Ursula Sonnewald

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.

Metabolic Trafficking between Neurons and Astrocytes: The Glutamate/Glutamine Cycle Revisited

Use of 13C-labeled precursors for the neuroactive amino acids glutamate and GABA as well as [U-13C]glutamate and glutamine combined with NMR spectroscopy has allowed detailed studies to be performed on cultured neurons and astrocytes yielding new information about synthesis and metabolism of these amino acids at the cellular level. Thus, it has become clear that astrocytes metabolize glutamate extensively through the tricarboxylic acid (TCA) cycle in a rather complex manner enabling the cells to generate lactate from malate. It has been shown that astrocytes can supply neurons not only with glutamine but also with TCA cycle constituents, lactate and alanine. Hence, an extended version of the glutamate/glutamine cycle is presented. Moreover, it has been demonstrated that citrate synthesized in astrocytes and released into the extracellular space can modify neuronal activity by regulating the Zn2+ concentration and thereby modulate N-methyl-D-aspartate receptor-mediated depolarization.

Direct demonstration by [13C]NMR spectroscopy that glutamine from astrocytes is a precursor for GABA synthesis in neurons.

Primary cultures of cerebral cortical astrocytes and neurons, as well as neurons growing on top of the astrocytes (sandwich co-cultures), were incubated with 1-[13C]glucose or 2-[13C]acetate and in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. [13C]NMR spectroscopy at 125 MHz was performed on perchloric acid extracts of the cells or on media collected from the cultures. In addition, the [13C/12C] ratios of the amino acids glutamine, glutamate and 4-aminobutyrate (GABA) were determined by gas chromatography/mass spectroscopy, showing a larger degree of labeling in GABA than in glutamate and glutamine from glucose. Glutamine and glutamate were predominantly labeled from acetate. A picture of cellular metabolism mainly regarding the tricarboxylic acid cycle and glycolysis was obtained. Due to the fact that acetate is not metabolized by neurons to any significant extent, it could be shown that precursors from astrocytes are incorporated into the GABA pool of neurons grown in co-culture with astrocytes. Spectra of media removed from these cultures revealed that likely precursor candidates for GABA were glutamine and citrate. The importance of glutamine is further substantiated by the finding that inhibition of glutamine synthetase, an enzyme present in astrocytes only, significantly decreased the labeling of GABA in co-cultures incubated with 2-[13C]acetate.

Trafficking between glia and neurons of TCA cycle intermediates and related metabolites

Net synthesis of the neurotransmitter amino acids glutamate and GABA can take place either from glutamine or from α‐ketoglutarate or another tricarboxylic acid (TCA) cycle intermediate plus an amino acid as donor of the amino group. Since neurons lack the enzymes glutamine synthetase and pyruvate carboxylase that are expressed only in astrocytes, trafficking of these metabolites must take place between neurons and astrocytes. Moreover, it is likely that astrocytes play an important role in maintaining the energy status in neurons supplying energy substrates, e.g., in the form of lactate. The role of trafficking of glutamine, TCA cycle constituents as well as the role of lactate as an energy source in neurons is discussed. Using [U‐13C]lactate and NMR spectroscopy, it is shown that lactate that can be produced in astrocytes can be taken up into neurons and metabolized through the TCA‐cycle leading to labeling of TCA cycle intermediates plus amino acids derived from these. The labeling pattern of glutamate and GABA indicates that C atoms from lactate remain in the cycle for several turns and that GABA formation may involve more than one glutamate pool, i.e., that compartmentation may exist. Additionally, a possible role of citrate as a chelator of Zn++ with regard to neuronal excitation is discussed. Astrocytes produce large quantities of citrate which by chelation of Zn++ alters the excitable state of neurons via regulation of N‐methyl‐D‐aspartate receptor activity. Thus, astrocytes may regulate neuronal activity at a number of different levels. GLIA 21:99–105, 1997.

Metabolism of [U-13C]glutamate in astrocytes studied by 13C NMR spectroscopy: incorporation of more label into lactate than into glutamine demonstrates the importance of the tricarboxylic acid cycle.

Abstract: Primary cultures of cerebral cortical astrocytes were incubated with [U‐13C]glutamate (0.5 mM) in modified Dulbeccos medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify 13C‐labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C‐3 position and two doublets in the C‐2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton‐decoupled 13C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C‐3 position not only showed the expected apparent triplet but also a doublet due to 13C incorporation into the C‐4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [13C]glutamate in the PCA extracts showed that 19% of the glutamate contained 12C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of 12C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C‐3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly.

Glutamate and Glutamine Metabolism and Compartmentation in Astrocytes

Metabolism of glutamate and glutamine in cultured mouse cerebral cortical astrocytes has been investigated using either radioactively labelled (14C) amino acids or 13C-labelled amino acids combined with NMR spectroscopy of cell extracts and lyophilyzed incubation media. Using [U-13C]glutamate it has been shown that in astrocytes exogenously supplied glutamate is primarily (70%) metabolized oxidatively through the tricarboxylic acid (TCA) cycle and to a lesser extent (30%) directly to glutamine. Glutamate metabolized in the TCA cycle is to a large extent recovered as lactate showing that the astrocyte-specific enzyme, malic enzyme is functionally active. Incubation with [U-14C]glutamine led to a higher specific radioactivity in glutamate than in glutamine. It could also be shown that glutamate and glutamine were metabolized differently to aspartate and alanine. These results taken together strongly suggest that glutamate/glutamine metabolism in astrocytes is compartmentalized and a model with multiple cytoplasmic and mitochondrial compartments of these amino acids is proposed.

Trafficking of Amino Acids Between Neurons and Glia In Vivo. Effects of Inhibition of Glial Metabolism by Fluoroacetate

Glial-neuronal interchange of amino acids was studied by 13C nuclear magnetic resonance spectroscopy of brain extracts from fluoroacetate-treated mice that received [1,2-13C]acetate and [1-13C]glucose simultaneously. [13C]Acetate was found to be a specific marker for glial metabolism even with the large doses necessary for nuclear magnetic resonance spectroscopy. Fluoroacetate, 100 mg/kg, blocked the glial, but not the neuronal tricarboxylic acid cycles as seen from the 13C labeling of glutamine, glutamate, and γ-aminobutyric acid. Glutamine, but not citrate, was the only glial metabolite that could account for the transfer of 13C from glia to neurons. Massive glial uptake of transmitter glutamate was indicated by the labeling of glutamine from [1-13C]glucose in fluoroacetate-treated mice. The C-3/C-4 enrichment ratio, which indicates the degree of cycling of label, was higher in glutamine than in glutamate in the presence of fluoroacetate, suggesting that transmitter glutamate (which was converted to glutamine after release) is associated with a tricarboxylic acid cycle that turns more rapidly than the overall cerebral tricarboxylic acid cycle.

Glial-neuronal interactions as studied by cerebral metabolism of [2-13C]acetate and [1-13C]glucose: an ex vivo 13C NMR spectroscopic study.

Abstract: Mice were injected intravenously with [2‐13C]‐acetate or [1‐13C]glucose and killed after 5, 15, or 30 min. Another group of animals was injected three times subcutaneously during 30 min with [2‐13C]acetate to achieve a steady‐state‐like situation. Brain extracts were analyzed by 13C NMR spectroscopy, and the percent enrichment of various carbon positions was calculated for amino acids, lactate, and glucose. Results obtained with [2‐13C]acetate, which is metabolized by glia and not by neurons, showed that glutamine originated from a glial tricarboxylic acid cycle (TCA cycle) that loses 65% of its intermediates per turn of the cycle. This TCA cycle was associated with pyruvate carboxylation, which may replenish virtually all of this loss, as seen from the labeling of glutamine from [1‐13C]glucose. From the C‐3/C‐4 labeling ratios in glutamine and glutamate and from the corresponding C‐3/C‐2 labeling ratio in GABA obtained with [2‐13C]acetate, it was concluded that the carbon skeleton of glutamine to some extent was passed through TCA cycles before glutamate and GABA were formed. Thus, astrocytically derived glutamine is not only a precursor for transmitter amino acids but is also an energy substrate for neurons in vivo. Furthermore, the neuronal TCA cycles may be control points in the synthesis of transmitter amino acids. Injection of [2‐13C]acetate led to a higher 13C enrichment of the C‐2 in glutamate and of the corresponding C‐4 in GABA than in the C‐3 of either compound. This could reflect cleavage of [2‐13C]‐citrate and formation of [3‐13C]oxaloacetate and acetyl‐CoA, i.e., the first step in fatty acid synthesis. [3‐13C]‐Oxaloacetate would, after entry into a TCA cycle, give the observed labeling of glutamate and GABA.

Utilization of Glutamine and of TCA Cycle Constituents as Precursors for Transmitter Glutamate and GABA

In the present review evidence is presented that (1) glutamine synthesis in astrocytes is essential for synthesis of GABA in neurons; (2) alpha-ketoglutarate in the presence of alanine (as an amino group donor) can replace glutamine as a precursor for synthesis of transmitter glutamate, but maybe not as a precursor for transmitter GABA; (3) differences exist in the intraneuronal metabolic pathways for utilization of alpha-ketoglutarate plus alanine and of glutamine, and (4) alanine also functions as a substrate for oxidative metabolism in glutamatergic neurons. It should be emphasized that the supply of precursors for transmitter glutamate and GABA in glutamatergic and GABAergic neurons depends on metabolic processes in astrocytes regardless whether glutamine or alpha-ketoglutarate plus L-alanine function as the transmitter precursors. The key reason that an interaction with astrocytes is essential is that both pyruvate carboxylase, the major enzyme in the brain for net synthesis of tricarboxylic acid cycle intermediates, and glutamine synthetase, the enzyme forming glutamine from glutamate, are specifically located in astrocytes, but not in neurons.

Metabolism of [U-13C5] glutamine in cultured astrocytes studied by NMR spectroscopy: first evidence of astrocytic pyruvate recycling.

Abstract: Metabolism of [U‐13C5]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify 13C‐labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U‐13C5]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U‐13C5]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl‐CoA. The present work demonstrates that this pathway is active, because [4,5‐13C2]glutamate was observed in astrocytes incubated with [U‐13C5]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono‐labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using 13CO2 produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.