Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons

We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca²⁺ during activation cause influx of Ca²⁺ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate–aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca²⁺ (i.e. independent of synaptic activity) on neuronal energy metabolism employing ¹³C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca²⁺ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca²⁺ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca²⁺-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca²⁺ signalling.     

How astrocytes feed hungry neurons

For years glucose was thought to constitute the sole energy substrate for neurons; it was believed to be directly provided to neurons via the extracellular space by the cerebral circulation. It was recently proposed that in addition to glucose, neurons might rely on lactate to sustain their activity. Therefore, it was demonstrated that lactate is a preferred oxidative substrate for neurons not only in vitro but also in vivo. Moreover, the presence of specific monocarboxylate transporters on neurons as well as on astrocytes is consistent with the hypothesis of a transfer of lactate from astrocytes to neurons. Evidence has been provided for a mechanism whereby astrocytes respond to glutamatergic activity by enhancing their glycolytic activity, resulting in increased lactate release. This is accomplished via the uptake of glutamate by glial glutamate transporters, leading to activation of the Na⁺/K⁺ ATPase and a stimulation of astrocytic glycolysis. Several recent observations obtained both in vitro and in vivo with different approaches have reinforced this view of brain energetics. Such an understanding might be critically important, not only because it forms the basis of some classical functional brain imaging techniques but also because several neurodegenerative diseases exhibit diverse alterations in energy metabolism.     

Refined Analysis of Brain Energy Metabolism Using In Vivo Dynamic Enrichment of 13C Multiplets

Carbon-13 nuclear magnetic resonance spectroscopy in combination with the infusion of ¹³C-labeled precursors is a unique approach to study in vivo brain energy metabolism. Incorporating the maximum information available from in vivo localized ¹³C spectra is of importance to get broader knowledge on cerebral metabolic pathways. Metabolic rates can be quantitatively determined from the rate of ¹³C incorporation into amino acid neurotransmitters such as glutamate and glutamine using suitable mathematical models. The time course of multiplets arising from ¹³C-¹³C coupling between adjacent carbon atoms was expected to provide additional information for metabolic modeling leading to potential improvements in the estimation of metabolic parameters.The aim of the present study was to extend two-compartment neuronal/glial modeling to include dynamics of ¹³C isotopomers available from fine structure multiplets in ¹³C spectra of glutamate and glutamine measured in vivo in rats brain at 14.1 T, termed bonded cumomer approach. Incorporating the labeling time courses of ¹³C multiplets of glutamate and glutamine resulted in elevated precision of the estimated fluxes in rat brain as well as reduced correlations between them.     

A new horizon of DNP technology: application to in-vivo 13C magnetic resonance spectroscopy and imaging

Dynamic nuclear polarization (DNP) is an emerging technique for increasing the sensitivity (>10,000-fold) of magnetic resonance spectroscopy and imaging (MRSI), in particularly for low-γ nuclei. DNP methodology is based on polarizing nuclear spins in an amorphous solid state at low temperature (ca. 1 K) through coupling of the nuclear spins with unpaired electron spins that are added to the sample via an organic free radical. In an amorphous solid state, the high electron spin polarization can be transferred to the nuclear spins by microwave irradiation. While this technique has been utilized in solid-state research for many years, it is only recently that dissolution methods and the required hardware have been developed to produce the high nuclear polarization provided by DNP to produce injectable hyperpolarized solutions suitable for in vivo studies. It has been applied to a number of ¹³C-labeled cell metabolites in biological systems and their real-time metabolic conversion has been imaged. This review focuses briefly on the DNP methodology and the significant molecules investigated to date in preclinical cancer models, in terms of their downstream metabolism in vivo or the biological processes that they can probe. In particular, conversion between hyperpolarized ¹³C-labeled pyruvate and lactate, catalyzed by lactate dehydrogenase, has been shown to have a number of potential applications such as diagnosis, staging tumor grade, and monitoring therapy response. Strategies for making this technique more viable to use in clinical settings have been discussed.     

Rat brain slices oxidize glucose at high rates: a (13)C NMR study

Since glucose is the main cerebral substrate, we have characterized the metabolism of various ¹³C glucose isotopomers in rat brain slices. For this, we have used our cellular metabolomic approach that combines enzymatic and carbon 13 NMR techniques with mathematical models of metabolic pathways. We identified the fate and the pathways of the conversion of glucose carbons into various products (pyruvate, lactate, alanine, aspartate, glutamate, GABA, glutamine and CO₂) and determined absolute fluxes through pathways of glucose metabolism. After 60 min of incubation, lactate and CO₂ were the main end-products of the metabolism of glucose which was avidly metabolized by the slices. Lactate was also used at high rates by the slices and mainly converted into CO₂. High values of flux through pyruvate carboxylase, which were similar with glucose and lactate as substrate, were observed. The addition of glutamine, but not of acetate, stimulated pyruvate carboxylation, the conversion of glutamate into succinate and fluxes through succinate dehydrogenase, malic enzyme, glutamine synthetase and aspartate aminotransferase. It is concluded that, unlike brain cells in culture, and consistent with high fluxes through PDH and enzymes of the tricarboxylic acid cycle, rat brain slices oxidized both glucose and lactate at high rates.     

MRS study of glutamate metabolism in cultured neurons/glia

[U-¹³C]Glutamate metabolism was studied in primary brain cell cultures. Cell extracts as well as redissolved lyophilized media were subjected to nuclear magnetic resonance spectroscopy in order to identify¹³C labeled metabolites. Both neurons and astrocytes metabolized glutamate extensively with¹³C label appearing in aspartate in all cultures. Additionally, GABA is synthesized in the GABAergic cortical neurons. Labeling of lactate and glutamine was prominent in medium from astrocytes, but not detectable in cerebral cortical neurons. Cerebellar granule neurons showed some labeling of lactate. Glutamate derived from the first turn of the tricarboxylic acid cycle (1,2,3-¹³C₃-isotopomer) is present in all cell types analyzed. However, glutamate derived from the second turn of the cycle was only detected in granule neurons. In astrocytes, the transaminase inhibitor aminooxyacetic acid not only abolished the appearance of aspartate, but also of the 1,2,3-¹³C₃-isotopomer of glutamate, thus showing that transmination is necessary for the conversion of 2-oxoglutarate to glutamate. The entry of glutamate into the tricarboxylic acid cycle was, however, not seriously impaired. 3-nitropropionic acid abolished the appearance of aspartate, the 1,2,3-¹³C₃-isotopomer of glutamate and lactate in cerebellar granule neurons. Special issue dedicated to Dr. Herman Bachelard.     

Glial Formation of Pyruvate and Lactate from TCA Cycle Intermediates: Implications for the Inactivation of Transmitter Amino Acids?

Abstract: Cerebral formation of lactate via the tricarboxylic acid (TCA) cycle was investigated through the labeling of lactate from [2-¹³C]acetate and [1-¹³C]glucose as shown by ¹³C NMR spectroscopy. In fasted mice that had received [2-¹³C]acetate intravenously, brain lactate C-2 and C-3 were labeled at 5, 15, and 30 min, reflecting formation of pyruvate and hence lactate from TCA cycle intermediates. In contrast, [1-¹³C]glucose strongly labeled lactate C-3, reflecting glycolysis, whereas lactate C-2 was weakly labeled only at 15 min. These data show that formation of pyruvate, and hence lactate, from TCA cycle intermediates took place predominantly in the acetate-metabolizing compartment, i.e., glia. The enrichment of total brain lactate from [2-¹³C]acetate reached ∼1% in both the C-2 and the C-3 position in fasted mice. It was calculated that this could account for 20% of the lactate formed in the glial compartment. In fasted mice, there was no significant difference between the labeling of lactate C-2 and C-3 from [2-¹³C]acetate, whereas in fed mice, lactate C-3 was more highly labeled than the C-2, reflecting adaptive metabolic changes in glia in response to the nutritional state of the animal. It is hypothesized that conversion of TCA cycle intermediates into pyruvate and lactate may be operative in the glial metabolism of extracellular glutamate and GABA in vivo. Given the vasodilating effect of lactate on cerebral vessels, which are ensheathed by astrocytic processes, conversion of glutamate and GABA into lactate could be one mechanism mediating increases in cerebral blood flow during nervous activity.     

Stable isotope-resolved metabolomic analysis of lithium effects on glial-neuronal metabolism and interactions

Despite the long-established therapeutic efficacy of lithium in the treatment of bipolar disorder (BPD), its molecular mechanism of action remains elusive. Newly developed stable isotope-resolved metabolomics (SIRM) is a powerful approach that can be used to elucidate systematically how lithium impacts glial and neuronal metabolic pathways and activities, leading ultimately to deciphering its molecular mechanism of action. The effect of lithium on the metabolism of three different ¹³C-labeled precursors ([U-¹³C]-glucose, ¹³C-3-lactate or ¹³C-2,3-alanine) was analyzed in cultured rat astrocytes and neurons by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS). Using [U-¹³C]-glucose, lithium was shown to enhance glycolytic activity and part of the Krebs cycle activity in both astrocytes and neurons, particularly the anaplerotic pyruvate carboxylation (PC). The PC pathway was previously thought to be active in astrocytes but absent in neurons. Lithium also stimulated the extracellular release of ¹³C labeled-lactate, -alanine (Ala), -citrate, and -glutamine (Gln) by astrocytes. Interrogation of neuronal pathways using ¹³C-3-lactate or ¹³C-2,3-Ala as tracers indicated a high capacity of neurons to utilize lactate and Ala in the Krebs cycle, particularly in the production of labeled Asp and Glu via PC and normal cycle activity. Prolonged lithium treatment enhanced lactate metabolism via PC but inhibited lactate oxidation via the normal Krebs cycle in neurons. Such lithium modulation of glycolytic, PC and Krebs cycle activity in astrocytes and neurons as well as release of fuel substrates by astrocytes should help replenish Krebs cycle substrates for Glu synthesis while meeting neuronal demands for energy. Further investigations into the molecular regulation of these metabolic traits should provide new insights into the pathophysiology of mood disorders and early diagnostic markers, as well as new target(s) for effective therapies.     

天冬酰胺内肽酶和胶质细胞在创伤性脑损伤中的激活

摘要 目的：创伤性脑损伤（traumatic brain injury, TBI）缺乏安全有效的治疗手段,亟须寻找新的干预靶点。天冬酰胺内肽酶 (asparaginyl endopeptidase, AEP)在免疫和神经系统疾病中起重要作用,本研究观察了小鼠TBI模型中AEP的激活和变化,探讨AEP对脑损伤和修复的意义。方法：控制性皮层撞击法在小鼠右脑半球制作TBI损伤,在造模后的不同时间点,测定受损脑组织内的乳酸含量和AEP的活性变化,免疫荧光化学染色观察TBI之后3天的胶质细胞活化,以及AEP在其中的表达。结果：TBI造成乳酸在受损脑组织内逐渐堆积,导致小胶质细胞和星形胶质细胞的反应性活化和增生,AEP的上调和激活出现在TBI的继发性脑损伤阶段,AEP在小胶质细胞和星形胶质细胞内均出现上调。结论：AEP有可能参与调控TBI引发的胶质细胞活化,在神经损伤和修复中发挥重要作用。     

Neuroprotective Effects of Ethyl Pyruvate on Brain Energy Metabolism after Ischemia-Reperfusion Injury: A 31P-Nuclear Magnetic Resonance Study

The neuroprotective effects of ethyl pyruvate (EP), a stable derivative of pyruvate, on energy metabolism of rat brain exposed to ischemia-reperfusion stress were investigated by ³¹P-nuclear magnetic resonance (³¹P-NMR) spectroscopy. Recovery level of phosphocreatine after ischemia was significantly greater when superfused with artificial cerebrospinal fluid (ACSF) with 2 mM EP than when superfused with ACSF without EP. EP was neuroprotective against ischemia only when administered before the ischemic exposure. Intracellular pH during ischemia was less acidic when superfused ahead of time with EP. EP did not show neuroprotective effects in neuron-rich slices pretreated with 100 μM fluorocitrate, a selective glial poison. It was suggested that both the administration of EP before ischemic exposure and the presence of astrocytes are required for EP to exert neuroprotective effects. We suggest the potential involvement of multiple mechanisms of action, such as less acidic intracellular pH, glial production of lactate, and radical scavenging ability. Special issue article in honor of Dr. Akitane Mori.     

Traumatic Brain Injury Down-Regulates Glial Glutamate Transporter (GLT-1 and GLAST) Proteins in Rat Brain

Abstract: Excess activation of NMDA receptors is felt to participate in secondary neuronal damage after traumatic brain injury (TBI). Increased extracellular glutamate is active in this process and may result from either increased release or decreased reuptake. The two high-affinity sodium-dependent glial transporters [glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST)] mediate the bulk of glutamate transport. We studied the protein levels of GLT-1 and GLAST in the brains of rats after controlled cortical impact-induced TBI. With use of subtype-specific antibodies, GLT-1 and GLAST proteins were quantitated by immunoblotting in the ipsilateral and contralateral cortex at 2, 6, 24, 72, and 168 h after the injury. Sham-operated rats served as control. TBI resulted in a significant decrease in GLT-1 (by 20–45%; p < 0.05) and GLAST (by 30–50%; p < 0.05) protein levels between 6 and 72 h after the injury. d -[³H]Aspartate binding also decreased significantly (by 30–50%; p < 0.05) between 6 and 72 h after the injury. Decreased glial glutamate transporter function may contribute to the increased extracellular glutamate that may mediate the excitotoxic neuronal damage after TBI. This is a first report showing altered levels of glutamate transporter proteins after TBI.     

Origins of the Extracellular Glutamate Released During Total Metabolic Blockade in the Immature Retina

Abstract: Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time-dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37°C with [³H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing ³H-labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [³H]acetate plus [U-¹⁴C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 ± 0.02 vs. 0.12 ± 0.01 µM for treated vs. control retina (means ± SEM), respectively], but was increased significantly at 15 (1.2 ± 0.26 µM) and 30 min (2.6 ± 0.22 µM). Total [³H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5- and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [³H]glutamate remained fairly constant, 731 ± 134 and 517 ± 82 dpm/nmol (means ± SEM) at 15 and 30 min, respectively. In contrast, ¹⁴C-labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of ¹⁴C-labeled extracellular glutamate decreased from 309 ± 87 dpm/nmol at 15 min to 42 ± 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na⁺-dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [³H]glutamate, constancy of the specific activity of extracellular [³H]glutamate, decrease in the specific activity of extracellular [¹⁴C]glutamate, and attenuation of release by prior loading with t-PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation.     

Astrocytic energetics during excitatory neurotransmission: What are contributions of glutamate oxidation and glycolysis?

Astrocytic energetics of excitatory neurotransmission is controversial due to discrepant findings in different experimental systems in vitro and in vivo. The energy requirements of glutamate uptake are believed by some researchers to be satisfied by glycolysis coupled with shuttling of lactate to neurons for oxidation. However, astrocytes increase glycogenolysis and oxidative metabolism during sensory stimulation in vivo, indicating that other sources of energy are used by astrocytes during brain activation. Furthermore, glutamate uptake into cultured astrocytes stimulates glutamate oxidation and oxygen consumption, and glutamate maintains respiration as well as glucose. The neurotransmitter pool of glutamate is associated with the faster component of total glutamate turnover in vivo, and use of neurotransmitter glutamate to fuel its own uptake by oxidation-competent perisynaptic processes has two advantages, substrate is supplied concomitant with demand, and glutamate spares glucose for use by neurons and astrocytes. Some, but not all, perisynaptic processes of astrocytes in adult rodent brain contain mitochondria, and oxidation of only a small fraction of the neurotransmitter glutamate taken up into these structures would be sufficient to supply the ATP required for sodium extrusion and conversion of glutamate to glutamine. Glycolysis would, however, be required in perisynaptic processes lacking oxidative capacity. Three lines of evidence indicate that critical cornerstones of the astrocyte-to-neuron lactate shuttle model are not established and normal brain does not need lactate as supplemental fuel: (i) rapid onset of hemodynamic responses to activation delivers oxygen and glucose in excess of demand, (ii) total glucose utilization greatly exceeds glucose oxidation in awake rodents during activation, indicating that the lactate generated is released, not locally oxidized, and (iii) glutamate-induced glycolysis is not a robust phenotype of all astrocyte cultures. Various metabolic pathways, including glutamate oxidation and glycolysis with lactate release, contribute to cellular energy demands of excitatory neurotransmission.     

Brain magnetic resonance study of Mecp2 deletion effects on anatomy and metabolism

Rett syndrome, a neurodevelopmental X-linked disorder, represents the most important genetic cause of severe mental retardation in the female population and results from a mutation in the gene encoding methyl-CpG-binding protein 2 (MECP2). We report here the first characterization of Mecp2-null mice, by in vivo magnetic resonance imaging and spectroscopy, delineating the cerebral phenotype associated with the lack of Mecp2. We performed a morphometric study that revealed a size reduction of the whole brain and of structures involved in cognitive and motor functions (cerebellum and motor cortex). Significant metabolic anomalies, including reduced N-acetylaspartate, myo-inositol, and glutamine plus glutamate, and increased choline levels were evidenced. These findings indicate that not only neuronal but also glial metabolism is affected in Mecp2-null mice. Furthermore, we uncovered an important reduction of brain ATP level, a hitherto undetected anomaly of energy metabolism that may reflect and contribute to cerebral injury and dysfunction.     

Limbic Structures Show Altered Glial–Neuronal Metabolism in the Chronic Phase of Kainate Induced Epilepsy

A better understanding is needed of how glutamate metabolism is affected in mesial temporal lobe epilepsy (MTLE). Here we investigated glial–neuronal metabolism in the chronic phase of the kainate (KA) model of MTLE. Thirteen weeks following systemic KA, rats were injected i.p. with [1-¹³C]glucose. Brain extracts from hippocampal formation, entorhinal cortex, and neocortex, were analyzed by ¹³C and ¹H magnetic resonance spectroscopy to quantify ¹³C labeling and concentrations of metabolites, respectively. The amount and ¹³C labeling of glutamate were reduced in the hippocampal formation and entorhinal cortex of epileptic rats. Together with the decreased concentration of NAA, these results indicate neuronal loss. Additionally, mitochondrial dysfunction was detected in surviving glutamatergic neurons in the hippocampal formation. In entorhinal cortex glutamine labeling and concentration were unchanged despite the reduced glutamate content and label, possibly due to decreased oxidative metabolism and conserved flux of glutamate through glutamine synthetase in astrocytes. This mechanism was not operative in the hippocampal formation, where glutamine labeling was decreased. In neocortex labeling and concentration of GABA were increased in epileptic rats, possibly representing a compensatory mechanism. The changes in the hippocampus might be of pathophysiological importance and merit further studies aiming at resolving metabolic causes and consequences of MTLE. Special issue article in honor of Dr. Frode Fonnum.     

Ca2+-Permeable Acid-sensing Ion Channels and Ischemic Brain Injury

Acidosis is a common feature of brain in acute neurological injury, particularly in ischemia where low pH has been assumed to play an important role in the pathological process. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain unclear. Recent studies have demonstrated that activation of Ca²⁺-permeable acid-sensing ion channels (ASIC1a) is largely responsible for acidosis-mediated, glutamate receptor-independent, neuronal injury. In cultured mouse cortical neurons, lowering extracellular pH to the level commonly seen in ischemic brain activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are permeable to Ca²⁺, and an activation of these channels induces increases in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i). Activation of ASICs with resultant [Ca²⁺]_i loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca²⁺ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing [Ca²⁺]_o. In focal ischemia, intracerebroventricular administration of ASIC1a blockers, or knockout of the ASIC1a gene protects brain from injury and does so more potently than glutamate antagonism. Furthermore, pharmacological blockade of ASICs has up to a 5 h therapeutic time window, far beyond that of glutamate antagonists. Thus, targeting the Ca²⁺-permeable acid-sensing ion channels may prove to be a novel neuroprotective strategy for stroke patients.     

Metabolic Effects of Blocking Lactate Transport in Brain Cortical Tissue Slices Using an Inhibitor Specific to MCT1 and MCT2

A novel inhibitor of lactate transport, AR-C122982, was used to study the effect of inhibiting the monocarboxylate transporters MCT1 and MCT2 on cortical brain slice metabolism. We studied metabolism of l-[3-¹³C]lactate, and d-[1-¹³C]glucose under a range of conditions. Experiments using l-[3-¹³C]lactate showed that the inhibitor AR-C122982 altered exchange of lactate. Under depolarizing conditions, net flux of label from d-[1-¹³C]glucose was barely altered by 10 or 100 nM AR-C122982. In the presence of AMPA or glutamate there were increases in net flux of label and metabolic pool sizes. These data suggest lactate may supply compartments in the brain not usually directly accessed by glucose. In general, it would appear that movement of lactate between cell types is not essential for metabolic activity, with the heavy metabolic workloads imposed being unaffected by inhibition of MCT1 and MCT2. Further experiments investigating the mechanism of operation of AR-C122982 are necessary to corroborate this finding.     

Excess exogenous pyruvate inhibits lactate dehydrogenase activity in live cells in an MCT1-dependent manner

Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-¹³C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1–dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-¹³C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.     

The impact of the glial spatial buffering on the K+ Nernst potential

Astrocytes play a critical role in CNS metabolism, regulation of volume and ion homeostasis of the interstitial space. Of special relevance is their clearance of K⁺ that is released by active neurons into the extracellular space. Mathematical analysis of a modified Nernst equation for the electrochemical equilibrium of neuronal plasma membranes, suggests that K⁺ uptake by glial cells is not only relevant during neuronal activity but also has a non-neglectable impact on the basic electrical membrane properties, specifically the resting membrane potential, of neurons and might be clinically valuable as a factor in the genetics and epigenetics of the epilepsy and tuberous sclerosis complex.     

Demonstration of Neuron-Glia Transfer of Precursors for Gaba Biosynthesis in a Co-Culture System of Dissociated Mouse Cerebral Cortex

Co-cultures of neurons and astrocytes were prepared from dissociated embryonic mouse cerebral cortex and cultured for 7 days. To investigate if these cultures may serve as a functional model system to study neuron-glia interaction with regard to GABA biosynthesis, the cells were incubated either in media containing [U-¹³C]glutamine (0.1, 0.3 and 0.5 mM) or 1 mM acetate plus 2.5 mM glucose plus 1 mM lactate. In the latter case one of the 3 substrates was uniformly ¹³C labeled. Cellular contents and ¹³C labeling of glutamate, GABA, aspartate and glutamine were determined in the cells after an incubation period of 2.5 h. The GABA biosynthetic machinery exhibited the expected complexity with regard to metabolic compartmentation and involvement of TCA cycle activity as seen in other culture systems containing GABAergic neurons. Metabolism of acetate clearly demonstrated glial synthesis of glutamine and its transfer to the neuronal compartment. It is concluded that this co-culture system serves as a reliable model in which functional and pharmacological aspects of GABA biosynthesis can be investigated. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella. An erratum to this article can be found at