Mitochondrial quinone redox states as a marker of mitochondrial metabolism.

Mitochondrial and thus cellular energetics are highly regulated both thermodynamically and kinetically. Cellular energetics is of prime importance in the regulation of cellular functions since it provides ATP for their accomplishment. However, cellular energetics is not only about ATP production but also about the ability to re-oxidize reduced coenzymes at a proper rate, such that the cellular redox potential remains at a level compatible with enzymatic reactions. However, this parameter is not only difficult to assess due to its dual compartmentation (mitochondrial and cytosolic) but also because it is well known that most NADH in the cells is bound to the enzymes. In this paper, we investigated the potential relevance of mitochondrial quinones redox state as a marker of mitochondrial metabolism and more particularly mitochondrial redox state. We were able to show that Q2 is an appropriate redox mediator to assess the mitochondrial quinone redox states. On isolated mitochondria, the mitochondrial quinone redox states depend on the mitochondrial substrate and the mitochondrial energetic state (phosphorylating or not phosphorylating). Last but not least, we show that the quinones redox state response allows to better understand the Krebs cycle functioning and respiratory substrates oxidation. Taken together, our results suggest that the quinones redox state is an excellent marker of mitochondrial metabolism.

The Warburg effect and mitochondrial oxidative phosphorylation: Friends or foes?

Cancer cells display an altered energy metabolism, which was proposed to be the root of cancer. This early discovery was done by O. Warburg who conducted one of the first studies of tumor cell energy metabolism. Taking advantage of cancer cells that exhibited various growth rates, he showed 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. In this review, we discuss of the origin of the decrease in cell respiratory rate, whether the Warburg effect is mandatory for an increased cell proliferation rate, the consequences of this effect on two major players of cell energy metabolism that are ATP and NADH, and the role of the microenvironment in the regulation of cellular respiration and metabolism both in cancer cell and in yeast.

Cell energy metabolism: An update.

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD+ molecules play a key role. Cell energy metabolism hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metabolism: glycolysis/fermentation and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodynamic and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metabolism will be reviewed using examples such as the Crabtree and the Warburg effect.

Respiratory complex III dysfunction in humans and the use of yeast as a model organism to study mitochondrial myopathy and associated diseases.

The bc1 complex or complex III is a central component of the aerobic respiratory chain in prokaryotic and eukaryotic organisms. It catalyzes the oxidation of quinols and the reduction of cytochrome c, establishing a proton motive force used to synthesize adenosine triphosphate (ATP) by the F1Fo ATP synthase. In eukaryotes, the complex III is located in the inner mitochondrial membrane. The genes coding for the complex III have a dual origin. While cytochrome b is encoded by the mitochondrial genome, all the other subunits are encoded by the nuclear genome. In this review, we compile an exhaustive list of the known human mutations and associated pathologies found in the mitochondrially-encoded cytochrome b gene as well as the fewer mutations in the nuclear genes coding for the complex III structural subunits and accessory proteins such as BCS1L involved in the assembly of the complex III. Due to the inherent difficulties of studying human biopsy material associated with complex III dysfunction, we also review the work that has been conducted to study the pathologies with the easy to handle eukaryotic microorganism, the yeast Saccharomyces cerevisiae. Phenotypes, biochemical data and possible effects due to the mutations are also discussed in the context of the known three-dimensional structure of the eukaryotic complex III. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

ACoM: A classification method for elementary flux modes based on motif finding.

Elementary flux mode analysis is a powerful tool for the theoretical study of metabolic networks. However, when the networks are complex, the determination of elementary flux modes leads to combinatorial explosion of their number which prevents from drawing simple conclusions from their analysis. To deal with this problem we have developed a method based on the Agglomeration of Common Motifs (ACoM) for classifying elementary flux modes. We applied this algorithm to describe the decomposition into elementary flux modes of the central carbon metabolism in Bacillus subtilis and of the yeast mitochondrial energy metabolism. ACoM helps to give biological meaning to the different elementary flux modes and to the relatedness between reactions. ACoM, which can be viewed as a bi-clustering method, can be of general use for sets of vectors with values 0, +1 or -1.

Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis.

Diversity of respiratory chain spectrophotometric assays may lead to difficult comparison of results between centers. The French network of mitochondrial diseases diagnostic centers undertook comparison of the results obtained with different protocols in the French diagnostic centers. The diversity of protocols was shown to have striking consequences, which prompted the network to undertake standardization and optimization of the protocols with respect to clinical diagnosis, i.e. high velocity while maintaining linear kinetics relative to time and enzyme concentration. Assays were set up on animal tissues and verified on control human muscle and fibroblasts. Influence of homogenization buffer and narrow range of optimal concentration of phosphate, substrate and tissue were shown. Experimental data and proposed protocols have been posted on a free access website. Their subsequent use in several diagnostic centers has improved consistency for all assays.

Pathway classification of TCA cycle.

The structural analysis of large metabolic networks exhibits a combinatorial explosion of elementary modes. A new method of classification has been developed [called aggregation around common motif (ACoM)], which groups elementary modes into classes with similar substructures. This method is applied to the tricarboxylic acid cycle and metabolite carriers. The analysis of this network evidences a great number of elementary flux modes (204) despite the low number of reactions (23). The ACoM is used to class these elementary modes in a low number of sets (8) with biological meanings.

Gradual alteration of mitochondrial structure and function by beta-amyloids: importance of membrane viscosity changes, energy deprivation, reactive oxygen species production, and cytochrome c release.

Intracellular amyloid beta-peptide (A beta) accumulation is considered to be a key pathogenic factor in sporadic Alzheimer's disease (AD), but the mechanisms by which it triggers neuronal dysfunction remain unclear. We hypothesized that gradual mitochondrial dysfunction could play a central role in both initiation and progression of sporadic AD. Thus, we analyzed changes in mitochondrial structure and function following direct exposure to increasing concentrations of A beta(1--42) and A beta(25--35) in order to look more closely at the relationships between mitochondrial membrane viscosity, ATP synthesis, ROS production, and cytochrome c release. Our results show the accumulation of monomeric A beta within rat brain and muscle mitochondria. Subsequently, we observed four different and additive modes of action of A beta, which were concentration dependent: (i) an increase in mitochondrial membrane viscosity with a concomitant decrease in ATP/O, (ii) respiratory chain complexes inhibition, (iii) a potentialization of ROS production, and (iv) cytochrome c release.

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery.

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.

Association of ATP synthase alpha-chain with neurofibrillary degeneration in Alzheimer's disease.

Amyloid deposits and neurofibrillary tangles (NFT) are the two hallmarks that characterize Alzheimer's disease (AD). In order to find the molecular partners of these degenerating processes, we have developed antibodies against insoluble AD brain lesions. One clone, named AD46, detects only NFT. Biochemical and histochemistry analyses demonstrate that the labeled protein accumulating in the cytosol of Alzheimer degenerating neurons is the alpha-chain of the ATP synthase. The cytosolic accumulation of the alpha-chain of ATP synthase is observed even at early stages of neurofibrillary degenerating process. It is specifically observed in degenerating neurons, either alone or tightly associated with aggregates of tau proteins, suggesting that it is a new molecular event related to neurodegeneration. Overall, our results strongly suggest the implication of the alpha-chain of ATP synthase in neurofibrillary degeneration of AD that is illustrated by the cytosolic accumulation of this mitochondrial protein, which belongs to the mitochondrial respiratory system. This regulatory subunit of the respiratory complex V of mitochondria is thus a potential target for therapeutic and diagnostic strategies.

Relationships between muscle mitochondrial metabolism and stress-induced corticosterone variations in rats.

In order to determine the effect of chronic and acute stress on muscle mitochondrial metabolism, two strains of rats were selected on the basis of their different hypothalamo-pituitary-adrenal (HPA) axis responses to different stressors [Spontaneous Hypertensive Rats (SHR) and Lewis rats]. For 8 weeks animals were stressed by daily exposure to either a novel environment (SHR: n=16, Lewis: n=16) or forced exercise (SHR: n=16, Lewis: n=16). An unstressed group was left undisturbed (SHR: n=5, Lewis: n=5). Half of the stressed animals (n=32) were submitted to an acute stress (1-h immobilization). The mitochondrial responses of plantaris muscle [cytochrome-c-oxidase (COX), citrate synthase and succinate dehydrogenase activities, the latter two being measured as indices of functional mitochondrial amount] in the presence of different physiological plasma corticosterone (CORT) concentrations were analyzed. The novel environment and forced exercise stress induced different levels of plasma CORT which were negatively correlated with the amount of functional mitochondria in the plantaris muscle. Therefore, a chronic intermittent stress is able to induce an increase in plasma CORT which may be related to deleterious changes in muscle mitochondrial metabolism. Lastly, the acute stress was not associated with a decrease in functional mitochondria but with an increase in COX activity. This suggests that the relationship between CORT and muscle mitochondrial metabolism depends both on the level and duration of endogenous glucocorticoids exposure.

Effect of 'binary mitochondrial heteroplasmy' on respiration and ATP synthesis: implications for mitochondrial diseases.

Respiratory-chain-complex subunits in mitochondria are encoded by nuclear or mitochondrial DNA. This property might have profound implications for the phenotypic expression of mutations affecting oxidative phosphorylation complexes. The aim of this paper is to study the importance of the origin of the mutation (nuclear or mitochondrial) on the expression of mitochondrial defects. We have therefore developed theoretical models illustrating three mechanisms of nuclear or mitochondrial DNA mutation giving rise to a deficiency in the respiratory-chain complex: (1) a partial deficiency, homogeneously distributed in all of the mitochondria; (2) a complete deficiency, only affecting some of the mitochondria ('binary mitochondrial heteroplasmy'); and (3) a partial deficiency, affecting only some of the mitochondria. We show that mutations affecting oxidative phosphorylation complexes will be expressed in different ways depending on their origins. Although the expression of nuclear or mitochondrial mutations is evidence of a biochemical threshold, we demonstrate that the threshold value depends on the origin and distribution of the mutation (homogeneous or not) and also on the energy demand of the tissue. This last prediction has been confirmed in an experimental model using hexokinase for the simulation of the energy demand and a variation in mitochondrial concentration. We also emphasize the possible role of 'binary mitochondrial heteroplasmy' in the expression of mitochondrial DNA mutations and thus the importance of the origin of the deficit (mutation) for the diagnosis or therapy of mitochondrial diseases.

Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics.

We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions. The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH. In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH. A stoichiometric conversion of precursors into threonine was observed. We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell. The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions. A response to threonine is only observed in a narrow concentration range from 0.23 to 2 mM.

An integrated study of threonine-pathway enzyme kinetics in Escherichia coli.

We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions.

Control of the threonine-synthesis pathway in Escherichia coli: a theoretical and experimental approach.

A computer simulation of the threonine-synthesis pathway in Escherichia coli Tir-8 has been developed based on our previous measurements of the kinetics of the pathway enzymes under near-physiological conditions. The model successfully simulates the main features of the time courses of threonine synthesis previously observed in a cell-free extract without alteration of the experimentally determined parameters, although improved quantitative fits can be obtained with small parameter adjustments. At the concentrations of enzymes, precursors and products present in cells, the model predicts a threonine-synthesis flux close to that required to support cell growth. Furthermore, the first two enzymes operate close to equilibrium, providing an example of a near-equilibrium feedback-inhibited enzyme. The predicted flux control coefficients of the pathway enzymes under physiological conditions show that the control of flux is shared between the first three enzymes: aspartate kinase, aspartate semialdehyde dehydrogenase and homoserine dehydrogenase, with no single activity dominating the control. The response of the model to the external metabolites shows that the sharing of control between the three enzymes holds across a wide range of conditions, but that the pathway flux is sensitive to the aspartate concentration. When the model was embedded in a larger model to simulate the variable demands for threonine at different growth rates, it showed the accumulation of free threonine that is typical of the Tir-8 strain at low growth rates. At low growth rates, the control of threonine flux remains largely with the pathway enzymes. As an example of the predictive power of the model, we studied the consequences of over-expressing different enzymes in the pathway.

What do mitochondrial diseases teach us about normal mitochondrial functions...that we already knew: threshold expression of mitochondrial defects.

This paper shows how metabolic control analysis (MCA) can help to explain two important features of mitochondrial diseases: (i) the existence of a threshold in the expression of the complex deficiencies on the respiratory flux or on ATP synthesis, i.e. the fact that it is necessary to have a large complex deficiency in order to observe a substantial decrease in these fluxes; (ii) the tissue specificity, i.e. the fact that all tissues are not affected, even if the complex deficiency is present in all of them. We also show the limits of MCA, particularly when considering the in vivo situation. However, MCA offers a new way to consider mitochondrial diseases. The fact that fluxes only slightly change, when a complex is affected, is done at the expense of great changes in intermediate metabolite concentrations; intermediate metabolites situated upstream from the deficient complex are more reduced, leading to a greater generation of free radicals. This could bring an explanation for the diseases observed in conditions where the mitochondrial rate of ATP synthesis is only slightly affected.

Simple models of threshold curves in the expression of inborn errors of metabolism: application to some experimental observations.

The expression of an enzymatic deficiency in a metabolic network can present a biochemical threshold. This threshold can be characterised thus: (1) a low activity of the enzyme can sustain a normal flux, but (2) a minute further decrease of its activity makes the flux collapse. We give simple mathematical models displaying such a behaviour, and we apply the models to some examples of oxidative phosphorylation dependency on respiratory chain complex deficiency.

Modification of mitochondrial metabolism in fibroblasts from mice with a skeletal muscle mutation (muscular dysgenesis). Evidence of embryonic communication between myoblasts and fibroblasts.

Muscle development during embryogenesis is a complex process involving many mechanisms. It requires a close communication among the different cellular types of the muscle, especially the fibroblasts and myoblasts. Indeed, any abnormality in one cell type might influence the differentiation of the other. Thus, any disturbance altering the metabolism of the myoblasts might lead to modifications in the fibroblasts. To study this phenomenon, we used the dysgenic mouse (mdg-"muscular dysgenesis") carrying a homozygous recessive lethal mutation expressed only in skeletal muscle cells. First, we found that fibroblasts isolated from such mutant muscle (and not from mutant skin tissue) and grown in culture exhibited an altered metabolism. Secondly, muscle fibroblasts showed a lower capacity for proliferation. We also observed that respiration and ATP synthesis of dysgenic muscle fibroblasts were deficient, while respiratory chain enzymatic activities were normal. Finally, intracellular [Ca2+] levels of dysgenic fibroblasts are 50% of those of normal fibroblasts. These results support the hypothesis that certain characteristics of fibroblasts are determined by the surrounding cellular environment during embryonic organogenesis, and that such modifications are stable when the fibroblasts are isolated in vitro. Since fibroblast differentiation was disrupted permanently, this suggests, in the case of myopathies, that the modified cells, surrounding the muscle tissue, could contribute to the muscle pathology. Synergistic activities of this type should be considered when studying the course of pathologies in different types of muscle diseases.

Functional mitochondrial heterogeneity in heteroplasmic cells carrying the mitochondrial DNA mutation associated with the MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes).

Most mitochondrial DNA (mtDNA) alterations associated with human disorders are heteroplasmic, i.e. mutant mtDNA molecules coexist with normal ones within the cell. We addressed the possibility of intermitochondrial exchanges through histologic analyses of cybrid clones with increasing proportion of the MELAS (A3243G) mtDNA transfer RNA point mutation. MtDNA-dependent cytochrome c oxidase activity and protein composition as well as mitochondrial membrane potential appeared heterogeneous in individual cells from clonal heteroplasmic cell populations on the basis of confocal and electron microscopy. The number of defective cells increased with increasing mutation load. We conclude that in the presence of a heteroplasmic mtDNA mutation in the cell type that we studied, intermitochondrial molecular exchanges cannot provide an efficient even distribution of the complementing molecules such as wild-type mtDNA, transfer RNA, or protein. Mitochondria in these heteroplasmic cells cannot, therefore, be considered a single functional unit.

Statistical analysis of mitochondrial pathologies in childhood: identification of deficiencies using principal component analysis.

Mitochondrial pathologies are a heterogeneous group of metabolic disorders that are frequently characterized by anomalies of oxidative phosphorylation, especially in the respiratory chain. The identification of these anomalies may involve many investigations, and biochemistry is a main tool. However, considering the whole set of biochemical data, the interpretation of the results by the traditionally used statistical methods remains complex and does not always lead to an unequivocal conclusion about the presence or absence of a respiratory chain defect. This arises from three main problems: (a) the absence of an a priori-defined control population, because the determination of the control values are derived from the whole set of investigated patients, (b) the small size of the population studied, (c) the large number of variables collected, each of which creates a wide variability. To cope with these problems, the principal component analysis (PCA) has been applied to the biochemical data obtained from 35 muscle biopsies of children suspected of having a mitochondrial disease. This analysis makes it possible for each respiratory chain complex to distinguish between different subsets within the whole population (normal, deficient, and, in between, borderline subgroups of patients) and to detect the most discriminating variables. PCA of the data of all complexes together showed that mitochondrial diseases in this population were mainly caused by multiple deficits in respiratory chain complexes. This analysis allows the definition of a new subgroup of newborns, which have high respiratory chain complex activity values. Our results show that the PCA method, which simultaneously takes into account all of the concerned variables, allows the separation of patients into subgroups, which may help clinicians make their diagnoses.