Nicole Averet

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.

Effects of CDP-choline on phospholipase A2 and cholinephosphotransferase activities following a cryogenic brain injury in the rabbit

Within the tissue surrounding the necrotic lesion, following a cryogenic injury of the brain, there is a definite activation of phospholipase A2 (at 2 and 4 hr post lesion) that accounts, at least in part, for the phospholipid breakdown. There is also an activation of cholinephosphotransferase (at 2 hr post lesion) that may correspond to an early process of phospholipid resynthesis. Oral CDP-choline in this model is able to completely inhibit the activation of phospholipase A2, but has no detectable effect on cholinephosphotransferase activity. The beneficial effect of CDP-choline might be explained by a prevention of destruction rather than by an enhancement of reconstruction of phospholipids.

Thyroid status is a key regulator of both flux and efficiency of oxidative phosphorylation in rat hepatocytes

Thyroid status is crucial in energy homeostasis, but despite extensive studies the actual mechanism by which it regulates mitochondrial respiration and ATP synthesis is still unclear. We studied oxidative phosphorylation in both intact liver cells and isolated mitochondria from in vivo models of severe not life threatening hyper- and hypothyroidism. Thyroid status correlated with cellular and mitochondrial oxygen consumption rates as well as with maximal mitochondrial ATP production. Addition of a protonophoric uncoupler, 2,4-dinitrophenol, to hepatocytes did not mimic the cellular energetic change linked to hyperthyroidism. Mitochondrial content of cytochrome oxidase, ATP synthase, phosphate and adenine nucleotide carriers were increased in hyperthyroidism and decreased in hypothyroidism as compared to controls. As a result of these complex changes, the maximal rate of ATP synthesis increased in hyperthyroidism despite a decrease in ATP/O ratio, while in hypothyroidism ATP/O ratio increased but did not compensate for the flux limitation of oxidative phosphorylation. We conclude that energy homeostasis depends on a compromise between rate and efficiency, which is mainly regulated by thyroid hormones.

NADH is specifically channeled through the mitochondrial porin channel in Saccharomyces cerevisiae

In many kinds of permeabilized cells, the restriction of metabolite diffusion by a mitochondrial porin “closed state” has been shown to control the respiration rate. However, since in isolated mitochondria the porin appears to be always “open,” the physiological relevance of a putative regulation via this channel status is now a subject of discussion. In Saccharomyces cerevisiae, in which some of the NADH dehydrogenase active sites are facing the intermembrane space, this regulatory mechanism might play an important role for the regulation of the cytosolic redox status. Using permeabilized spheroplasts from wild-type and porin-deficient mutant, we show that the NADH produced in the cytosol is channeled to the mitochondrial NADH dehydrogenases through a metabolic network involving the porin channel. Thus, the control exerted by the porin (i.e., “open” or “closed” state) seems to be determined through its participation or not in organized metabolic networks.

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.

Modifications of Synaptosomal Na+‐K+‐ATPase Activity During Vasogenic Brain Edema in the Rabbit

Abstract: This study investigates the functioning of synaptosomal ouabain‐sensitive Na+‐K+‐ATPase in cold‐induced edema. During vasogenic brain edema development, the enzyme affinities for Na+ and K+ are progressively decreased paralleling the increase in the tissue water content, whereas maximal velocity of the reaction is not changed. On the basis of these data, it is likely that Na+‐K+‐ATPase impairment accounts for the intracellular uptake of water in this model of edema.

Redistribution of the flux-control coefficients in mitochondrial oxidative phosphorylations in the course of brain edema

This work describes the control exerted by dicarboxylate carrier and succinate dehydrogenase activities on the oxidative phosphorylations in rabbit brain mitochondria as an edema develops. Vasogenic edema leads to an uncompetitive inhibition of succinate dehydrogenase activity and to a large decrease of oxidative phosphorylations linked to succinate utilisation. Naftidrofuryl treatment in vivo restores both a high succinate dehydrogenase activity and a normal respiratory rate. In order to quantify the control of oxidative phosphorylations by the succinate dehydrogenase step, we applied the control analysis (Kacser, H. and Burns, J.A. (1973) in Rate Control of Biological Processes (Davies, D.D., ed.), pp. 65-104, Cambridge University Press, London; Heinrich, R. and Rapoport, T.A. (1974) Eur. J. Biochem. 42, 89-95). By using two inhibitors, one (phenylsuccinate) acting only on the dicarboxylate carrier and another (malonate) acting on both the dicarboxylate carrier and the succinate dehydrogenase, a method was developed to calculate the control coefficients of these two steps. The main result is that in mitochondria isolated from normal tissue succinate dehydrogenase exerted no control, but in the course of edema this enzymatic step became a controlling one: a transition from zero to a high control coefficient (0.5) was observed from the onset of intracellular edema for the threshold value of water/dry-weight tissue of 4.6.

The Early Phase of Cryogenic Lesions: An Experimental Model of Seizures Updated

Summary: : Following cryogenic lesions in 64 rabbits, epileptic activity and gray matter edema were correlatively investigated during the initial phase (23 h). Indexes were developed to allow a quantitative assessment of epileptic activity. The indexes demonstrated that the early phase of cryogenic epilepsy is a very rapid phenomenon during the first 4 h postlesion. Epileptic activity precedes brain edema, and in individual animals, there is a significant positive correlation between the total amount of epileptic activity and the total amount of edema. The relationship between epileptic activity and brain edema are most likely interrelated at the level of pathophysiological mechanisms and several mechanisms can be postulated.

Attempts to induce asymmetrical reconstitution of the sarcoplasmic reticulum calcium transporting system

Abstract The main purpose of this work was to explore the conditions allowing the most efficient reconstitution of the sarcoplasmic reticulum calcium transporting system, displaying the same morphology as in the corresponding native membranes. The method used was based on the centrifugation of the solubilized-in-Triton X-100 Ca2+-stimulated adenosine triphosphatase through a sucrose gradient containing Tween 80 and preformed small lipid vesicles. The morphology of the reconstituted material was followed by freeze-fracture electron microscopy. The results presented in this paper show that, under appropriate experimental conditions, a large part of the reconstituted material appears to be very similar to the native sarcoplasmic reticulum.

Relationships between age-dependent changes in the effect of almitrine on H+-ATPase/ATPsynthase and the pattern of membrane fatty acid composition

The effects of almitrine on ATPase/ATPsynthase previously described in beef heart mitochondria (Rigoulet et al. (1990) Biochim. Biophys. Acta 1018, 91-97) are also observed in liver mitochondria isolated from rats older than 7 weeks. In contrast, in rats younger than 5 weeks, almitrine at the same concentration has no effect on the ATPase/ATPsynthase complex. This age-dependent action of almitrine is well correlated with age-dependent modifications of two fatty acids: linoleic and docosahexaenoic acids. The possibility of a change in H+/ATP stoichiometry of the ATPase/ATPsynthase induced by almitrine seems related to more general modifications of membrane properties during growth of the rat.