Véronique Nogueira

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.

mTORC1 Hyperactivity Inhibits Serum Deprivation-Induced Apoptosis via Increased Hexokinase II and GLUT1 Expression, Sustained Mcl-1 Expression, and Glycogen Synthase Kinase 3β Inhibition

ABSTRACT The current concept is that Tsc-deficient cells are sensitized to apoptosis due to the inhibition of Akt activity by the negative feedback mechanism induced by the hyperactive mTORC1. Unexpectedly, however, we found that Tsc1/2-deficient cells exhibit increased resistance to serum deprivation-induced apoptosis. mTORC1 hyperactivity contributes to the apoptotic resistance of serum-deprived Tsc1/2-deficient cells in part by increasing the growth factor-independent expression of hexokinase II (HKII) and GLUT1. mTORC1-mediated increase in hypoxia-inducible factor 1α (HIF1α) abundance, which occurs in the absence of serum in normoxic Tsc2-deficient cells, contributes to these changes. Increased HIF1α abundance in these cells is attributed to both an increased level and the sustained translation of HIF1α mRNA. Sustained glycogen synthase kinase 3β inhibition and Mcl-1 expression also contribute to the apoptotic resistance of Tsc2-deficient cells to serum deprivation. The inhibition of mTORC1 activity by either rapamycin or Raptor knockdown cannot resensitize these cells to serum deprivation-induced apoptosis because of elevated Akt activity that is an indirect consequence of mTORC1 inhibition. However, the increased HIF1α abundance and the maintenance of Mcl-1 protein expression in serum-deprived Tsc2−/− cells are dependent largely on the hyperactive eIF4E in these cells. Consistently, the reduction of eIF4E levels abrogates the resistance of Tsc2−/− cells to serum deprivation-induced apoptosis.

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.

Mitochondrial Adaptation to in vivo Polyunsaturated Fatty Acid Deficiency: Increase in Phosphorylation Efficiency

Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (ΔΨ), mitochondrial and cytosolic ATP/ADP–Pi ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP–Pi ratio being maintained despite the lower ΔΨ. This is achieved by (1) decreasing mitochondrial Pi accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.

2,4 DINITROPHENOL-UNCOUPLING EFFECT ON DELTA PSI IN LIVING HEPATOCYTES DEPENDS ON REDUCING-EQUIVALENT SUPPLY

Mitochondrial uncouplers, such as 2,4 dinitrophenol (DNP), increase the cellular respiration by decreasing mitochondrial membrane potential (delta psi). We show that this respiratory effect can be transient or even prevented in isolated liver cells depending on the exogenous substrate used (dihydroxyacetone vs. octanoate or proline). Moreover the decrease in ATP/ADP ratio induced by DNP is partially restored by addition of octanoate or proline. By using rhodamine 123 (Rh123) monitored by flow cytometry in living hepatocytes, we were able to follow in time delta psi in such DNP-uncoupled cells incubated with various substrates. The ability of this method to evaluate delta psi changes was assessed by using myxothiazol (3.6 microM), an inhibitor of the b-c1 complex of the respiratory chain which decreased delta psi (65%), or oligomycin (6 microg/ml), an inhibitor of the F0F1-ATPase which increased it (50%). Although DNP induced a dose-dependent decrease of delta psi, we found that octanoate or proline addition prevented such effect. We propose that octanoate or proline may counteract the uncoupling effect of DNP by providing a high supply of reducing equivalents to the respiratory chain.

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.