Mitochondrial Oxidation of the Cytoplasmic Reducing Equivalents at the Onset of Oxidant Stress in the Isoproterenol-Induced Rat Myocardial Infarction

We have developed and characterized a model of isoproterenol (ISO)-induced myocardial necrosis, identifying three stages of cardiac damage: a pre-infarction (0–12 h), infarction (24 h), and post-infarction period (48–96 h). Using this model, we have previously found alterations in calcium homeostasis and their relationship with oxidant stress in mitochondria, which showed deficient oxygen consumption and coupled ATP synthesis. Therefore, the present study was aimed at assessing the mitochondrial ability to transport and oxidize cytoplasmic reducing equivalents (NADH), correlating the kinetic parameters of the malate-aspartate shuttle, oxidant stress, and mitochondrial functionality. Our results showed only discreet effects during the cardiotoxic ISO action on the endogenous malate-aspartate shuttle activity, suggesting that endogenous mitochondrial NADH oxidation capacity (Nohl dehydrogenase) was not affected by the cellular stress. On the contrary, the reconstituted system showed significant enhancement in maximal capacity of the malate-aspartate shuttle activity only at later times (post-infarction period), probably as a compensatory part of cardiomyocytes’ response to the metabolic and functional consequences of the infarcted tissue. Therefore, these findings support the notion that heart damage associated with myocardial infarction suffers a set of sequential biochemical and metabolic modifications within cardiomyocytes, where mitochondrial activity, controlling the redox state, could play a relevant role.

Malate-Aspartate Shuttle Plays an Important Role in LPS-Induced Neuroinflammation of Mice Due to its Effect on STAT3 Phosphorylation

Neuroinflammation is a key pathological factor in numerous neurological disorders. Cumulating evidence has indicated critical roles of NAD+/NADH metabolism in multiple major diseases, while the role of malate-aspartate shuttle (MAS) - a major NADH shuttle - in inflammation has remained unclear. In this study we investigated the roles of MAS in LPS-induced neuroinflammation both in vivo and in vitro. Immunofluorescence staining, Western blot assay and Real-time PCR assays were conducted to determine the activation of Iba-1, the protein levels of iNOS and COX2 and the mRNA levels of IL-1β, IL-6, and TNF-α in vivo, showing that both pre-treatment and post-treatment of aminooxyacetic acid (AOAA) - an MAS inhibitor - profoundly decreased the LPS-induced neuroinflammation in mice. BV2 microglia was also used as a cellular model to investigate the mechanisms of this finding, in which such assays as Western blot assay and nitrite assay. Our study further indicated that AOAA produced its effects on LPS-induced microglial activation by its effects on MAS: Pyruvate treatment reversed the effects of AOAA on the cytosolic NAD+/NADH ratio, which also restored the LPS-induced activation of the AOAA-treated microglia. Moreover, the lactate dehydrogenase (LDH) inhibitor GSK2837808A blocked the effects of pyruvate on the AOAA-produced decreases in both the cytosolic NAD+/NADH ratio and LPS-induced microglial activation. Our study has further suggested that AOAA produced inhibition of LPS-induced microglial activation at least partially by decreasing STAT3 phosphorylation. Collectively, our findings have indicated AOAA as a new and effective drug for inhibiting LPS-induced neuroinflammation. Our study has also indicated that MAS is a novel mediator of LPS-induced neuroinflammation due to its capacity to modulate LPS-induced STAT3 phosphorylation, which has further highlighted a critical role of NAD+/NADH metabolism in inflammation.

“A feed-forward Ca2+-dependent mechanism boosting glycolysis and OXPHOS by activating Aralar-malate-aspartate shuttle, upon neuronal stimulation”

SummaryCalcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In cortical neurons using glucose as only fuel, activation by NMDA, which elicits a strong workload dependent on Na+ entry, stimulates glucose uptake, glycolysis, pyruvate and lactate production, and OXPHOS in a Ca2+-dependent way. We find that Ca2+-upregulation of glycolysis, pyruvate levels and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the Ca2+regulated mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). Ca2+-activation of MAS increases pyruvate production, which directly fuels workload-stimulated respiration. Also it stimulates glycolysis. MCU silencing had no effect indicating that none of these processes required mitochondrial Ca2+. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We also find that cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+ flow maintaining basal cell bioenergetics in which Ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ release, and in which MCU does not participate. The results reveal that in neurons using glucose MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.

Deficiency of malate-aspartate shuttle component SLC25A12 induces pulmonary metastasis

Background Aspartate biosynthesis and its delivery to the cytosol can be crucial for tumor growth in vivo. However, the impact of intracellular aspartate levels on metastasis has not been studied. We previously described that loss-of-aspartate glutamate carrier 1 (SLC25A12 or AGC1), an important component of the malate-aspartate shuttle, impairs cytosolic aspartate levels, NAD+/NADH ratio, mitochondrial respiration, and tumor growth. Here, we report the impact of AGC1-knockdown on metastasis. Results Low AGC1 expression correlates with worse patient prognosis in many cancers. AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis following subcutaneous or intravenous injections, respectively. On the other hand, conventional in vitro metastasis assays show no indication of increased metastasis capacity of AGC1-knockdown cells. Conclusion This study highlights that certain branches of metabolism impact tumor growth and tumor metastasis differently. In addition, it also argues that commonly known metastasis indicators, including EMT genes, cell migration, or colony formation, do not always reflect metastatic capacity in vivo.

ACTIVITY OF MALATE-ASPARTATE SHUTTLE ENZYMES OF BULLS AND BOARS REPRODUCTIVE ORGANS AND EPIDIDIMAL SPERMATOZOA

The activity of malate-aspartate shuttle enzymes in the reproductive organs and epididimal sperm of bulls and boars was studied. The research was conducted on bulls of the Ukrainian black-spotted dairy breed (n = 5; aged 14 - 16 months) and boars of the Great White breed (n = 3; aged 10 - 12 months). After slaughter, the testes and testicular appendages were removed and the spermatozoa were washed with 0.9% sodium chloride solution. Testicular and epididimal tissues were homogenized and centrifuged. Aspartate aminotransferase (AST), malate dehydrogenase (MDG) and protein concentration were determined in the supernatant and epididimal sperm. The specie and tissue specificity of aspartate aminotransferase and malate dehydrogenase activity have been established. In particular, in bulls, the activity of AST in the tissues of the testis, head and body of the epididymis of bulls is almost the same (116.5 - 118.3 nmol/min×mg of protein) and on 18.5 - 19.7% (р<0,05) higher in the tail of the appendix. In this case, MDG activity in the reproductive organs shows wavy changes: in the testicular tissue is high (1.96 ± 0.15 nmol / min × mg of protein), and in the epididymis: in the head is reduced (3.5 times ; p <0,001), increased in the body (p <0,05) and decreased again in the tail. In the sperm of bulls, the activity of AST from the head of the appendix - 69.3 ± 8.06 nmol / min × mg of protein, from the body - increased (p <0.05) and again (p <0.001) decreased from the tail of the appendix. MDG activity in sperm from the head of the epididymis was 0.50 ± 0.04 nmol/min×mg of protein, lower by 38.0% (p <0.05) from the body and 50.0% (p <0.001) higher in the tail. In boars, the activity of AST in testicular tissue - 20.3 ± 5.22 nmol / min × mg of protein and higher in the appendix: 45.6% - in the head, 59.0% - in the body and 64.0 % - in the tail. Similarly, the activity of MDG in testicular tissue was 0.22 ± 0.02 nmol / min × mg of protein, increases by 40.6% in the head and remained at the same level in the body of the appendix, and in the tail tissue above 2, 5 - 2.7 times (p <0.001). In bovine germ cells, the activity of AST and MDG from the body of the appendix was, respectively, 102.3 ± 6.1 and 13.20 ± 0.15 nmol / min × mg of protein, lowered twice (p <0,001) and 10.7 % in the head and more than four times (p <0,001) and 61,2% (p <0,001) in the tail of the appendix.

C/EBPβ-LIP dually activates glycolysis and the malate aspartate shuttle to maintain NADH/NAD+ homeostasis

Oncogene-induced metabolic reprograming supports cell growth and proliferation. Yet, it also links cancer cell survival to certain metabolic pathways and nutrients. In order to synthesise amino acids and nucleotides de novo for growth and proliferation, cancer cells depend on glycolysis, the cytoplasmic oxidation of glucose, which generates necessary metabolic intermediates and ATP. During glycolysis, NAD+ is used as the oxidizing agent and is thereby reduced into NADH. To ensure high glycolysis rates and maintain NADH/NAD+ homeostasis, cytoplasmic NAD+ has to be regenerated. The mitochondria are the major sites of NADH reoxidation into NAD+ where NADH-derived electrons enter the electron transport chain for ATP production. Since NADH/NAD+ cannot cross membranes, the malate-aspartate shuttle (MAS) or the glycerol-3-phosphate shuttle (GPS) are used as intermediate electron carriers. In addition, cytoplasmic NAD+ is generated by NADH-electron transfer to pyruvate, reducing it to lactate (the Warburg effect). NADH/NAD+ homeostasis plays a pivotal role in cancer cell survival, but our knowledge about the involved regulatory mechanisms is still limited. Here, we show that the proto-oncogenic transcription factor C/EBPβ-LIP stimulates both glycolysis and the MAS. Inhibition of glycolysis with ongoing C/EBPβ-LIP-induced MAS activity results in NADH depletion and apoptosis that can be rescued by inhibiting either the MAS or other NADH-consuming processes. Therefore, beyond the discovery of C/EBPβ-LIP as a dual activator of glycolysis and the MAS, this study indicates that simultaneous inhibition of glycolysis and lowering of the NADH/NAD+ ratio may be considered to treat cancer.

The Impact of Acetyl-CoA and Aspartate Shortages on the N-Acetylaspartate Level in Different Models of Cholinergic Neurons

N-acetylaspartate is produced by neuronal aspartate N-acetyltransferase (NAT8L) from acetyl-CoA and aspartate. In cholinergic neurons, acetyl-CoA is also utilized in the mitochondrial tricarboxylic acid cycle and in acetylcholine production pathways. While aspartate has to be shared with the malate–aspartate shuttle, another mitochondrial machinery together with the tricarboxylic acid cycle supports the electron transport chain turnover. The main goal of this study was to establish the impact of toxic conditions on N-acetylaspartate production. SN56 cholinergic cells were exposed to either Zn2+ overload or Ca2+ homeostasis dysregulation and male adult Wistar rats’ brains were studied after 2 weeks of challenge with streptozotocin-induced hyperglycemia or daily theophylline treatment. Our results allow us to hypothesize that the cholinergic neurons from brain septum prioritized the acetylcholine over N-acetylaspartate production. This report provides the first direct evidence for Zn2+-dependent suppression of N-acetylaspartate synthesis leading to mitochondrial acetyl-CoA and aspartate shortages. Furthermore, Zn2+ is a direct concentration-dependent inhibitor of NAT8L activity, while Zn2+-triggered oxidative stress is unlikely to be significant in such suppression.

Loss of malate-aspartate shuttle component SLC25A12 induces pulmonary metastasis

BackgroundAspartate biosynthesis and its delivery to the cytosol can be crucial for tumor growth in vivo. However, the impact of aspartate synthesis on metastasis has not been studied. We previously described that loss-of-aspartate glutamate carrier 1 (SLC25A12 or AGC1), an important component of the malate-aspartate shuttle, impairs cytosolic aspartate levels, NAD+/NADH ratio, mitochondrial respiration, and tumor growth. Here, we report the impact of AGC1-knockdown on metastasis.ResultsAGC1 expression is positively correlated with worse patient prognosis in many cancers. AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis following subcutaneous or intravenous injections, respectively. On the other hand, conventional in vitro metastasis assays show no indication of increased metastasis capacity of AGC1-knockdown cells.ConclusionThis study highlights that certain branches of metabolism impact tumor growth and tumor metastasis differently. In addition, it also argues that commonly known metastasis indicators, including EMT genes, cell migration, or colony formation do not always reflect the metastatic capacity in vivo.