Annette Brand

Multinuclear NMR studies on the energy metabolism of glial and neuronal cells

In this multinuclear NMR study myo-inositol is identified as a glia-specific marker for in vivo NMR studies. The unusually high inositol concentration may participate in the osmoregulatory system in astrocytes. Primary astrocytes also synthesize and export high amounts of hypotaurine, an intermediate of taurine synthesis. Taurine--another osmolyte--is synthesized from cysteine by astrocytes but not by primary neurons. Taurine as well as hypotaurine is accumulated by neurons from the extracellular medium. 13C NMR labelling results with 2-13C pyruvate indicate a considerable contribution of the anaplerotic pathway in primary neurons from rat. The activity is only half of the activity in primary astrocytes. The ratio of pyruvate carboxylase/malic enzyme activity versus pyruvate dehydrogenase activity reflects the degree of maturation. The 13C isotopomer ratio of glutamate and glutamine is different for pure astrocyte cultures. Therefore, the different isotopomer ratios of glutamate to glutamine obtained from intact brain studies alone do not prove TCA cycle compartimentation in the brain. Finally, the PCr/ATP ratio in primary astrocytes is 3 times higher than in primary neurons. This has to be considered in case of recovery from ischemic insults.

Docosahexaenoic acid abundance in the brain: a biodevice to combat oxidative stress.

Abstract Docosahexaenoic acid (DHA) (22:6) is a polyunsaturated fatty acid of the n−3 series which is believed to be a molecular target for lipid peroxides (LPO) formation. Its ubiquitous nature in the nervous tissue renders it particularly vulnerable to oxidative stress, which is high in brain during normal activity because of high oxygen consumption and generation of reactive oxygen species (ROS). Under steady state conditions potentially harmful ROS and LPO are maintained at low levels due to a strong antioxidant defense mechanism, which involves several enzymes and low molecular weight reducing compounds. The present review emphasizes a paradox: a discrepancy between the expected high oxidability of the DHA molecule due to its high degree of unsaturation and certain experimental results which would indicate no change or even decreased lipid peroxidation when brain tissue is supplied or enriched with DHA. The following is a critical review of the experimental data relating DHA levels in the brain to lipid peroxidation and oxidative damage there. A neuroprotective role for DHA, possibly in association with the vinyl ether (VE) linkage of plasmalogens (pPLs) in combating free radicals is proposed.

NMR spectroscopic study on the metabolic fate of [3-13C]alanine in astrocytes, neurons, and cocultures: Implications for glia-neuron interactions in neurotransmitter metabolism

Nuclear magnetic resonance (NMR) spectroscopy and biochemical assays were used to study the fate of [3‐13C]alanine in astrocytes, neurons, and cocultures. 1H‐ and 13C‐NMR analysis of the media demonstrated a high and comparable uptake of [3‐13C]alanine by the cells. Thereafter, alanine is transaminated predominantly to [3‐13C]pyruvate, from which the 13C‐label undergoes different metabolic pathways in astrocytes and neurons: Lactate is almost exclusively synthesized in astrocytes, while in neurons and cocultures labeled neurotransmitter amino acids are formed, i.e., glutamate and γ‐aminobutyric acid (GABA). A considerable contribution of the anaplerotic pathway is observed in cocultures, as concluded from the ratio (C‐2–C‐3)/C‐4 of labeled glutamine. Analysis of the multiplet pattern of glutamate isotopomers indicates carbon scrambling through the TCA cycle and the use of alanine also as energy substrate in neurons. In cocultures, astrocyte‐deduced lactate and unlabeled exogenous carbon substrates contribute to glutamate synthesis and dilute the [2‐13C]acetyl‐CoA pool by 30%. The coupling of neuronal activity with shuttling of tricarboxylic acid (TCA) cycle‐derived metabolites between astrocytes and neurons is concluded from the use of [4‐13C]‐monolabeled glutamate leaving the first TCA cycle turn already for glutamine and GABA synthesis, as well as from the labeling pattern of extracellular glutamine. Further evidence of a metabolic interaction between astrocytes and neurons is obtained, as alanine serves as a carbon and nitrogen carrier through the synthesis and regulated release of lactate from astrocytes for use by neurons. Complementary to the glutamine‐glutamate cycle in the brain, a lactate‐alanine shuttle between astrocytes and neurons would account for the nitrogen exchange of the glutamatergic neurotransmitter cycle in mammalian brain. GLIA 32:286–303, 2000.

Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione

Abstract: The metabolism of [2‐13C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13C NMR spectroscopy. After a 24‐h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2‐13C]glycine, cell extracts and incubation media were analyzed for 13C‐labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2‐13C]glycine, the amount of de novo synthesized [2‐13C]creatine was two‐fold increased, and in addition, 13C‐labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2‐13C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13C‐labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2‐13C]serine, [3‐13C]serine, and [2,3‐13C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.

Metabolism of Cysteine in Astroglial Cells: Synthesis of Hypotaurine and Taurine

Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia‐rich primary cultures obtained from brains of neonatal Wistar rats using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by 1H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3‐13C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with 13C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3‐13C]cysteine. The presence of [1‐13C]hypotaurine and [1‐13C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3‐13C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3‐13C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.

Changes in organic solutes, volume, energy state, and metabolism associated with osmotic stress in a glial cell line: A multinuclear NMR study

Diffusion-weighted in vivo1H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbeccos modified Eagles medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM.1H-,13C-and31P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo31P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.

Lipid constituents in oligodendroglial cells alter susceptibility to H2O2‐induced apoptotic cell death via ERK activation

The present work examines the effect of membrane lipid composition on activation of extracellular signal‐regulated protein kinases (ERK) and cell death following oxidative stress. When subjected to 50 µm docosahexaenoic acid (DHA, 22 : 6 n‐3), cellular phospholipids of OLN 93 cells, a clonal line of oligodendroglia origin low in DHA, were enriched with this polyunsaturated fatty acid. In the presence of 1 mmN,N‐dimethylethanolamine (dEa) a new phospholipid species analog was formed in lieu of phosphatidylcholine. Exposure of DHA‐enriched cells to 0.5 mm H2O2, caused sustained activation of ERK up to 24 h. At this time massive apoptotic cell death was demonstrated by ladder and TUNEL techniques. H2O2‐induced stress applied to dEa or DHA/dEa co‐supplemented cells showed only a transient ERK activation and no cell death after 24 h. Moreover, while ERK was rapidly translocated into the nucleus in DHA‐enriched cells, dEa supplements completely blocked ERK nuclear translocation. This study suggests that H2O2‐induced apoptotic cell death is associated with prolonged ERK activation and nuclear translocation in DHA‐enriched OLN 93 cells, while both phenomena are prevented by dEa supplements. Thus, the membrane lipid composition ultimately modulates ERK activation and translocation and therefore can promote or prevent apoptotic cell death.

Evaluation of the effects of immunosuppressants on neuronal and glial cells in vitro by multinuclear magnetic resonance spectroscopy.

The use of the undecapeptide cyclosporine and the macrolide tacrolimus as immunosuppressants in transplantation medicine and for the therapy of immune diseases often provokes side effects, among the most important one is neurotoxicity. Changes in the cellular metabolism of glial cells (C6 rat glioma), neuronal cells (N1E-115 mouse neuroblastoma) and primary glia cells (isolated from rats) after addition of cyclosporine and tacrolimus were investigated using 1H-, 13C- and 31P-NMR spectroscopy in vitro. Cells were exposed to various concentrations of the drugs from 3 h to 42 days. The immunosuppressants (cyclosporine IC50 : 55 mumol/l; tacrolimus IC50 : 47 mumol/l) inhibited cell proliferation in a concentration- and time-dependent fashion. Multinuclear NMR studies of PCA extracts of drug-treated cells showed a significant deterioration in the energy status (a decreasing level of PCr : -46 +/- 11%; an increasing NDP/NTP ratio: +136 +/- 4% and an increasing level of Pi : +248 +/- 15%; mean +/- standard deviation). It also showed decreasing concentrations of major cell metabolites like NAA (-59 +/- 12%) in neuroblastoma cells and myo-inositol (-47 +/- 6%) in glia cells compared with untreated controls. Immunosuppressive treatment caused a large reduction of taurine (-36 +/- 12%) and glutamate (-68 +/- 10%) in all cell cultures, whereas intermediates of phospholipid biosynthesis (PE: +59 +/- 13%; PC: +127 +/- 27%;) and breakdown (GPE: +215 +/- 24%; GPC: +245 +/- 17%) increased. No significant differences were observed between the two immunosuppressants. The toxic effects of immunosuppressants on cell cultures are in line with MRI studies of brain oedema observed in patients under immunosuppressive treatment.

OXIDATIVE STRESS-INDUCED METABOLIC ALTERATIONS IN RAT BRAIN ASTROCYTES STUDIED BY MULTINUCLEAR NMR SPECTROSCOPY

Oxidative stress in cultured astrocytes exerted by 30‐min treatment with 50–200 μM H2O2 caused time‐ and concentration‐ dependent effects on cellular metabolism. These changes were accompanied by alterations in cellular morphology. Using 31P nuclear magnetic resonance (NMR) spectroscopy, the data demonstrate that the energy status of the cells was greatly affected directly after the stress, as indicated by the loss of high energy phosphates, i.e., phosphocreatine (PCr) and nucleoside triphosphates (NTP). Oxidative stress also involves a dysregulation of the osmotic control in astrocytes, which is accompanied by a dramatic loss of myo‐inositol, taurine, and hypotaurine, as monitored by 1H and 13C NMR spectroscopy. While the energy state of the cells was essentially restored during a 7‐hr recovery period, the changes in osmolyte concentrations lasted longer and went on throughout the recovery period. Even after 24‐hr recovery, organic osmolyte concentrations were still below the control levels. 13C NMR spectra of astrocyte cell extracts also demonstrated an enhanced glucose metabolism via the pentose phosphate pathway (PPP) and a reduced glycolysis. Additionally, the appearance of 13C glutamate points to a distortion of glutamine synthetase (GS), leading to the accumulation of glutamate. Glycolysis as well as GS activity were back to control levels after 7 hr recovery. Thus, in contrast to the energy metabolism, osmoregulatory processes and complex glucose metabolism was impaired not only directly after oxidative stress, but occurred with a later onset during a 2‐hr recovery period, and cells only slowly recovered during the next 24 hr. J. Neurosci. Res. 58: 576–585, 1999.

Multinuclear NMR Spectroscopy Studies on NH4Cl-Induced Metabolic Alterations and Detoxification Processes in Primary Astrocytes and Glioma Cells

Glutamine synthesis, the major pathway of ammonia detoxification, and the intracellular concentration of organic osmolytes in primary astrocytes and F98 glioma cells were investigated with multinuclear magnetic resonance spectroscopy. Acute exposure to ammonia (3 h incubation with NH4Cl) raised the concentration of glutamine and other amino acids, such as glutamate and aspartate, and decreased myo-inositol, hypotaurine, and taurine concentrations. The loss of these osmolytes was partially reversed by co-treatment with the glutamine synthetase inhibitor, methionine sulphoximine. Glutamate, the precursor of glutamine, is provided by stimulated anaplerotic flux via pyruvate carboxylase and glutamate dehydrogenase activity. Thus, the glutamine increase and myo-inositol decrease observed by in vivo magnetic resonance spectroscopy on patients with hepatic encephalopathy may be due to the disturbed osmoregulation in astrocytes caused by accumulation of glutamine and the subsequent loss of organic osmolytes.