an energy source for these cells. Since transport into the astrocytes may play an important role in regulating nutrient use by individuals types of brain cells, we investigated the uptake of L-[U-14C]lactate by primary cultures of rat brain astrocytes. Measurement of the net uptake suggested two carrier-mediated mechanisms and an Eadie-Hofstee type plot of the data supported this conclusion revealing 2 Km values of 0.49 and 11.38 mM and Vmax values of 16.55 and 173.84 nmol/min/mg protein, respectively. The rate of uptake was temperature dependent and was 3-fold higher at pH 6.2 than at 7.4, but was 50% less at pH 8.2. Although the lactate uptake carrier systems in astrocytes appeared to be labile when incubated in phosphate buffered saline for 20 minutes, the uptake process exhibited an accelerative exchange mechanism. In addition, lactate uptake was altered by several metabolic inhibitors and effectors. Potassium cyanide and alpha-cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, alpha-ketoisocaproate, and 3-hydroxybutyrate at 5 and 10 mM greatly attenuated the rate of lactate uptake. These results suggest that the availability of lactate as an energy source is regulated in part by a biphasic transport system in primary astrocytes.

It is well documented that the brain preferentially utilizes alternative substrates for energy during brain development; however, less is known about the use of these substrates by synaptic terminals. The present study compared the rates of ¹⁴CO₂ production from 1 mM D-[6-¹⁴C]glucose, L-[U-¹⁴C]glutamine, D-3-hydroxy[3-¹⁴C]butyrate, L-[U-¹⁴C]lactate and L-[U-¹⁴C]malate by synaptic terminals isolated from 17- to 18-day-old and 7- to 8-week-old rat brain. The rates of ¹⁴CO₂ production from glucose, glutamine, 3-hydroxybutyrate, lactate and malate were 8.55 ± 0.78, 25.90 ± 4.58, 42.28 ± 3.54, 48.42 ± 2.09, and 9.31 ± 1.61 nmol/h/mg protein (mean ± SEM), respectively, in synaptic terminals isolated from 17- to 18-day-old rat brain and 12.95 ± 1.64, 30.62 ± 4.19, 16.09 ± 2.62,40.33 ± 6.77, and 8.25 ± 1.69 nmol/ h/mg protein (mean ± SEM), respectively, in synaptic terminals isolated from 7- to 8-week-old rat brain. In competition studies using unlabelled added substrates, the addition of 3-hydroxybutyrate, lactate or glutamine greatly decreased the rate of ¹⁴CO₂ production from labelled glucose. Added unlabelled glucose increased the rate of ¹⁴CO₂ production from 3-hydroxybutyrate in synaptic terminals from 7- to 8-week-old rat brain, but had no effect on ¹⁴CO₂ production from any other substrates. Lactate also increased ¹⁴CO₂ production from 3-hydroxybutyrate at 7–8 weeks, whereas the addition of 3-hydroxybutyrate decreased ¹⁴CO₂ production from lactate only in synaptic terminals from 17- to 18-day-old rat brain. None of the added substrates altered the rate of ¹⁴CO₂ production from labelled glutamine or malate suggesting that these substrates are metabolized in relatively distinct compartments within synaptic terminals. Overall the data demonstrate that synaptic terminals from both weanling and adult rat brain can utilize a variety of substrates for energy. In addition, the competition studies demonstrate that the interactions of substrates change with age and suggest that there are multiple compartments of energy metabolism (or tricarboxylic acid cycle activity) in isolated synaptic terminals.

Since lactate released by glial cells may be a key substrate for energy in neurons, the kinetics for the uptake of L-[U-¹⁴C]lactate by cortical synaptic terminals from 7- to 8-week-old rat brain were determined. Lactate uptake was temperature-dependent, and increased by 64.9% at pH 6.2, and decreased by 43.4% at pH 8.2 relative to uptake at pH 7.3. Uptake of monocarboxylic acids was saturable with increasing substrate concentration. Eadie-Hofstee plots of the data gave evidence of two carrier-mediated uptake mechanisms with a high-affinity K_m of 0.66 mM and V_max of 3.66 mM for pyruvate, and a low-affinity system with a K_m of 9.9 mM for both lactate and pyruvate and V_max values of 16.6 and 23.1 nmol/30 s/mg protein for lactate and pyruvate, respectively. Saturable uptake was seen in the presence of 10 mM α-cyano-4-hydroxycinnamate. Lactate transport by synaptic terminals was much more sensitive to inhibition by sulfhydryl reagents than transport in astrocytes. Addition of 0.5 and 2 mM mersalyl decreased the uptake of 1 mM lactate by synaptic terminals by 59.3 and 66.37%, respectively. Pyruvate moderately decreased lactate transport, whereas 3-hydroxybutyrate had little effect. Quercetin, an inhibitor of lactate release, had little effect on the content of ¹⁴C lactate in synaptic terminals, supporting the concept that the majority of lactate produced within brain is from glial cells. Oxidation of L-[U-¹⁴C]lactate by synaptosomes was saturable, and yielded a K_m of 1.23 mM and a V_max of 116 nmol/h/mg protein. Overall the studies show that synaptic terminals from adult brain have a high capacity for transport and oxidation of lactate, consistent with the proposed role for this compound in metabolic trafficking in brain. Furthermore, the data provide kinetic evidence of two carrier-mediated mechanisms for monocarboxylic acid transport by synaptosomes and demonstrate that uptake of lactate by synaptic terminals is regulated differently than transport by astrocytes. Uptake of lactate by synaptic terminals also has differences from the systems described for neurons.

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