Arne Schousboe

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.

Comparison of Lactate and Glucose Metabolism in Cultured Neocortical Neurons and Astrocytes Using 13C-NMR Spectroscopy

In cerebral cortical neurons, synthesis of the tricarboxylic acid (TCA) cycle-derived amino acids, glutamate and aspartate as well as the neurotransmitter of these neurons, γ-aminobutyrate (GABA), was studied incubating the cells in media containing 0.5 mM [U-13C]glucose in the absence or presence of glutamine (0.5 mM). Lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance (NMR) spectroscopy and HPLC. The present findings were compared to results previously obtained using 1.0 mM [U-13C]lactate as the labeled substrate for the neurons. Regardless of the amino acids studied, incubation periods of 1 and 4 h resulted in identical amounts of 13C incorporated. Furthermore, the metabolism of lactate was studied under analogous conditions in cultured cerebral cortical astrocytes. The incorporation of 13C from lactate into glutamate was much lower in the astrocytes than in the neurons. In cerebral cortical neurons the total amount of 13C in GABA, glutamate and aspartate was independent of the labeled substrate. The enrichment in glutamate and aspartate was, however, higher in neurons incubated with lactate. Thus, lactate appears to be equivalent to glucose with regard to its access to the TCA cycle and subsequent labeling of glutamate, aspartate and GABA. It should be noted, however, that incubation with lactate in place of glucose led to lower cellular contents of glutamate and aspartate. The presence of glutamine affected the metabolism of glucose and lactate differently, suggesting that the metabolism of these substrates may be compartmentalized.

Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission.

The fine tuning of both glutamatergic and GABAergic neurotransmission is to a large extent dependent upon optimal function of astrocytic transport processes. Thus, glutamate transport in astrocytes is mandatory to maintain extrasynaptic glutamate levels sufficiently low to prevent excitotoxic neuronal damage. In GABA synapses hyperactivity of astroglial GABA uptake may lead to diminished GABAergic inhibitory activity resulting in seizures. As a consequence of this the expression and functional activity of astrocytic glutamate and GABA transport is regulated in a number of ways at transcriptional, translational and post-translational levels. This opens for a number of therapeutic strategies by which the efficacy of excitatory and inhibitory neurotransmission may be manipulated.

Differential roles of alanine in GABAergic and glutamatergic neurons

Studies in different preparations of neurons and astrocytes of alanine transport and activities of its metabolizing enzyme alanine aminotransferase have led to the proposal that this amino acid is preferentially synthesized in astrocytes and transferred from the astrocytic to the neuronal compartment. From a functional point of view this may well be the case in a GABAergic synapse since theoretically alanine can be utilized as a metabolic fuel in GABAergic neurons where the GABA shunt is operating. Thus, a metabolic scheme is proposed, according to which alanine catabolism is coupled to the TCA cycle where the GABA shunt replaces the alpha-ketoglutarate dehydrogenase/succinyl CoA synthetase reactions. In a glutamatergic synapse in which the large demand for synthesis of neurotransmitter glutamate leads to a large production of ammonia, it is possible that alanine could play a completely different role. Hence, experimental evidence is reviewed suggesting that alanine may serve as a carrier of ammonia nitrogen from the neuronal compartment to the astrocytic compartment using a flux of lactate in the opposite direction to account for transfer of the C-3 carbon skeleton.

NMR spectroscopic study of cell cultures of astrocytes and neurons exposed to hypoxia: Compartmentation of astrocyte metabolism

Primary cultures of murine cerebral cortical astrocytes or cerebellar granule neurons were exposed to 7 h of hypoxia (3 h in some cases). The culture medium was analyzed at the end of the hypoxic or normoxic period by 1H NMR spectroscopy and intracellular components were analyzed as perchloric acid extracts by 31P and 1H NMR spectroscopy. Lactate production in astrocytes increased only marginally, whereas high energy phosphate concentrations were reduced, during 7 h of hypoxia and after 17 h of reoxygenation. After 3 h of hypoxia full recovery was possible during reoxygenation. Citrate and glutamine secretion was reduced or unchanged, respectively, during 7 h of hypoxia. Succinate secretion was only observed during normoxia, whereas pyruvate was secreted during hypoxia. Cerebellar granule neurons were more efficient in increasing glycolysis and were, therefore, more resistant to the effects of hypoxia than astrocytes. In the neurons lactate production was doubled and no effects on levels of high energy phosphates were seen after 7 h of hypoxia. Astrocytes were reoxygenated for 17 h after hypoxia or normoxia in a medium containing [2-13C]acetate in order to access if astrocytes were still capable of supplying neurons with essential precursors. The media were subsequently analyzed by 13C NMR spectroscopy. After shorter periods of hypoxia (3 h) full recovery was possible. Citrate and glutamine production remained however decreased during reoxygenation after 7 h of hypoxia. 13C incorporation into glutamine was greatly reduced but that into citrate was unchanged. These results suggest that under the present conditions, neurons are more efficient than astrocytes in switching the energy metabolism from aerobic to anaerobic glycolysis and that astrocytes may suffer long term damage to mitochondria from longer periods of hypoxia. Furthermore, evidence is presented for the existence of several TCA cycles within astrocytes based on labeling ratios. During normoxia the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively.

Role of Astrocytes in Glutamate Homeostasis

Glutamate which is one of the most abundant amino acids in the central nervous system (CNS) plays two prominent roles in brain function as an important metabolite coupling tricarboxylic acid (TCA) cycle and amino acid metabolism and as the major excitatory neurotransmitter (see Schousboe and Frandsen, 1995). The latter function is fine tuned by homeostatic mechanisms which during pathological conditions such as energy failure may be easily impaired leading to overexposure of glutamate receptors and subsequent neuronal damage, a phenomenon termed excitotoxicity (Lucas and Newhouse, 1957; Olney et al., 1971; Lipton and Rosenberg, 1994; Schousboe and Frandsen, 1995). The mechanisms responsible for the maintenance of extracellular glutamate concentrations within a very narrow physiological range involve control of its release and uptake as well as its intracellular metabolism. The present review shall deal with these aspects with the main emphasis on glutamate uptake and metabolism. It has been generally accepted for a number of years that astrocytes play a very important role in these processes (see, Schousboe, 1981) and therefore emphasis will be placed on a discussion of the role of astrocytes in glutamate homeostasis.

NEW ASPECTS OF LACTATE METABOLISM : IGF-I AND INSULIN REGULATE MITOCHONDRIAL FUNCTION IN CULTURED BRAIN CELLS DURING NORMOXIA AND HYPOXIA

Using 13C nuclear magnetic resonance spectroscopy in combination with conventional biochemical techniques, effects of insulin and IGF-I on energy metabolism and cell viability were studied in cerebral cortical neurons, astrocytes and cocultures thereof during normoxia and hypoxia. Lactate dehydrogenase leakage was used to monitor the cytoprotective effects of IGF-I and insulin. Thus, during normoxia both peptides decreased LDH leakage from neurons. During hypoxia, however, this protection was only observed when insulin was present. Interestingly, neurons showed much less LDH leakage during hypoxia than astrocytes or cocultures. A possible explanation could be an increased glycolysis in neurons. Thus, lactate production and glucose consumption were increased severalfold in neurons during hypoxia whereas astrocytes and cocultures only showed a slight increase. Both insulin and IGF-I increased glucose metabolism during normoxia in astrocytes but not in neurons, whereas during hypoxia this increase was less pronounced. Using [1-13C]glucose it could be demonstrated that production of lactate from mitochondrial precursors was, in the presence of insulin or IGF-I, down regulated in astrocytes but increased in neurons during normoxia. This route for lactate production was not used during hypoxia and incorporation into the C-3 position of lactate approached the theoretical maximum of 50%.

Characterization of the Substrate-Binding Site in GABA Transporters

γ-Aminobutyric acid (GABA) neurotransmission is characterized by the restricted expression of the GABA-synthesizing enzyme glutamate decarboxylase (GAD), the GABA receptors, and the GABA transporters in GABAergic synapses consisting of a presynaptic nerve ending, a postsynaptic entity, and surrounding astrocytes (1). Additionally, the presynaptic nerve ending is characterized by the presence of the vesicles expressing the vesicular GABA transporter (2,3). GABA catabolism, on the other hand, is not restricted to GABAergic synapses because the primary metabolic enzyme GABA transaminase is widely distributed not only in the central nervous system (CNS), but also in many other tissues, including the liver (4,5). Among the different proteins involved in these processes, the receptors, transporters, and the GABA transaminase are all capable of recognizing, and binding the GABA molecule, each with a unique affinity and stringency regarding specificity (6–8). Considering the high degree of flexibility of the GABA molecule, it is not surprising that GABA may be recognized by these different proteins in distinctly different conformations. Thus, the receptors (GABAA) and the transporters have been known for several years to bind GABA in a more extended and somewhat folded conformation, respectively (see KrogsgaardLarsen et al. [9]). Actually, in agreement with this, the GABA analogs of restricted conformation guvacine, nipecotic acid, THPO, and isoguvacine, isonipecotic acid, and THIP (Fig. 1) can be divided in two groups, each reflecting the GABA molecule in a folded and elongated conformation. The three folded analogs are specific ligands for GABA-transporters, whereas the three latter analogs specifically bind to the GABAA receptors with no affinity for the transporters. Hence, these GABA analogs have served as important lead structures in the design of new analogs interacting with either one or the other of the macromolecules constituting important functional components of the GABA synapses (9). The present review is aimed at a further characterization of the molecular structures, which are recognized by the cloned GABA transporters as well as neurons and astrocytes expressing these transporters. For a number of years, it has been a challenge to explain on a rational pharmacological basis how these two cell types can exhibit completely different pharmacological properties regarding inhibitors of GABA transporters, considering the fact that at least the most abundant transporters are expressed in each of these cell types (7,10). This aspect will be discussed extensively in this chapter.

13C and 1H MRS of Cultured Neurons and Glia

Magnetic resonance spectroscopy (MRS) can be used to probe cellular metabolism. This chapter describes how 1H MRS can be used to detect cellular marker substances and to quantitatively determine amounts of metabolites secreted by different cell types. 13C MRS is a valuable tool for investigation of metabolic pathways. Thus, metabolism of [1-13C]glucose, [2-13C]acetate, [U-13C]glutamate, or [U-13C]glutamine has been studied in cerebral cultures of astrocytes and neurons and in co-cultures thereof. The effects of hypoxia on metabolism are also described.