Intercellular metabolic compartmentation in the brain: past, present and future

The first indication of 'metabolic compartmentation' in brain was the demonstration that glutamine after intracisternal [14C]glutamate administration is formed from a compartment of the glutamate pool that comprises at most one-fifth of the total glutamate content in the brain. This pool, which was designated 'the small compartment,' is now known to be made up predominantly or exclusively of astrocytes, which accumulate glutamate avidly and express glutamine synthetase activity, whereas this enzyme is absent from neurons, which eventually were established to constitute 'the large compartment.' During the following decades, the metabolic compartment concept was refined, aided by emerging studies of energy metabolism and glutamate uptake in cellularly homogenous preparations and by the histochemical observations that the two key enzymes glutamine synthetase and pyruvate carboxylase are active in astrocytes but absent in neurons. It is, however, only during the last few years that nuclear magnetic resonance (NMR) spectroscopy, assisted by previously obtained knowledge of metabolic pathways, has allowed accurate determination in the human brain in situ of actual metabolic fluxes through the neuronal tricarboxylic acid (TCA) cycle, the glial, presumably mainly astrocytic, TCA cycle, pyruvate carboxylation, and the 'glutamate-glutamine cycle,' connecting neuronal and astrocytic metabolism. Astrocytes account for 20% of oxidative metabolism of glucose in the human brain cortex and accumulate the bulk of neuronally released transmitter glutamate, part of which is rapidly converted to glutamine and returned to neurons in the glutamate-glutamine cycle. However, one-third of released transmitter glutamate is replaced by de novo synthesis of glutamate from glucose in astrocytes, suggesting that at steady state a corresponding amount of glutamate is oxidatively degraded. Net degradation of glutamate may not always equal its net production from glucose and enhanced glutamatergic activity, occurring during different types of cerebral stimulation, including the establishment of memory, may be associated with increased de novo synthesis of glutamate. This process may contribute to a larger increase in glucose utilization rate than in rate of oxygen consumption during brain activation. The energy yield in astrocytes from glutamate formation is strongly dependent upon the fate of the generated glutamate.     

Biophysical basis of brain activity: implications for neuroimaging

In vivo 13C magnetic resonance spectroscopy (MRS) studies of the brain have quantitatively assessed rates of glutamate-glutamine cycle (Veye) and glucose oxidation (CMRGle(ox)) by detecting 13C label turnover from glucose to glutamate and glutamine. Contrary to expectations from in vitro and ex vivo studies, the in vivo 13C-MRS results demonstrate that glutamate recycling is a major metabolic pathway, inseparable from its actions of neurotransmission. Furthermore, both in the awake human and in the anesthetized rat brain, Veye and CMRGle(ox) are stoichiometrically related, where more than two thirds of the energy from glucose oxidation supports events associated with glutamate neurotransmission. The high energy consumption of the brain measured at rest and its quantitative relation to neurotransmission reflects a sizeable activity level for the resting brain. The high activity of the non-stimulated brain, as measured by cerebral metabolic rate of oxygen use (CMRO2), establishes a new neurophysiological basis of cerebral function that leads to reinterpreting functional imaging data because the large baseline signal is commonly discarded in cognitive neuroscience paradigms. Changes in energy consumption (delta CMRO2%) can also be obtained from magnetic resonance imaging (MRI) experiments, using the blood oxygen level-dependent (BOLD) image contrast, provided that all the separate parameters contributing to the functional MRI (fMRI) signal are measured. The BOLD-derived delta CMRO2% when compared with alterations in neuronal spiking rate (delta v%) during sensory stimulation in the rat reveals a stoichiometric relationship, in good agreement with 13C-MRS results. Hence fMRI when calibrated so as to provide delta CMRO2% can provide high spatial resolution evaluation of neuronal activity. Our studies of quantitative measurements of changes in neuroenergetics and neurotransmission reveal that a stimulus does not provoke an arbitrary amount of activity in a localized region, rather a total level of activity is required where the increment is inversely related to the level of activity in the non-stimulated condition. These biophysical experiments have established relationships between energy consumption and neuronal activity that provide novel insights into the nature of brain function and the interpretation of fMRI data.     

Cerebral metabolism and consciousness

In vivo 13C magnetic resonance spectroscopy studies of the brain have measured rates of glutamate-glutamine cycle (Vcyc) and glucose oxidation (CMRglc(ox)) by detecting 13C label turnover from glucose to glutamate and glutamine. In both the awake human and in the anesthetized rat brains Vcyc and CMRglc(ox) are stoichiometrically related, and form a major pathway in which approximately 80% of the energy from glucose oxidation supports events associated with glutamate neurotransmission. The high energy consumption of the brain at rest and its quantitative usage for neurotransmission reflect a high level of neuronal activity for the non-stimulated brain. This high activity supports a reinterpretation of functional imaging data, e.g., where the large baseline signal has commonly been discarded. Independent measurements of energy consumption (delta CMRO2%) obtained from calibrated fMRI equaled percentage changes in neuronal spiking rate (delta nu %) measured by electrodes during sensory stimulation at two depths of anesthesia. These quantitative biophysical relationships between energy consumption and neuronal activity provide novel insights into the nature of brain function. The high resting brain activity is proposed to include the global interactions constituting the subjective aspects of consciousness. Anesthesia by lowering the total firing rates correlates with the loss of consciousness. These results, which measure the localized neuronal response and distinguish inputs of peripheral neurons from inputs of neurons from other brain regions, fit comfortably into the neuronal scheme of a global workspace proposed by Dehaene and Changeux.     

Chronic acetyl-L-carnitine alters brain energy metabolism and increases noradrenaline and serotonin content in healthy mice

Acetyl-L-carnitine (ALCAR), the short-chain ester of carnitine, is a common dietary supplement readily available in health food stores, claimed to improve energy levels and muscle strength. ALCAR has numerous effects on brain and muscle metabolism, protects against neurotoxic insults and may be an effective treatment for certain forms of depression. However, little is known about the effect of chronic ALCAR supplementation on the brain metabolism of healthy mice. Here, we investigated ALCAR's effect on cerebral energy and neurotransmitter metabolism after supplementing the drinking water of mice with ALCAR for 25 days, providing a daily dose of about 0.5 g/kg. Thereafter the animals were injected with [1-(13)C]glucose, and (13)C incorporation into and levels of various metabolites were quantified in extracts of the hippocampal formation (HF) and cortex using (1)H- and (13)C-nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC). Increased glucose levels were detected in both regions together with a decreased amount of [3-(13)C]lactate, but no alterations in incorporation of (13)C derived from [1-(13)C]glucose into the amino acids glutamate, GABA and glutamine. These findings are consistent with decreased metabolism of glucose to lactate but not via the TCA cycle. Higher amounts of the sum of adenosine nucleotides, phosphocreatine and the phosphocreatine/creatine ratio found in the cortex of ALCAR-treated mice are indicative of increased energy levels. Furthermore, ALCAR supplementation increased the levels of the neurotransmitters noradrenaline in the HF and serotonin in cortex, consistent with ALCAR's potential efficacy for depressive symptoms. Other ALCAR-induced changes observed included reduced amounts of GABA in the HF and increased myo-inositol. In conclusion, chronic ALCAR supplementation decreased glucose metabolism to lactate, resulted in increased energy metabolite and altered monoamine neurotransmitter levels in the mouse brain.     

In vivo 13C NMR studies of compartmentalized cerebral carbohydrate metabolism

Localized 13C nuclear magnetic resonance (NMR) spectroscopy provides a unique window for studying cerebral carbohydrate metabolism through, e.g. the completely non-invasive measurement of cerebral glucose and glycogen metabolism. In addition, label incorporation into amino acid neurotransmitters such as glutamate (Glu), GABA and aspartate can be measured providing information on Krebs cycle flux and oxidative metabolism. Given the compartmentation of key enzymes such as pyruvate carboxylase and glutamine synthetase, the detection of label incorporation into glutamine indicated that neuronal and glial metabolism can be measured in vivo. The purpose of this paper is to provide a critical overview of these recent advances into measuring compartmentation of brain energy metabolism using localized in vivo 13C NMR spectroscopy. The studies reviewed herein showed that anaplerosis is significant in brain, as is oxidative ATP generation in glia and the rate of glial glutamine synthesis attributed to the replenishment of the neuronal Glu pool and that brain glycogen metabolism is slow under resting conditions. This new modality promises to provide a new investigative tool to study aspects of normal and diseased brain hitherto unaccessible, such as the interplay between glutamatergic action, glucose and glycogen metabolism during brain activation, and the derangements thereof in patients with hepatic encephalopathy, neurodegenerative diseases and diabetes.     

Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet C NMR of the adult mouse brain

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the ¹³C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-¹³C₂]glucose (oxidized by both neurons and glia) and [1,2-¹³C₂]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by ¹³C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of ¹³C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The ¹³C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-¹³C]acetate relative to [1,6-¹³C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.     

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.     

In situ 3D magnetic resonance metabolic imaging of microwave-irradiated rodent brain: a new tool for metabolomics research

The rapid elevation in rat brain temperature achieveable with focused beam microwave irradiation (FBMI) leads to a permanent inactivation of enzymes, thereby minimizing enzyme-dependent post-mortem metabolic changes. An additional characteristic of FBMI is that the NMR properties of the tissue are close to those of the in vivo condition and remain so for at least 12 h. These features create an opportunity to develop magnetic resonance spectroscopy and imaging on microwave-irradiated samples into a technique with a resolution, coverage and sensitivity superior to any experiment performed directly in vivo . Furthermore, when combined with pre-FBMI infusion of ¹³C-labeled substrates, like [1-¹³C]-glucose, the technique can generate maps of metabolic fluxes, like the tricarboxylic acid and glutamate-glutamine neurotransmitter cycle fluxes at an unprecedented spatial resolution.     

Regional cerebral metabolism in mouse under chronic manganese exposure: implications for manganism

Chronic manganese (Mn) exposure in rodents, non-human primates and humans has been linked to Parkinson's disease like condition known as Manganism. Mn being a cofactor for many enzymes in brain has been known to be accumulated in various regions differentially and thus exert toxic effect upon chronic overexposure. In present study, neuropathology of Manganism was investigated by evaluating regional neuronal and astroglial metabolism in mice under chronic Mn exposure. Male C57BL6 mice were treated with MnCl(2) (25 mg/kg, i.p.) for 21 days. Cerebral metabolism was studied by co-infusing [U-(13)C(6)]glucose and [2-(13)C]acetate, and monitoring (13)C labeling of amino acids in brain tissue extract using (1)H-[(13)C] and (13)C-[(1)H]-NMR spectroscopy. Glutamate, choline, N-acetyl aspartate and myo-inositol were found to be reduced in thalamus and hypothalamus indicating a loss in neuronal and astroglial cells due to Mn neurotoxicity. Reduced labeling of Glu(C4) from [U-(13)C(6)]glucose and [2-(13)C]acetate indicates an impairment of glucose oxidation by glutamatergic neurons and glutamate-glutamine neurotransmitter cycle in cortex, striatum, thalamus-hypothalamus and olfactory bulb with chronic Mn exposure. Additionally, reduced labeling of Gln(C4) from [2-(13)C]acetate indicates a decrease in acetate oxidation by astroglia in the same regions. However, GABAergic function was alleviated only in thalamus-hypothalamus. Our findings indicate that chronic Mn impairs excitatory (glutamatergic) function in the majority of regions of brain while inhibitory (GABAergic) activity is perturbed only in basal ganglia.     

Neuron–Astrocyte Interactions,Pyruvate Carboxylation and the Pentose Phosphate Pathway in the Neonatal Rat Brain

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-¹³C]glucose and [1,2-¹³C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-¹³C]glucose and were sacrificed 30 min later. Brain extracts were analysed using ¹H- and ¹³C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-¹³C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-¹³C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-¹³C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.     

Comparison of Glutamate Turnover in Nerve Terminals and Brain Tissue During [1,6-<Superscript>13</Superscript>C<Subscript>2</Subscript>]Glucose Metabolism in Anesthetized Rats

The ¹³C turnover of neurotransmitter amino acids (glutamate, GABA and aspartate) were determined from extracts of forebrain nerve terminals and brain homogenate, and fronto-parietal cortex from anesthetized rats undergoing timed infusions of [1,6-¹³C₂]glucose or [2-¹³C]acetate. Nerve terminal ¹³C fractional labeling of glutamate and aspartate was lower than those in whole cortical tissue at all times measured (up to 120 min), suggesting either the presence of a constant dilution flux from an unlabeled substrate or an unlabeled (effectively non-communicating on the measurement timescale) glutamate pool in the nerve terminals. Half times of ¹³C labeling from [1,6-¹³C₂]glucose, as estimated by least squares exponential fitting to the time course data, were longer for nerve terminals (Glu_C4, 21.8 min; GABA_C2 21.0 min) compared to cortical tissue (Glu_C4, 12.4 min; GABA_C2, 14.5 min), except for Asp_C3, which was similar (26.5 vs. 27.0 min). The slower turnover of glutamate in the nerve terminals (but not GABA) compared to the cortex may reflect selective effects of anesthesia on activity-dependent glucose use, which might be more pronounced in the terminals. The ¹³C labeling ratio for glutamate-C4 from [2-¹³C]acetate over that of ¹³C-glucose was twice as large in nerve terminals compared to cortex, suggesting that astroglial glutamine under the ¹³C glucose infusion was the likely source of much of the nerve terminal dilution. The net replenishment of most of the nerve terminal amino acid pools occurs directly via trafficking of astroglial glutamine.     

An update on the role of brain glutamine synthesis and its relation to cell-specific energy metabolism in the hyperammonemic brain: further studies using NMR spectroscopy

Ammonia is a neurotoxin that is implicated in the pathogenesis of hepatic encephalopathy due to acute and chronic liver failure. However, its relation to neurological damage and brain edema is poorly understood. During the last decades, it has been the prevailing hypothesis that an osmotic disturbance induced by the astrocytic accumulation of glutamine leads to brain edema. However, various findings are at variance with this hypothesis. The present review will discuss: (a) correlation of ammonia with encephalopathy and brain edema in HE; (b) glutamine synthesis and astrocyte swelling; (c) glutamine synthesis and the glutamine-cycle: relation to brain energy metabolism; (d) glutamine synthesis and the glutamate-glutamine cycle and its relation to anaplerotic activity; (e) evidence favouring the "glutamine hypothesis"; (f) evidence contradicting the "glutamine hypothesis"; (g) glutamine synthesis and osmoregulation; (h) glutamine synthesis in chronic liver failure; (i) impaired brain energy metabolism in acute liver failure (ALF) and its relation to astrocytic glutamine synthesis. Taken together, the precise role of glutamine in the development of brain edema in ALF remains unclear. Astrocytic changes due to glutamine accumulation may lead secondarily to effects on brain energy metabolism. However, the relation between impaired energy metabolism and glutamine accumulation has not been well established. It is noteworthy that no single biochemical factor appears to be responsible for the many symptoms of HE. For example, brain glutamine accumulation and low-grade brain edema occur in chronic liver failure (CLF) suggesting common mechanisms are responsible for the neurological dysfunction in CLF and ALF. Recent NMR spectroscopic studies have provided considerably new information in this area. Future NMR studies using the stable isotope 13C may be useful in the study of the dynamics of brain metabolism in patients with ALF so as to better elucidate the precise role of glutamine accumulation and of glutamine-independent components to brain edema in ALF.     

Neuron-astrocyte interactions in the regulation of brain energy metabolism: a focus on NMR spectroscopy

An adequate and timely production of ATP by brain cells is of cardinal importance to support the major energetic cost of the rapid processing of information via synaptic and action potentials. Recently, evidence has been accumulated to support the view that the regulation of brain energy metabolism is under the control of an intimate dialogue between astrocytes and neurons. In vitro studies on cultured astrocytes and in vivo studies on rodents have provided evidence that glutamate and Na(+) uptake in astrocytes is a key triggering signal regulating glucose use in the brain. With the advent of NMR spectroscopy, it has been possible to provide experimental evidence to show that energy consumption is mainly devoted to glutamatergic neurotransmission and that glutamate-glutamine cycling is coupled in a approximately 1 : 1 molar stoichiometry to glucose oxidation, at least in the cerebral cortex. This improved understanding of neuron-astrocyte metabolic interactions offers the potential for developing novel therapeutic strategies for many neurological disorders that include a metabolic deficit.     

13C Nuclear Magnetic Resonance Evidence for γ-Aminobutyric Acid Formation via Pyruvate Carboxylase in Rat Brain: A Metabolic Basis for Compartmentation0

The compartmentation of amino acid metabolism is an active and important area of brain research. 13C labeling and 13C nuclear magnetic resonance (NMR) are powerful tools for studying metabolic pathways, because information about the metabolic histories of metabolites can be determined from the appearance and position of the label in products. We have used 13C labeling and 13C NMR in order to investigate the metabolic history of gamma-aminobutyric acid (GABA) and glutamate in rat brain. [1-13C]Glucose was infused into anesthetized rats and the 13C labeling patterns in GABA and glutamate examined in brain tissue extracts obtained at various times after infusion of the label. Five minutes after infusion, most of the 13C label in glutamate appeared at the C4 position; at later times, label was also present at C2 and C3. This 13C labeling pattern occurs when [1-13C]glucose is metabolized to pyruvate by glycolysis and enters the pool of tricarboxylic acid (TCA) intermediates via pyruvate dehydrogenase. The label exchanges into glutamate from the TCA cycle pool through glutamate transaminases or dehydrogenase. After 30 min of infusion, approximately 10% of the total 13C in brain extracts appeared in GABA, primarily (greater than 80%) at the amino carbon (C4), indicating that the GABA detected is labeled through pyruvate carboxylase. The different labeling patterns observed for glutamate and GABA show that the large detectable glutamate pool does not serve as the precursor to GABA. Our NMR data support previous experiments suggesting compartmentation of metabolism in brain, and further demonstrate that GABA is formed from a pool of TCA cycle intermediates derived from an anaplerotic pathway involving pyruvate carboxylase.     

Measurements of the anaplerotic rate in the human cerebral cortex using 13C magnetic resonance spectroscopy and [1-13C] and [2-13C] glucose

Recent studies in rodent and human cerebral cortex have shown that glutamate-glutamine neurotransmitter cycling is rapid and the major pathway of neuronal glutamate repletion. The rate of the cycle remains controversial in humans, because glutamine may come either from cycling or from anaplerosis via glial pyruvate carboxylase. Most studies have determined cycling from isotopic labeling of glutamine and glutamate using a [1-(13)C]glucose tracer, which provides label through neuronal and glial pyruvate dehydrogenase or via glial pyruvate carboxylase. To measure the anaplerotic contribution, we measured (13)C incorporation into glutamate and glutamine in the occipital-parietal region of awake humans while infusing [2-(13)C]glucose, which labels the C2 and C3 positions of glutamine and glutamate exclusively via pyruvate carboxylase. Relative to [1-(13)C]glucose, [2-(13)C]glucose provided little label to C2 and C3 glutamine and glutamate. Metabolic modeling of the labeling data indicated that pyruvate carboxylase accounts for 6 +/- 4% of the rate of glutamine synthesis, or 0.02 micromol/g/min. Comparison with estimates of human brain glutamine efflux suggests that the majority of the pyruvate carboxylase flux is used for replacing glutamate lost due to glial oxidation and therefore can be considered to support neurotransmitter trafficking. These results are consistent with observations made with arterial-venous differences and radiotracer methods.     

Metabolic changes in undifferentiated and differentiated human colon adenocarcinoma cells studied by multinuclear magnetic resonance spectroscopy

Aspects of energetic and intermediary metabolism were studied in a colon adenocarcinoma cell line (HT29) by multinuclear magnetic resonance spectroscopy. Experiments were carried out on the HT29-D4 clone, which was isolated by limit dilution techniques. This clone, usually undifferentiated (D4-UD), can be maintained in a differentiated state (D4-D) in a glucose-free medium. Metabolic data were obtained by NMR analysis of perchloric acid extracts from D4-UD and D4-D cells. Phosphorus-31 and proton NMR spectra showed the presence of a large amount of choline and phosphorylcholine in the differentiated state (400% and 200%, respectively, of the levels found in D4-UD cells). Other differences appeared in the content of phosphocreatine (absent in D4-D cells) and myoinositol (absent in D4-UD cells). Carbon-13 spectra were recorded from perchloric acid extracts of cells incubated with [1-13C]-labeled glucose or [2-13C]-labeled acetate. The data indicated that both types of cells metabolize glucose through the glycolytic pathway to give lactate, but only D4-D cells were able to store glucose as glycogen at a very high level. A mathematical analysis of fluxes through the tricarboxylic acid (TCA) cycle was developed on the basis of models derived from previous 14C tracer studies. The model was based on the steady-state labeling of glutamate carbons by the 13C isotope and gave the fraction of labeled acetyl-Coa entering the TCA cycle, and the activity y of anaplerotic reactions relative to the flux through the citrate synthetase reaction. The data indicated that y greater than 0.3 in all cases. Only 15% and 30% of labeled acetyl CoA entered the TCA cycle in D4-UD and D4-D cells, respectively, under labeled glucose incubation: these values were significantly different upon labeled acetate feeding, reaching 55% for D4-UD cells and 85% for D4-D cells. The main result of this study is that the process of differentiation of HT29 cells is correlated with a large increase in the activity of oxidative metabolism.     

MRL/lpr mice have alterations in brain metabolism as shown with [1H-13C] NMR spectroscopy

Cerebral glucose metabolism and cerebral blood flow are altered in patients with lupus who have neuropsychiatric manifestations. However, the dynamics of changes in glucose metabolism remain unclear. The present study was undertaken using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy to determine the rates of incorporation of glucose into amino acids and lactate via cell-specific pathways in mice with lupus. In the well-established MRL/lpr lupus mouse model, 24-week-old mice had a significant increase of 30-80% (P<0.001) in total brain glutamine, glutamate and lactate concentrations, while alanine, aspartate, N-acetyl aspartate (NAA) and gamma-aminobutyric acid (GABA) remained unchanged as compared to the congenic MRL+/+control mice. Although succinate concentration was increased in lupus brain, it did not reach statistical significance. Furthermore, 13C isotopomer analysis showed a selective increase of de novo synthesis of lactate from [1-(13)C] glucose through glycolysis resulting in 1.5-fold increased fractional 13C enrichments in lactate in MRL/lpr mice. [4-(13)C] Glutamate, which is synthesized mainly by the neuronal pyruvate dehydogenase, was selectively increased, while [2-(13)C] and [3-(13)C] GABA synthesis were decreased by 25% compared to controls. In accordance with the total concentrations, aspartate synthesis remained unaltered in brains of lupus mice, while alanine synthesis was elevated, indicating increased utilization of alanine. Creatine was unchanged in MRL/lpr mice as compared to controls. An interesting finding was a significant increase (158%, P<0.005) in choline concentration in MRL/lpr mice while the myo-inositol concentration remained the same in both groups. Furthermore a significant increase in total brain water content was observed, indicative of possible edema. In conclusion, the cumulative effect of increased brain lactate synthesis, altered glucose metabolism and intracellular glutamine accumulation could be an important mechanism causing brain pathology in SLE. The alteration in metabolites could alter downstream pathways and cause neurological dysfunction. Future NMR spectroscopic studies using stable isotopes and real-time measurements of metabolic rates, along with levels of metabolites in plasma and cerebrospinal fluid, could be valuable in the elucidation of the cerebral metabolic consequences of systemic lupus erythematosis (SLE) in humans.     

13C and 1H nuclear magnetic resonance study of glycogen futile cycling in strains of the genus Fibrobacter

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. 13C nuclear magnetic resonance (NMR) analysis of [1-13C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by 13C NMR by following the simultaneous metabolism of labeled [13C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH2 of succinate and the CH3 of acetate when the resting cells were metabolizing [1-13C]glucose and unlabeled glycogen were precisely quantified by using 13C-filtered spin-echo difference 1H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.     

Glucose metabolism of adult Schistosoma japonicum as revealed by nuclear magnetic resonance spectroscopy with D-[13C6]glucose

Excretory end-products of adult Schistosoma japonicum, fed D-[13C6]glucose in vitro under aerobic and anaerobic conditions, were studied using 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy. The glucose in the medium is degraded to produce lactate and alanine aerobically and succinate and acetate as well as lactate and alanine anaerobically. Succinate and acetate have not been previously recorded as excretory products resulting from the metabolism of glucose for schistosomes. The presence of [13C3] and [2,3-13C2]lactate, and [1,2,2'-13C3] and [2,2'-13C2]succinate as end-products suggests that a partial reversed tricarboxylic acid (TCA) cycle is active in adult Schistosoma japonicum under anaerobic conditions. The physiological role of this pathway in adult schistosomes remains obscure.     

Metabolism of [1-(13)C)glucose and [2-(13)C]acetate in the hypoxic rat brain.

The effects of hypoxia on the metabolism of the central nervous system were investigated in rats submitted to a low oxygen atmosphere (8% O(2); 92% N(2)). [1-(13)C]glucose and [2-(13)C]acetate were used as substrates, this latter being preferentially metabolized by glial cells. After 1-h substrate infusion, the incorporation of 13C in brain metabolites was determined by NMR spectroscopy. Under hypoxia, an important hyperglycemia was noted. As a consequence, when using labeled glucose, the specific enrichment of brain glucose C1 was lower (48.2+/-5.1%) than under normoxia (66.9+/-2.5%). However, relative to this specific enrichment, the (13)C incorporation in amino acids was increased under hypoxia. This suggested primarily a decreased exchange between blood and brain lactate. The glutamate C2/C4 enrichment ratio was higher under hypoxia (0.62+/-0.01) than normoxia (0.51+/-0.06), indicating a lower glutamate turnover relative to the neuronal TCA cycle activity. The glutamine C2/C4 enrichment ratio was also higher under hypoxia (0.87+/-0.07 instead of 0.65+/-0.11), indicating a new balance in the contributions of different carbon sources at the acetyl-CoA level. When using [2-(13)C]acetate as substrate, no difference in glutamine enrichment appeared under hypoxia, whereas a significant decrease in glutamate, aspartate, alanine and lactate enrichments was noted. This indicated a lower trafficking between astrocytes and neurons and a reduced tricarboxylic acid cycle intermediate recycling of pyruvate.