Niels Westergaard

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.

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.

Evaluation of the importance of transamination versus deamination in astrocytic metabolism of [U- 13C] glutamate

Glutamate metabolism was studied in primary cultures of cerebral cortical astrocytes to determine the significance of transamination for the oxidative metabolism of glutamate. Cultures were incubated with [U‐13C]glutamate (0.5 mM) in the presence and absence of the transaminase inhibitor aminooxyacetic acid (AOAA) and in some cases with methionine sulfoximine, an inhibitor of glutamine synthetase. Perchloric acid extracts of the cells as well as redissolved lyophilized incubation media were subjected to nuclear magnetic resonance spectroscopy to identify 13C‐labeled metabolites. Additionally, biochemical analyses were performed to quantify amino acids, lactate, citrate, and ammonia. Glutamine released into the medium and intracellular glutamate were labeled uniformly to a large extent, but the C‐3 position showed not only the expected apparent triplet but also a doublet due to 12C incorporation into the C‐4 and C‐5 positions. Incorporation of 12C into the C‐4 and C‐5 positions of glutamate and glutamine as well as labeling of lactate, citrate, malate, and aspartate could only arise via metabolism of [U‐13C]glutamate through the tricarboxylic acid (TCA) cycle. Entry of the carbon skeleton of glutamate into the TCA cycle must proceed via 2‐oxoglutarate. This conversion can occur as a transamination or an oxidative deamination. After blocking transamination with AOAA, metabolism of glutamate through the TCA cycle was still taking place since lactate labeling was only slightly reduced. Glutamate and glutamine synthesis from 2‐oxoglutarate could, however, not be detected under this condition. It therefore appears that while glutamate dehydrogenase is important for glutamate degradation, glutamate biosynthesis occurs mainly as a transamination.

Transport of L-[3H]arginine in cultured neurons : characteristics and inhibition by nitric oxide synthase inhibitors

Abstract: To investigate whether transport of l‐arginine into neurons plays an integral role in the metabolism of the novel neuromodulator nitric oxide, the uptake of l‐[3H]arginine was characterized in cultured neurons of mouse cerebellum and cerebral cortex (neocortex). The transport of l‐[3H]arginine was saturable and monophasic into both types of neurons with an apparent Km of 100 μM and Vmax of 2.6 nmol/min/mg of protein. The transport process was stereospecific, and the pattern of inhibition by basic l‐amino acids was consistent with a “y+” transport system. The potencies with regard to inhibition of l‐arginine uptake of the two inhibitors of nitric oxide synthase, NG‐monomethyl‐l‐arginine and NG‐amino‐l‐arginine (IC50 values of 120 and 65 μM, respectively), suggest that these nitric oxide synthase inhibitors may also reduce formation of nitric oxide and cyclic GMP via a block of arginine uptake. Overall, these data indicate that uptake of l‐arginine could regulate its intracellular concentration and, consequently, its availability for synthesis of nitric oxide.

Inhibition of glycogenolysis in primary rat hepatocytes by 1,4-dideoxy-1,4-imino-D-arabinitol

1,4-Dideoxy-1,4-imino-d-arabinitol (DAB) was identified previously as a potent inhibitor of both the phosphorylated and non-phosphorylated forms of glycogen phosphorylase (EC 2.4.1.1). In the present study, the effects of DAB were investigated in primary cultured rat hepatocytes. The transport of DAB into hepatocytes was dependent on time and DAB concentration. The rate of DAB transport was 192 pmol/min per mg of protein per mM DAB(medium-concentration). In hepatocytes, DAB inhibited basal and glucagon-stimulated glycogenolysis with IC(50) values of 1.0+/-0.3 and 1.1+/-0.2 microM, respectively. The primary inhibitory effect of DAB on glycogenolysis was shown to be due to inhibition of glycogen phosphorylase but, at higher concentrations of DAB, inhibition of the debranching enzyme (4-alpha-glucanotransferase, EC 2.4.1.25) may have an effect. No effects on glycogen synthesis were observed, demonstrating that glycogen recycling does not occur in cultured hepatocytes under the conditions tested. Furthermore, DAB had no effects on phosphorylase kinase, the enzyme responsible for phosphorylation and thereby activation of glycogen phosphorylase, or on protein phosphatase 1, the enzyme responsible for inactivation of glycogen phosphorylase through dephosphorylation.

Uptake, Release, and Metabolism of Citrate in Neurons and Astrocytes in Primary Cultures

Abstract: Synthesis, uptake, release, and oxidative metabolism of citrate were investigated in neurons and astrocytes cultured from cerebral cortex or cerebellum. In addition, the possible role of citrate as a donor of the carbon skeleton for biosynthesis of neurotransmitter glutamate was studied. All cell types expressed the enzyme citrate synthase at a high activity, the cerebellar granule neurons containing the enzyme at a higher activity than that found in the astrocytes from the two brain regions or the cortical neurons. Saturable citrate uptake could not be detected in any of the cell types, but the astrocytes, and, in particular, those of cerebellar origin, had a very active de novo synthesis and release of citrate (∼70 nmol × h−1× mg of protein−1). The rate of release of citrate from neurons was <5% of this value. Using [14C]citrate it could be shown that citrate was oxidatively metabolized to 14CO2 at a modest rate (∼1 nmol × n−1× mg−1 of protein) with slightly higher rates in astrocytes compared with neurons. Experiments designed to investigate the ability of exogenously supplied citrate to serve as a precursor for synthesis of transmitter glutamate in cerebellar granule neurons failed to demonstrate this. Rather than citrate serving this purpose it may be suggested that astrocytically released citrate may regulate the extracellular concentration of Ca2+ and Mg2+ by chelation, thereby modulating neuronal excitability.