Michel Rigoulet

Dimethylbiguanide Inhibits Cell Respiration via an Indirect Effect Targeted on the Respiratory Chain Complex I

We report here a new mitochondrial regulation occurring only in intact cells. We have investigated the effects of dimethylbiguanide on isolated rat hepatocytes, permeabilized hepatocytes, and isolated liver mitochondria. Addition of dimethylbiguanide decreased oxygen consumption and mitochondrial membrane potential only in intact cells but not in permeabilized hepatocytes or isolated mitochondria. Permeabilized hepatocytes after dimethylbiguanide exposure and mitochondria isolated from dimethylbiguanide pretreated livers or animals were characterized by a significant inhibition of oxygen consumption with complex I substrates (glutamate and malate) but not with complex II (succinate) or complex IV (N,N,N′,N′-tetramethyl-1,4-phenylenediamine dihydrochloride (TMPD)/ascorbate) substrates. Studies using functionally isolated complex I obtained from mitochondria isolated from dimethylbiguanide-pretreated livers or rats further confirmed that dimethylbiguanide action was located on the respiratory chain complex I. The dimethylbiguanide effect was temperature-dependent, oxygen consumption decreasing by 50, 20, and 0% at 37, 25, and 15 °C, respectively. This effect was not affected by insulin-signaling pathway inhibitors, nitric oxide precursor or inhibitors, oxygen radical scavengers, ceramide synthesis inhibitors, or chelation of intra- or extracellular Ca2+. Because it is established that dimethylbiguanide is not metabolized, these results suggest the existence of a new cell-signaling pathway targeted to the respiratory chain complex I with a persistent effect after cessation of the signaling process.

Mitochondrial ROS Generation and Its Regulation: Mechanisms Involved in H2O2 Signaling

Mitochondria are the main source of reactive oxygen species in the cell. These reactive oxygen species have long been known as being involved in oxidative stress. This is a review of the mechanisms involved in reactive oxygen species generation by the respiratory chain and some of the dehydrogenases and the control by thermodynamic and kinetic constraints. Mitochondrial ROS produced at the level of the bc1 complex as well at the level of complex I are discussed. It was recognized more than a decade ago that they can also function as signaling molecules. This signaling role will be developed both in terms of mechanism and in terms of mitochondrial ROS signaling. The notion that hydrogen peroxide acts not only as a damaging oxidant but also as a signaling molecule was proposed more than a decade ago. Hydrogen peroxide signaling can be either direct (oxidation of its target) or indirect (involving peroxiredoxins, for example). The consequences of ROS signaling on crucial biologic processes such as cell proliferation and differentiation are discussed.

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 gut2Δ 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.

Mitochondrial ROS Metabolism: Modulation by Uncoupling Proteins

Most of the oxygen consumed by aerobic organisms is reduced to water by the enzyme cytochrome c oxidase in the terminal reaction of the mitochondrial respiratory chain. A significant proportion of the oxygen molecules are converted to superoxide anion radicals by complexes I and III via a nonenzymatic process. A cascade of enzymes, some of them inside the mitochondria themselves, scavenges superoxide anions in order to protect cells from oxidative damage induced by reactive oxygen species (ROS). Unfortunately, the quantification of the fluxes of mitochondrial ROS inside living cells is currently almost impossible, and this in turn limits our knowledge. Presently, the involvement of mitochondrial ROS can only be demonstrated by indirect strategies and among them knockout techniques are the most convincing. The yield of superoxide generation and subsequently ROS production depend mostly on oxygen concentration but can be efficiently modulated by mitochondrial uncoupling. This role could be assumed in part by one of the Uncoupling Proteins (UCPs). These proteins have coenzyme Q as an obligatory partner and we present here the hypothesis of UCPs as a crucial element of the respiratory chain. ROS have been mostly involved in degenerative processes including ageing. More recently, numerous studies point out the role of ROS as true intracellular second messengers. A putative role of mitochondrial ROS as the sensing element of energy metabolism is discussed here. We propose that UCPs could play a central role in modulation of ROS‐dependent signalling pathways and metabolic sensing via the modulation of ROS generation.

Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae

Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpd1p, and the mitochondrial glycerol-3-phosphate dehydrogenase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supramolecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed.

Mitochondrial Oxidative Phosphorylation Is Regulated by Fructose 1,6-Bisphosphate A POSSIBLE ROLE IN CRABTREE EFFECT INDUCTION?

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the “Crabtree effect.” We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into “Crabtree-positive,” where the Crabtree effect occurs, and “Crabtree-negative,” where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

Inhibition of preadipocyte proliferation by mitochondrial reactive oxygen species

Preadipocytes are present and can proliferate to increase fat mass throughout adult life. The importance of mitochondria in these cells has never been investigated, although we recently reported that mitochondrial oxidative metabolism is non‐negligible in white preadipocytes. Mitochondrial reactive oxygen species generation is intimately associated with respiratory chain function. An increasing number of reports support their role as signalling molecules. The aim of this work was to study the effects of mitochondrial reactive oxygen species on proliferation of white preadipocytes. Rotenone and oligomycin, inhibitors of complex I and of ATP synthase respectively, increased H2O2 and inhibited cell growth of preadipocytes (without inducing necrosis or apoptosis). These effects were partly prevented by addition of radical scavengers. A chemical uncoupler had opposite effects on reactive oxygen species generation and cell growth. Propofol, which inhibits complex I but also scavenges free radicals, had effects similar to those of the uncoupler on both parameters. Thus, mitochondrial reactive oxygen species can influence development of adipose tissue by affecting the size of the white preadipocyte pool.

Modern trends in biothermokinetics

Proceedings of the Fifth International Biothermokinetics Meeting held in Bordeaux-Bombannes, France, September 1992. The volume is divided into seven sections: thermodynamics and kinetics of transport processes and biological energy transduction; modeling of cell processes with applications to biote

Control of oxidative phosphorylations in yeast mitochondria. Role of the phosphate carrier

This work describes the control of ATP synthesis and O2 consumption as a function of external inorganic phosphate (Pi) concentration at steady states which are characterised by a high external (ADPATP) ratio. As a function of external Pi concentration both fluxes vary closely with each other and have a biphasic behaviour characterised by a rapid increase below 2 mM and a slow increase above this concentration. Nevertheless, the control of the fluxes is not always the same. The adenine nucleotide carrier exhibits no control in either flux whatever the external Pi concentration is. Cytochrome c oxidase is always a controlling step in both fluxes. At low Pi concentration, the proton leak controls both fluxes, but at high Pi concentration Pi transport takes the place of proton reentry as the other main controlling step. The steep threshold in external Pi concentration for which the exchange between the controlling steps occurs strongly depends on the proton permeability of the membrane. In ATP synthesis flux, proton permeability exerts a negative control (branched pathway) although the control is positive on the O2 consumption flux. In accordance with the summation theorem, this property implies that the controls must be very different at some other steps of the two fluxes.

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.”