No description available.

Hypoxia-inducible factor- 1α (HIF-1α) is a transcription factor that directs many of the cellular responses to hypoxia. In these studies, we have used a mouse model containing a cardiac-specific, oxygen-stabilized, doxycycline (Dox)-off regulated HIF-1α transgene to probe the role of HIF-1α in cardioprotection. Hearts used in these studies were derived from wild-type (WT), noninduced (Non-I), and 2 day (2D) and 6 day (6D) Dox-deprived mice. Whereas HIF-1α protein is undetectable in WT mice, it is present in heart tissue of "noninduced" transgenic mice, presumably because of leakiness of the promoter construct. In mice denied Dox for 2 or 6 days, HIF-1α is overexpressed to a much greater extent than Non-I or WT animals, as expected. WT and HIF-1α- expressing hearts (Non-I, 2D and 6D induced) were subjected to 30 min of ischemia, and functional recovery was measured upon reperfusion. Recovery of preischemic left ventricular developed pressure was 14% for WT, 67% for Non-I hearts, 64% for 2D-induced, and 62% for 6D-induced hearts. 6D-induced HIF hearts have increased preischemic glycogen reserves, higher glycogen synthase protein levels, and significantly higher lactic acid release during ischemia. 6D-induced HIF hearts were also better able to maintain ATP levels during ischemia compared with WT and Non-I hearts. Interestingly, Non-I hearts showed no significant increase in glycogen reserves, glycolytic flux, or greater ATP preservation during ischemia and yet were protected to a similar extent as the 6D-induced hearts. Finally, the mitochondrial membrane potential of isolated adult myocytes was monitored during anoxia or treatments with cyanide and 2-deoxyglucose. HIF-1α expression was shown to protect mitochondrial polarization during both stress treatments. Taken together these data indicate that, while HIF-1α expression in heart does induce increases in compensatory glycolytic capacity, these changes are not necessarily required for cardioprotection, at least in this model of ischemic stress.

Hypoxia-inducible factor- 1α (HIF-1α) is a transcription factor that directs many of the cellular responses to hypoxia. In these studies, we have used a mouse model containing a cardiac-specific, oxygen-stabilized, doxycycline (Dox)-off regulated HIF-1α transgene to probe the role of HIF-1α in cardioprotection. Hearts used in these studies were derived from wild-type (WT), noninduced (Non-I), and 2 day (2D) and 6 day (6D) Dox-deprived mice. Whereas HIF-1α protein is undetectable in WT mice, it is present in heart tissue of "noninduced" transgenic mice, presumably because of leakiness of the promoter construct. In mice denied Dox for 2 or 6 days, HIF-1α is overexpressed to a much greater extent than Non-I or WT animals, as expected. WT and HIF-1α- expressing hearts (Non-I, 2D and 6D induced) were subjected to 30 min of ischemia, and functional recovery was measured upon reperfusion. Recovery of preischemic left ventricular developed pressure was 14% for WT, 67% for Non-I hearts, 64% for 2D-induced, and 62% for 6D-induced hearts. 6D-induced HIF hearts have increased preischemic glycogen reserves, higher glycogen synthase protein levels, and significantly higher lactic acid release during ischemia. 6D-induced HIF hearts were also better able to maintain ATP levels during ischemia compared with WT and Non-I hearts. Interestingly, Non-I hearts showed no significant increase in glycogen reserves, glycolytic flux, or greater ATP preservation during ischemia and yet were protected to a similar extent as the 6D-induced hearts. Finally, the mitochondrial membrane potential of isolated adult myocytes was monitored during anoxia or treatments with cyanide and 2-deoxyglucose. HIF-1α expression was shown to protect mitochondrial polarization during both stress treatments. Taken together these data indicate that, while HIF-1α expression in heart does induce increases in compensatory glycolytic capacity, these changes are not necessarily required for cardioprotection, at least in this model of ischemic stress.

Cobalamin must be metabolized intracellularly in order to bind two enzymes: methionine synthase in cytoplasm and methylmalonyl-CoA mutase in mitochondria. Defects in this process cause different inborn errors of cobalamin metabolism (cblA-cblG and mut). A previous study described a cobalamin-binding protein, in addition to methylmalonyl-CoA mutase, in crude mitochondrial fractions. The amount of [57Co]cobalamin bound to this protein was increased in cblB, mut and cblD variant2 cell lines, compared to control cell lines. In the present study, this protein was identified as transcobalamin (TC). Mitochondrial fractions from a cblB cell line were incubated with anti-TC antibodies, which precipitated the cobalamin-bound protein. Analysis of mitochondrial and cytoplasmic fractions isolated from a chloroquine-incubated cblF cell line showed that isolated mitochondrial fractions contain lysosomal material, suggesting that the identified TC is lysosomal. Quantification of cobalamin-bound TC levels in whole cell extracts showed significant increases in cblB and mut groups compared to control cell lines.

No description available.

La résistance des cellules cancéreuses à la chimiothérapie est une cause majeure de l’échec thérapeutique. Des études suggèrent qu’une adaptation du métabolisme énergétique pourrait jouer un rôle dans cette résistance. Ce travail de thèse montre que la résistance des cellules cancéreuses mammaires MCF-7dox à la doxorubicine est associée à une diminution de l’activité du complexe I de la chaîne respiratoire mitochondriale et à un métabolisme des cardiolipines (CL) particulier (diminution de la quantité de CL et augmentation de la quantité de MLCL, la forme immature des CL). Nos résultats montrent aussi que les mitochondries des cellules MCF-7dox expriment deux pompes d’efflux ATP-dépendantes (BCRP et MRP1) qui participent à limiter la quantité de doxorubicine accumulée dans ces mitochondries. De plus, l’activité de ces deux transporteurs dépend partiellement de l’ATP mitochondrial dont l’efficacité de synthèse est améliorée dans les cellules MCF-7dox. D’autre part, nous montrons que l’effet sensibilisant du DHA à la doxorubicine dans les cellules MCF-7dox implique un stress oxydant mitochondrial et s’accompagne d’une diminution de l’efficacité de la synthèse d’ATP. / Resistance of cancer cells to chemotherapy is a major cause of treatment failure. Studies have suggested that an adaptation of energy metabolism may play a role in the development of this resistance. The present work shows that resistance of the breast cancer cell line MCF-7dox to doxorubicin is associated with decreased activity of the mitochondrial respiratory chain complex I and particularly altered cardiolipin (CL) metabolism, (decreased CL levels and increased MLCL levels, the immature form of the CL). Our results also show that mitochondria from MCF-7dox cells express two ATP-dependent efflux pumps (BCRP and MRP1) limiting the accumulation of doxorubicin in these mitochondria. In addition, the activity of these two transporters is partially dependent on mitochondrial ATP synthesis which efficiency is improved in MCF-7dox cells. Moreover, we show that the sensitizing effect of DHA to doxorubicin in MCF-7dox cells is regulated by mitochondrial oxidative stress and is accompanied by a decrease in ATP synthesis efficiency.

L’anorexie mentale (AN), un trouble du comportement alimentaire multifactoriel, se traduit par une perte de poids. La sévère dénutrition retrouvée dans l’AN est associée à des altérations métaboliques induisant une dérégulation de l’axe intestin cerveau. Les mécanismes physiopathologiques sont encore mal connus. Le travail de cette thèse était de mieux appréhender les dysfonctions de l’axe intestin cerveau en évaluant le métabolisme protéique de divers tissus (hypothalamus, côlon et estomac) dans un modèle murin d’anorexie par une approche protéomique. Le premier travail a permis de mieux caractériser le modèle d’anorexie nommé activity-based anorexia (ABA) en fonction du sexe. Puis les différentes analyses protéomiques ont permis de constater une adaptation tissu dépendant des mécanismes régulant l’équilibre énergétique, avec une activité cérébrale potentiellement augmentée au détriment des fonctions digestives. Chez les souris femelles ABA, il a été constaté une augmentation d’expression de protéines mitochondriales au niveau de l’hypothalamus et à l’inverse, une diminution du métabolisme protéino-énergétique au niveau colique avec un rôle de la voie de signalisation mTOR. L’autophagie était augmentée dans ces deux tissus. Ensuite, nous avons démontré un ralentissement de la vidange gastrique secondaire à la dénutrition, et l’analyse protéomique a permis de constater une augmentation du stress oxydant au niveau de l’antre des souris ABA femelles. Ces altérations peuvent contribuer aux troubles fonctionnels gastro intestinaux. En conclusion, nos études soulignent des mécanismes d’adaptation tissu dépendants dans l’anorexie, qui devront être ultérieurement approfondis. / Anorexia nervosa, a multifactorial eating disorder, is a major public health problem and results in a severe body weight loss. The severe malnutrition observed in anorectic patients is associated with metabolic alterations inducing disturbance of the gut-brain axis. However, involved mechanisms remained poorly understood. The aim of the present thesis was to better understand the alterations of the gut-brain axis in the activity-based anorexia (ABA) model by evaluating the protein metabolism of various tissues (hypothalamus, colon and stomach) by proteomic approach. Firstly, we have better characterized the response to ABA model according to sex. Then, different proteomic analyses were performed using female C57BL/6 mice. Our results revealed a tissue-dependent adaptation of protein and energy metabolism with an increased hypothalamic activity and a decrease in the gastrointestinal tract. Indeed, ABA mice exhibited an increased expression of proteins involved in mitochondrial metabolism at the level of the hypothalamus, and conversely a decrease of proteins involved in protein and energy metabolism in colonic mucosa with a key role of the mTOR signaling pathway. Both in hypothalamus and colon, autophagy was increased. We were also able to show that gastric emptying was delayed in ABA mice that is mainly due to malnutrition. In addition, proteomic analysis revealed an increase in gastric oxidative stress in female ABA mice. These alterations may contribute to the gastrointestinal functional disorders frequently described in anorexia nervosa. In conclusions, our study underlined tissue-dependent adaptive metabolic process during anorexia that should be further explored.

Mitochondrial disorders, with incidence 1:5000 live births children, are one of the most common metabolic diseases. Clinically, it is heterogeneous group of disorders caused by mutations in more than 250 genes. Diagnostic of patients with suspected mitochondrial disorder relies on broad spectrum of biochemical analysis. One of them is a measurement of Mitochondrial Energy Generating Capacity (MEGC). The principle of MEGC analysis is measuring oxidations rate of 14 C - labeled substrates in 10 different incubations. These incubations contain [1-14 C]pyruvate, [U-14 C]malate or [1,4-14 C]succinate, donors and acceptors of Acetyl-CoA and inhibitors of TCA cycle. The results of MEGC analysis provide a variety of information about mitochondrial energy metabolism (MEM) of individual in particular tissue. In diagnostic of patients with suspected mitochondrial disorder is MEGC routinely determined in skeletal muscle. The aim of this study is to optimize MEGC analysis for its use in cultures skin fibroblasts. In sum, MEGC analysis was performed in 23 patients with primary deficiency of oxidative phosphorylation (OXPHOS), in 7 patients with secondary deficiency of OXPHOS and in 15 controls cell lines. The results of MEGC in cultured skin fibroblasts were then compared with results of spectrophotometric...