New evidence of a mitochondrial genetic background paradox: Impact of the J haplogroup on the A3243G mutation

International audienceBackground: The A3243G mutation in the tRNALeu gene (UUR), is one of the most common pathogenic mitochondrial DNA (mtDNA) mutations in France, and is associated with highly variable and heterogeneous disease phenotypes. To define the relationships between the A3243G mutation and mtDNA backgrounds, we determined the haplogroup affiliation of 142 unrelated French patients – diagnosed as carriers of the A3243G mutation – by control-region sequencing and RFLP survey of their mtDNAs. Results: The analysis revealed 111 different haplotypes encompassing all European haplogroups, indicating that the 3243 site might be a mutational hot spot. However, contrary to previous findings, we observed a statistically significant underepresentation of the A3243G mutation on haplogroup J in patients (p = 0.01, OR = 0.26, C.I. 95%: 0.08–0.83), suggesting that might be due to a strong negative selection at the embryo or germ line stages. Conclusion: Thus, our study supports the existence of mutational hotspot on mtDNA and a "haplogroup J paradox," a haplogroup that may increase the expression of mtDNA pathogenic mutations, but also be beneficial in certain environmental contexts

Ubiquinone Analogs: A Mitochondrial Permeability Transition Pore-Dependent Pathway to Selective Cell Death

International audienceBACKGROUND: Prolonged opening of the mitochondrial permeability transition pore (PTP) leads to cell death. Various ubiquinone analogs have been shown to regulate PTP opening but the outcome of PTP regulation by ubiquinone analogs on cell fate has not been studied yet. METHODOLOGY/PRINCIPAL FINDINGS: The effects of ubiquinone 0 (Ub(0)), ubiquinone 5 (Ub(5)), ubiquinone 10 (Ub(10)) and decyl-ubiquinone (DUb) were studied in freshly isolated rat hepatocytes, cultured rat liver Clone-9 cells and cancerous rat liver MH1C1 cells. PTP regulation by ubiquinones differed significantly in permeabilized Clone-9 and MH1C1 cells from that previously reported in liver mitochondria. Ub(0) inhibited PTP opening in isolated hepatocytes and Clone-9 cells, whereas it induced PTP opening in MH1C1 cells. Ub(5) did not affect PTP opening in isolated hepatocytes and MH1C1 cells, but it induced PTP opening in Clone-9 cells. Ub(10) regulated PTP in isolated hepatocytes, whereas it did not affect PTP opening in Clone-9 and MH1C1 cells. Only DUb displayed the same effect on PTP regulation in the three hepatocyte lines tested. Despite such modifications in PTP regulation, competition between ubiquinones still occurred in Clone-9 and MH1C1 cells. As expected, Ub(5) induced a PTP-dependent cell death in Clone-9, while it did not affect MH1C1 cell viability. Ub(0) induced a PTP-dependent cell death in MH1C1 cells, but was also slightly cytotoxic in Clone-9 by an oxidative stress-dependent mechanism. CONCLUSIONS/SIGNIFICANCE: We found that various ubiquinone analogs regulate PTP in different ways depending on the cell studied. We took advantage of this unique property to develop a PTP opening-targeted strategy that leads to cell death specifically in cells where the ubiquinone analog used induces PTP opening, while sparing the cells in which it does not induce PTP opening

Stress exposure alters brain mRNA expression of the genes involved in insulin signalling, an effect modified by a high fat/high fructose diet and cinnamon supplement

In occidental societies, high fat and high sugar diets often coincide with episodes of stress. The association is likely to modify brain energy control. Brain insulin signalling is rarely studied in stressed individuals consuming high fat diets. Furthermore the effects of cinnamon supplement are not known in these conditions. Therefore, we exposed rats, over a 12-week period, to a control (C) or a high fat/high fructose (HF/HFr) diet that induces peripheral insulin resistance. A cinnamon supplement (C+CN and HF/HFr +CN) was added or not. After diet exposure, one group of rats was exposed to a 30-min restraint followed by a 10-min open-field test, their combination featuring a moderate stressor, the other rats staying unstressed in their home cages. The insulin signalling in hippocampus and frontal cortex was studied through the mRNA expression of the following genes: insulin receptor (Ir), insulin receptor substrate (Irs1), glucose transporters (Glut1 and Glut3), glycogen synthase (Gys1) and their modulators, Akt1 and Pten. In C rats, stress enhanced the expression of Ir, Irs1, Glut1, Gys1 and Akt1 mRNA. In C+CN rats, stress induced an increase in Pten but a decrease in Gys1 mRNA expression. In HF/HFr rats, stress was associated with an increase in Pten mRNA expression. In HF/HFr+CN rats, stress increased Pten mRNA expression but also decreased Gys1 mRNA expression. This suggests that a single moderate stress favours energy refilling mechanisms, an effect blunted by a previous HF/HFr diet and cinnamon supplement

L'énolase spécifique du neurone comme marqueur des tumeurs pulmonaires neuroendocrines humaines : étude clinique et expérimentale

Neuroendocrine tumors constitute a heterogeneous group that represents nearly 30% of lung cancers. Despite recent therapeutic advances, their prognosis remains poor and cures exceptional. We have investigated ways to recognize and evaluate these tumors in a clinical study - on patients with small cell lung carcinoma (SCLC) and experimental - on athymic (Nude) mice transplanted with human tumor tissue.Neuron-specific enolase (NSE) is a recognized marker of tumors of neuroendocrine origin. We have shown that the electrophoretic separation of enolase isoenzymes on tissue extracts represents a specific method of recognition of the neuroendocrine character of pulmonary tumors. In the patients' serum, this biochemical method is compatible with the requirements of a clinical use as an index of the evolution of the disease. In particular, it allows early visualization of the efficacy of chemotherapy. The serum activity results were compared with the corresponding results of an enzyme immunoassay that we have developed. The study of other tumor enzymatic markers (creatine kinase and lactate dehydrogenase) led us to establish energy metabolism profiles that differentiate highly malignant neuroendocrine lung tumors (SCLC) from low proliferative carcinoid and non -neuroendocrine tumors.We have demonstrated the validity of heterotransplantation on Nude mice as a model to study the chemoresistance of SCLC: by means of the electrophoretic profile of enolase we have visualized: on the one hand the conservation of the human character and on the other hand the maintenance of the level of neuroendocrine differentiation and of the metabolic activity of the transplanted tumors.Les tumeurs neuroendocrines constituent un groupe hétérogène qui représente près de 30% des cancers du poumon. Malgré les progrès thérapeutiques récents leur pronostic reste sombre et les guérisons exceptionnelles. Nous avons recherché des moyens de reconnaissance et d'évaluation de ces tumeurs dans le cadre d'une étude clinique -.sur des patients porteurs de carcinomes pulmonaires à petites cellules (C.P.P.C.)et expérimentale - sur des souris athymiques (Nude) greffées avec du tissu tumoral humain.L'énolase spécifique du neurone (NSE) est un marqueur reconnu des tumeurs d'origine neuroendocrine. Nous avons montré que la séparation électrophorétique des isoenzymes de l'énolase, sur les extraits tissulaires représentait une méthode spécifique de reconnaissance ducaractère neuroendocrine des tumeurs pulmonaires. Sur le sérum des patients, cette méthode biochimique est compatible avec les exigences d'une utilisation clinique comme indice d' évolution de la maladie. Elle permet notamment de visualiser précocement l'efficacité de la chimiothérapie. Les résultats d'activité sérique ont été comparés aux résultats correspondants d'un dosage immunoenzymatique que nous avons mis au point. L'étude d'autres marqueurs enzymatiques tumoraux (créatine kinase et lactate déshydrogénase)nous a conduits à établir des profils du métabolisme énergétique qui différencient- les tumeurs pulmonaires neuroendocrines hautement malignes (C.P.P.C.) des.carcinoides, peu prolifératifs et des tumeurs non neuroendocrines.Nous avons démontré la validité de l'hétérotransplantation sur souris Nude comme modèle d'étude de la chimiorésistance des C.P.P.C.: grâce au profil électrophorétique de l'énolase nous avons visualisé : d'une part la conservation du caractère humain et d'autre part le maintien du niveau de différenciation neuroendocrine et de l'activité métabolique des tumeurs greffées

Opening of the mitochondrial permeability transition pore induces reactive oxygen species production at the level of the respiratory chain complex I.

International audienceWe have investigated the consequences of permeability transition pore (PTP) opening on the rate of production of reactive oxygen species in isolated rat liver mitochondria. We found that PTP opening fully inhibited H(2)O(2) production when mitochondria were energized both with complex I or II substrates. Because PTP opening led to mitochondrial pyridine nucleotide depletion, H(2)O(2) production was measured again in the presence of various amounts of NADH. PTP opening-induced H(2)O(2) production began when NADH concentration was higher than 50 microm and reached a maximum at over 300 microm. At such concentrations of NADH, the maximal H(2)O(2) production was 4-fold higher than that observed when mitochondria were permeabilized with the channel-forming antibiotic alamethicin, indicating that the PTP opening-induced H(2)O(2) production was not due to antioxidant depletion. Moreover, PTP opening decreased rotenone-sensitive NADH ubiquinone reductase activity, whereas it did not affect the NADH FeCN reductase activity. We conclude that PTP opening induces a specific conformational change of complex I that (i) dramatically increases H(2)O(2) production so long as electrons are provided to complex I, and (ii) inhibits the physiological pathway of electrons inside complex I. These data allowed the identification of a novel consequence of permeability transition that may partly account for the mechanism by which PTP opening induces cell death

Choosing the right substrate.

International audienceCarbohydrate and fatty acids are major energetic substrates, although amino acid oxidation also permits ATP synthesis. Among several major metabolic differences between lipids and carbohydrate (activation, transport, effect of insulin, etc.), two are of particular importance when considering energy metabolism of critically ill patients: the yield of ATP synthesis and the response to uncoupling. (I) Oxidative phosphorylation yield is higher when NADH is the electron donor (three coupling sites: complex 1, 3 and 4) as compared to FADH2 (two coupling sites: complex 3 and 4). Since the ratio NADH/FADH2 is higher for glycolysis as compared to beta-oxidation, the stoichiometry of ATP synthesis to oxygen consumption is also higher. Lipid oxidation provides more ATP than carbohydrate, but it requires more oxygen per mole of ATP synthesized. (II) The ratio of NADH oxidation versus FADH, oxidation depends on the proton motive force, and lowering proton motive force by uncoupling favours FADH2 oxidation, i.e. lipids versus carbohydrate. In conclusion, lipid oxidation provides a high rate of ATP synthesis even during a mild uncoupling state, but at a high rate of oxygen consumption. If oxygen availability is limited, the major metabolic adaptation to increase the efficiency is represented by a switch from lipid oxidation to glucose oxidation

How valid is the concept of antioxidants and cell injury?

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Determination of mitochondrial reactive oxygen species: methodological aspects.

International audienceThe generation of Reactive Oxygen Species (ROS) as by-products in mitochondria Electron Transport Chain (ETC) has long been admitted as the cost of aerobic energy metabolism with oxidative damages as consequence. The purpose of this methodological review is to present some of the most widespread methods of ROS generation and to underline the limitations as well as some problems, identified with some experiments as examples, in the interpretation of such results. There is now no doubt that besides their pejorative role, ROS are involved in a variety of cellular processes for the continuous adaptation of the cell to its environment. Because ROS metabolism is a complex area (low production, instability of species, efficient antioxidant defense system, several places of production...) bias, variances and limitations in ROS measurements must be recognized in order to avoid artefactual conclusions, and especially to improve our understanding of physiological and pathophysiological mechanisms of such phenomenon

Mitochondrial NADH redox potential impacts the reactive oxygen species production of reverse Electron transfer through complex I

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