Wieland Willker

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.

Rapid uptake and degradation of glycine by astroglial cells in culture: Synthesis and release of serine and lactate

Free glycine is known to have vital functions in the mammalian brain, where it serves mainly as both neurotransmitter and neuromodulator. Despite its importance, little is known about the metabolic pathways of glycine synthesis and degradation in the central nervous system. In this study, the pathway of glycine metabolism in astroglia‐rich primary cultures from rat brain was examined. The cells were allowed to degrade glycine in the presence of [U‐14C]glycine, [U‐13C]glycine or [15N]glycine. The resulting intra‐ and extracellular metabolites were analyzed both by high‐performance liquid chromatography and by 13C/15N nuclear magnetic resonance spectroscopy. Glycine was rapidly consumed in a process obeying first‐order kinetics. The initial glycine consumption rate was 0.47 nmol per mg protein. The half‐life of glycine radiolabel in the incubation medium was shorter than that of glycine mass. This suggests that glycine is produced from endogenous sources and released simultaneously with glycine uptake and metabolism. As the main metabolites of the glycine carbon skeleton in astroglia‐rich primary cultures from rat brain, serine and lactate were released during glycine consumption. The main metabolite containing the glycine amino nitrogen was glutamine. To establish a metabolic pathway from glycine to serine in neural tissue, homogenates of rat brain and of neural primary cultures were assayed for their content of serine hydroxymethyltransferase (SHMT) and glycine cleavage system (GCS). SHMT activity was present in homogenates of rat brain as well as of astroglia‐rich and neuron‐rich primary cultures, whereas GCS activity was detectable only in homogenates of rat brain and astroglia‐rich primary culture. Of the two known SHMT isoenzymes, only the mitochondrial form was found in rat brain homogenate. It is proposed that, in neural tissue, glycine is metabolized by the combined action of SHMT and the GCS. Owing to the absence of the GCS from neurons, astrocytes appear to be the only site of this part of glycine metabolism in brain. However, neurons are able to utilize as energy source the lactate formed by astroglial cells in this metabolic pathway. GLIA 27:239–248, 1999.

Subfield-specific loss of hippocampal N-acetyl aspartate in temporal lobe epilepsy.

Purpose: In patients with mesial temporal lobe epilepsy (MTLE) it remains an unresolved issue whether the interictal decrease in N‐acetyl aspartate (NAA) detected by proton magnetic resonance spectroscopy (1H‐MRS) reflects the epilepsy‐associated loss of hippocampal pyramidal neurons or metabolic dysfunction.

Metabolic progression markers of neurodegeneration in the transgenic G93A-SOD1 mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. Visualizing corresponding metabolic changes in the brain of patients with ALS with proton magnetic resonance spectroscopy (1H‐MRS) may provide surrogate markers for an early disease detection, for monitoring the progression and for evaluating a treatment response. The primary objective of our study was to evaluate whether modifications in MR metabolite levels occur before clinical disease onset, and whether these changes are directly linked to a distinct spatial progression pattern in the CNS. Therefore, age‐dependent alterations in the cerebral and spinal metabolic profile in the mouse model of ALS overexpressing the mutated human G93A‐superoxide dismutase 1 (G93A‐SOD1) were determined by high‐resolution MRS of tissue extracts at 14.1 Tesla. Both non‐transgenic mice (control mice) and transgenic mice overexpressing the non‐mutated human SOD1 (tg‐SOD1) served as controls. In the spinal cord of G93A‐SOD1 mice significantly decreased levels of N‐acetyl aspartate were already detected 34 days postpartum, i.e. about 60 days before the average disease onset caused by motor neuron decline. In addition, glutamine and γ‐aminobutyric acid concentrations were significantly diminished at Day 75, which is still in the presymptomatic phase of the disease. These metabolic changes were further progressive in the course of the disease and started to involve the brainstem at Day 75. Overall, high‐resolution 1H‐MRS allows a sensitive spatial and temporal metabolite profiling in the presymptomatic phase of ALS even before significant neuronal cell loss occurs.

A 1H/13C inverse 2D method for the analysis of the polyamines putrescine, spermidine and spermine in cell extracts and biofluids †

The polyamines putrescine, spermidine and spermine are involved in the regulation of various metabolic processes. It is therefore desirable to detect and quantify the polyamines with NMR. We present the proton and carbon assignments for all polyamine signals obtained from PCA extracts of F98 glioma cells with high resolution using a semi‐selective HSQC 2D‐experiment. The biosynthesis of the polyamines in cell culture was examined using the labeled substrates [U‐13 C]glucose and [U‐13 C]glutamate. In such studies the high resolution of the semi‐selective HSQC experiment at very high magnetic fields (14–19 T) allows the analysis of carbon‐carbon couplings, and isotopomer patterns. The different effects of osmotic stress on the concentrations of polyamines and amino acids are also reported.

Accurate measurement of homonuclear coupling constants using JHH-TOCSY

The homonuclear JHH coupling constant is one of the most important NMR parameters. In complex molecules with quite long relaxation times, E.COSY (1) in combination with the DISCO procedure (2) seems to be the best homonuclear method for quantifying J couplings. Other homonuclear methods are z-filtered COSY (3)) J-6 spectroscopy (4)) and selective excitation (5). These methods fail, however, in molecules with short spin-spin relaxation times where the linewidth is in the range of coupling constants. For these situations, some triple-resonance methods have been proposed recently (6-8), which can be used for 15Nand “Ccuenriched peptides and proteins. Their pulse sequences include a heteronuclear coherence transfer from “N to 13C. Additionally the magnetization transfer using a 13C spin lock works well for completely 13C-enriched molecules (9). In these spectra, the signal is split in Fi by the ‘JCH coupling constant, whereas the 3J(HN-H~) coupling constant can be extracted from the displacement in F2. From these methods, precisely estimated coupling constants are obtained, and they are, in distinction to E.COSY, independent of the linewidth. Until now no similar method has been proposed that could be used for molecules at natural abundance. For this purpose, we have developed two new pulse sequences shown in Figs. la and 1 b, called JHH-TOCSY. Sequence 1 a is a H,H correlation and sequence 1 b is a H,X correlation. As for the methods mentioned above, these sequences provide the JHH splitting for XH groups only, whereas XH2 groups show no splitting. For overlapping XH and XH2 groups, and for spectral simplification, XH selection may be used as shown in Fig. 1 c. Figure 1 d shows the 30 extension of sequence 1 a with and without XH selection. JHH-TOCSY is well suited to molecules at natural abundance or for 13Cor “N-monolabeled compounds. In comparison with the triple-resonance methods, the sequences are much shorter, because there is no need for the long 1 /( 2 JCN) delay. The evolution of the magnetization after 2, is as follows (only the relevant operators are shown); cf. Fig. la:

Metabolism of [U-13C]Leucine in Cultured Astroglial Cells

Leucine is rapidly metabolized in astroglial primary cultures. Therefore, it is considered as valuable fuel in brain energy metabolism. Only few of the leucine metabolites generated and released by astroglial cells have been identified. Therefore, a more detailed study was performed analyzing by NMR techniques the 13C-labeled metabolites, which were released by astroglial primary cultures during the degradation of [U-13C]leucine. Confirming a former radioactive study this analysis revealed 13C-labeled 2-oxoisocaproate and ketone bodies. Additionally, 13C-labeled alanine, citrate, glutamine, lactate and succinate were identified. Their detailed isotopomer analysis proves that 13C-labeled acetyl-CoA enters the tricarboxylic acid cycle, that intermediates with a characteristic 13C-labeling pattern can be withdrawn at several positions of the cycle, and that in the case of lactate and alanine there appears to be a participation of an active phosphoenolpyruvate carboxykinase and/or malic enzyme pathway. Thus, astroglial cells generate and release into the extracellular fluid not only the leucine catabolites 2-oxoisocaproate and ketone bodies, but also several tricarboxylic acid cycle dependent metabolites.

Adaptation of Cellular Metabolism to Anisosmotic Conditions in a Glial Cell Line, as Assessed by 13C-NMR Spectroscopy

13C-NMR spectroscopy of perchloric acid and lipid extracts of F98 glioma cells showed that volume-regulatory processes under anisosmotic conditions were accompanied by marked alterations in cellular metabolism. Production of alanine, glutamate, and glycine from [U-13C]-glucose is decreased under hypotonic stress and is oppositely increased under hypertonic stress. In contrast, degradation of these molecules is raised under hypotonic conditions and reduced under hypertonic conditions. Furthermore, phospholipid synthesis is decreased under hypertonic stress and increased under hypotonic stress. Obviously, glial metabolism is directed under hypertonic conditions to maintain a high level of small, osmotically active molecules, whereas under hypotonic conditions molecular fragments are increasingly incorporated into the phospholipids and so do not contribute to the osmotic pressure. The latter is evoked by the activation of membrane synthesis process to compensate for stretching and/or damaging of the membranes due to cell swelling.

Determination of de novo synthesized amino acids in cellular proteins revisited by 13C NMR spectroscopy

13C nuclear magnetic resonance spectroscopy was used to determine the absolute amounts of de novo synthesized amino acids in both the perchloric acid extracts and the hydrolyzed protein fractions of F98 glioma cells incubated for 2 h with 5 mmol/l [U‐13C]glucose. 13C NMR spectra of the hydrolyzed protein fraction revealed a marked incorporation of 13C‐labelled alanine, aspartate and glutamate into the proteins of F98 cells within the incubation period. Additionally, small amounts of 13C‐labelled glycine, proline and serine could unambiguously be identified in the protein fraction. Astonishingly, approximately equal amounts of 13C‐labelled glutamate and aspartate were incorporated into the cellular proteins, although the cytosolic steady‐state concentration of aspartate was below 13C NMR detectability. Hypertonic stress decreased the incorporation of 13C‐labelled amino acids into the total protein, albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. On the other hand, hypotonic stress increased the amount of 13C‐labelled proline incorporated into the cellular proteins even though the cytosolic concentration of 13C‐labelled proline was largely decreased. Apparently, hypoosmotic conditions stimulate the synthesis of proteins or peptides with a high proline content. The results show that already after 2 h of incubation with [U‐13C]glucose there is a pronounced flux of 13C label into the cellular proteins, which is usually disregarded if cytosolic fluids are examined only. This means that calculations of metabolic fluxes based on 13C NMR spectroscopic data obtained from perchloric acid extracts of cells or tissues and also from in vivo measurements consider only the labelled ‘NMR visible’ cytosolic metabolites, which may have to be corrected for fast label flowing off into other compartments.

Lipid oxidation in blood plasma of patients with neurological disorders

Nuclear magnetic resonance (NMR) spectra of blood plasma lipids from lyophilized plasma samples from patients with neurological disorders stored for several weeks in an evacuated exsiccator show characteristic differences compared to freshly lyophilized plasma samples. The main differences concern the unsaturated fatty acids, e.g., the extent of unsaturation and their structural composition. The total amount of double bond signals of unsaturated fatty acids are noticeably reduced in intensity and new signals arise from conjugated double bonds. These signals can be assigned to keto-octadecadienoic acid (KODE) or hydroxy-octadecadienoic acid (HODE). The proton and carbon NMR chemical shifts and their structural assignment to the main molecular components are given. Whereas the KODE and HODE signals occur only as storage artifacts in the spectra, we have found small amounts of 9,11-octadecadienoic acid also in fresh blood plasma of controls. Its concentration is about 60 microM. In two-dimensional H,H total correlation spectroscopy spectra also a very low amount (6-7 microM) of 13-HODE can be detected.