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M Rigoulet

Publications and source records attributed to M Rigoulet.

At least 37 records · Page 2Linked to original sources
PubMed1999

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.

Aldehyde Oxidoreductases↗
PubMed1999

Activation and deactivation of F0F1-ATPase in yeast mitochondria.

The regulation of membrane-bound proton F0F1 ATPase by the protonmotive force and nucleotides was studied in yeast mitochondria. Activation occurred in whole mitochondria and the ATPase activity was measured just after disrupting the membranes with Triton X-100. Deactivation occurred either in whole mitochondria uncoupled with FCCP, or in disrupted membranes. No effect of Triton X-100 on the ATPase was observed, except a slow reactivation observed only in the absence of MgADP. Both AMPPNP and ATP increased the ATPase deactivation rate, thus indicating that occupancy of nucleotidic sites by ATP is more decisive than catalytic turnover for this process. ADP was found to stimulate the energy-dependent ATPase activation. ATPase deactivated at the same rate in uncoupled and disrupted mitochondria This suggests that deactivation is not controlled by rebinding of some soluble factor, like IF1, but rather by the conversion of the F1.IF1 complex into an inactive form.

Adenosine Diphosphate↗
PubMed1999

ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa.

The role of the nuclear-encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit VIa-null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP-regulating sites were characterized with respect to their location and their kinetic effect: (a) intra-mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of ATP on the wild-type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated by subunit VIa. However, at physiological ionic strength (i.e. approximately 200 mM), activation by ATP was absent but inhibition was not impaired; (b) in ethanol-respiring mitochondria, when the electron flux was modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild-type) or inhibition (null-mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of cytochrome oxidase on respiratory flux, decreased (wild-type) or increased (null-mutant) in the presence of ATP; (d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild-type but not for the mutant subunit. Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration. This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP.

Adenosine Triphosphate↗
PubMed1999

[Cellular energy metabolism: physiologic and pathologic aspects].

Cellular homeostasis requires permanent energy production and consumption. Adenosine triphosphate (ATP) is the major energy component for the cell. Its synthesis occurs mainly in mitochondria where the oxidative phosphorylations realise the coupling between oxygen consumption and phosphorylation of adenosine diphosphate. The anaerobic production of ATP plays an important role in the intermediary metabolism. The enzymatic complexes of the mitochondrial respiratory chain are energy transducers acting as proton pumps. In cardiomyocytes, the phosphocreatine circuit allows a substrate channelling between mitochondria and myofibrils. This metabolic compartmentation explains the difficulties of studying energetic metabolism in the beating heart and the lack of correlation between cardiac function and the usual energy parameters. Mitochondria are a potential site of action of anaesthetic agents. Lipophilic local anaesthetics impair cellular energy metabolism and mitochondrial ATP production. Such effects could be associated with toxic effects of these molecules. NMR or near-infrared spectroscopy are non invasive techniques for monitoring energetic metabolism in vivo. Clinical applications are developed for analysing brain, muscle or cardiac function in physiological and pathological conditions.

Adenosine Triphosphate↗
PubMed1998

Flux-force relationships in intact cells: a helpful tool for understanding the mechanism of oxidative phosphorylation alterations?

On isolated mitochondria, numerous studies of the relationships between fluxes and their associated forces have led to the description of some properties of the oxidative phosphorylation pathway. However whether such an approach can be applied to understanding the actual situation in intact living cells needs further consideration. In this study on isolated hepatocytes, we describe the dependence of the respiratory rate on the three thermodynamic forces linked to oxidative phosphorylation (i.e. the redox span over the respiratory chain, the electrical potential difference across the inner mitochondrial membrane and the free energy of ATP synthesis reaction). Even if this description is phenomenological and some objections may be raised regarding the relevance of such a bulk-phase force estimation, we present some results showing that the study of flux-force relationships in intact cells may be a helpful approach for understanding the mechanisms by which oxidative phosphorylation activity is changed.

Adenosine Triphosphate↗
PubMed1998

The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae.

Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism. Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions. Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle. We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2 delta mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P. To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction. The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle. However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol. Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g. the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane. By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1.2, whereas the shuttle gave low rates but a high P/O ratio of 1.7. Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2 delta strain proved dependent on GPD1 but not on GPD2.

Aerobiosis↗
PubMed1998

Characterization of the yeast mitochondria unselective channel: a counterpart to the mammalian permeability transition pore?

Large and unselective permeabilities through the inner membrane of yeast mitochondria have been observed for more than 20 years, but the characterization of these permeabilities, leading to hypothesize the existence of a large-conductance unselective channel in yeast inner mitochondrial membrane, was done only recently by several groups. This channel has been tentatively identified as a yeast counterpart to the mammalian permeability transition pore, the crucial role of which is now well-documented in physiopathological phenomena, such as Ca2+ homeostasis, ischemic damages, or programmed cell death. The aim of this review is to make a point on the known characteristics of this yeast mitochondrial unselective channel (YMUC) and to analyze whether or not it can be considered as a "yeast permeability transition pore."

Adenosine Triphosphate↗
PubMed1998

Energetic and morphological plasticity of C6 glioma cells grown on 3-D support; effect of transient glutamine deprivation.

The energetic metabolism of rat C6 glioma cells has been investigated as a function of the proliferative and differentiation states under three-dimensional (3-D) growing conditions on microcarrier beads. First, the transient deprivation of glutamine from the culture medium induced a marked decrease in the growth rate and a differentiation of C6 cells through the oligodendrocytic phenotype. Second, the respiratory capacity of the C6 cells during short-term subcultures with or without glutamine continuously declined as a function of the cell density, in part due to the mitochondrial content decrease. During the transition from the early exponential to the plateau growth phase in glutamine-containing medium, the oxygen consumption rate per single cell decreased concomitantly with a decrease in the glucose consumption and lactate production rates. This phenomenon led to a sixfold decrease in the total ATP production flux, without significantly affecting the cellular ATP/ADP ratio, thus indicating that some ATP-consuming processes were simultaneously suppressed during C6 proliferation. In glutamine-free medium, the cellular ATP/ADP ratio transiently increased due to growth arrest and to a reduced ATP turnover. Moreover, the results indicated that glutamine is not an essential respiratory substrate for rat C6 glioma under short-term glutamine deprivation. Worth noting was the high contribution of the mitochondrial oxidative phosphorylation toward the total ATP synthesis (about 80%), regardless of the proliferation or the differentiation status of the C6 cells.

Animals↗
PubMed1998

Quantitative analysis of some mechanisms affecting the yield of oxidative phosphorylation: dependence upon both fluxes and forces.

The purpose of this work was to show how the quantitative definition of the different parameters involved in mitochondrial oxidative phosphorylation makes it possible to characterize the mechanisms by which the yield of ATP synthesis is affected. Three different factors have to be considered: (i) the size of the different forces involved (free energy of redox reactions and ATP synthesis, proton electrochemical difference); (ii) the physical properties of the inner mitochondrial membrane in terms of leaks (H+ and cations); and finally (iii) the properties of the different proton pumps involved in this system (kinetic properties, regulation, modification of intrinsic stoichiometry). The data presented different situations where one or more of these parameters are affected, leading to a different yield of oxidative phosphorylation. (1) By manipulating the actual flux through each of the respiratory chain units at constant protonmotive force in yeast mitochondria, we show that the ATP/O ratio decreases when the flux increases. Moreover, the highest efficiency was obtained when the respiratory rate was low and almost entirely controlled by the electron supply. (2) By using almitrine in different kinds of mitochondria, we show that this drug leads to a decrease in ATP synthesis efficiency by increasing the H+/ATP stoichiometry ofATP synthase (Rigoulet M et al. Biochim Biophys Acta 1018: 91-97, 1990). Since this enzyme is reversible, it was possible to test the effect of this drug on the reverse reaction of the enzyme i.e. extrusion of protons catalyzed by ATP hydrolysis. Hence, we are able to prove that, in this case, the decrease in efficiency of oxidative phosphorylation is due to a change in the mechanistic stoichiometry of this proton pump. To our knowledge, this is the first example of a modification in oxidative phosphorylation yield by a change in mechanistic stoichiometry of one of the proton pumps involved. (3) In a model of polyunsaturated fatty acid deficiency in rat, it was found that non-ohmic proton leak was increased, while ohmic leak was unchanged. Moreover, an increase in redox slipping was also involved, leading to a complex picture. However, the respective role of these two mechanisms may be deduced from their intrinsic properties. For each steady state condition, the quantitative effect of these two mechanisms in the decrease of oxidative phosphorylation efficiency depends on the values of different fluxes or forces involved. (4) Finally the comparison of the thermokinetic data in view of the three dimensional-structure of some pumps (X-ray diffraction) also gives some information concerning the putative mechanism of coupling (i.e. redox loop or proton pump) and their kinetic control versus regulation of mitochondrial oxidative phosphorylation.

ATP Synthetase Complexes↗
PubMed1998

Oxidative phosphorylation in intact hepatocytes: quantitative characterization of the mechanisms of change in efficiency and cellular consequences.

Two mechanisms may affect the yield of the oxidative phosphorylation pathway in isolated mitochondria: (i) a decrease in the intrinsic coupling of the proton pumps (H+/2e- or H+/ATP), and (ii) an increase in the inner membrane conductance (proton or cation leak). Hence three kinds of modifications can occur and each of them have been characterized in isolated rat liver mitochondria (see preceding chapter by Rigoulet et al.). In intact isolated hepatocytes, these modifications are linked to specific patterns of bioenergetic parameters, i.e. respiratory flux, mitochondrial redox potential, DY, and phosphate potential. (1) The increase in H+/ATP stoichiometry of the mitochondrial ATP synthase, as induced by almitrine [20], leads to a decrease in mitochondrial and cytosolic ATP/ADP ratios without any change in the protonmotive force nor in the respiratory rate or redox potential. (2) In comparison to carbohydrate, octanoate metabolism by beta-oxidation increases the proportion of electrons supplied at the second coupling site of the respiratory chain. This mimics a redox slipping. Octanoate addition results in an increased respiratory rate and mitochondrial NADH/NAD ratio while protonmotive force and phosphate potential are almost unaffected. The respiratory rate increase is associated with a decrease in the overall apparent thermodynamic driving force (2deltaE'o - ndeltap) which confirms the 'redox-slipping-like' effect. (3) An increase in proton conductance as induced by the protonophoric uncoupler 2,4-dinitrophenol (DNP) leads to a decrease, as expected, in the mitochondrial NADH/NAD and ATP/ ADP ratios and in deltapsi while respiratory rate is increased. Thus, each kind of modification (proton leak, respiratory chain redox slipping or increase in H+/ATP stoichiometry of ATPase) is related to a specific set of bioenergetic parameters in intact cells. Moreover, these patterns are in good agreement with the data found in isolated mitochondria. From this work, we conclude that quantitative analysis of four bioenergetic parameters (respiration rate, mitochondrial NADH/ NAD ratio, protonmotive force and mitochondrial phosphate potential) gives adequate tools to investigate the mechanism by which some alterations may affect the yield of the oxidative phosphorylation pathway in intact cells.

2,4-Dinitrophenol↗
PubMed1998

Yeast mitochondrial metabolism: from in vitro to in situ quantitative study.

In this work, we first compared yeast mitochondrial oxidative metabolism at different levels of organization: whole cells (C), spheroplasts (S), permeabilized spheroplasts (PS) or isolated mitochondria (M). At present, S are more suitable for use than C for biochemical techniques such as fast extraction of metabolites and permeabilization. We show here that respiratory rates of S with various substrates are similar to C, which demonstrate that they are adapted to yeast bioenergetic studies. It appeared from ethanol metabolism +/- NAD+ or NADH respiratory rates on PS that ethanol metabolism was largely cytosolic; moreover, the activity of NADH dehydrogenase was lesser in the case of PS than in S. By comparing PS and M, the biggest difference concerned the respiratory rates of pyruvate and pyruvate-malate, which were much lower for M. Thus mitochondria preparation caused an unidentified loss involved directly in pyruvate metabolism. When the respiratory rate was lowered as a consequence of a high kinetic control of oxidative activity upstream from the respiratory chain, a similar correlation between the increase in ATP/O and decrease in respiratory rate was observed. So, the intrinsic uncoupling of proton pumps is not a particularity of M. Secondly, we demonstrate the existence of a mechanism of retarded diffusion in yeast similar to that already observed in permeabilized mammalian cells for ADP. Such a mechanism also occurs in yeast for several respiratory substrates: the K0.5 for each substrate toward the respiration rate in PS always exceeds that for M. It is proposed that such a discrepancy is due to a restriction of metabolite movement across the outer mitochondrial membrane in permeabilized cells, i.e. regulation of the substrate permeability through porin channels. In the porin-deficient yeast mutant, the K0.5 for NADH is not significantly different in either M or PS and is comparable to that of the parent strain PS. This result confirms that this retarded diffusion is essentially due to the opening-closing of the porin channel.

Adenosine Triphosphate↗
PubMed1998

Energetics of swelling in isolated hepatocytes: a comprehensive study.

Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.

Animals↗
PubMed1997

Cytosolic NAD+ content strictly depends on ATP concentration in isolated liver cells.

By focusing on the question of the thermodynamic relationships involved in the regulation of biological energy conversion, bioenergetic studies usually consider the free pyridine and adenine nucleotide rather than their total pools, in either cytosol or mitochondria. In this study, we report a new observation that, at steady state, nicotinamide nucleotide content is increased by a rise in the ATP content of the whole cell under physiological conditions. It is a straight line relationship when only NAD+ and ATP are considered. When regarding the compartmentation of this phenomenon, it appears that the linear relationship between [NAD+] and [ATP] occurs only in the cytosol. Such a dependence could be a supplementary mechanism of regulation between various metabolic pathways in the liver cell.

Adenosine Triphosphate↗
PubMed1997

Control of oxidative phosphorylation in rat liver mitochondria: effect of ionic media.

The aim of this work was to compare oxidative phosphorylation activity and its kinetic control on isolated rat liver mitochondria in either various ionic or sucrose isoosmotic media. Whatever the ionic medium, state 3 and uncoupled state respiratory rates were higher in ionic than in sucrose media, although state 4 respiration rate remained constant. Moreover, under isoosmotic conditions, the salt concentration necessary for half state 3 stimulation depends on the cation involved: for inorganic cations, these K0.5 values increased, as did the absolute value of hydratation enthalpy. The ATP/O ratio did not vary in any medium and matrix volume was about 20% increased in ionic media. JO2 versus delta p relationships were left-shifted in ionic media compared to sucrose medium: for the same respiratory rate, the protonmotive force maintained was lesser in ionic media. However, the relationship between JO2 and delta p is unique whatever the ionic medium under study. In ionic media compared to the sucrose medium, kinetic control was increased on one of the protonmotive force generating systems (cytochrome c oxidase) and decreased on one of the protonmotive force dissipating systems (adenine nucleotide translocator), even if the fluxes increased.

Animals↗
PubMed1997

The yield of oxidative phosphorylation is controlled both by force and flux.

Dissipation of energy during oxidative phosphorylation may be due to two distinct mechanisms: passive permeability to protons and/or cations (leak) or decrease in the efficiency of some proton pumps (slip). Whatever the mechanism involved, it is admitted that the wastage depends on the protonmotive force. However, the most relevant question in physiology is to determine whether other factors contribute or not to this efficiency. By comparing phosphorylating (high respiratory flux) or non phosphorylating (low respiratory flux) states at similar protonmotive force, we have shown that the wastage is higher in phosphorylating than in non-phosphorylating conditions. This strongly argues for the fact that the flux of oxidative phosphorylation is an important parameter in the control of the yield of this major energetic pathway.

Adenosine Triphosphate↗
PubMed1997

Regulation of intracellular chloride concentration in rat lactotrophs: possible role of mitochondria.

Increasing evidence is accumulating for the involvement of chloride ions in the stimulus-secretion coupling of pituitary cells. We show that the mean intracellular chloride concentration [Cl-]i of rat lactotroph cells maintained in culture is high, close to 60 mM (59.4 mM), using the Cl- sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), coupled with whole-cell patch-clamp recordings. We demonstrate that this high level is correlated with the presence of mitochondrial stores of Cl- as shown by the release of Cl- in response to various metabolic inhibitors. We determine that CCP (50 microM) induces a mean [Cl-]i increase of 15.8+/-5.8 mM, using combined electrophysiology and microspectrofluorimetry methods. These data strongly suggest that cell metabolism, including the mitochondrial function, modulate [Cl-]i.

Animals↗
PubMed1997

Response of isolated rat liver mitochondria to variation of external osmolarity in KCl medium: regulation of matrix volume and oxidative phosphorylation.

When isolated rat liver mitochondria are incubated in KCI medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K+/H+ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and deltamuH+-dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD+ ratio, state 3 respiratory rate, and deltamuH+, while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.

Animals↗