Time-dependence of the contribution of pyruvate carboxylase versus pyruvate dehydrogenase to rat brain glutamine labelling from [1-(13) C]glucose metabolism

[1-(13) C]glucose metabolism in the rat brain was investigated after intravenous infusion of the labelled substrate. Incorporation of the label into metabolites was analysed by NMR spectroscopy as a function of the infusion time: 10, 20, 30 or 60 min. Specific enrichments in purified mono- and dicarboxylic amino acids were determined from (1) H-observed/(13) C-edited and (13) C-NMR spectroscopy. The relative contribution of pyruvate carboxylase versus pyruvate dehydrogenase (PC/PDH) to amino acid labelling was evaluated from the enrichment difference between either C2 and C3 for Glu and Gln, or C4 and C3 for GABA, respectively. No contribution of pyruvate carboxylase to aspartate, glutamate or GABA labelling was evidenced. The pyruvate carboxylase contribution to glutamine labelling varied with time. PC/PDH decreased from around 80% after 10 min to less than 30% between 20 and 60 min. This was interpreted as reflecting different labelling kinetics of the two glutamine precursor glutamate pools: the astrocytic glutamate and the neuronal glutamate taken up by astrocytes through the glutamate-glutamine cycle. The results are discussed in the light of the possible occurrence of neuronal pyruvate carboxylation. The methods previously used to determine PC/PDH in brain were re-evaluated as regards their capacity to discriminate between astrocytic (via pyruvate carboxylase) and neuronal (via malic enzyme) pyruvate carboxylation.     

Nitrogen metabolism in cultured cotyledon explants of Pinus radiata during de novo organogenesis

Nitrogen metabolism was investigated under shoot-forming (SF) and non-shoot-forming (NSF) conditions in cultured cotyledon explants of Pinus radiata by following the incorporation of [¹⁴C]-l,2-acetate into various metabolites. Early in culture, the lipid fraction contained the most ¹⁴C; however, this percentage decreased in favor of increased label in the amphoteric fraction. Label in the amphoteric fraction of SF cultures decreased by day 21 but plateaued in NSF cultures at this time. Radioactive labeling of the principle nitrogen metabolites, glutamate and glutamine, which made up the majority of the amphoteric fraction, paralleled labeling patterns in the amphoteric fraction. Percentage label in glutamate remained at similar levels throughout the 21-day culture period for both SF and NSF cultures. Specific activity of glutamate (kBq mg^-1) was significantly greater during promeristemoid formation in SF compared to that in NSF tissues. Glutamine labeling increased during shoot bud initiation in SF cultures, but dropped to lower levels during shoot bud development. In contrast, in NSF cultures, there was a continual and substantial increase in glutamine labeling throughout the 21-day culture period. These trends were similar when the specific activities of glutamine were determined, as there was a continual decrease from culture initiation to the end of shoot bud differentiation in SF cultures. In NSF cultures, in contrast, specific activity of glutamine increased substantially from day 5 to 21 relative to that in SF cultures. The nitrogen assimilation enzymes glutamate synthase and glutamine synthase increased in activity from day 0 to 21 for both SF and NSF tissues. Enzyme activities for glutamate dehydrogenase were similar in both treatments to day 10 in culture but subsequently diverged, with activities in NSF cultures being substantially greater than those of SF cultures by day 21. Taken together, labeling and enzyme data indicate that nitrogen metabolism is enhanced during culture, especially in SF tissues at the time of promeristemoid formation, and in non-organ-forming tissue senescence-like metabolism was exhibited later in culture.     

In vivo quantification of neuro‐glial metabolism and glial glutamate concentration using 1H‐[13C] MRS at 14.1T

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo ¹³C NMR spectroscopy coupled with the infusion labeled glial‐specific substrate, such as acetate. In this study, we infused alpha‐chloralose anesthetized rats with [2‐¹³C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using ¹H‐[¹³C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two‐compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (V_g) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (V_NT) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (V_PC) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (V_x^g and V_xⁿ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations.

     

Mild reduction in the activity of the α-ketoglutarate dehydrogenase complex elevates GABA shunt and glycolysis

Diminished energy metabolism and reduced activity of brain α-ketoglutarate dehydrogenase complex (KGDHC) occur in a number of neurodegenerative diseases. The relation between diminished KGDHC activity and altered energy metabolism is unknown. The present study tested whether a reduction in the KGDHC activity would alter cellular metabolism by comparing metabolism of [U-¹³C]glucose in a human embryonic kidney cell line (E2k100) to one in which the KGDHC activity was about 70% of control (E2k67). After a 2 h incubation of the cells with [U-¹³C]glucose, the E2k67 cells showed a greater increase in ¹³C labeling of alanine compared with the E2k100 cells. This suggested an increase in glycolysis. Furthermore, an increase in labeled lactate after 12 h incubation supported the suggestion of an increased glycolysis in the E2k67 cells. Increased GABA shunt in the E2k67 cells was indicated by increased ¹³C labeling of GABA at both 2 and 12 h compared with the control cells. GABA concentration as determined by HPLC was also increased in the E2k67 cells compared with the control cells. However, the GABA shunt was not sufficient to normalize metabolism in the E2k67 cells compared with control at 2 or 12 h. However, by 24 h metabolism had normalized (i.e. labeling was similar in E2k67 and E2k100). Thus, the data are consistent with an enhanced glycolysis and GABA shunt in response to a mild reduction in KGDHC. Our findings indicate that a mild change in KGDHC activity can lead to large changes in metabolism. The changes may maintain normal energy metabolism but make the cells more vulnerable to perturbations such as occur with oxidants.     

Amino acid and purine release in rat brain following temporary middle cerebral artery occlusion

Excitatory amino acid release and neurotoxicity in the ischemic brain may be reduced by endogenously released adenosine which can modulate both glutamate or aspartate release and depress neuronal excitability. The present study reports on the patterns of release of glutamate and aspartate; the inhibitory amino acids GABA and glycine; and of the purine catabolites adenosine and inosine from the rat parietal cerebral cortex during 20 and 60 min periods of middle cerebral artery (MCA) occlusion followed by reperfusion. Aspartate and glutamate efflux into cortical superfusates rose steadily during the period of ischemia and tended to increase even further during the subsequent 40 min of reperfusion. GABA release rose during ischemia and declined during reperfusion, whereas glycine efflux was relatively unchanged during both ischemia and reperfusion. Adenosine levels in cortical superfusates rose rapidly at the onset of ischemia and then declined even though MCA occlusion was continued. Recovery to pre-occulusion levels was rapid following reperfusion. Inosine efflux also increased rapidly, but its decline during reperfusion was slower than that of adenosine.     

In vivo neurochemical profiling of rat brain by 1H-[13C] NMR spectroscopy: cerebral energetics and glutamatergic/GABAergic neurotransmission

The quantification of excitatory and inhibitory neurotransmission and the associated energy metabolism is crucial for a proper understanding of brain function. Although the detection of glutamatergic neurotransmission in vivo by ¹³C NMR spectroscopy is now relatively routine, the detection of GABAergic neurotransmission in vivo has remained elusive because of the low GABA concentration and spectral overlap. Using ¹H-[¹³C] NMR spectroscopy at high magnetic field in combination with robust spectral modeling and the use of different substrates, [U-¹³C₆]-glucose and [2-¹³C]-acetate, it is shown that GABAergic, as well as glutamatergic neurotransmitter fluxes can be detected non-invasively in rat brain in vivo .     

Direct determination of the N-acetyl-L-aspartate synthesis rate in the human brain by (13)C MRS and [1-(13)C]glucose infusion

A non-invasive (13)C magnetic resonance spectroscopy (MRS) technique is described for the determination of the N-acetyl-L-aspartate (NAA) synthesis rate, V(NAA), in the human brain in vivo. In controls, the mean V(NAA) was 9.2 +/- 3.9 nmol/min/g. In Canavan disease, where [NAA] is increased (p < 0.001) and [aspartate] is deceased (p < 0.001), V(NAA) was significantly reduced to 3.6 +/- 0.1 nmol/min/g (p < 0.001). These rates are in close agreement with the activity of the biosynthetic enzyme measured in vitro in animals, and with the rate of urinary excretion of NAA in human subjects with Canavan disease. The present result is consistent with the regulation of NAA synthesis by the activity of a single enzyme, L-aspartate-N-acetyltransferase, in vivo, and with its control in Canavan disease by limited substrate supply and/or product inhibition. The (13)C MRS technique provides the means for further determination of abnormal rates of neuronal NAA synthesis among neurological disorders in which low cerebral [NAA] has been identified.     

Nickel-induced changes in carbon metabolism in wheat shoots

In this study, we analyzed the toxic effect of Ni during the development of wheat shoots. Typical developmental alterations in carbon metabolism-related parameters reflecting changes associated with the transition of the seedlings from heterotrophic to autotrophic metabolism were observed in the control shoots between the 1st and the 4th days. Adverse effects of 50 and 100 μM Ni became evident starting from the 4th day of growth on the metal-containing media. We found that Ni-induced stimulation of phosphoenolpyruvate carboxylase (PEPC) activity coincided with decrease in the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) level and with declines in net photosynthetic rate (P_N) and stomatal conductance (g_s). Application of Ni resulted in increased activities of several dehydrogenases: glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (NADP-ICDH) and malate dehydrogenase (NADH-MDH). In contrast, the activities of malic enzymes (NADP-ME and NAD-ME) decreased due to Ni stress. Treatment with Ni led to accumulation of glucose and declined concentration of sucrose as well as considerable increases in concentrations of malic and citric acids. Our results indicate that Ni stress redirects the carbon metabolism of developing wheat shoots to provide carbon skeletons for synthesis of amino acids and organic acids as well as to supply reducing power to sustain normal metabolic processes and to support defense mechanisms against oxidative stress.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-¹³C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this long-distance signal-transduction pathway. Received: 24 April 1996 / Accepted: 18 July 1996     

Aspirin-like drugs inhibit arachidonic acid metabolism via lipoxygenase and cyclo-oxygenase in rat neutrophils from carrageenan pleural exudates

Aspirin-like drugs inhibit the metabolism of arachidonic acid via lipoxygenase and cyclo-oxygenase in rat neutrophils from carrageenan pleural exudates. These non-steroidal anti-inflammatory drugs inhibit the formation of 11-hydroxy- and 15-hydroxy-eicosatetraenoic acid (HETE) as well as prostaglandins. In addition, the concentration- and time-dependent irreversible inhibition of lipoxygenase by aspirin and indomethacin parallels closely the patterns observed for inhibition of cyclo-oxygenase. The results suggest that some common steps may exist for the synthesis of HETE and prostaglandins from arachidonic acid in rat neutrophils. The ability of aspirin-like drugs to inhibit the formation of the chemotactic hydroxy-fatty acids may contribute to their anti-inflammatory activity.     

Compartmentation of Lactate Originating from Glycogen and Glucose in Cultured Astrocytes

Brain glycogen metabolism was investigated by employing isofagomine, an inhibitor of glycogen phosphorylase. Cultured cerebellar and neocortical astrocytes were incubated in medium containing [U-¹³C]glucose in the absence or presence of isofagomine and the amounts and percent labeling of intra- and extracellular metabolites were determined by mass spectrometry (MS). The percent labeling in glycogen was markedly decreased in the presence of isofagomine. Surprisingly, the percent labeling of intracellular lactate was also decreased demonstrating the importance of glycogen turnover. The decrease was limited to the percent labeling in the intracellular pool of lactate, which was considerably lower compared to that observed in the medium in which it was close to 100%. These findings indicate compartmentation of lactate derived from glycogenolysis and that derived from glycolysis. Inhibiting glycogen degradation had no effect on the percent labeling in citrate. However, the percent labeling of extracellular glutamine was slightly decreased in neocortical astrocytes exposed to isofagomine, indicating an importance of glycogen turnover in the synthesis of releasable glutamine. In conclusion, the results demonstrate that glycogen in cultured astrocytes is continuously synthesized and degraded. Moreover, it was found that lactate originating from glycogen is compartmentalized from that derived from glucose, which lends further support to a compartmentalized metabolism in astrocytes. Special issue dedicated to Dr. Bernd Hamprecht.     

Lipid metabolism in mammalian tissues and its control by retinoic acid

Evidence has accumulated that specific retinoids impact on developmental and biochemical processes influencing mammalian adiposity including adipogenesis, lipogenesis, adaptive thermogenesis, lipolysis and fatty acid oxidation in tissues. Treatment with retinoic acid, in particular, has been shown to reduce body fat and improve insulin sensitivity in lean and obese rodents by enhancing fat mobilization and energy utilization systemically, in tissues including brown and white adipose tissues, skeletal muscle and the liver. Nevertheless, controversial data have been reported, particularly regarding retinoids' effects on hepatic lipid and lipoprotein metabolism and blood lipid profile. Moreover, the molecular mechanisms underlying retinoid effects on lipid metabolism are complex and remain incompletely understood. Here, we present a brief overview of mammalian lipid metabolism and its control, introduce mechanisms through which retinoids can impact on lipid metabolism, and review reported activities of retinoids on different aspects of lipid metabolism in key tissues, focusing on retinoic acid. Possible implications of this knowledge in the context of the management of obesity and the metabolic syndrome are also addressed. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Glutamate dehydrogenase in brain mitochondria: do lipid modifications and transient metabolon formation influence enzyme activity?

Metabolism of glutamate, the primary excitatory neurotransmitter in brain, is complex and of paramount importance to overall brain function. Thus, understanding the regulation of enzymes involved in formation and disposal of glutamate and related metabolites is crucial to understanding glutamate metabolism. Glutamate dehydrogenase (GDH) is a pivotal enzyme that links amino acid metabolism and TCA cycle activity in brain and other tissues. The allosteric regulation of GDH has been extensively studied and characterized. Less is known about the influence of lipid modifications on GDH activity, and the participation of GDH in transient heteroenzyme complexes (metabolons) that can greatly influence metabolism by altering kinetic parameters and lead to channeling of metabolites. This review summarizes evidence for palmitoylation and acylation of GDH, information on protein binding, and information regarding the participation of GDH in transient heteroenzyme complexes. Recent studies suggest that a number of other proteins can bind to GDH altering activity and overall metabolism. It is likely that these modifications and interactions contribute additional levels of regulation of GDH activity and glutamate metabolism.     

Quantitative assessment of neurochemical changes in a rat model of long-term alcohol consumption as detected by in vivo and ex vivo proton nuclear magnetic resonance spectroscopy

The aim of present study was to quantitatively investigate the neurochemical profile of the frontal cortex region in a rat model of long-term alcohol consumption, by using in vivo proton magnetic resonance spectroscopy (¹H-MRS) at 4.7 T and ex vivo¹H high-resolution magic angle spinning (HR-MAS) technique at 11.7 T. Twenty male rats were divided into two groups and fed a liquid diet for 10 weeks. After 10 weeks, in vivo¹H MRS spectra were acquired from the frontal cortex brain region. After in vivo¹H MRS experiments, all animals were sacrificed and 20 frontal cortex tissue samples were harvested. All tissue examinations were performed with the 11.7 T HR-MAS spectrometer and high-resolution spectra were acquired. The in vivo and ex vivo spectra were quantified as absolute metabolite concentrations and normalized ratios of total signal-intensity (i.e., metabolites_Norm), respectively. The absolute quantifications of in vivo spectra showed significantly higher glycerophosphocholine plus phosphocholine (GPC + PCh) and lower myo-inositol (mIns) concentrations in ethanol-treated rats compared to controls. The quantifications of ex vivo spectra showed significantly higher PCh_Norm, Cho_Norm and tCho_Norm, and lower GPC_Norm and mIns_Norm ratio levels in ethanol-treated rats compared to controls. Our findings suggest that reduced mIns concentrations caused by the long-term alcohol consumption may lead to hypo-osmolarity syndrome and astrocyte hyponatremia. In addition, increased choline-containing compound concentrations may reflect an increased cell turnover rate of phosphatidylcholine and other phospholipids, indicating an adaptive mechanism. Therefore, these results might be utilized as key markers in chronic alcohol intoxication metabolism.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

A combination of ischemic preconditioning and allopurinol protects against ischemic injury through a nitric oxide-dependent mechanism

This study examined the cytoprotective mechanisms of a combination of ischemic preconditioning (IPC) and allopurinol against liver injury caused by ischemia/reperfusion (I/R). Allopurinol (50 mg/kg) was intraperitoneally administered 18 and 1 h before sustained ischemia. A rat liver was preconditioned by 10 min of ischemia, followed by 10 min of reperfusion, and then subjected to 90 min of ischemia, followed by 5 h of reperfusion. Rats were pretreated with adenosine deaminase (ADA), 3,7-dimethyl-1-[2-propargyl]-xanthine (DMPX), and N-nitro-l-arginine methyl ester (l-NAME) before IPC. Hepatic nitrite and nitrate and eNOS protein expression levels were increased by the combination of IPC and allopurinol. This increase was attenuated by ADA, DMPX, and l-NAME. I/R induced an increase in alanine aminotransferase activity, whereas it decreased the hepatic glutathione level. A combination of IPC and allopurinol attenuated these changes, which were abolished by ADA, DMPX, and l-NAME. The increase in the liver wet weight-to-dry weight ratio after I/R was attenuated by the combination of IPC and allopurinol. In contrast, hepatic bile flow was decreased after I/R, which was attenuated by the combination of IPC and allopurinol. These changes were restored by l-NAME. I/R induced a decrease in the level of mitochondrial dehydrogenase, whereas it increased mitochondrial swelling. A combination of IPC and allopurinol attenuated these changes, which were restored by ADA, DMPX, and l-NAME. Our findings suggest that a combination of IPC and allopurinol reduces post-ischemic hepatic injury by enhancing NO generation.