Respiratory Chain Inhibitors Research Articles

ATP Restoration by ATP-Deprived Cultured Primary Astrocytes

A high cellular concentration of adenosine triphosphate (ATP) is essential to fuel many important functions of brain astrocytes. Although cellular ATP depletion has frequently been reported for astrocytes, little is known on the metabolic pathways that contribute to ATP restoration by ATP-depleted astrocytes. Incubation of cultured primary rat astrocytes in glucose-free buffer for 60 min with the mitochondrial uncoupler BAM15 lowered the cellular ATP content by around 70%, the total amount of adenosine phosphates by around 50% and the adenylate energy charge (AEC) from 0.9 to 0.6. Testing for ATP restoration after removal of the uncoupler revealed that the presence of glucose as exclusive substrate allowed the cells to restore within 6 h around 80% of the initial ATP content, while coapplication of adenosine plus glucose enabled the cells to fully restore their initial ATP content within 60 min. A rapid but incomplete and transient ATP restoration was found for astrocytes that had been exposed to adenosine alone. This restoration was completely prevented by application of the pyruvate uptake inhibitor UK5099, the respiratory chain inhibitor antimycin A or by the continuous presence of BAM15. However, the presence of these compounds strongly accelerated the release of lactate from the cells, suggesting that the ribose moiety of adenosine can serve as substrate to fuel some ATP restoration via mitochondrial metabolism. Finally, the adenosine-accelerated ATP restoration in glucose-fed astrocytes was inhibited by the presence of the adenosine kinase inhibitor ABT-702. These data demonstrate that astrocytes require for a rapid and complete ATP restoration the presence of both glucose as substrate and adenosine as AMP precursor.

Facilitating Intracellular Electron Bifurcation by Mediating Flavin-Based Extracellular and Transmembrane Electron Transfer: A Novel Role of Pyrogenic Carbon in Dark Fermentation for Hydrogen Production.

Pyrogenic carbon is considered an enhancer to H2-yielding dark fermentation (DF), but little is known about how it regulates extracellular electron transfer (EET) and influences transmembrane respiratory chains and intracellular metabolisms. This study addressed these knowledge gaps and demonstrated that wood waste pyrogenic carbon (biochar) could significantly improve the DF performance; e.g., addition of pyrogenic carbon produced by pyrolysis at 800 °C (PC800) increased H2 yield by 369.7%. Biochemical quantification, electrochemical analysis, and electron respiratory chain inhibition tests revealed that PC800 promoted the extracellular flavin-based electron transfer process and further activated the acceleration of the transmembrane electron transfer. Comparative metagenome/metatranscriptome analyses indicated that the flavin-containing Rnf complex was the potential transmembrane respiratory enzyme associated with PC800-mediated EET. Based on NADH/NAD+ circulation, the promoted Rnf complex could stimulate the functions of the electron bifurcating Etf/Bcd complex and startup of glycolysis. The promoted Etf/Bcd could further contribute to balance the NADH/NAD+ level for glycolytic reactions and meanwhile provide reduced ferredoxin for group A1 [FeFe]-hydrogenases. This proton-energy-linked mechanism could achieve coupling production of ATP and H2. This study verified the important roles of pyrogenic carbon in mediating EET and transmembrane/intracellular pathways and revealed the crucial roles of electron bifurcation in DF for hydrogen production.

Pomalidomide sensitizes lung cancer cells to TRAIL/CDDP-induced apoptosis via directly targeting electron transfer flavoprotein alpha subunit

Immunomodulatory drugs (IMiDs) represented by thalidomide exhibit benefits when combined with other chemotherapeutic drugs for patients with lung cancer, which inspired the exploration of combining pomalidomide with another agent to treat lung cancer as it is more potent than thalidomide. However, the drugs that can be combined with pomalidomide to benefit patients and related mechanisms remain unclear. Here, we performed a proteomic analysis based on the streptavidin pull-down to identify the potential target of pomalidomide in non-small cell lung cancer (NSCLC). In this work, electron transfer flavoprotein alpha subunit (ETFA), an important enzyme involved in electron transport in the respiratory chains was identified as a crucial cellular target of pomalidomide in NCI-H460 cells. Using apoptosis model and combination analyses, we found that pomalidomide directly targeted ETFA, and increased ATP generation, thereby significantly promoting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Specific knockdown of ETFA could effectively eliminate the promoting effect of pomalidomide on energy production. Furthermore, respiratory chain inhibitors can effectively block cell apoptosis induced by TRAIL and pomalidomide. These results suggested that pomalidomide may promote apoptosis by facilitating energy production by targeting ETFA and thus enhanced the anticancer effects of chemotherapeutic drugs. It is noteworthy that pomalidomide noticeably increased the anticancer efficacy of cisplatin (CDDP) in NCI-H460 xenograft model with the main mechanisms by inducing apoptosis. Collectively, our data not only provide new insights into the anticancer mechanisms of pomalidomide but also reflect translational prospects of combining pomalidomide with CDDP for NSCLC treatment.

A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy.MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures

Astrocyte-derived pyruvate is considered to have neuroprotective functions. In order to investigate the processes that are involved in astrocytic pyruvate release, we used primary rat astrocyte cultures as model system. Depending on the incubation conditions and medium composition, astrocyte cultures established extracellular steady state pyruvate concentrations in the range between 150 µM and 300 µM. During incubations for up to 2 weeks in DMEM culture medium, the extracellular pyruvate concentration remained almost constant for days, while the extracellular lactate concentration increased continuously during the incubation into the millimolar concentration range as long as glucose was present. In an amino acid-free incubation buffer, glucose-fed astrocytes released pyruvate with an initial rate of around 60 nmol/(h × mg) and after around 5 h an almost constant extracellular pyruvate concentration was established that was maintained for several hours. Extracellular pyruvate accumulation was also observed, if glucose had been replaced by mannose, fructose, lactate or alanine. Glucose-fed astrocyte cultures established similar extracellular steady state concentrations of pyruvate by releasing pyruvate into pyruvate-free media or by consuming excess of extracellular pyruvate. Inhibition of the monocarboxylate transporter MCT1 by AR-C155858 lowered extracellular pyruvate accumulation, while inhibition of mitochondrial pyruvate uptake by UK5099 increased the extracellular pyruvate concentration. Finally, the presence of the uncoupler BAM15 or of the respiratory chain inhibitor antimycin A almost completely abolished extracellular pyruvate accumulation. The data presented demonstrate that cultured astrocytes establish a transient extracellular steady state concentration of pyruvate which is strongly affected by modulation of the mitochondrial pyruvate metabolism.

Chronic and acute exposure to rotenone reveals distinct Parkinson's disease-related phenotypes in human iPSC-derived peripheral neurons

Peripheral autonomic nervous system (P-ANS) dysfunction is a critical non-motor phenotype of Parkinson's disease (PD). The majority of PD cases are sporadic and lack identified PD-associated genes involved. Epidemiological and animal model studies suggest an association with pesticides and other environmental toxins. However, the cellular mechanisms underlying toxin induced P-ANS dysfunctions remain unclear. Here, we mapped the global transcriptome changes in human induced pluripotent stem cell (iPSC) derived P-ANS sympathetic neurons during inhibition of the mitochondrial respiratory chain by the PD-related pesticide, rotenone. We revealed distinct transcriptome profiles between acute and chronic exposure to rotenone. In the acute stage, there was a down regulation of specific cation channel genes, known to mediate electrophysiological activity, while in the chronic stage, the human P-ANS neurons exhibited dysregulation of anti-apoptotic and Golgi apparatus-related pathways. Moreover, we identified the sodium voltage-gated channel subunit SCN3A/Nav1.3 as a potential biomarker in human P-ANS neurons associated with PD. Our analysis of the rotenone-altered coding and non-coding transcriptome of human P-ANS neurons may thus provide insight into the pathological signaling events in the sympathetic neurons during PD progression.

Mitochondrial Esterase Activity Measured at the Single Organelle Level by Nano-flow Cytometry.

Monitoring mitochondrial esterase activity is crucial not only for investigating mitochondrial metabolism but also for assessing the effectiveness of mitochondrial-targeting prodrugs. However, accurately detecting esterase activity within mitochondria poses challenges due to its ubiquitous presence in cells and the uncontrolled localization of fluorogenic probes. To overcome this hurdle and reveal variations among different mitochondria, we isolated mitochondria and preserved their activity and functionality in a buffered environment. Subsequently, we utilized a laboratory-built nano-flow cytometer in conjunction with an esterase-responsive calcein-AM fluorescent probe to measure the esterase activity of individual mitochondria. This approach enabled us to investigate the influence of temperature, pH, metal ions, and various compounds on the mitochondrial esterase activity without any interference from other cellular constituents. Interestingly, we observed a decline in the mitochondrial esterase activity following the administration of mitochondrial respiratory chain inhibitors. Furthermore, we found that mitochondrial esterase activity was notably higher in the presence of a high concentration of ATP compared to that of ADP and AMP. Additionally, we noticed a correlation between elevated levels of complex IV and increased mitochondrial esterase activity. These findings suggest a functional connection between the mitochondrial respiratory chain and mitochondrial esterase activity. Moreover, we detected an upsurge in mitochondrial esterase activity during the early stages of apoptosis, while cellular esterase activity decreased. This highlights the significance of analyzing enzyme activity within specific organelle subregions. In summary, the integration of a nano-flow cytometer and fluorescent dyes introduces a novel method for quantifying mitochondrial enzyme activity with the potential to uncover the alterations and unique functions of other mitochondrial enzymes.

Definition of the Neurotoxicity-Associated Metabolic Signature Triggered by Berberine and Other Respiratory Chain Inhibitors.

To characterize the hits from a phenotypic neurotoxicity screen, we obtained transcriptomics data for valinomycin, diethylstilbestrol, colchicine, rotenone, 1-methyl-4-phenylpyridinium (MPP), carbaryl and berberine (Ber). For all compounds, the concentration triggering neurite degeneration correlated with the onset of gene expression changes. The mechanistically diverse toxicants caused similar patterns of gene regulation: the responses were dominated by cell de-differentiation and a triggering of canonical stress response pathways driven by ATF4 and NRF2. To obtain more detailed and specific information on the modes-of-action, the effects on energy metabolism (respiration and glycolysis) were measured. Ber, rotenone and MPP inhibited the mitochondrial respiratory chain and they shared complex I as the target. This group of toxicants was further evaluated by metabolomics under experimental conditions that did not deplete ATP. Ber (204 changed metabolites) showed similar effects as MPP and rotenone. The overall metabolic situation was characterized by oxidative stress, an over-abundance of NADH (>1000% increase) and a re-routing of metabolism in order to dispose of the nitrogen resulting from increased amino acid turnover. This unique overall pattern led to the accumulation of metabolites known as biomarkers of neurodegeneration (saccharopine, aminoadipate and branched-chain ketoacids). These findings suggest that neurotoxicity of mitochondrial inhibitors may result from an ensemble of metabolic changes rather than from a simple ATP depletion. The combi-omics approach used here provided richer and more specific MoA data than the more common transcriptomics analysis alone. As Ber, a human drug and food supplement, mimicked closely the mode-of-action of known neurotoxicants, its potential hazard requires further investigation.

Inhibition of the mitochondrial respiratory chain reduces catecholamine‑stimulated lipolysis via increasing lactate production in 3T3‑L1 adipocytes.

Adipose tissue serves a significant role in the regulation of energy metabolism in the body. The re‑esterification of the fatty acids generated during lipolysis is critical for efficient lipolysis. However, the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis is not yet fully understood. The present study aimed to assess the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis. 3T3‑L1 adipocytes were incubated with mitochondrial respiratory chain inhibitors, oligomycin A or rotenone, during isoproterenol stimulation; and glycerol, glucose and lactate concentrations in the medium were measured. Western blot analysis was performed to examine the phosphorylation levels of cAMP‑dependent protein kinase A (PKA). The results showed that inhibition of mitochondrial ATP synthesis decreased catecholamine‑stimulated lipolysis without affecting PKA signaling. The inhibition of mitochondrial respiration increased glucose uptake and lactate production, indicating that a large amount of glucose taken up into the cell was preferentially used for ATP production rather than for re‑esterification. In conclusion, the results of the present study suggested that the energy state during lipolysis may influence lipolytic activity by suppressing fatty acid re‑esterification.

Modulation of Cellular Levels of Adenosine Phosphates and Creatine Phosphate in Cultured Primary Astrocytes

Adenosine triphosphate (ATP) is the main energy currency of all cells, while creatine phosphate (CrP) is considered as a buffer of high energy-bond phosphate that facilitates rapid regeneration of ATP from adenosine diphosphate (ADP). Astrocyte-rich primary cultures contain ATP, ADP and adenosine monophosphate (AMP) in average specific contents of 36.0 ± 6.4 nmol/mg, 2.9 ± 2.1 nmol/mg and 1.7 ± 2.1 nmol/mg, respectively, which establish an adenylate energy charge of 0.92 ± 0.04. The average specific cellular CrP level was found to be 25.9 ± 10.8 nmol/mg and the CrP/ATP ratio was 0.74 ± 0.28. The specific cellular CrP content, but not the ATP content, declined with the age of the culture. Absence of fetal calf serum for 24 h caused a partial loss in the cellular contents of both CrP and ATP, while application of creatine for 24 h doubled the cellular CrP content and the CrP/ATP ratio, but did not affect ATP levels. In glucose-deprived astrocytes, the high cellular ATP and CrP contents were rapidly depleted within minutes after application of the glycolysis inhibitor 2-deoxyglucose and the respiratory chain inhibitor antimycin A. For those conditions, the decline in CrP levels always preceded that of ATP contents. In contrast, incubation of glucose-fed astrocytes for up to 30 min with antimycin A had little effect on the high cellular ATP content, while the CrP level was significantly lowered. These data demonstrate the importance of cellular CrP for maintaining a high cellular ATP content in astrocytes during episodes of impaired ATP regeneration.

Exploring biomarkers of premature ovarian insufficiency based on oxford nanopore transcriptional profile and machine learning

Premature ovarian insufficiency (POI) is a reproductive endocrine disorder characterized by infertility and perimenopausal syndrome, with a highly heterogeneous genetic etiology and its mechanism is not fully understood. Therefore, we utilized Oxford Nanopore Technology (ONT) for the first time to characterize the full-length transcript profile, and revealed biomarkers, pathway and molecular mechanisms for POI by bioinformatics analysis and machine learning. Ultimately, we identified 272 differentially expressed genes, 858 core genes, and 25 hub genes by analysis of differential expression, gene set enrichment, and protein–protein interactions. Seven candidate genes were identified based on the intersection features of the random forest and Boruta algorithm. qRT-PCR results indicated that COX5A, UQCRFS1, LCK, RPS2 and EIF5A exhibited consistent expression trends with sequencing data and have potential as biomarkers. Additionally, GSEA analysis revealed that the pathophysiology of POI is closely associated with inhibition of the PI3K-AKT pathway, oxidative phosphorylation and DNA damage repair, as well as activation of inflammatory and apoptotic pathways. Furthermore, we emphasize that downregulation of respiratory chain enzyme complex subunits and inhibition of oxidative phosphorylation pathways play crucial roles in the pathophysiology of POI. In conclusion, our utilization of long-read sequencing has refined the annotation information within the POI transcriptional profile. This valuable data provides novel insights for further exploration into molecular regulatory networks and potential biomarkers associated with POI.

Unique Properties of Apicomplexan Mitochondria.

Apicomplexan parasites constitute more than 6,000 species infecting a wide range of hosts. These include important pathogens such as those causing malaria and toxoplasmosis. Their evolutionary emergence coincided with the dawn of animals. Mitochondrial genomes of apicomplexan parasites have undergone dramatic reduction in their coding capacity, with genes for only three proteins and ribosomal RNA genes present in scrambled fragments originating from both strands. Different branches of the apicomplexans have undergone rearrangements of these genes, with Toxoplasma having massive variations in gene arrangements spread over multiple copies. The vast evolutionary distance between the parasite and the host mitochondria has been exploited for the development of antiparasitic drugs, especially those used to treat malaria, wherein inhibition of the parasite mitochondrial respiratory chain is selectively targeted with little toxicity to the host mitochondria. We describe additional unique characteristics of the parasite mitochondria that are being investigated and provide greater insights into these deep-branching eukaryotic pathogens.

Viability of HepG2 and MCF-7 cells is not correlated with mitochondrial bioenergetics

Alterations in metabolism are a hallmark of cancer. It is unclear if oxidative phosphorylation (OXPHOS) is necessary for tumour cell survival. In this study, we investigated the effects of severe hypoxia, site-specific inhibition of respiratory chain (RC) components, and uncouplers on necrotic and apoptotic markers in 2D-cultured HepG2 and MCF-7 tumour cells. Comparable respiratory complex activities were observed in both cell lines. However, HepG2 cells exhibited significantly higher oxygen consumption rates (OCR) and respiratory capacity than MCF-7 cells. Significant non-mitochondrial OCR was observed in MCF-7 cells, which was insensitive to acute combined inhibition of complexes I and III. Pre-treatment of either cell line with RC inhibitors for 24–72 h resulted in the complete abolition of respective complex activities and OCRs. This was accompanied by a time-dependent decrease in citrate synthase activity, suggesting mitophagy. High-content automated microscopy recordings revealed that the viability of HepG2 cells was mostly unaffected by any pharmacological treatment or severe hypoxia. In contrast, the viability of MCF-7 cells was strongly affected by inhibition of complex IV (CIV) or complex V (CV), severe hypoxia, and uncoupling. However, it was only moderately affected by inhibition of complexes I, II, and III. Cell death in MCF-7 cells induced by inhibition of complexes II, III, and IV was partially abrogated by aspartate. These findings indicate that OXPHOS activity and viability are not correlated in these cell lines, suggesting that the connection between OXPHOS and cancer cell survival is dependent on the specific cell type and conditions.

Commentary: Inhibitors of mitochondrial respiratory chain in the treatment of type 2 diabetes

Commentary: Inhibitors of mitochondrial respiratory chain in the treatment of type 2 diabetes

Ginkgo Biloba and Long COVID: In Vivo and In Vitro Models for the Evaluation of Nanotherapeutic Efficacy.

Coronavirus infections are neuroinvasive and can provoke injury to the central nervous system (CNS) and long-term illness consequences. They may be associated with inflammatory processes due to cellular oxidative stress and an imbalanced antioxidant system. The ability of phytochemicals with antioxidant and anti-inflammatory activities, such as Ginkgo biloba, to alleviate neurological complications and brain tissue damage has attracted strong ongoing interest in the neurotherapeutic management of long COVID. Ginkgo biloba leaf extract (EGb) contains several bioactive ingredients, e.g., bilobalide, quercetin, ginkgolides A-C, kaempferol, isorhamnetin, and luteolin. They have various pharmacological and medicinal effects, including memory and cognitive improvement. Ginkgo biloba, through its anti-apoptotic, antioxidant, and anti-inflammatory activities, impacts cognitive function and other illness conditions like those in long COVID. While preclinical research on the antioxidant therapies for neuroprotection has shown promising results, clinical translation remains slow due to several challenges (e.g., low drug bioavailability, limited half-life, instability, restricted delivery to target tissues, and poor antioxidant capacity). This review emphasizes the advantages of nanotherapies using nanoparticle drug delivery approaches to overcome these challenges. Various experimental techniques shed light on the molecular mechanisms underlying the oxidative stress response in the nervous system and help comprehend the pathophysiology of the neurological sequelae of SARS-CoV-2 infection. To develop novel therapeutic agents and drug delivery systems, several methods for mimicking oxidative stress conditions have been used (e.g., lipid peroxidation products, mitochondrial respiratory chain inhibitors, and models of ischemic brain damage). We hypothesize the beneficial effects of EGb in the neurotherapeutic management of long-term COVID-19 symptoms, evaluated using either in vitro cellular or in vivo animal models of oxidative stress.

Evidence for the feasibility of transmembrane proton gradient regulating oxytetracycline extracellular biodegradation mediated by biosynthesized palladium nanoparticles

Extracellular biodegradation is a promising technology for removing antibiotics and repressing the spread of resistance genes, but the strategy is limited by the low extracellular electron transfer (EET) efficiency of microorganisms. In this work, biogenic Pd0 nanoparticles (bio-Pd0) were introduced in cells in situ to enhance oxytetracycline (OTC) extracellular degradation and the effects of transmembrane proton gradient (TPG) on EET and energy metabolism mediated by bio-Pd0 were investigated. The results indicated that the intracellular OTC concentration gradually decreased with increase in pH due to the simultaneous decreases of OTC adsorption and TPG-dependent OTC uptake. On the contrary, the efficiency of OTC biodegradation mediated by bio-Pd0@B. megaterium showed a pH-dependent increase. The negligible intracellular OTC degradation, the high dependence of OTC biodegradation on respiration chain and the results on enzyme activity and respiratory chain inhibition experiments showed that NADH-dependent (rather than FADH2-dependent) EET process mediated by substrate-level phosphorylation modulated OTC biodegradation due to high energy storage and proton translocation capacity. Moreover, the results showed that altering TPG is an efficient approach to improve EET efficiency, which can be attributed to the increased NADH generation by the TCA cycle, enhanced transmembrane electron output efficiency (as evidenced by increased intracellular electron transfer system (IETS) activity, the negative shift of onset potential, and enhanced one-electron transfer through bound flavin) and stimulation of substrate-level phosphorylation energy metabolism catalyzed by succinic thiokinase (STH) under low TPG conditions. The results of structural equation model that OTC biodegradation was directly and positively modulated by the net outward proton flux as well as STH activity, and indirectly regulated by TPG through NADH level and IETS activity confirmed the previous findings. This study provides a new perspective for engineering microbial EET and application of bioelectrochemistry processes in bioremediation.

Monitoring live mitochondrial metabolism in real-time using NMR spectroscopy.

Investigation of mitochondrial metabolism is gaining increased interest owing to the growing recognition of the role of mitochondria in health and numerous diseases. Studies of isolated mitochondria promise novel insights into the metabolism devoid of confounding effects from other cellular organelles such as cytoplasm. This study describes the isolation of mitochondria from mouse skeletal myoblast cells (C2C12) and the investigation of live mitochondrial metabolism in real-time using isotope tracer-based NMR spectroscopy. [3-13 C1 ]pyruvate was used as the substrate to monitor the dynamic changes of the downstream metabolites in mitochondria. The results demonstrate an intriguing phenomenon, in which lactate is produced from pyruvate inside the mitochondria and the results were confirmed by treating mitochondria with an inhibitor of mitochondrial pyruvate carrier (UK5099). Lactate is associated with health and numerous diseases including cancer and, to date, it is known to occur only in the cytoplasm. The insight that lactate is also produced inside mitochondria opens avenues for exploring new pathways of lactate metabolism. Further, experiments performed using inhibitors of the mitochondrial respiratory chain, FCCP and rotenone, show that [2-13 C1 ]acetyl coenzyme A, which is produced from [3-13 C1 ]pyruvate and acts as a primary substrate for the tricarboxylic acid cycle in mitochondria, exhibits a remarkable sensitivity to the inhibitors. These results offer a direct approach to visualize mitochondrial respiration through altered levels of the associated metabolites.

Mitochondria Profoundly Influence Apolipoprotein E Biology.

Mitochondria can trigger Alzheimer's disease (AD)-associated molecular phenomena, but how mitochondria impact apolipoprotein E (APOE; apoE) is not well known. Consider whether and how mitochondrial biology influences APOE and apoE biology. We measured APOE expression in human SH-SY5Y neuronal cells with different forms of mitochondrial dysfunction including total, chronic mitochondrial DNA (mtDNA) depletion (ρ0 cells); acute, partial mtDNA depletion; and toxin-induced mitochondrial dysfunction. We further assessed intracellular and secreted apoE protein levels in the ρ0 cells and interrogated the impact of transcription factors and stress signaling pathways known to influence APOE expression. SH-SY5Y ρ0 cells exhibited a 65-fold increase in APOE mRNA, an 8-fold increase in secreted apoE protein, and increased intracellular apoE protein. Other models of primary mitochondrial dysfunction including partial mtDNA-depletion, toxin-induced respiratory chain inhibition, and chemical-induced manipulations of the mitochondrial membrane potential similarly increased SH-SY5Y cell APOE mRNA. We explored potential mediators and found in the ρ0 cells knock-down of the C/EBPα and NFE2L2 (Nrf2) transcription factors reduced APOE mRNA. The activity of two mitogen-activated protein kinases, JNK and ERK, also strongly influenced ρ0 cell APOE mRNA levels. Primary mitochondrial dysfunction either directly or indirectly activates APOE expression in a neuronal cell model by altering transcription factors and stress signaling pathways. These studies demonstrate mitochondrial biology can influence the biology of the APOE gene and apoE protein, which are implicated in AD.

Endogenous Energy Stores Maintain a High ATP Concentration for Hours in Glucose-Depleted Cultured Primary Rat Astrocytes

Adenosine triphosphate (ATP) is the central energy currency of all cells. Cultured primary rat astrocytes contain a specific cellular ATP content of 27.9 ± 4.7 nmol/mg. During incubation in a glucose- and amino acid-free incubation buffer, this high cellular ATP content was maintained for at least 6 h, while within 24 h the levels of ATP declined to around 30% of the initial value without compromising cell viability. In contrast, cells exposed to 1 mM and 5 mM glucose maintained the initial high cellular ATP content for 24 and 72 h, respectively. The loss in cellular ATP content observed during a 24 h glucose-deprivation was fully prevented by the presence of glucose, fructose or mannose as well as by the mitochondrial substrates lactate, pyruvate, β-hydroxybutyrate or acetate. The high initial specific ATP content in glucose-starved astrocytes, was almost completely abolished within 30 min after application of the respiratory chain inhibitor antimycin A or the mitochondrial uncoupler BAM-15, while these inhibitors lowered in glucose-fed cells the ATP content only to 60% (BAM-15) and 40% (antimycin A) within 5 h. Inhibition of the mitochondrial pyruvate carrier by UK5099 alone or of mitochondrial fatty acid uptake by etomoxir alone hardly affected the high ATP content of glucose-deprived astrocytes during an incubation for 8 h, while the co-application of both inhibitors depleted cellular ATP levels almost completely within 5 h. These data underline the importance of mitochondrial metabolism for the ATP regeneration of astrocytes and demonstrate that the mitochondrial oxidation of pyruvate and fatty acids strongly contributes to the maintenance of a high ATP concentration in glucose-deprived astrocytes.

β-lapachone-mediated WST1 Reduction as Indicator for the Cytosolic Redox Metabolism of Cultured Primary Astrocytes

Electron cycler-mediated extracellular reduction of the water-soluble tetrazolium salt 1 (WST1) is frequently used as tool for the determination of cell viability. We have adapted this method to monitor by determining the extracellular WST1 formazan accumulation the cellular redox metabolism of cultured primary astrocytes via the NAD(P)H-dependent reduction of the electron cycler β-lapachone by cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1). Cultured astrocytes that had been exposed to β-lapachone in concentrations of up to 3 µM remained viable and showed an almost linear extracellular accumulation of WST1 formazan for the first 60 min, while higher concentrations of β-lapachone caused oxidative stress and impaired cell metabolism. β-lapachone-mediated WST1 reduction was inhibited by the NQO1 inhibitors ES936 and dicoumarol in a concentration-dependent manner, with half-maximal inhibition observed at inhibitor concentrations of about 0.3 µM. β-lapachone-mediated WST1 reduction depended strongly on glucose availability, while mitochondrial substrates such as lactate, pyruvate or ketone bodies allowed only residual β-lapachone-mediated WST1 reduction. Accordingly, the mitochondrial respiratory chain inhibitors antimycin A and rotenone hardly affected astrocytic WST1 reduction. Both NADH and NADPH are known to supply electrons for reactions catalysed by cytosolic NQO1. Around 60% of the glucose-dependent β-lapachone-mediated WST1 reduction was prevented by the presence of the glucose-6-phosphate dehydrogenase inhibitor G6PDi-1, while the glyceraldehyde-3-phosphate dehydrogenase inhibitor iodoacetate had only little inhibitory potential. These data suggest that pentose phosphate pathway-generated NADPH, and not glycolysis-derived NADH, is the preferred electron source for cytosolic NQO1-catalysed reductions in cultured astrocytes.