Joseph H. Stevenson

Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes

Abstract: 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.

Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: differences revealed using aminooxyacetate.

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]glucose, L-[U-14C]lactate, D-3-hydroxy[3-14C]butyrate, L-[U-14C]glutamine 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential distribution of the enzymes glutamate dehydrogenase and aspartate aminotransferase in cortical synaptic mitochondria contributes to metabolic compartmentation in cortical synaptic terminals.

There have been numerous studies on the activity and localization of aspartate aminotransferase (AAT) and glutamate dehydrogenase (GDH) in brain tissue. However, there is still a controversy as to the specific roles and relative importance of these enzymes in glutamate and glutamine metabolism in astrocytes and neurons or synaptic terminals. There are many reports documenting GDH activity in synaptic terminals, yet the misconception that it is a glial enzyme persists. Furthermore, there is evidence that this tightly regulated enzyme may have an increased role in synaptic metabolism in adverse conditions such as low glucose and hyperammonemia that could compromise synaptic function. In the present study, we report high activity of both AAT and GDH in mitochondrial subfractions from cortical synaptic terminals. The relative amount of GDH/AAT activity was higher in SM2 mitochondria, compared to SM1 mitochondria. Such a differential distribution of enzymes can contribute significantly to the compartmentation of metabolism. There is evidence that the metabolic capabilities of the SM1 and SM2 subfractions of synaptic mitochondria are compatible with the compartments A and B of neuronal metabolism proposed by Waagepetersen et al. (1998b. Dev. Neurosci. 20, 310-320).

Transport ofl-lactate by cultured rat brain astrocytes

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 ofl-[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 α-cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, α-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.

New Insights into the Compartmentation of Glutamate and Glutamine in Cultured Rat Brain Astrocytes

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.

Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells.

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.

Energy metabolism in cortical synaptic terminals from weanling and mature rat brain: evidence for multiple compartments of tricarboxylic acid cycle activity.

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.

Regulation of mitochondrial and cytosolic malic enzymes from cultured rat brain astrocytes

Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is converted to pyruvate and CO2 via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37°C in the presence of 0.5 mM malate was 4.15±0.47 and 11.61±0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean±SEM, n=18–19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. α-Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.

THE METABOLISM OF MALATE BY CULTURED RAT BRAIN ASTROCYTES

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of14CO2 production froml-[U-14C]malate in primary cultures of rat brain astrocytes. The14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased14CO2 production from 0.01 mM and 0.5 mM malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, α-ketoglutarate and succinate decreased14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, α-cyano-4-hydroxycinnamate and ouabain. Both the biphasic kinetics and the differential action of many of the effectors on the14CO2 production from 0.01 mM and 0.5 mM malate provide evidence for the presence of more than one pool of malate metabolism in cultured rat brain astrocytes.

Estimation of free tryptophan in plasma. A simplified spectrofluorometric micromethod

Abstract Plasma tryptophan can be extracted from spots on filter paper with dilute ethanol and estimated directly by spectrofluorometry in alkaline medium. The use of an inert support simplifies the required deproteinization step permitting good recovery of the free amino acid. This procedure enables the assay of a large number of samples in a short period. Results obtained for normal fasting adults are in agreement with previously published values.