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Monocarboxylate transporters (MCTs) constitute a family of 14 members among which MCT1–4 facilitate the passive transport of monocarboxylates such as lactate, pyruvate and ketone bodies together with protons across cell membranes. Their anchorage and activity at the plasma membrane requires interaction with chaperon protein such as basigin/CD147 and embigin/gp70. MCT1–4 are expressed in different tissues where they play important roles in physiological and pathological processes. This review focuses on the brain and on cancer. In the brain, MCTs control the delivery of lactate, produced by astrocytes, to neurons, where it is used as an oxidative fuel. Consequently, MCT dysfunctions are associated with pathologies of the central nervous system encompassing neurodegeneration and cognitive defects, epilepsy and metabolic disorders. In tumors, MCTs control the exchange of lactate and other monocarboxylates between glycolytic and oxidative cancer cells, between stromal and cancer cells and between glycolytic cells and endothelial cells. Lactate is not only a metabolic waste for glycolytic cells and a metabolic fuel for oxidative cells, but it also behaves as a signaling agent that promotes angiogenesis and as an immunosuppressive metabolite. Because MCTs gate the activities of lactate, drugs targeting these transporters have been developed that could constitute new anticancer treatments. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells., Highlights • Insulin acutely improves mitochondrial function of skeletal muscle cells. • Mitochondrial proton leak of skeletal muscle cells is attenuated by insulin. • Insulin sensitises anaerobic glycolysis of skeletal muscle cells to glucose. • Bioenergetic insulin effects offer novel assays for cellular insulin sensitivity.

Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M.

Skeletal muscle insulin resistance in obesity associates with mitochondrial dysfunction, but the causality of this association is controversial. This review evaluates mitochondrial models of nutrient-induced muscle insulin resistance. It transpires that all models predict that insulin resistance arises as a result of imbalanced cellular bioenergetics. The nature and precise origin of the proposed insulin-numbing molecules differ between models but all species only accumulate when metabolic fuel supply outweighs energy demand. This observation suggests that mitochondrial deficiency in muscle insulin resistance is not merely owing to intrinsic functional defects, but could instead be an adaptation to nutrient-induced changes in energy expenditure. Such adaptive effects are likely because muscle ATP supply is fully driven by energy demand. This market-economic control of myocellular bioenergetics offers a mechanism by which insulin-signalling deficiency can cause apparent mitochondrial dysfunction, as insulin resistance lowers skeletal muscle anabolism and thus dampens ATP demand and, consequently, oxidative ATP synthesis.

Background Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C 10 ) spacer yields a fluorescent uncoupler, coined mitoFluo, that selectively accumulates in energized mitochondria (Denisov et al., Chem.Commun. 2014). Methods Proton-transport activity of mitoFluo was tested in liposomes reconstituted with bacteriorhodopsin. To examine the uncoupling action on mitochondria, we monitored mitochondrial membrane potential in parallel with oxygen consumption. Neuro- and nephroprotecting activity was detected by a limb-placing test and a kidney ischemia/reperfusion protocol, respectively. Results We compared mitoFluo properties with those of its newly synthesized analog having a short (butyl) spacer (C 4 -mitoFluo). MitoFluo, but not C 4 -mitoFluo, caused collapse of mitochondrial membrane potential resulting in stimulation of mitochondrial respiration. The dramatic difference in the uncoupling activity of mitoFluo and C 4 -mitoFluo was in line with the difference in their protonophoric activity on a lipid membrane. The accumulation of mitoFluo in mitochondria was more pronounced than that of C 4 -mitoFluo . MitoFluo decreased the rate of ROS production in mitochondria. MitoFluo was effective in preventing consequences of brain trauma in rats: it suppressed trauma-induced brain swelling and reduced a neurological deficit. Besides, mitoFluo attenuated acute kidney injury after ischemia/reperfusion in rats. Conclusions A long alkyl linker was proved mandatory for mitoFluo to be a mitochondria- targeted uncoupler. MitoFluo showed high protective efficacy in certain models of oxidative stress-related diseases. General significance MitoFluo is a candidate for developing therapeutic and fluorescence imaging agents to treat brain and kidney pathologies.

This study explores the V600BRAF-MITF-PGC-1α axis and compares metabolic and functional changes occurring in primary and metastatic V600BRAF melanoma cell lines. V600BRAF mutations in homo/heterozygosis were found to be correlated to high levels of pERK, to downregulate PGC-1α/β, MITF and tyrosinase activity, resulting in a reduced melanin synthesis as compared to BRAFwt melanoma cells. In this scenario, V600BRAF switches on a metabolic reprogramming in melanoma, leading to a decreased OXPHOS activity and increased glycolytic ATP, lactate, HIF-1α and MCT4 levels. Furthermore, the induction of autophagy and the presence of ER stress markers in V600BRAF metastatic melanoma cells suggest that metabolic adaptations of these cells occur as compensatory survival mechanisms. For the first time, we underline the role of peIF2α as an important marker of metastatic behaviour in melanoma. Our results suggest the hypothesis that inhibition of the glycolytic pathway, inactivation of peIF2α and a reduction of basal autophagy could be suitable targets for novel combination therapies in a specific subgroup of metastatic melanoma.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is biochemically characterized by tissue accumulation of octanoic (OA), decanoic (DA) and cis-4-decenoic (cDA) acids, as well as by their carnitine by-products. Untreated patients present episodic encephalopathic crises and biochemical liver alterations, whose pathophysiology is poorly known. We investigated the effects of OA, DA, cDA, octanoylcarnitine (OC) and decanoylcarnitine (DC) on critical mitochondrial functions in rat brain and liver. DA and cDA increased resting respiration and diminished ADP- and CCCP-stimulated respiration and complexes II-III and IV activities in both tissues. The data indicate that these compounds behave as uncouplers and metabolic inhibitors of oxidative phosphorylation. Noteworthy, metabolic inhibition was more evident in brain as compared to liver. DA and cDA also markedly decreased mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded brain and liver mitochondria. The reduction of Ca(2+) retention capacity was more pronounced in liver and totally prevented by cyclosporine A and ADP, as well as by ruthenium red, demonstrating the involvement of mitochondrial permeability transition (mPT) and Ca(2+). Furthermore, cDA induced lipid peroxidation in brain and liver mitochondria and increased hydrogen peroxide formation in brain, suggesting the participation of oxidative damage in cDA-induced alterations. Interestingly, OA, OC and DC did not alter the evaluated parameters, implying lower toxicity for these compounds. Our results suggest that DA and cDA, in contrast to OA and medium-chain acylcarnitines, disturb important mitochondrial functions in brain and liver by multiple mechanisms that are possibly involved in the neuropathology and liver alterations observed in MCAD deficiency.

Glucose intolerance in C57Bl/6 mice has been associated with mutations in the nicotinamide nucleotide transhydrogenase (Nnt) gene. It has been proposed that the absence of NNT from mitochondria leads to increased mitochondrial reactive oxygen species production and subsequent activation of uncoupling protein 2 (UCP2). Activation of UCP2 has been suggested to uncouple electron transport from ATP synthesis in pancreatic beta cell mitochondria thereby decreasing glucose tolerance due to decreased insulin secretion through lower ATP/ADP ratios. The hypothesis tested in this paper is that UCP2 function is required for the dysregulation of glucose homeostasis observed in NNT ablated mice. Single and double Nnt and Ucp2 knockout mouse lines were used to measure glucose tolerance, whole animal energy balance and biochemical characteristics of mitochondrial uncoupling. As expected, glucose tolerance was diminished in mice lacking NNT. This was independent of UCP2 as it was observed either in the presence or absence of UCP2. The range of metabolic parameters examined in the mice and the proton conductance of isolated mitochondria remained unaltered in this double NNT and UCP2 knockout model. Ablation of UCP2 did not itself affect glucose tolerance and therefore previous observations of increased glucose tolerance of mice lacking UCP2 were not confirmed. We conclude that the decreased glucose tolerance in Nnt knockout mice observed in our experiments does not require UCP2.

Cells infected by the hepatitis C virus (HCV) are characterized by endoplasmic reticulum stress, deregulation of the calcium homeostasis and unbalance of the oxido-reduction state. In this context, mitochondrial dysfunction proved to be involved and is thought to contribute to the outcome of the HCV-related disease. Here, we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists of a release of Ca2+ from the endoplasmic reticulum, followed by uptake into mitochondria. This causes successive mitochondrial alterations comprising generation of reactive oxygen and nitrogen species and impairment of the oxidative phosphorylation. A progressive adaptive response results in an enhancement of the glycolytic metabolism sustained by up-regulation of the hypoxia inducible factor. Pathogenetic implications of the model are discussed.

Mitochondrial dysfunction has been associated with obesity-related muscle insulin resistance, but the causality of this association is controversial. The notion that mitochondrial oxidative capacity may be insufficient to deal appropriately with excessive nutrient loads is for example disputed. Effective mitochondrial capacity is indirectly, but largely determined by ATP-consuming processes because skeletal muscle energy metabolism is mostly controlled by ATP demand. Probing the bioenergetics of rat and human myoblasts in real time we show here that the saturated fatty acid palmitate lowers the rate and coupling efficiency of oxidative phosphorylation under conditions it causes insulin resistance. Stearate affects the bioenergetic parameters similarly, whereas oleate and linoleate tend to decrease the rate but not the efficiency of ATP synthesis. Importantly, we reveal that palmitate influences how oxidative ATP supply is used to fuel ATP-consuming processes. Direct measurement of newly made protein demonstrates that palmitate lowers the rate of de novo protein synthesis by more than 30%. The anticipated decrease of energy demand linked to protein synthesis is confirmed by attenuated cycloheximide-sensitivity of mitochondrial respiratory activity used to make ATP. This indirect measure of ATP turnover indicates that palmitate lowers ATP supply reserved for protein synthesis by at least 40%. This decrease is also provoked by stearate, oleate and linoleate, albeit to a lesser extent. Moreover, palmitate lowers ATP supply for sodium pump activity by 60-70% and, in human cells, decreases ATP supply for DNA/RNA synthesis by almost three-quarters. These novel fatty acid effects on energy expenditure inform the 'mitochondrial insufficiency' debate.

Merely considered as a toxic gas in the past, hydrogen sulfide (H2S) is currently viewed as the third 'gasotransmittee in addition to nitric oxide (NO) and carbon monoxide (CO), playing a key signalling role in human (patho)physiology. H2S can either act as a substrate or, similarly to CO and NO, an inhibitor of mitochondrial respiration, in the latter case by targeting cytochrome c oxidase (CcOX). The impact of H2S on mitochondrial energy metabolism crucially depends on the bioavailability of this gaseous molecule and its interplay with the other two gasotransmitters. The H2S-producing human enzyme cystathionine beta-synthase (CBS), sustaining cellular bioenergetics in colorectal cancer cells, plays a role in the interplay between gasotransmitters. The enzyme was indeed recently shown to be negatively modulated by physiological concentrations of CO and NO, particularly in the presence of its allosteric activator S-adenosyl-L-methionine (AdoMet). These newly discovered regulatory mechanisms are herein reviewed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. (C) 2016 Elsevier B.V. All rights reserved.

Plasma membrane potassium channels importantly contribute to maintain ion homeostasis across the cell membrane. The view is emerging that also those residing in intracellular membranes play pivotal roles for the coordination of correct cell function. In this review we critically discuss our current understanding of the nature and physiological tasks of potassium channels in organelle membranes in both animal and plant cells, with a special emphasis on their function in the regulation of photosynthesis and mitochondrial respiration. In addition, the emerging role of potassium channels in the nuclear membranes in regulating transcription will be discussed. The possible functions of endoplasmic reticulum-, lysosome- and plant vacuolar membrane-located channels are also referred to. Altogether, experimental evidence obtained with distinct channels in different membrane systems points to a possible unifying function of most intracellular potassium channels in counterbalancing the movement of other ions including protons and calcium and modulating membrane potential, thereby fine-tuning crucial cellular processes. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-7, 2016', edited by Prof. Paolo Bernardi.

Mitochondrial energy in cardiac cells has been reported to be channeled into the cytosol through the intermembrane contact sites formed by the adenine nucleotide translocator, creatine kinase and VDAC. Computational analysis performed in this study showed a high probability of the outer membrane potential (OMP) generation coupled to such a mechanism of energy channeling in respiring mitochondria. OMPs, positive inside, calculated at elevated concentrations of creatine are high enough to restrict ATP release from mitochondria, to significantly decrease the apparent K(m,ADP) for state 3 respiration and to maintain low concentrations of Ca(2+) in the mitochondrial intermembrane space. An inhibition by creatine of Ca(2+)-induced swelling of isolated mitochondria and other protective effects of creatine reported in the literature might be explained by generated positive OMP. We suggest that VDAC-creatine kinase-dependent generation of OMP represents a novel physiological factor controlling metabolic state of mitochondria, cell energy channeling and resistance to death.

Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance.

Mitochondria play a key role in energy metabolism, hosting the machinery for oxidative phosphorylation, the most efficient cellular pathway for generating ATP. A major checkpoint in this process is the transport of pyruvate produced by cytosolic glycolysis into the mitochondrial matrix, which is accomplished by the recently identified mitochondrial pyruvate carrier (MPC). As the gatekeeper for pyruvate entry into mitochondria, the MPC is thought to be of fundamental importance in establishing the metabolic programming of a cell. This is especially relevant in the context of the aerobic glycolysis, also known as the Warburg effect, which is a hallmark in many types of cancer, and MPC loss of function promotes cancer growth. Moreover, mitochondrial pyruvate uptake is needed for efficient hepatic gluconeogenesis and the regulation of blood glucose levels. In this review we discuss recent advances in our knowledge of the MPC, and we argue that it may offer a promising target in diseases like cancer and type 2 diabetes. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

In the mitochondrial inner membrane, oxidative phosphorylation generates ATP via the operation of several multimeric enzymes. The proton-pumping Complex I (NADH:ubiquinone oxidoreductase) is the first and most complicated enzyme required in this process. Complex I is an L-shaped enzyme consisting of more than 40 subunits, one FMN molecule and eight Fe-S clusters. In recent years, genetic and proteomic analyses of Complex I mutants in various model systems, including plants, have provided valuable insights into the assembly of this multimeric enzyme. Assisted by a number of key players, referred to as "assembly factors", the assembly of Complex I takes place in a sequential and modular manner. Although a number of factors have been identified, their precise function in mediating Complex I assembly still remains to be elucidated. This review summarizes our current knowledge of plant Complex I composition and assembly derived from studies in plant model systems such as Arabidopsis thaliana and Chlamydomonas reinhardtii. Plant Complex I is highly conserved and comprises a significant number of subunits also present in mammalian and fungal Complexes I. Plant Complex I also contains additional subunits absent from the mammalian and fungal counterpart, whose function in enzyme activity and assembly is not clearly understood. While 14 assembly factors have been identified for human Complex I, only two proteins, namely GLDH and INDH, have been established as bona fide assembly factors for plant Complex I. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

Osteoarthritis is the most common joint disorder with increasing prevalence due to aging of the population. Its multi-factorial etiology includes oxidative stress and the overproduction of reactive oxygen species, which regulate intracellular signaling processes, chondrocyte senescence and apoptosis, extracellular matrix synthesis and degradation along with synovial inflammation and dysfunction of the subchondral bone. As disease-modifying drugs for osteoarthritis are rare, targeting the complex oxidative stress signaling pathways would offer a valuable perspective for exploration of potential therapeutic strategies in the treatment of this devastating disease.

The present study shows that in isolated mitochondria and myoblast cultures depletion of cAMP, induced by sAC inhibition, depresses both ATP synthesis and hydrolysis by the FOF1 ATP synthase (complex V) of the oxidative phosphorylation system (OXPHOS). These effects are accompanied by the decrease of the respiratory membrane potential, decreased level of FOF1 connecting subunits and depressed oligomerization of the complex. All these effects of sAC inhibition are prevented by the addition of the membrane-permeant 8-Br-cAMP. These results show, for the first time, that cAMP promotes ATP production by complex V and prevents, at the same time, its detour to a mitochondrial membrane leak conductance, which is involved in cell death.

The content and composition of cardiolipin (CL) is critical for preservation of mitochondrial oxidative phosphorylation (OXPHOS) and inner membrane integrity. Tafazzin (Taz) is an enzyme responsible for remodeling of immature CL containing mixed acyl groups into the mature tetralinoleyl form (C18:2)4-CL. We hypothesized that acquired defects in Taz in the mature heart would impact remodeling of CL and augment cardiac injury. The role of acquired Taz deficiency was studied using the inducible Taz knockdown (TazKD) mouse. Taz-specific shRNA is induced by doxycycline (DOX). One day of DOX intake decreased Taz mRNA in the heart to 20% vs. DOX-treated WT. Knockdown was initiated at an adult age and was stable during long term feeding. CL phenotype was assessed by (C18:2)4-CL content and was reduced 40% vs. WT at two months of DOX. TazKD showed increased production of reactive oxygen species and increased susceptibility to permeability transition pore opening at baseline. However, OXPHOS measured using the rate of oxygen consumption was unchanged in the setting of acquired Taz deficiency. Infarct size, measured in isolated buffer-perfused Langendorff hearts following 25min. Stop flow ischemia and 60min. Reperfusion was not altered in TazKD hearts. Thus, impaired Taz-function with onset at adult age does not enhance susceptibility to ischemia-reperfusion injury.

Objective Oxidised low density lipoprotein (oxLDL) contributes to atherosclerosis, whereas high density lipoprotein (HDL) is known to be atheroprotective due, at least in part, to its ability to remove oxidised lipids from oxLDL. The molecular details of the lipid transfer process are not fully understood. We aimed to identify major oxidised lipid species of oxLDL and investigate their transfer upon co-incubation with HDL with varying levels of oxidation. Approach and results A total of 14 major species of oxidised phosphatidylcholine and oxidised cholesteryl ester from oxLDL were identified using an untargeted mass spectrometry approach. HDL obtained from pooled plasma of normolipidemic subjects (N = 5) was oxidised under mild and heavy oxidative conditions. Non-oxidised (native) HDL and oxidised HDL were co-incubated with oxLDL, re-isolated and lipidomic analysis was performed. Lipoprotein surface lipids, oxidised phosphatidylcholines and oxidised cholesterols (7-ketocholesterol and 7β-hydroxycholesterol), but not internal oxidised cholesteryl esters, were effectively transferred to native HDL. Saturated and monounsaturated lyso-phosphatidylcholines were also transferred from the oxLDL to native HDL. These processes were attenuated when HDL was oxidised under mild and heavy oxidative conditions. The impaired capacities were accompanied by an increase in a ratio of sphingomyelin to phosphatidylcholine and a reduction in phosphatidylserine content in oxidised HDL, both of which are potentially important regulators of the oxidised lipid transfer capacity of HDL. Conclusions Our study has revealed the differential transfer efficiency of surface and internal oxidised lipids from oxLDL and their acceptance onto HDL. These capacities were modulated when HDL was itself oxidised.

Anodic bioelectrodes for biofuel cells are more complex than cathodic bioelectrodes for biofuel cells, because laccase and bilirubin oxidase can individually catalyze four electron reduction of oxygen to water, whereas most anodic enzymes only do a single two electron oxidation of a complex fuel (i.e. glucose oxidase oxidizing glucose to gluconolactone while generating 2 electrons of the total 24 electrons), so enzyme cascades are typically needed for complete oxidation of the fuel. This review article will discuss the lessons learned from natural metabolic pathways about multi-step oxidation and how those lessons have been applied to minimal or artificial enzyme cascades. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and cellular maintenance processes. A crucial parameter for growth evaluation is its yield, i.e. the efficiency of the transformation processes. The yeast Candida utilis is of peculiar interest since its mitochondria exhibit a complex I that is proposed to pump protons but also an external NADH dehydrogenase that do not pump protons. Here, we show that in C. utilis cells grown on non-fermentable media, growth yield is 30% higher as compared to that of Saccharomyces cerevisiae that do not exhibit a complex I. Moreover, ADP/O determination in C. utilis shows that electrons coming from internal NADH dehydrogenase go through proton pumping complex I, whereas electrons coming from external NADH dehydrogenases do not go through proton pumping complex I. Furthermore, we show that electron competition strictly depends on extra-mitochondrial NADH concentration, i.e. the higher the extra-mitochondrial NADH concentration, the higher the competition process with a right way for electrons coming from external NADH dehydrogenases. Such a complex regulation in C. utilis allows an increase in growth yield when cytosolic NADH is not plentiful but still favors the cytosolic NADH re-oxidation at high NADH, favoring biomass generation metabolic pathways.

Cellular senescence entails an irreversible cell-cycle arrest characterised by drastic cytomorphological and metabolic changes. In recent years, the implications of cellular senescence in physiological and pathological settings, such as ageing and cancer, have gained firm ground. It is, therefore, important to understand the mechanisms underpinning the establishment and maintenance of senescence. Age-dependent alterations in cellular metabolic processes are greatly driven by changes in mitochondrial function and homeostasis. Classically, mitochondrial dysfunction has been implicated in cellular senescence mainly by promoting oxidative damage-induced cell-cycle arrest; however, emerging data suggests that other mitochondrial-dependent factors play an important role in the induction of senescent phenotypes. Here we review the role of mitochondrial homeostatic mechanisms, mitochondrial metabolites and ROS generation in the signalling pathways leading to the induction and maintenance of cellular senescence and discuss how this may contribute to the ageing process. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.

Oxygen reduction by cytochrome ba 3 oxidase from Thermus thermophilus was studied by stopped-flow and microsecond freeze-hyperquenching analyzed with UV-Vis and EPR spectroscopy. In the initial phase, the low-spin heme b 560 is rapidly and almost completely oxidized ( k obs > 33,000 s − 1 ) whereas Cu A remains nearly fully reduced. The internal equilibrium between Cu A and heme b 560 with forward and reverse rate constants of 4621 s − 1 and 3466 s − 1 , respectively, indicates a ~ 7.5 mV lower midpoint potential for Cu A compared to heme b 560 . The formation of the oxidized enzyme is relatively slow (693 s − 1 ). In contrast to the Paracoccus denitrificans cytochrome aa 3 oxidase, where in the last phase of the oxidative half cycle a radical from the strictly conserved Trp272 is formed, no radical is formed in the cytochrome ba 3 oxidase. Mutation of the Trp229, the cytochrome ba 3 oxidase homologue to the Trp272, did not abolish the activity, again in contrast to the Paracoccus cytochrome aa 3 oxidase. Differences in the proton pumping mechanisms of Type A and Type B oxidases are discussed in view of the proposed role of the strictly conserved tryptophan residue in the mechanism of redox-linked proton pumping in Type A oxidases. In spite of the differences between the Type A and Type B oxidases, we conclude that protonation of the proton-loading site constitutes the major rate-limiting step in both catalytic cycles.

Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,β-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.

Background During maturation and storage, spermatozoa generate substantial amounts of reactive oxygen species (ROS) and are thus forced to cope with an increasingly oxidative environment that is both needed and detrimental to their biology. Such a janus-faceted intermediate needs to be tightly controlled and this is done by a wide array of redox enzymes. These enzymes not only have to prevent unspecific modifications of essential cellular biomolecules by quenching undesired ROS, but they are also required and often directly involved in critical protein modifications. Scope of review The present review is conceived to present an update on what is known about critical roles of redox enzymes, whereby special emphasis is put on the family of glutathione peroxidases, which for the time being presents the best characterized tasks during gametogenesis. Major conclusions We therefore demonstrate that understanding the function of (seleno)thiol-based oxidases/reductases is not a trivial task and relevant knowledge will be mainly gained by using robust systems, as exemplified by several (conditional) knockout studies. We thus stress the importance of using such models for providing unequivocal evidence in the molecular understanding of redox regulatory mechanisms in sperm maturation. General significance ROS are not merely detrimental by-products of metabolism and their proper generation and usage by specific enzymes is essential for vital functions as beautifully exemplified during male gametogenesis. As such, lessons learnt from thiol-based oxidases/reductases in male gametogenesis could be used as a general principle for other organs as it is most likely not only restricted to this developmental phase. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

Spinocerebellar ataxia type 7 (SCA7) is one of the nine neurodegenerative disorders caused by expanded polyglutamine (polyQ) domains. Common pathogenic mechanisms, including bioenergetics defects, have been suggested for these so called polyQ diseases. However, the exact molecular mechanism(s) behind the metabolic dysfunction is still unclear. In this study we identified a previously unreported mechanism, involving disruption of p53 and NADPH oxidase 1 (NOX1) activity, by which the expanded SCA7 disease protein ATXN7 causes metabolic dysregulation. The NOX1 protein is known to promote glycolytic activity, whereas the transcription factor p53 inhibits this process and instead promotes mitochondrial respiration. In a stable inducible PC12 model of SCA7, p53 and mutant ATXN7 co-aggregated and the transcriptional activity of p53 was reduced, resulting in a 50% decrease of key p53 target proteins, like AIF and TIGAR. In contrast, the expression of NOX1 was increased approximately 2 times in SCA7 cells. Together these alterations resulted in a decreased respiratory capacity, an increased reliance on glycolysis for energy production and a subsequent 20% reduction of ATP in SCA7 cells. Restoring p53 function, or suppressing NOX1 activity, both reversed the metabolic dysfunction and ameliorated mutant ATXN7 toxicity. These results hence not only enhance the understanding of the mechanisms causing metabolic dysfunction in SCA7 disease, but also identify NOX1 as a novel potential therapeutic target in SCA7 and possibly other polyQ diseases.

Neoadjuvant, adjuvant or definitive fractionated radiation therapy are implemented in first line anti-cancer treatment regimens of many tumor entities. Ionizing radiation kills the tumor cells mainly by causing double strand breaks of their DNA through formation of intermediate radicals. Survival of the tumor cells depends on both, their capacity of oxidative defense and their efficacy of DNA repair. By damaging the targeted cells, ionizing radiation triggers a plethora of stress responses. Among those is the modulation of ion channels such as Ca2+-activated K+ channels or Ca2+-permeable nonselective cation channels belonging to the super-family of transient receptor potential channels. Radiogenic activation of these channels may contribute to radiogenic cell death as well as to DNA repair, glucose fueling, radiogenic hypermigration or lowering of the oxidative stress burden. The present review article introduces these channels and summarizes our current knowledge on the mechanisms underlying radiogenic ion channel modulation. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

Background Mitochondria are multifunctional organelles that not only serve as cellular energy stores but are also actively involved in several cellular stress responses, including apoptosis. In addition, mitochondria themselves are also continuously challenged by stresses such as reactive oxygen species (ROS), an inevitable by-product of oxidative phosphorylation. To exert various functions against these stresses, mitochondria must be equipped with appropriate stress responses that monitor and maintain their quality. Scope of review Interestingly, increasing evidence indicates that mitochondrial proteolysis has important roles in mitochondrial and cellular stress responses. In this review, we summarize current advances in mitochondrial proteolysis-mediated stress responses. Major conclusions Mitochondrial proteases do not only function as surveillance systems of protein quality control by degrading unfolded proteins but also regulate mitochondrial stress responses by processing specific mitochondrial proteins. General significance Studies on the regulation of mitochondrial proteolysis-mediated stress responses will provide the novel mechanistic insights into the stress response research fields.

Background It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown. Methods The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens. Results Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine. Conclusion Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective. General significance The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens.

Tumor cells undergo metabolic rewiring from oxidative phosphorylation towards aerobic glycolysis to maintain the increased anabolic requirements for cell proliferation. It is widely accepted that specific expression of the M2 type pyruvate kinase (PKM2) in tumor cells contributes to this aerobic glycolysis phenotype. To date, researchers have uncovered myriad forms of functional regulation for PKM2, which confers a growth advantage on the tumor cells to enable them to adapt to various microenvironmental signals. Here the richness of our understanding on the modulations and functions of PKM2 in tumor progression is reviewed, and some new insights into the paradoxical expression and functional differences of PKM2 in distinct cancer types are offered.

Macrophage polarization elicits various metabolic alterations which in turn influence the polarized phenotype. Activation of glycolytic metabolism accompanies and supports macrophage pro-inflammatory M1 polarization. In contrast, M2 polarization of murine macrophages in response to the Th2 cytokine interleukin-4 (IL-4) was linked to the up-regulation of mitochondrial oxidative metabolism and fatty acid oxidation (FAO), which was necessary for coining an IL-4-polarized phenotype. Here we investigated whether similar mechanisms operate in human macrophages stimulated with IL-4. IL-4 causes only moderate changes of mitochondrial oxidative metabolism and FAO, correlating with an unaltered expression of peroxisome proliferator-activated receptor-γ coactivator 1 α/β (PGC-1α/β), the master transcriptional regulators of mitochondrial biogenesis. Furthermore, attenuating FAO had no effect on IL-4-induced polarization-associated gene expression. Apparently, FAO is dispensable for IL-4-induced polarization of human macrophages, pointing to fundamental differences in the metabolic requirements of macrophage phenotype alterations between mice and humans.

Background Methylene blue (MB) is used for various clinical purposes, including chromoendoscopy and methemoglobinemia treatment. However, MB induces tumors of pancreatic islets and small intestine in experimental animals. This finding raises a possibility that MB induces carcinogenicity in these organs via light-independent mechanisms, although MB is known to cause light-dependent DNA damage. Methods We investigated the mechanism of MB-induced DNA damage using 32P-5′-end-labeled DNA fragments of human tumor-relevant genes. We investigated the redox reaction of MB by UV–visible spectrometry. Results MB induced DNA damage at the 5′-A CG -3′ sequence, a hot spot of the p53 gene, in the presence of NADH and Cu(II). DNA damage was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator. MB induced DNA damage at every nucleotide in the presence of NADH and Fe(III)-ethylenediaminetetraacetic acid, which was inhibited by OH scavengers and catalase. MB significantly increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine, an oxidative DNA lesion, in the presence of NADH and metal ions. UV–visible spectrometry revealed that the absorbance of oxidized form of MB at 668 nm was decreased by NADH, and the addition of metal ions attenuated the spectral change. Conclusions MB undergoes NADH-dependent reduction followed by metal ion-mediated reoxidation. Reduced metal ions [Cu(I) and Fe(II)] interact with H2O2, generated during the redox reaction, to produce Cu(I)OOH and OH that cause DNA damage, respectively. These findings suggest that metal-mediated DNA damage contributes to MB-mediated carcinogenesis. General significance This study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.

Cytochrome bd is a prokaryotic respiratory quinol:02 oxidoreductase, phylogenetically unrelated to the extensively studied heme-copper oxidases (HCOs). The enzyme contributes to energy conservation by generating a proton motive force, though working with a lower energetic efficiency as compared to HCOs. Relevant to patho-physiology, members of the bd-family were shown to promote virulence in some pathogenic bacteria, which makes these enzymes of interest also as potential drug targets. Beyond its role in cell bioenergetics, cytochrome bd accomplishes several additional physiological functions, being apparently implicated in the response of the bacterial cell to a number of stress conditions. Compelling experimental evidence suggests that the enzyme enhances bacterial tolerance to oxidative and nitrosative stress conditions, owing to its unusually high nitric oxide (NO) dissociation rate and a notable catalase activity; the latter has been recently documented inane of the two bd-type oxidases of Escherichia coli. Current knowledge on cytochrome bd and its reactivity with O-2, NO and H2O2 is summarized in this review in the light of the hypothesis that the preferential (over HCOs) expression of cytochrome bd in pathogenic bacteria may represent a strategy to evade the host immune attack based on production of NO and reactive oxygen species (ROS). This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. (C) 2014 Elsevier B.V. All rights reserved.

A study is presented on the expression of mitochondrial oxidative phosphorylation complexes in exponentially growing and serum-starved, quiescent human fibroblast cultures. The functional levels of respiratory complexes I and III and complex V (adenosine triphosphate (ATP) synthase) were found to be severely depressed in serum-starved fibroblasts. The depression of oxidative phosphorylation system (OXPHOS) complexes was associated with reduced levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and the down-stream nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factors (TFAM). In serum-starved fibroblasts decrease of the catalytic activity of AMP cyclic dependent protein kinase (PKA) and phosphorylation of cAMP response element-binding protein (CREB), the transcription coactivator of the PGC-1α gene, was found. Hydroxytyrosol prevented the decline in the expression of the PGC-1α transcription cascade of OXPHOS complexes in serum-starved fibroblast cultures. The positive effect of HT was associated with activation of PKA and CREB phosphorylation. These results show involvement of PKA, CREB and PGC-1α in the regulation of OXPHOS in cell transition from the replicating to the quiescent state.

Oxidative phosphorylation (OXPHOS) is the main source of energy in eukaryotic cells. This process is performed by means of electron flow between four enzymes, of which three are proton pumps, in the inner mitochondrial membrane. The energy accumulated in the proton gradient over the inner membrane is utilized for ATP synthesis by a fifth OXPHOS complex, ATP synthase. Four of the OXPHOS protein complexes associate into stable entities called respiratory supercomplexes. This review summarises the current view on the arrangement of the electron transport chain in mitochondrial cristae. The functional role of the supramolecular organisation of the OXPHOS system and the factors that stabilise such organisation are highlighted. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.

Background Most neurodegenerative diseases are associated with mitochondrial dysfunction. In humans, mutations in mitochondrial genes result in a range of phenotypic outcomes which do not correlate well with the underlying genetic cause. Other neurodegenerative diseases are caused by mutations that affect the function and trafficking of lysosomes, endosomes and autophagosomes. Many of the complexities of these human diseases can be avoided by studying them in the simple eukaryotic model Dictyostelium discoideum . Scope of review This review describes research using Dictyostelium to study cytopathological pathways underlying a variety of neurodegenerative diseases including mitochondrial, lysosomal and vesicle trafficking disorders. Major conclusions Generalised mitochondrial respiratory deficiencies in Dictyostelium produce a consistent pattern of defective phenotypes that are caused by chronic activation of a cellular energy sensor AMPK (AMP-activated protein kinase) and not ATP deficiency per se . Surprisingly, when individual subunits of Complex I are knocked out, both AMPK-dependent and AMPK-independent, subunit-specific phenotypes are observed. Many nonmitochondrial proteins associated with neurological disorders have homologues in Dictyostelium and are associated with the function and trafficking of lysosomes and endosomes. Conversely, some genes associated with neurodegenerative disorders do not have homologues in Dictyostelium and this provides a unique avenue for studying these mutated proteins in the absence of endogeneous protein. General significance Using the Dictyostelium model we have gained insights into the sublethal cytopathological pathways whose dysregulation contributes to phenotypic outcomes in neurodegenerative disease. This work is beginning to distinguish correlation, cause and effect in the complex network of cross talk between the various organelles involved. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research

It has been suggested that human mitochondrial variants influence maximal oxygen uptake (VO2max). Whether mitochondrial respiratory capacity per mitochondrion (intrinsic activity) in human skeletal muscle is affected by differences in mitochondrial variants is not known. We recruited 54 males and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present.

Increasing evidence reveals a large dependency of epithelial cancer cells on oxidative phosphorylation (OXPHOS) for energy production. In this study we tested the potential of epigallocatechin-3-gallate (EGCG), a natural polyphenol known to target mitochondria, in inducing OXPHOS impairment and cell energy deficit in human epitheliod (REN cells) and biphasic (MSTO-211H cells) malignant pleural mesothelioma (MMe), a rare but highly aggressive tumor with high unmet need for treatment. Due to EGCG instability that causes H2O2 formation in culture medium, the drug was added to MMe cells in the presence of exogenous superoxide dismutase and catalase, already proved to stabilize the EGCG molecule and prevent EGCG-dependent reactive oxygen species formation. We show that under these experimental conditions, EGCG causes the selective arrest of MMe cell growth with respect to normal mesothelial cells and the induction of mitochondria-mediated apoptosis, as revealed by early mitochondrial ultrastructure modification, swelling and cytochrome c release. We disclose a novel mechanism by which EGCG induces apoptosis through the impairment of mitochondrial respiratory chain complexes, particularly of complex I, II and ATP synthase. This induces a strong reduction in ATP production by OXPHOS, that is not adequately counterbalanced by glycolytic shift, resulting in cell energy deficit, cell cycle arrest and apoptosis. The EGCG-dependent negative modulation of mitochondrial energy metabolism, selective for cancer cells, gives an important input for the development of novel pharmacological strategies for MMe.

International audience; Aging is accompanied by the gradual deterioration of cell functions. Particularly, mitochondrial dysfunction, associated with an accumulation of damaged proteins, is of key importance due to the central role of these organelles in cellular metabolism. However, the detailed molecular mechanisrps involved in such impairment have not been completely elucidated. In the present study, proteomic analyses looking at both changes at the expression level as well as to glycative modifications of the mitochondrial proteome were performed. Two-dimensional difference gel electrophoresis analysis revealed 16 differentially expressed proteins with aging. Thirteen exhibited a decreased expression and are crucial enzymes related to OXPHOS chain complex I/V components, TCA cycle or fatty add beta-oxidation reaction. On the other hand, 2 enzymes involved in fatty acid beta-oxidation cycle were increased in aged mitochondria. Immunodetection and further identification of glycated proteins disclosed a set of advanced glycation endproduct-modified proteins, including 6 enzymes involved in the fatty acid beta-oxidation process, and 2 enzymes of the TCA/urea cycles. A crucial antioxidant enzyme, catalase, was among the most strongly glycated proteins. In addition, several AGE-damaged enzymes (aldehyde dehydrogenase 2, medium chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA dehydrogenase) exhibited a decreased activity with age. Taken together, these data suggest that liver mitochondria in old rats suffer from a decline in their capacity for energy production, due to (i) decreased expression of OXPHOS complex I/V components and (ii) glycative damage to key fatty acid beta-oxidation and TCA/urea cycle enzymes.

Robust anaerobic metabolism plays a causative role in the origin of cancer cells; however, the oncogenic metabolic genes, factors, pathways, and networks in genesis of tumor-initiating cells (TICs) have not yet been systematically summarized. In addition, the mechanisms of oncogenic metabolism in the genesis of TICs are enigmatic. In this review, we discussed multiple cancer metabolism-related genes (MRGs) that are overexpressed in TICs and are responsible for inducing pluripotent stem cells. Moreover, we summarized that oncogenic metabolic genes and onco-metabolites induce metabolic reprogramming, which switches normal mitochondrial oxidative phosphorylation to cancer anaerobic metabolism, triggers epigenetic, genetic, and environmental alterations, drives the generation of TICs, and boosts the development of cancer. Importantly, cancer metabolism is controlled by positive and negative metabolic regulators. Positive oncogenic metabolic regulators, including key oncogenic metabolic genes, onco-metabolites, hypoxia, and an acidic environment, promote oncogenic metabolic reprogramming and anaerobic metabolism. However, dysfunction of negative metabolic regulators, including defects in p53, PTEN, and LKB1-AMPK-mTOR pathways, enhances cancer metabolism. Loss of the metabolic balance results in oncogenic metabolic reprogramming, genesis of TICs, and tumorigenesis. Collectively, this review provides new insight into the role and mechanism of these oncogenic metabolisms in the genesis of TICs and tumorigenesis. Accordingly, targeting key oncogenic genes, onco-metabolites, pathways, networks, and the acidic cancer microenvironment appears to be an attractive strategy for novel anti-tumor treatment.

Background Targeting multiple aspects of cellular metabolism, such as both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), has the potential to improve cancer therapeutics. Berberine (BBR), a widely used traditional Chinese medicine, exerts its antitumor effects by inhibiting OXPHOS. 2-Deoxy- d -glucose (2-DG) targets aerobic glycolysis and demonstrates potential anticancer effects in the clinic. We hypothesized that BBR in combination with 2-DG would be more efficient than either agent alone against cancer cell growth. Methods The effects of BBR and 2-DG on cancer cell growth were evaluated using the Sulforhodamine B (SRB) method. Cell death was detected with the PI uptake assay, and Western blot, Q-PCR and luciferase reporter assays were used for signaling pathway detection. An adenovirus system was used for gene overexpression. Results BBR combined with 2-DG synergistically enhanced the growth inhibition of cancer cells in vitro. Further mechanistic studies showed that the combination drastically enhanced ATP depletion and strongly disrupted the unfolded protein response (UPR). Overexpressing GRP78 partially prevented the cancer cell inhibition induced by both compounds. Conclusions Here, we report for the first time that BBR and 2-DG have a synergistic effect on cancer cell growth inhibition related to ATP energy depletion and disruption of UPR. General significance Our results propose the potential use of BBR and 2-DG in combination as an anticancer treatment, reinforcing the hypothesis that targeting both aerobic glycolysis and OXPHOS provides more effective cancer therapy and highlighting the important role of UPR in the process.

Background Selenoprotein W (SelW) was thought to play an antioxidant role in mammals. Because chicken SelW has no cysteine (Cys) at the residue 37 (Cys37) that is required for the presumed antioxidant function in mammals, this study was conducted to determine whether chicken SelW possessed the same function. Methods Small interfering RNAs (siRNAs) technology was applied to suppress the SelW expression in chicken embryonic myoblasts. Thereafter, these myoblasts were treated with different concentrations of H2O2 and assayed for cell viability, apoptosis rate, reactive oxygen species (ROS) status, and expression levels of apoptosis-related genes and proteins (Bax, Bcl-2, and caspase-3). Results Silencing of the myoblast SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H2O2. While the knockout down of SelW up-regulated Bax and caspase-3 and down-regulated Bcl-2, the induced oxidative injuries were alleviated by treatment with a ROS scavenger, N-acetyl- l -cysteine (NAC). Conclusion Chicken SelW protected embryonic myoblasts against cell apoptosis mediated by endogenous and exogenous H2O2. General significance Chicken SelW possesses antioxidant function similar to the mammalian homologues despite the lack of Cys37 in the peptide.

A common set of functional characteristics of cancer cells is that cancer cells consume a large amount of glucose, maintain high rate of glycolysis and convert a majority of glucose into lactic acid even in the presence of oxygen compared to that of normal cells (Warburg's Effects). In addition, cancer cells exhibit substantial alterations in several energy metabolism pathways including glucose transport, tricarboxylic acid (TCA) cycle, glutaminolysis, mitochondrial respiratory chain oxidative phosphorylation and pentose phosphate pathway (PPP). In the present work, we focused on reviewing the current knowledge about the dysregulation of the proteins/enzymes involved in the key regulatory steps of glucose transport, glycolysis, TCA cycle and glutaminolysis by several oncogenes including c-Myc and hypoxia inducible factor-1 (HIF-1) and tumor suppressor, p53, in cancer cells. The dysregulation of glucose transport and energy metabolism pathways by oncogenes and lost functions of the tumor suppressors have been implicated as important biomarkers for cancer detection and as valuable targets for the development of new anticancer therapies.

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.

Background Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus , a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus . This study aims to clarify the role of V. parahaemolyticus SODs against ROS. Methods V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured. Results Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance. Conclusion VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses. General significance The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.