Labeling Pattern Of Glutamate Research Articles

Effects of Adrenergic Agents on Intracellular Ca2+ Homeostasis and Metabolism of Glucose in Astrocytes with an Emphasis on Pyruvate Carboxylation, Oxidative Decarboxylation and Recycling: Implications for Glutamate Neurotransmission and Excitotoxicity

Glucose and glycogen are essential sources of energy for maintaining glutamate homeostasis as well as glutamatergic neurotransmission. The metabolism of glycogen, the location of which is confined to astrocytes, is affected by norepinephrine (NE), and hence, adrenergic signaling in the astrocyte might affect glutamate homeostasis with implications for excitatory neurotransmission and possibly excitotoxic neurodegeneration. In order to study this putative correlation, cultured astrocytes were incubated with 2.5 mM [U-(13)C]glucose in the presence and absence of NE as a time course for 1 h. Employing mass spectrometry, labeling in intracellular metabolites was determined. Moreover, the involvement of Ca(2+) in the noradrenergic response was studied. In unstimulated astrocytes, the labeling pattern of glutamate, aspartate, malate and citrate confirmed important roles for pyruvate carboxylation and oxidative decarboxylation in astrocytic glucose metabolism. Importantly, pyruvate carboxylation was best visualized at 10 min of incubation. The abundance and pattern of labeling in lactate and alanine indicated not only an extensive activity of malic enzyme (initial step for pyruvate recycling) but also a high degree of compartmentalization of the pyruvate pool. Stimulating with 1 μM NE had no effect on labeling patterns and glycogen metabolism, whereas 100 μM NE increased glutamate labeling and decreased labeling in alanine, the latter supposedly due to dilution from degradation of non-labeled glycogen. It is suggested that further experiments uncovering the correlation between adrenergic and glutamatergic pathways should be performed in order to gain further insight into the role of astrocytes in brain function and dysfunction, the latter including excitotoxicity.

Energy Contribution of Octanoate to Intact Rat Brain Metabolism Measured by13C Nuclear Magnetic Resonance Spectroscopy

Glucose is the dominant oxidative fuel for brain, but studies have indicated that fatty acids are used by brain as well. We postulated that fatty acid oxidation in brain could contribute significantly to overall energy usage and account for non-glucose-derived energy production. [2,4,6,8-13C4]octanoate oxidation in intact rats was determined by nuclear magnetic resonance spectroscopy. We found that oxidation of 13C-octanoate in brain is avid and contributes approximately 20% to total brain oxidative energy production. Labeling patterns of glutamate and glutamine were distinct, and analysis of these metabolites indicated compartmentalized oxidation of octanoate in brain. Examination of liver and blood spectra revealed that label from 13C-octanoate was incorporated into glucose and ketones, which enabled calculation of its overall energy contribution to brain metabolism: glucose (predominantly unlabeled) and 13C-labeled octanoate can account for the entire oxidative metabolism of brain. Additionally, flux through anaplerotic pathways relative to tricarboxylic acid cycle flux (Y) was calculated to be 0.08 +/- 0.039 in brain, indicating that anaplerotic flux is significant and should be considered when assessing brain metabolism. Y was associated with the glutamine synthesis compartment, consistent with the view that anaplerotic flux occurs primarily in astrocytes.

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. © 1997 Wiley-Liss, Inc.

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 alpha-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.

Accurate Determination of13C Enrichments in Nonprotonated Carbon Atoms of Isotopically Enriched Amino Acids by1H Nuclear Magnetic Resonance

A method for the accurate determination of13C enrichments in nonprotonated carbon atoms of organic compounds that makes use of unresolved13C satellites of proton(s) bonded to the vicinal carbon atom was developed. Using glutamate as a model molecule, this1H nuclear magnetic resonance (NMR) inverse spin-echo difference spectroscopy method was calibrated for inversion efficiency and relaxation effects which were then shown to cause only a minor loss of the measured13C satellite amplitude (2% for glutamate C-1 and 7% for glutamate C-5). The determination of13C enrichments in nonprotonated glutamate carbon atoms by this method was shown to be more precise than13C NMR. As a first application, a [5-13C]glucose labeling experiment withCorynebacterium glutamicumASK1 was performed. The labeling patterns of glutamate and arginine extracted from cellular protein were determined using the newly developed method and standard1H NMR with and without broadband13C decoupling. Determination of the13C enrichment in C-5 of glutamate and arginine, respectively, by the two methods showed good agreement. From the deduced labeling pattern of 2-oxoglutarate, anin vivocarbon flux distribution within the central metabolism ofC. glutamicumASK1 was calculated. Thus, the relative flux toward oxaloacetate via the tricarboxylic acid cycle enzyme malate dehydrogenase was determined as 45%, whereas that via anaplerotic C3carboxylation was determined as 55%.

Carbohydrate and Amino Acid Metabolism in the Ectomycorrhizal Ascomycete Sphaerosporella brunnea during Glucose Utilization

Nuclear magnetic resonance spectroscopy was utilized to study the metabolism of [1-(13)C]glucose in mycelia of the ectomycorrhizal ascomycete Sphaerosporella brunnea. The main purpose was to assess the biochemical pathways for the assimilation of glucose and to identify the compounds accumulated during glucose assimilation. The majority of the (13)C label was incorporated into mannitol, while glycogen, trehalose and free amino acids were labeled to a much lesser extent. The high enrichment of the C1/C6 position of mannitol indicated that the polyol was formed via a direct route from absorbed glucose. Randomization of the (13)C label was observed to occur in glucose and trehalose leading to the accumulation of [1,6-(13)C]trehalose and [1,6-(13)C]glucose. This suggests that the majority of the glucose carbon used to form trehalose was cycled through the metabolically active mannitol pool. The proportion of label entering the free amino acids represented 38% of the soluble (13)C after 6 hours of continuous glucose labeling. Therefore, amino acid biosynthesis is an important sink of assimilated carbon. Carbon-13 was incorporated into [3-(13)C]alanine and [2-(13)C]-, [3-(13)C]-, and [4-(13)C]glutamate and glutamine. From the analysis of the intramolecular (13)C enrichment of these amino acids, it is concluded that [3-(13)C]pyruvate, arising from [1-(13)C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase, and pyruvate carboxylase (or phosphoenolpyruvate carboxykinase). Intramolecular (13)C labeling patterns of glutamate and glutamine were similar and are consistent with the operation of the Krebs cycle. There is strong evidence for (a) randomization of the label on C2 and C3 positions of oxaloacetate via malate dehydrogenase and fumarase, and (b) the dual biosynthetic and respiratory role of the citrate synthase, aconitase, and isocitrate dehydrogenase reactions. The high flux of carbon through the carboxylation (presumably pyruvate carboxylase) step indicates that CO(2) fixation is an important component of the carbon metabolism in S. brunnea, and it is likely that this anaplerotic role is particularly prevalent during NH(4) (+) assimilation. The most relevant information resulting from this investigation is (a) the occurrence of the mannitol cycle, (b) a large part of the trehalose pool is synthesized after the cycling of glucose-carbon through the mannitol cycle, and (c) pyruvate (or phosphoenolpyruvate) carboxylation plays an important role in the primary metabolism of glucose-fed mycelia.