Malate-aspartate Shuttle Research Articles

Cytosolic Ca2+ oversees the MASs production of pyruvate for the mitochondrial market

It is generally accepted that mitochondrial Ca2+ controls the pace of mitochondrial bioenergetics and thus ATP production. Szibor et al. challenge this paradigm, proposing that the balance between ATP consumption and production depends on mitochondrial pyruvate supply via the malate-aspartate shuttle (MAS) and is controlled by cytosolic Ca2+.

Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply

Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca2+). Current models assume that cytosolic Ca2+ regulates workload and that mitochondrial Ca2+ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca2+ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca2+, but not mitochondrial matrix Ca2+, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate-aspartate shuttle (MAS)-dependent substrate supply in OXPHOS responses to changing Ca2+ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from WT and MCU KO mice and the isolated working rat heart. Our results indicate that extramitochondrial Ca2+ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca2+ accounts for the remaining 15%. Of note, the complete MAS, as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the “mitochondrial gas pedal.” Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.

Succinate accumulation induces mitochondrial reactive oxygen species generation and promotes status epilepticus in the kainic acid rat model

Though succinate accumulation is associated with reactive oxygen species (ROS) production and neuronal injury, which play critical roles in epilepsy, it is unclear whether succinate accumulation contributes to the onset of epilepsy or seizures. We sought to investigate changes in succinate, oxidative stress, and mito-SOX levels, as well as mitophagy and neuronal change, in different status epilepticus (SE) rat models. Our results demonstrate that KA-induced SE was accompanied by increased levels of succinate, oxidative stress, and mito-SOX, as well as mitophagy and neuronal degeneration. The similarly increased levels of succinate, oxidative stress, and mito-SOX were also found in pilocarpine-induced SE. Moreover, the reduction of succinate accumulation by the inhibition of succinate dehydrogenase (SDH), malate/aspartate shuttle (MAS), or purine nucleotide cycle (PNC) served to reduce succinate, oxidative stress, and mito-SOX levels, thereby preventing oxidative stress-related neuronal damage and lessening seizure severity. Interestingly, simulating succinate accumulation with succinic acid dimethyl ester may induce succinate accumulation and increased oxidative stress and mito-SOX levels, as well as behavior and seizures in electroencephalograms similar to those observed in rats exposed to KA. Our results indicate that succinate accumulation may contribute to the increased oxidative stress/mitochondrial ROS levels, neuronal degeneration, and SE induced by KA administration. Furthermore, we found that succinate accumulation was mainly due to the inverse catalysis of SDH from fumarate, which was supplemented by the MAS and PNC pathways. These results reveal new insights into the mechanisms underlying SE and that reducing succinate accumulation may be a clinically useful therapeutic target in SE.

Ischemic Neuroprotectant PKCε Restores Mitochondrial Glutamate Oxaloacetate Transaminase in the Neuronal NADH Shuttle after Ischemic Injury.

The preservation of mitochondrial function is a major protective strategy for cerebral ischemic injuries. Previously, our laboratory demonstrated that protein kinase C epsilon (PKCε) promotes the synthesis of mitochondrial nicotinamide adenine dinucleotide (NAD+). NAD+ along with its reducing equivalent, NADH, is an essential co-factor needed for energy production from glycolysis and oxidative phosphorylation. Yet, NAD+/NADH are impermeable to the inner mitochondrial membrane and their import into the mitochondria requires the activity of specific shuttles. The most important neuronal NAD+/NADH shuttle is the malate-aspartate shuttle (MAS). The MAS has been implicated in synaptic function and is potentially dysregulated during cerebral ischemia. The aim of this study was to determine if metabolic changes induced by PKCε preconditioning involved regulation of the MAS. Using primary neuronal cultures, we observed that the activation of PKCε enhanced mitochondrial respiration and glycolysis in vitro. Conversely, inhibition of the MAS resulted in decreased oxidative phosphorylation and glycolytic capacity. We further demonstrated that activation of PKCε increased the phosphorylation of key components of the MAS in rat brain synaptosomal fractions. Additionally, PKCε increased the enzyme activity of glutamic oxaloacetic transaminase 2 (GOT2), an effect that was dependent on the import of PKCε into the mitochondria and phosphorylation of GOT2. Furthermore, PKCε activation was able to rescue decreased GOT2 activity induced by ischemia. These findings reveal novel protective targets and mechanisms against ischemic injury, which involves PKCε-mediated phosphorylation and activation of GOT2 in the MAS.

Mitochondrial movement in Aralar/Slc25a12/AGC1 deficient cortical neurons

The elevated energy demands in the brain are fulfilled mainly by glucose catabolism. In highly polarized neurons, about 10–50% of mitochondria are transported along microtubules using mitochondrial-born ATP to locations with high energy requirements. In this report, we have investigated the impact of Aralar deficiency on mitochondrial transport in cultured cortical neurons. Aralar/slc25a12/AGC1 is the neuronal isoform of the aspartate-glutamate mitochondrial carrier, a component of the malate-aspartate shuttle (MAS) which plays an important role in redox balance, which is essential to maintain glycolytic pyruvate supply to neuronal mitochondria. Using live imaging microscopy we observed that the lack of Aralar does not affect the number of moving mitochondria nor the Ca2+-induced stop, the only difference being a 10% increase in mitochondrial velocity in Aralar deficient neurons. Therefore, we evaluated the possible fuels used in each case by studying the relative contribution of oxidative phosphorylation and glycolysis to mitochondrial movement using specific inhibitors. We found that the ATP synthase inhibitor oligomycin caused a smaller inhibition of mitochondrial movement in Aralar-KO than control neurons, whereas the glycolysis inhibitor iodoacetate had similar effects in neurons from both genotypes. In line with these findings, the decrease in cytosolic ATP/ADP ratio caused by oligomycin was more pronounced in control than in Aralar-KO neurons, but no differences were observed with iodoacetate. Oligomycin effect was reverted by aralar re-expression in knock out cultures. As mitochondrial movement is not reduced in Aralar-KO neurons, these results suggest that these neurons may use an additional pathway for mitochondria movement and ATP/ADP ratio maintenance.

Skeletal muscle PGC-1\u03b11 reroutes kynurenine metabolism to increase energy efficiency and fatigue-resistance

The coactivator PGC-1α1 is activated by exercise training in skeletal muscle and promotes fatigue-resistance. In exercised muscle, PGC-1α1 enhances the expression of kynurenine aminotransferases (Kats), which convert kynurenine into kynurenic acid. This reduces kynurenine-associated neurotoxicity and generates glutamate as a byproduct. Here, we show that PGC-1α1 elevates aspartate and glutamate levels and increases the expression of glycolysis and malate-aspartate shuttle (MAS) genes. These interconnected processes improve energy utilization and transfer fuel-derived electrons to mitochondrial respiration. This PGC-1α1-dependent mechanism allows trained muscle to use kynurenine metabolism to increase the bioenergetic efficiency of glucose oxidation. Kat inhibition with carbidopa impairs aspartate biosynthesis, mitochondrial respiration, and reduces exercise performance and muscle force in mice. Our findings show that PGC-1α1 activates the MAS in skeletal muscle, supported by kynurenine catabolism, as part of the adaptations to endurance exercise. This crosstalk between kynurenine metabolism and the MAS may have important physiological and clinical implications.

The Response to Stimulation in Neurons and Astrocytes.

The brain uses mainly glucose as fuel with an index of glucose to oxygen utilization close to 6, the maximal index if all glucose was completely oxidized. However, this high oxidative index, contrasts with the metabolic traits of the major cell types in the brain studied in culture, neurons and astrocytes, including the selective use of the malate-aspartate shuttle (MAS) in neurons and the glycerol-phosphate shuttle in astrocytes. Metabolic interactions among these cell types may partly explain the high oxidative index of the brain. In vivo, neuronal activation results in a decrease in the oxygen glucose index, which has been attributed to a stimulation of glycolysis and lactate production in astrocytes in response to glutamate uptake (astrocyte-neuron lactate shuttle, ANLS). Recent findings indicate that this is accompanied with a stimulation of pyruvate formation and astrocyte respiration, indicating that lactate formation is not the only astrocytic response to neuronal activation. ANLS proposes that neurons utilize lactate produced by neighboring astrocytes. Indeed, neurons can use lactate to support an increase in respiration with different workloads, and this depends on the Ca2+ activation of MAS. However, whether this activation operates in the brain, particularly at high stimulation conditions, remains to be established.

Abstract WP337: The Ischemic Neuroprotectant Protein Kinase C Epsilon, Phosphorylates Enzymes in Neuronal NADH Shuttle and Regulates Mitochondrial Respiration and Glycolysis

Background: Among PKCε-mediated potential protective mechanisms in ischemic preconditioning, one target that we surmise is crucial for neuroprotection is the malate-aspartate shuttle (MAS). MAS is the most important NAD + /NADH shuttle importing reducing equivalents (NADH) generated by glycolysis from the cytosol into the mitochondria, and thus, tightly controls neuronal respiration. We hypothesized that PKCε regulates MAS in the brain and mediates neuronal mitochondrial respiration. Method: Phosphoproteomics analysis and immunoprecipitation (IP) were used to determine PKCε-induced phosphorylation of MAS components. Synaptosomes from cortices of naïve SD rats were treated with Tat-conjugated ΨεRACK (selective PKCε activator) or control Tat peptide and sent for phosphoproteomics analysis. To confirm the predicted phosphorylation, IP was performed. SD rats were treated with ΨεRACK (i.p.) or Tat and sacrificed 48hrs later. Proteins from rat cortical synaptosomes were immunoprecipitated with phosphoserine/threonine/tyrosine and immunoblotted for predicted phosphorylated proteins. Once phosphorylation was determined, we evaluated energy metabolism in primary neurons. Neuronal cultures were obtained from embryonic pups cortices and treated with ΨεRACK or Tat on DIV 7-10. Cellular respiration and glycolytic function were evaluated 48hrs after treatment using Seahorse XF Analyzer. Result: Phosphoproteomics analysis and IP results revealed increased phosphorylation of key MAS components: the mitochondrial aspartate aminotransferase (GOT2) and the oxoglutarate carrier (OGC). After PKCε activation, GOT2 and OGC exhibited serine phosphorylation increased by 2.14 fold (P < 0.05, n=4-5), and 1.74 fold (P < 0.01, n=4-5), respectively. In addition, in primary neurons, ΨεRACK treatment increased the basal and maximum cellular respiration by 0.13 (P < 0.05, n=4), and 0.22 fold (P < 0.01, n=4), respectively. The basal and maximum glycolysis were increased by 0.14, and 0.21 fold (P<0.05, n=4), respectively. Conclusion: Our data strongly suggest that PKCε regulates MAS, and increases neuronal metabolism by promoting cellular respiration and glycolysis. These changes may be linked to our observed PKCε-induced ischemic tolerance in the brain.

Changes in the Serum Metabolome of Acute Myocardial Ischemia Rat Pretreatment with Electroacupuncture

Myocardial infarction (MI), the most common symptom is chest pain, occurs when blood flow decreases or stops to a part of the heart, causing damage to the heart muscle. Electroacupuncture pretreatment (EP) is a recent observation which has been shown to induce ischemic tolerance like the ischemia preconditioning, suggesting that EP may be a promising preventive strategy for individual susceptibility to MI. This study investigated mechanisms that underlie the effect of EP on MI through the use of gas chromatography-mass spectrometry (GC-MS)-based metabolic profiling. Male Sprague-Dawley rats were randomly divided to receive or not receive three days of EP at PC6 (Neiguan). Then on the fourth day, each group was further divided to undergo mock surgery or MI, induced by ligation of the left anterior descending coronary artery. After 24h, the blood samples and hearts were collected for the follow-up research. The results showed that treatment by EP significantly reduced the levels of CK-MB, cTnT, AST, and MDH in serum and decreased myocardial infarction area. According to GC-MS-based serum metabolic profiling and analysis, a total of 636 characteristic peaks were identified, including 158 known and 478 unknown metabolites. MI caused comprehensive metabolic changes in glycolysis-related metabolites, malate-aspartate shuttle (MAS) metabolites, and purine metabolites with anti-oxidant functions, while EP reversed more than half of the differential metabolic changes, mainly affecting amino acid and energy metabolism, especially the glutamate metabolism and MAS. In a word, our findings suggest that EP exerts its cardioprotective effect on MI by regulating amino acid and energy metabolisms. Meanwhile, GC-MS-based metabolomics provided a powerful way to characterize the metabolic features of MI, with and without EP, and thereby improved our understanding of the effect and mechanisms of EP.

Mitochondrial Membrane Potential Regulates Nuclear Gene Expression in Macrophages Exposed to Prostaglandin E2

Metabolic engagement is intrinsic to immune cell function. Prostaglandin E2 (PGE2) has been shown to modulate macrophage activation, yet how PGE2 might affect metabolism is unclear. Here, we show that PGE2 caused mitochondrial membrane potential (Δψm) to dissipate in interleukin-4-activated (M(IL-4)) macrophages. Effects on Δψm were a consequence of PGE2-initiated transcriptional regulation of genes, particularly Got1, in the malate-aspartate shuttle (MAS). Reduced Δψm caused alterations in the expression of 126 voltage-regulated genes (VRGs), including those encoding resistin-like molecule α (RELMα), a key marker of M(IL-4) cells, and genes that regulate the cell cycle. The transcription factor ETS variant 1 (ETV1) played a role in the regulation of 38% of the VRGs. These results reveal ETV1 as a Δψm-sensitive transcription factor and Δψm as a mediator of mitochondrial-directed nuclear gene expression.

Molecular basis for increased lactate formation in the Müller glial cells of retina

Müller glial cells are highly metabolic active cells that compensate for the high energy demand of retinal neurons. It has been believed that glucose provides the energy needs by the complete oxidation within Müller cells. However, numerous studies indicated that glial cells convert the majority of glucose to lactate, which may serve as an energy source for neurons. It is still not well understood why within glia, glucose is not completely oxidized under aerobic glycolysis conditions. The aspartate glutamate carrier (AGC) is a major component of the malate-aspartate shuttle (MAS) responsible for transporting the reducing equivalent of glycolysis to the mitochondria for the complete oxidation of glucose. Here, we report the absence of AGC within Müller glial cells which impairs the ability to oxidize glucose. We investigated the expression and localization of AGC and its isoforms (aralar and citrin) in the intact rat retina. We also analyzed the expression and regulation of AGC and its metabolic activity within cultured Müller cells (TR-MUL). The results suggest that AGC and its isoforms seem to be neuronal, with no or low expression within Müller cells of the intact retina. The study of cultured Müller cells suggests a very low expression of AGC and a decreased metabolic activity of the carrier especially under cell differentiation conditions due to low serum and hydrocortisone treatments. Thus, these data give a molecular explanation of increased levels of lactate formation due to a lack of AGC in the retina by Müller glial cells.

Acetyl-CoA from inflammation-induced fatty acids oxidation promotes hepatic malate-aspartate shuttle activity and glycolysis.

Hepatic metabolic syndrome is associated with inflammation, as inflammation stimulates the reprogramming of nutrient metabolism and hepatic mitochondria-generated acetyl-CoA, but how acetyl-CoA affects the reprogramming of nutrient metabolism, especially glucose and fatty acids, in the condition of inflammation is still unclear. Here, we used an acute inflammation model in which pigs were injected with lipopolysaccharide (LPS) and found that hepatic glycolysis and fatty acid oxidation are both promoted. Acetyl-proteome profiling of LPS-infected pigs liver showed that inflammatory stress exacerbates the acetylation of mitochondrial proteins. Both mitochondrial glutamate oxaloacetate transaminase 2 (GOT2) and malate dehydrogenase 2 (MDH2) were acetylated, and the malate-aspartate shuttle (MAS) activity was stimulated to maintain glycolysis. With the use of 13C-carbon tracing in vitro, acetyl-CoA was found to be mainly supplied by lipid-derived fatty acid oxidation rather than glucose-derived pyruvate oxidative decarboxylation, while glucose was mainly used for lactate production in response to inflammatory stress. The results of the mitochondrial experiment showed that acetyl-CoA directly increases MDH2 and, in turn, the GOT2 acetylation level affects MAS activity. Treatment with palmitate in primary hepatocytes from LPS-injected pigs increased the hepatic production of acetyl-CoA, pyruvate, and lactate; MAS activity; and hepatic MDH2 and GOT2 hyperacetylation, while the deficiency of long-chain acetyl-CoA dehydrogenase resulted in the stabilization of these parameters. These observations suggest that acetyl-CoA produced by fatty acid oxidation promotes MAS activity and glycolysis via nonenzymatic acetylation during the inflammatory stress response.

Integration between Glycolysis and Glutamate-Glutamine Cycle Flux May Explain Preferential Glycolytic Increase during Brain Activation, Requiring Glutamate.

The 1988 observation by Fox et al. (1988) that brief intense brain activation increases glycolysis (pyruvate formation from glucose) much more than oxidative metabolism has been abundantly confirmed. Specifically glycolytic increase was unexpected because the amount of ATP it generates is much smaller than that formed by subsequent oxidative metabolism of pyruvate. The present article shows that preferential glycolysis can be explained by metabolic processes associated with activation of the glutamate-glutamine cycle. The flux in this cycle, which is essential for production of transmitter glutamate and GABA, equals 75% of brain glucose utilization and each turn is associated with utilization of ~1 glucose molecule. About one half of the association between cycle flux and glucose metabolism occurs during neuronal conversion of glutamine to glutamate in a process similar to the malate-aspartate shuttle (MAS) except that glutamate is supplied from glutamine, not formed from α-ketoglutarate (αKG) as during operation of conventional MAS. Regular MAS function is triggered by one oxidative process in the cytosol during glycolysis causing NAD+ reduction to NADH. Since NADH cannot cross the mitochondrial membrane (MEM) for oxidation NAD+ is re-generated by conversion of cytosolic oxaloacetate (OAA) to malate, which enters the mitochondria for oxidation and in a cyclic process regenerates cytosolic OAA. Therefore MAS as well as the “pseudo-MAS” necessary for neuronal glutamate formation can only operate together with cytosolic reduction of NAD+ to NADH. The major process causing NAD+ reduction is glycolysis which therefore also must occur during neuronal conversion of glutamine to glutamate and may energize vesicular glutamate uptake which preferentially uses glycolytically derived energy. Another major contributor to the association between glutamate-glutamine cycle and glucose utilization is the need for astrocytic pyruvate to generate glutamate. Although some oxidative metabolism occurs during glutamate formation it is only one half of that during normal tricarboxylic acid (TCA) cycle function. Glutamate’s receptor stimulation leads to potassium ion (K+) release and astrocytic uptake, preferentially fueled by glycolysis and followed by release and neuronal re-accumulation. The activation-induced preferential glycolysis diminishes with continued activation and is followed by an increased ratio between oxidative metabolism and glycolysis, reflecting oxidation of generated glutamate and accumulated lactate.

ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate.

ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain invivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells.

Pre-ischaemic mitochondrial substrate constraint by inhibition of malate-aspartate shuttle preserves mitochondrial function after ischaemia-reperfusion.

Pre-ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against ischaemia-reperfusion injury. The underlying mechanism remains unknown. We examined whether transient inhibition of the MAS during ischaemia and early reperfusion by AOA treatment could prevent mitochondrial damage at later reperfusion. The AOA treatment preserved mitochondrial respiratory capacity with reduced mitochondrial oxidative stress during late reperfusion to the same extent as ischaemic preconditioning (IPC). However, AOA treatment, but not IPC, reduced the myocardial interstitial concentration of tricarboxylic acid cycle intermediates at the onset of reperfusion. The results obtained in the present study demonstrate that metabolic regulation by inhibition of the MAS at the onset of reperfusion may be beneficial for the preservation of mitochondrial function during late reperfusion in an IR-injured heart. Mitochondrial dysfunction plays a central role in ischaemia-reperfusion (IR) injury. Pre-ischaemic administration of aminooxyacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against IR injury, although the underlying mechanism remains unknown. We hypothesized that a transient inhibition of the MAS during ischaemia and early reperfusion could preserve mitochondrial function at later phase of reperfusion in the IR-injured heart to the same extent as ischaemic preconditioning (IPC), which is a well-validated cardioprotective strategy against IR injury. In the present study, we show that pre-ischaemic administration of AOA preserved mitochondrial complex I-linked state 3 respiration and fatty acid oxidation during late reperfusion in IR-injured isolated rat hearts. AOA treatment also attenuated the excessive emission of mitochondrial reactive oxygen species during state 3 with complex I-linked substrates during late reperfusion, which was consistent with reduced oxidative damage in the IR-injured heart. As a result, AOA treatment reduced infarct size after reperfusion. These protective effects of MAS inhibition on the mitochondria were similar to those of IPC. Intriguingly, the protection of mitochondrial function by AOA treatment appears to be different from that of IPC because AOA treatment, but not IPC, downregulated myocardial tricarboxilic acid (TCA)-cycle intermediates at the onset of reperfusion. MAS inhibition thus preserved mitochondrial respiratory capacity and decreased mitochondrial oxidative stress during late reperfusion in the IR-injured heart, at least in part, via metabolic regulation of TCA cycle intermediates in the mitochondria at the onset of reperfusion.

The cardioprotective effect of sildenafil is mediated by the activation of malate dehydrogenase and an increase in the malate-aspartate shuttle in cardiomyocytes

Recent evidence has shown the cardioprotective effect of PDE5 inhibition in myocardial ischemia/reperfusion injury, heart failure and cardiac hypertrophy. To investigate the biochemical changes that occur during PDE5 inhibition in cardiac cells, this study assessed the metabolic profile of the HL1 cell line, a murine atrial cell line with adult cardiomyocyte properties. After one hour of treatment with sildenafil, glycolysis was moderately but selectively stimulated, unlike the pentose phosphate pathway and the Krebs cycle. Moreover, malate and a-Ketoglutarate accumulated, paralleled by a decrease in aspartate and glutamate. Interestingly, increased activity of malate dehydrogenase (MDH) was also detected in these cells after sildenafil treatment. Thus, we hypothesized that sildenafil stimulates the malate-aspartate shuttle (MAS) with the final effect of transferring electrons and protons from glycolysis-derived cytosolic NADH into the matrix for use by the electron transport chain, using malate as an electron carrier. Through this metabolic modification, sildenafil may counteract what is often observed in ischemia, i.e. reduced MAS flux as well as a dramatic acceleration of glycolysis, which switches to lactate production. Additionally, the results observed in HL1 cells were also found in isolated mouse hearts. The documented metabolic alteration in cardiomyocytes upon treatment with sildenafil occurred by stimulating cGMP production, which did not activate PKG (cGMP-PKG signaling), since the addition of DT-2, a PKG inhibitor, did not block malate accumulation and increased MDH activity. Conversely, the addition of chelerythrine, a PKC inhibitor, counteracted both malate accumulation and MAS activation, supporting previous evidence that, upon the addition of sildenafil, some PKC isoforms may be implicated in cardioprotection (cGMP-PKC signaling). Interestingly, an increase in cGMP, driven by sildenafil, another cGMP stimulator such as nitroprusside (SNP), or a C-type natriuretic peptide (CNP) which does not inhibit PDE5, led to MAS stimulation and increased MDH activity.

Malate-aspartate shuttle inhibitor aminooxyacetic acid leads to decreased intracellular ATP levels and altered cell cycle of C6 glioma cells by inhibiting glycolysis

NADH shuttles, including malate-aspartate shuttle (MAS) and glycerol-3-phosphate shuttle, can shuttle the reducing equivalents of cytosolic NADH into mitochondria. It is widely accepted that the major function of NADH shuttles is to increase mitochondrial energy production. Our study tested the hypothesis that the novel major function of NADH shuttles in cancer cells is to maintain glycolysis by decreasing cytosolic NADH/NAD+ ratios. We found that AOAA, a widely used MAS inhibitor, led to decreased intracellular ATP levels, altered cell cycle and increased apoptosis and necrosis of C6 glioma cells, without affecting the survival of primary astrocyte cultures. AOAA also decreased the glycolytic rate and the levels of extracellular lactate and pyruvate, without affecting the mitochondrial membrane potential of C6 cells. Moreover, the toxic effects of AOAA were completely prevented by pyruvate treatment. Collectively, our study has suggested that AOAA may be used to selectively decrease glioma cell survival, and the major function of MAS in cancer cells may be profoundly different from its major function in normal cells: The major function of MAS in cancer cells is to maintain glycolysis, instead of increasing mitochondrial energy metabolism.

The regulation of neuronal mitochondrial metabolism by calcium.

Calcium signalling is fundamental to the function of the nervous system, in association with changes in ionic gradients across the membrane. Although restoring ionic gradients is energetically costly, a rise in intracellular Ca(2+) acts through multiple pathways to increase ATP synthesis, matching energy supply to demand. Increasing cytosolic Ca(2+) stimulates metabolite transfer across the inner mitochondrial membrane through activation of Ca(2+) -regulated mitochondrial carriers, whereas an increase in matrix Ca(2+) stimulates the citric acid cycle and ATP synthase. The aspartate-glutamate exchanger Aralar/AGC1 (Slc25a12), a component of the malate-aspartate shuttle (MAS), is stimulated by modest increases in cytosolic Ca(2+) and upregulates respiration in cortical neurons by enhancing pyruvate supply into mitochondria. Failure to increase respiration in response to small (carbachol) and moderate (K(+) -depolarization) workloads and blunted stimulation of respiration in response to high workloads (veratridine) in Aralar/AGC1 knockout neurons reflect impaired MAS activity and limited mitochondrial pyruvate supply. In response to large workloads (veratridine), acute stimulation of respiration occurs in the absence of MAS through Ca(2+) influx through the mitochondrial calcium uniporter (MCU) and a rise in matrix [Ca(2+) ]. Although the physiological importance of the MCU complex in work-induced stimulation of respiration of CNS neurons is not yet clarified, abnormal mitochondrial Ca(2+) signalling causes pathology. Indeed, loss of function mutations in MICU1, a regulator of MCU complex, are associated with neuromuscular disease. In patient-derived MICU1 deficient fibroblasts, resting matrix Ca(2+) is increased and mitochondria fragmented. Thus, the fine tuning of Ca(2+) signals plays a key role in shaping mitochondrial bioenergetics.

Malate-Aspartate Shuttle Inhibitor Aminooxyacetate Acid Induces Apoptosis and Impairs Energy Metabolism of Both Resting Microglia and LPS-Activated Microglia.

NADH shuttles mediate the transfer of the reducing equivalents of cytosolic NADH into mitochondria. Cumulating evidence has suggested that malate-aspartate shuttle (MAS), one of the two types of NADH shuttles, plays significant roles in such biological processes as glutamate synthesis in neurons. However, there has been no information regarding the roles of NADH shuttle in the survival and energy metabolism of microglia. In current study, using microglial BV2 cells as a cellular model, we determined the roles of MAS in the survival and energy metabolism of microglia by using aminooxyacetate acid (AOAA)-a widely used MAS inhibitor. Our study has suggested that AOAA can effectively inhibit the MAS activity of the cells. We also found that AOAA can induce both early- and late-stage apoptosis of resting microglia and lipopolysaccharides (LPS)-activated microglia. AOAA also induced mitochondrial depolarization, increases in the cytosolic Ca(2+) concentrations, and decreases in the intracellular ATP levels. Moreover, our study has excluded the possibility that the major nonspecific effect of AOAA-inhibition of GABA transaminase-is involved in theses effects of AOAA. Collectively, our study has provided first information suggesting significant roles of MAS in the survival and energy metabolism in both resting microglia and LPS-activated microglia.

L-malate enhances the gene expression of carried proteins and antioxidant enzymes in liver of aged rats.

Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. To investigate the antioxidant mechanism of L-malate in the mitochondria, we analyzed the change in gene expression of two malate-aspartate shuttle (MAS)-related carried proteins (AGC, aspartate/glutamate carrier and OMC, oxoglutarate/malate carrier) in the inner mitochondrial membrane, and three antioxidant enzymes (CAT, SOD, and GSH-Px) in the mitochondria. The changes in gene expression of these proteins and enzymes were examined by real-time RT-PCR in the heart and liver of aged rats treated with L-malate. L-malate was orally administered in rats continuously for 30 days using a feeding atraumatic needle. We found that the gene expression of OMC and GSH-Px mRNA in the liver increased by 39 % and 38 %, respectively, in the 0.630 g/kg L-malate treatment group than that in the control group. The expression levels of SOD mRNA in the liver increased by 39 %, 56 %, and 78 % in the 0.105, 0.210, and 0.630 g/kg L-malate treatment groups, respectively. No difference were observed in the expression levels of AGC, OMC, CAT, SOD, and GSH-Px mRNAs in the heart of rats between the L-malate treatment and control groups. These results predicted that L-malate may increase the antioxidant capacity of mitochondria by enhancing the expression of mRNAs involved in the MAS and the antioxidant enzymes.