The Warburg Effect in Yeast: Repression of Mitochondrial Metabolism Is Not a Prerequisite to Promote Cell Proliferation.

O. Warburg conducted one of the first studies on tumor energy metabolism. His early discoveries pointed out that cancer cells display a decreased respiration and an increased glycolysis proportional to the increase in their growth rate, suggesting that they mainly depend on fermentative metabolism for ATP generation. Warburg's results and hypothesis generated controversies that are persistent to this day. It is thus of great importance to understand the mechanisms by which cancer cells can reversibly regulate the two pathways of their energy metabolism as well as the functioning of this metabolism in cell proliferation. Here, we made use of yeast as a model to study the Warburg effect and its eventual function in allowing an increased ATP synthesis to support cell proliferation. The role of oxidative phosphorylation repression in this effect was investigated. We show that yeast is a good model to study the Warburg effect, where all parameters and their modulation in the presence of glucose can be reconstituted. Moreover, we show that in this model, mitochondria are not dysfunctional, but that there are fewer mitochondria respiratory chain units per cell. Identification of the molecular mechanisms involved in this process allowed us to dissociate the parameters involved in the Warburg effect and show that oxidative phosphorylation repression is not mandatory to promote cell growth. Last but not least, we were able to show that neither cellular ATP synthesis flux nor glucose consumption flux controls cellular growth rate.

"Labile" heme critically regulates mitochondrial biogenesis through the transcriptional co-activator Hap4p in Saccharomyces cerevisiae.

Heme (iron protoporphyrin IX) is a well-known prosthetic group for enzymes involved in metabolic pathways such as oxygen transport and electron transfer through the mitochondrial respiratory chain. However, heme has also been shown to be an important regulatory molecule (as "labile" heme) for diverse processes such as translation, kinase activity, and transcription in mammals, yeast, and bacteria. Taking advantage of a yeast strain deficient for heme production that enabled controlled modulation and monitoring of labile heme levels, here we investigated the role of labile heme in the regulation of mitochondrial biogenesis. This process is regulated by the HAP complex in yeast. Using several biochemical assays along with EM and epifluorescence microscopy, to the best of our knowledge, we show for the first time that cellular labile heme is critical for the post-translational regulation of HAP complex activity, most likely through the stability of the transcriptional co-activator Hap4p. Consequently, we found that labile heme regulates mitochondrial biogenesis and cell growth. The findings of our work highlight a new mechanism in the regulation of mitochondrial biogenesis by cellular metabolites.

Microwell array integrating nanoelectrodes for coupled opto-electrochemical monitorings of single mitochondria.

Chips composed of microwell arrays integrating nanoelectrodes (OptoElecWell) were developed to achieve dual optical and electrochemical detections on isolated biological entities. Each array consists in 106 microwells of 6 µm diameter × 5.2 µm height each, with a transparent bottom surface for optical observations, a platinum nano-ring electrode at its half-height for in situ electrochemistry, and a top open surface to inject solutions. Then, populations of individual mitochondria isolated from yeasts (Saccharomyces cerevisiae) were let to sediment on the array and be trapped within microwells. The trapping efficiency reached 20% but owing to the large number of microwells on the platform, hundreds of them could be filled simultaneously by single mitochondria. This allowed to follow up their individual energetic status based on fluorescence microscopy of their endogenous NADH. Simultaneously, the array of interconnected Pt nanoelectrodes in the microwells was used to monitor in situ variations of dioxygen consumed by all mitochondria captured in the device. Mitochondrial bioenergetics were modulated sequentially using respiratory chain-ATP synthase substrates (ethanol and ADP) and inhibitor (antimycin A). Overall, we show how two complementary analytical approaches, fluorescence and electrochemical detections, can be coupled for a multi-parametric monitoring of mitochondrial activities, with a resolution ranging from a small population (whole device) to the single mitochondrion level (unique well).

Mitochondrial NADH redox potential impacts the reactive oxygen species production of reverse Electron transfer through complex I.

There is substantial evidence that Reactive Oxygen Species (ROS) play a major part in cell functioning. Although their harmfulness through oxidative stress is well documented, their role in signaling and sensing as an oxidative signal still needs to be investigated. In most cells, the mitochondrial Electron Transport Chain (ETC) is the primary source of ROS. The production of ROS by reverse electron transfer through complex I has been demonstrated both in an experimental context but also in many pathophysiological situations. Therefore, understanding the mechanisms that regulate this ROS production is of great interest to control its harmful effects. We used nigericin, Pi and valinomycin as tools to modulate the pH gradient (∆pH) and the membrane potential (∆Ψ) of the protonmotive force (∆p) in liver and muscle mitochondria to accurately determine how these parameters control the ROS production. We show that a high ∆Ψ is the "sine qua none" condition for ROS production from the reverse electron transfer (RET) through the complex I. However, a high ∆Ψ is not the only condition governing ROS production. Indeed, using tools that modulate the mitochondrial NADH level, we also demonstrate that ROS production is directly related to the mitochondrial redox potential when the membrane potential is almost stable.

The role of glycolysis-derived hexose phosphates in the induction of the Crabtree effect.

Evidence for the Crabtree effect was first reported by H. Crabtree in 1929 and is defined as the glucose-induced decrease of cellular respiratory flux. This effect was observed in tumor cells and was not detected in most non-tumor cells. A number of hypotheses on the mechanism underlying the Crabtree effect have been formulated. However, to this day, no consensual mechanism for this effect has been described. In a previous study on isolated mitochondria, we have proposed that fructose-1,6-bisphosphate (F1,6bP), which inhibits the respiratory chain, induces the Crabtree effect. Using whole cells from the yeast Saccharomyces cerevisiae as a model, we show here not only that F1,6bP plays a key role in the process but that glucose-6-phosphate (G6P), a hexose that has an effect opposite to that of F1,6bP on the regulation of the respiratory flux, does as well. Thus, these findings reveal that the Crabtree effect strongly depends on the ratio between these two glycolysis-derived hexose phosphates. Last, in silico modeling of the Crabtree effect illustrated the requirement of an inhibition of the respiratory flux by a coordinated variation of glucose-6-phosphate and fructose-1,6-bisphosphate to fit the respiratory rate decrease observed upon glucose addition to cells. In summary, we conclude that two glycolysis-derived hexose phosphates, G6P and F1,6bP, play a key role in the induction of the Crabtree effect.

PDMS microwells for multi-parametric monitoring of single mitochondria on a large scale: a study of their individual membrane potential and endogenous NADH.

Microwell arrays have been developed to monitor simultaneously, and on a large scale, multiple metabolic responses of single mitochondria. Wells of 50 to 1000 µm-diameter were prepared based on easy structuration of thin polydimethylsiloxane layers (PDMS; 100 µm thickness). Their surface treatment with oxygen plasma allowed the immobilization in situ and observation with time of populations of single isolated mitochondria. Their metabolic activities could be monitored individually by fluorescence microscopy under several activation/inhibition conditions. We measured the concomitant variations of two main metabolic parameters - the endogenous NADH level and the internal membrane potential difference Δψ owing to a cationic fluorescent probe (TMRM) - at energized, uncoupled and inhibited stages of the mitochondrial respiratory chain. Microwell arrays allowed analyses on large populations, and consequently statistical studies with a single organelle resolution. Thus, we observed rapid individual polarizations and depolarizations of mitochondria following their supply with the energetic substrate, while an averaged global polarization (increase of TMRM fluorescence within mitochondria) and NADH increase were detected for the whole population. In addition, statistical correlation studies show that the NADH content of all mitochondria tends toward a metabolic limit and that their polarization-depolarization ability is ubiquitous. These results demonstrate that PDMS microwell platforms provide an innovative approach to better characterize the individual metabolic status of isolated mitochondria, possibly as a function of their cell or organ origin or in different physio-pathological situations.

The Crabtree and Warburg effects: Do metabolite-induced regulations participate in their induction?

The Crabtree and Warburg effects are two well-known deviations of cell energy metabolism that will be described herein. A number of hypotheses have been formulated regarding the molecular mechanisms leading to these cellular energy metabolism deviations. In this review, we will focus on the emerging notion that metabolite-induced regulations participate in the induction of these effects. All throughout this review, it should be kept in mind that no regulatory mechanism is exclusive and that it may vary in cancer cells owing to different cell types or oncogenic background. 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.

Proton pumping complex I increases growth yield in Candida utilis.

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.

Electrochemical monitoring of the early events of hydrogen peroxide production by mitochondria.

Mitochondria consume oxygen in the respiratory chain and convert redox energy into ATP. As a side process, they produce reactive oxygen species (ROS), whose physiological activities are still not understood. However, current analytical methods cannot be used to monitor mitochondrial ROS quantitatively and unambiguously. We have developed electrochemical biosensors based on peroxidase-redox polymer-modified electrodes, providing selective detection of H2O2 with nanomolar sensitivity, linear response over five concentration decades, and fast response time. The release of H2O2 by mitochondria was then monitored under phosphorylating or inhibited respiration conditions. We report the detection of two concomitant regimes of H2O2 release: large fluxes (hundreds of nM) under complex III inhibition, and bursts of a few nM immediately following mitochondria activation. These unprecedented bursts of H2O2 are assigned to the role of mitochondria as the hub of redox signaling in cells.

The role of mitochondrial biogenesis and ROS in the control of energy supply in proliferating cells.

In yeast, there is a constant growth yield during proliferation on non-fermentable substrate where the ATP generated originates from oxidative phosphorylation. This constant growth yield is due to a tight adjustment between the growth rate and the cellular mitochondrial amount. We showed that this cellular mitochondrial amount is strictly controlled by mitochondrial biogenesis. Moreover, the Ras/cAMP pathway is the cellular signaling pathway involved in the regulation of mitochondrial biogenesis, with a direct relationship between the activity of this pathway and the cellular amount of mitochondria. The cAMP protein kinase Tpk3p is the catalytic subunit specifically involved in the regulation of mitochondrial biogenesis through regulation of the mitochondrial ROS production. An overflow of mitochondrial ROS decreases mitochondrial biogenesis through a decrease in the transcriptional co-activator Hap4p, which can be assimilated to mitochondria quality control. Moreover, the glutathione redox state is shown as being an intermediate in the regulation of mitochondrial biogenesis. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Optical microwell array for large scale studies of single mitochondria metabolic responses.

Microsystems based on microwell arrays have been widely used for studies on single living cells. In this work, we focused on the subcellular level in order to monitor biological responses directly on individual organelles. Consequently, we developed microwell arrays for the entrapment and fluorescence microscopy of single isolated organelles, mitochondria herein. Highly dense arrays of 3-µm mean diameter wells were obtained by wet chemical etching of optical fiber bundles. Favorable conditions for the stable entrapment of individual mitochondria within a majority of microwells were found. Owing to NADH auto-fluorescence, the metabolic status of each mitochondrion was analyzed at resting state (Stage 1), then following the addition of a respiratory substrate (Stage 2), ethanol herein, and of a respiratory inhibitor (Stage 3), antimycin A. Mean levels of mitochondrial NADH were increased by 29% and 35% under Stages 2 and 3, respectively. We showed that mitochondrial ability to generate higher levels of NADH (i.e., its metabolic performance) is not correlated either to the initial energetic state or to the respective size of each mitochondrion. This study demonstrates that microwell arrays allow metabolic studies on populations of isolated mitochondria with a single organelle resolution.

The mitochondrial genome impacts respiration but not fermentation in interspecific Saccharomyces hybrids.

In eukaryotes, mitochondrial DNA (mtDNA) has high rate of nucleotide substitution leading to different mitochondrial haplotypes called mitotypes. However, the impact of mitochondrial genetic variant on phenotypic variation has been poorly considered in microorganisms because mtDNA encodes very few genes compared to nuclear DNA, and also because mitochondrial inheritance is not uniparental. Here we propose original material to unravel mitotype impact on phenotype: we produced interspecific hybrids between S. cerevisiae and S. uvarum species, using fully homozygous diploid parental strains. For two different interspecific crosses involving different parental strains, we recovered 10 independent hybrids per cross, and allowed mtDNA fixation after around 80 generations. We developed PCR-based markers for the rapid discrimination of S. cerevisiae and S. uvarum mitochondrial DNA. For both crosses, we were able to isolate fully isogenic hybrids at the nuclear level, yet possessing either S. cerevisiae mtDNA (Sc-mtDNA) or S. uvarum mtDNA (Su-mtDNA). Under fermentative conditions, the mitotype has no phenotypic impact on fermentation kinetics and products, which was expected since mtDNA are not necessary for fermentative metabolism. Alternatively, under respiratory conditions, hybrids with Sc-mtDNA have higher population growth performance, associated with higher respiratory rate. Indeed, far from the hypothesis that mtDNA variation is neutral, our work shows that mitochondrial polymorphism can have a strong impact on fitness components and hence on the evolutionary fate of the yeast populations. We hypothesize that under fermentative conditions, hybrids may fix stochastically one or the other mt-DNA, while respiratory environments may increase the probability to fix Sc-mtDNA.

Monitoring metabolic responses of single mitochondria within poly(dimethylsiloxane) wells: study of their endogenous reduced nicotinamide adenine dinucleotide evolution.

It is now demonstrated that mitochondria individually function differently because of specific energetic needs in cell compartments but also because of the genetic heterogeneity within the mitochondrial pool-network of a cell. Consequently, understanding mitochondrial functioning at the single organelle level is of high interest for biomedical research, therefore being a target for analyticians. In this context, we developed easy-to-build platforms of milli- to microwells for fluorescence microscopy of single isolated mitochondria. Poly(dimethylsiloxane) (PDMS) was determined to be an excellent material for mitochondrial deposition and observation of their NADH content. Because of NADH autofluorescence, the metabolic status of each mitochondrion was analyzed following addition of a respiratory substrate (stage 2), ethanol herein, and a respiratory inhibitor (stage 3), Antimycin A. Mean levels of mitochondrial NADH were increased by 32% and 62% under stages 2 and 3, respectively. Statistical studies of NADH value distributions evidenced different types of responses, at least three, to ethanol and Antimycin A within the mitochondrial population. In addition, we showed that mitochondrial ability to generate high levels of NADH, that is its metabolic performance, is not correlated either to the initial energetic state or to the respective size of each mitochondrion.

Mitochondrial energetic metabolism-some general principles.

In nonphotosynthetic organisms, mitochondria are the power plant of the cell, emphasizing their great potentiality for adenosine triphosphate (ATP) synthesis from the redox span between nutrients and oxygen. Also of great importance is their role in the maintenance of the cell redox balance. Even though crystallographic structures of respiratory complexes, ATP synthase, and ATP/adenosine diphosphate (ADP) carrier are now quite well known, the coupling between ATP synthesis and cell redox state remains a controversial issue. In this review, we will present some of the processes that allow a modular coupling between ATP synthesis and redox state. Furthermore, we will present some theoretical approaches of this highly integrated system.

Dietary polyphenols preconditioning protects 3T3-L1 preadipocytes from mitochondrial alterations induced by oxidative stress.

Numerous studies indicate that an increase in reactive oxygen species (ROS) significantly affects white adipose tissue biology and leads to an inflammatory profile and insulin resistance, which could contribute to obesity-associated diabetes and cardiovascular diseases. Mitochondria play a key role in adipose tissue energy metabolism and constitute the main source of cellular ROS such as H(2)O(2). Polyphenols constitute the most abundant antioxidants provided by the human diet. Indeed, they are widely distributed in fruits, vegetables and some plant-derived beverages such as coffee and tea. Thus, the biological effects of dietary polyphenols that may increase the antioxidant capacity of the body against obesity-induced oxidative stress are of high interest. Here, we studied the capacity of polyphenols to modulate the impact of oxidative stress on the mitochondria of preadipocytes, which are important cells governing the adipose tissue development for energy homeostasis. Whereas H(2)O(2) treatment induces a proliferation arrest associated with an increase in mitochondrial content in 3T3-L1 preadipocytes, preconditioning with some major dietary polyphenols totally or partially protects the cells against oxidative stress consequences. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

cAMP-induced mitochondrial compartment biogenesis: role of glutathione redox state.

Cell fate and proliferation are tightly linked to the regulation of the mitochondrial energy metabolism. Hence, mitochondrial biogenesis regulation, a complex process that requires a tight coordination in the expression of the nuclear and mitochondrial genomes, has a major impact on cell fate and is of high importance. Here, we studied the molecular mechanisms involved in the regulation of mitochondrial biogenesis through a nutrient-sensing pathway, the Ras-cAMP pathway. Activation of this pathway induces a decrease in the cellular phosphate potential that alleviates the redox pressure on the mitochondrial respiratory chain. One of the cellular consequences of this modulation of cellular phosphate potential is an increase in the cellular glutathione redox state. The redox state of the glutathione disulfide-glutathione couple is a well known important indicator of the cellular redox environment, which is itself tightly linked to mitochondrial activity, mitochondria being the main cellular producer of reactive oxygen species. The master regulator of mitochondrial biogenesis in yeast (i.e. the transcriptional co-activator Hap4p) is positively regulated by the cellular glutathione redox state. Using a strain that is unable to modulate its glutathione redox state (Δglr1), we pinpoint a positive feedback loop between this redox state and the control of mitochondrial biogenesis. This is the first time that control of mitochondrial biogenesis through glutathione redox state has been shown.

Importance of mitochondrial dynamin-related protein 1 in hypothalamic glucose sensitivity in rats.

AIMS: Hypothalamic mitochondrial reactive oxygen species (mROS)-mediated signaling has been recently shown to be involved in the regulation of energy homeostasis. However, the upstream signals that control this mechanism have not yet been determined. Here, we hypothesize that glucose-induced mitochondrial fission plays a significant role in mROS-dependent hypothalamic glucose sensing. RESULTS: Glucose-triggered translocation of the fission protein dynamin-related protein 1 (DRP1) to mitochondria was first investigated in vivo in hypothalamus. Thus, we show that intracarotid glucose injection induces the recruitment of DRP1 to VMH mitochondria in vivo. Then, expression was transiently knocked down by intra-ventromedial hypothalamus (VMH) DRP1 siRNA (siDRP1) injection. 72 h post siRNA injection, brain intracarotid glucose induced insulin secretion, and VMH glucose infusion-induced refeeding decrease were measured, as well as mROS production. The SiDRP1 rats decreased mROS and impaired intracarotid glucose injection-induced insulin secretion. In addition, the VMH glucose infusion-induced refeeding decrease was lost in siDRP1 rats. Finally, mitochondrial function was evaluated by oxygen consumption measurements after DRP1 knock down. Although hypothalamic mitochondrial respiration was not modified in the resting state, substrate-driven respiration was impaired in siDRP1 rats and associated with an alteration of the coupling mechanism. INNOVATION AND CONCLUSION: Collectively, our results suggest that glucose-induced DRP1-dependent mitochondrial fission is an upstream regulator for mROS signaling, and consequently, a key mechanism in hypothalamic glucose sensing. Thus, for the first time, we demonstrate the involvement of DRP1 in physiological regulation of brain glucose-induced insulin secretion and food intake inhibition. Such involvement implies DRP1-dependent mROS production.

Acute mitochondrial actions of glitazones on the liver: a crucial parameter for their antidiabetic properties.

BACKGROUND/AIMS: Glitazones are synthetic insulin-sensitizing drugs which act as agonists of peroxisome proliferator-activated receptor gamma (PPARγ). However, TZDs action does not exclude independent PPARγ-activation effects. Remarkably, direct mitochondrial action of these agents has not been fully studied yet. METHODS: Oxygen consumption rates (JO(2)) were measured using a Clark-type oxygen electrode in intact hepatocytes and isolated liver mitochondria. Mitochondrial reactive oxygen species (ROS) production was quantified by fluorescence assay. Moreover, activities of mitochondrial respiratory chain complex I, II and III were spectrometrically determined. RESULTS: Pioglitazone and rosiglitazone inhibited JO(2) in liver cells and mitochondria. This inhibition affected the state 3 of respiration (in the presence of ADP) and the uncoupled state (after addition of dinitrophenol). Moreover, these agents dramatically reduced mitochondrial ROS production in all situations tested. We also demonstrated that both glitazones specifically inhibited the activities of complex I and complex III, by 50% and 35% respectively. Additionally, they do not modify neither the oxidative phosphorylation yield nor the permeability transition pore opening. CONCLUSIONS: Pioglitazone and rosiglitazone reduce both respiration intensity and ROS production, acutely and by a probable PPARγ-independent way, through inhibition of complex I and III activities. This new finding could positively contribute to their anti-diabetic properties.

Control of mitochondrial volume by mitochondrial metabolic water.

It is well-known that mitochondrial volume largely controls mitochondrial functioning. We investigate whether metabolic water produced by oxidative phosphorylation could be involved in mitochondrial volume regulation. We modulated the generation of this water in liver mitochondria and assess their volume by two independent techniques. In liver mitochondria, the mitochondrial volume was specifically decreased when no water was produced independently of energetic parameters and uncoupling activity. In all other conditions associated with water generation, there was no significant change in mitochondrial metabolic volume. Altogether these data demonstrate that mitochondrial volume is regulated, independently of energetic status, by the mitochondrial metabolic water that acts as a signal.