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 dimethylbiguanidepretreated livers or rats further confirmed that dimethylbiguanide action was located on the respiratory chain complexI.Thedimethylbiguanideeffectwastemperaturedependent, 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 Ca 2؉ . 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.Mitochondria are intracellular organelles devoted mainly to energy metabolism (ATP production) that also play a pivotal role in the onset of cell death (1, 2). The regulation of such functions is essential and has been well characterized in isolated mitochondria, whereas much less is known in intact cells. Short term regulation of intact cell respiration has been established with Ca 2ϩ and is related to the Ca 2ϩ -dependent mitochondrial dehydrogenases that regulate the supply of substrates to the respiratory chain (3). It has been reported that lipopolysaccharide plus interferon-␥ can persistently inhibit respiratory chain complex IV in intact astrocytes (4) and that activation of glutamate receptors induces a persistent inhibition of complexes II, III, and IV in intact neurons (5). Both inhibitions can be prevented by nitric-oxide synthase inhibitors. Furthermore, it has been shown that prolonged direct exposure to nitric oxide (NO) 1 in intact J774 cells leads to a persistent inhibition of respiratory chain complex I, whereas inhibition of complex IV was reversible (6).Dimethylbiguanide (metformin) is an oral antihyperglycemic drug widely used in the treatment of type-II diabetes (7-10), the action mechanism of which remains largely unknown (see Refs. 11 and 12 for review). Dimethylbiguanide inhibits hepatic gluconeogenesis, possibly through a decrease in the cytosolic ATP/ADP ratio (13). Although it has been long known that big...

Carbonylated proteins were visualized in single cells of the budding yeast Saccharomyces cerevisiae, revealing that they accumulate with replicative age. Furthermore, carbonylated proteins were not inherited by daughter cells during cytokinesis. Mother cells of a yeast strain lacking the sir2 gene, a life-span determinant, failed to retain oxidatively damaged proteins during cytokinesis. These findings suggest that a genetically determined, Sir2p-dependent asymmetric inheritance of oxidatively damaged proteins may contribute to free-radical defense and the fitness of newborn cells.

During the last decades a considerable amount of research has been focused on cancer. Recently, tumor cell metabolism has been considered as a possible target for cancer therapy. It is widely accepted that tumors display enhanced glycolytic activity and impaired oxidative phosphorylation (Warburg effect). Therefore, it seems reasonable that disruption of glycolysis might be a promising candidate for specific anti-cancer therapy. Nevertheless, the concept of aerobic glycolysis as the paradigm of tumor cell metabolism has been challenged, as some tumor cells exhibit high rates of oxidative phosphorylation. Mitochondrial physiology in cancer cells is linked to the Warburg effect. Besides, its central role in apoptosis makes this organelle a promising "dual hit target" to selectively eliminate tumor cells. From a metabolic point of view, the fermenting yeast Saccharomyces cerevisiae and tumor cells share several features. In this paper we will review these common metabolic properties as well as the possible origins of the Crabtree and Warburg effects.

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.

Mitochondrial reactive oxygen species (ROS) production was investigated in mitochondria extracted from liver of rats treated with or without metformin, a mild inhibitor of respiratory chain complex 1 used in type 2 diabetes. A high rate of ROS production, fully suppressed by rotenone, was evidenced in non-phosphorylating mitochondria in the presence of succinate as a single complex 2 substrate. This ROS production was substantially lowered by metformin pretreatment and by any decrease in membrane potential (Delta Phi(m)), redox potential (NADH/NAD), or phosphate potential, as induced by malonate, 2,4-dinitrophenol, or ATP synthesis, respectively. ROS production in the presence of glutamate-malate plus succinate was lower than in the presence of succinate alone, but higher than in the presence of glutamate-malate. Moreover, while rotenone both increased and decreased ROS production at complex 1 depending on forward (glutamate-malate) or reverse (succinate) electron flux, no ROS overproduction was evidenced in the forward direction with metformin. Therefore, we propose that reverse electron flux through complex 1 is an alternative pathway, which leads to a specific metformin-sensitive ROS production.

Recent reports emphasize the importance of mitochondria in white adipose tissue biology. In addition to their crucial role in energy homeostasis, mitochondria are the main site of reactive oxygen species generation. When moderately produced, they function as physiological signaling molecules. Thus, mitochondrial reactive oxygen species trigger hypoxia-dependent gene expression. Therefore the present study tested the implication of mitochondrial reactive oxygen species in adipocyte differentiation and their putative role in the hypoxiadependent effect on this differentiation. Pharmacological manipulations of mitochondrial reactive oxygen species generation demonstrate a very strong and negative correlation between changes in mitochondrial reactive oxygen species and adipocyte differentiation of 3T3-F442A preadipocytes. Moreover, mitochondrial reactive oxygen species positively and specifically control expression of the adipogenic repressor CHOP-10/ GADD153. Hypoxia (1% O 2 ) strongly increased reactive oxygen species generation, hypoxia-inducible factor-1 and CHOP-10/GADD153 expression, and inhibited adipocyte differentiation. All of these hypoxia-dependent effects were partly prevented by antioxidants. By using hypoxia-inducible factor-1␣ (HIF-1␣)-deficient mouse embryonic fibroblasts, HIF-1␣ was shown not to be required for hypoxia-mediated CHOP-10/GADD153 induction. Moreover, the comparison of hypoxia and CoCl 2 effects on adipocyte differentiation of wild type or HIF-1␣ deficient mouse embryonic fibroblasts suggests the existence of at least two pathways dependent or not on the presence of HIF-1␣. Together, these data demonstrate that mitochondrial reactive oxygen species control CHOP-10/GADD153 expression, are antiadipogenic signaling molecules, and trigger hypoxia-dependent inhibition of adipocyte differentiation.White adipose tissue is the main energy store in adult mammals and displays great plasticity according to the energy needs of the organism. Adipocyte differentiation results from a subtle balance of sequential and interdependent transcription factors expression that activate or inhibit promoters of adipogenic genes.

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.

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.