A disfunção mitocondrial pode ser um mecanismo chave associado à ocorrência de doenças metabólicas como o diabetes. Neste contexto, é importante obeservar os mecanismos envolvidos nesse processo. MicroRNAs (miRs) são conhecidos por regular a expressão de genes em vários processos fisiológicos, incluindo o metabolismo de glicose e ácidos graxos, biogênese mitocondrial, proliferação, diferenciação e morte celular no músculo esquelético. Usando análise \"in silico\" (Sfold2.2) identificamos 219 microRNAs que, potencialmente, se ligam à região 3 \'UTR do PGC-1?, um gene envolvido na biogênese mitocondrial e no metabolismo de glicose. Dos 219 candidatos, encontramos um alto valor de energia livre de hibridização entre o microRNA miR-696 e PGC-1? (-29,8 kcal / mol), sugerindo que o miR-696 poderia estar envolvido na regulação negativa do PGC-1? resultando em disfunção mitocondrial. Consistente com esta hipótese, observamos que a expressão do miR-696 apresentou-se aumentada nos músculos esqueléticos de dois modelos de camundongos com diabetes: camundongos diabéticos induzidos por STZ e camundongos alimentados com dieta hiperlipídica. Para compreender se o miR-696 regula a disfunção mitocondrial utilizamos células musculares C2C12 expostas a uma alta dose de ácido palmítico (700 µM) durante 24 horas, o que causou uma redução na expressão de genes mitocondriais, bem como no consumo de oxigênio. Vale destacar que a inibição do miR-696 através da transfecção de oligonucleotídeos antisenso (ASO) preveniu, parcialmente, a perda da função mitocondrial de células C2C12 tratadas com ácido palmítico. Curiosamente, não houve nenhuma alteração nos níveis de miR-696 em modelos envolvidos com a proteína AMPK, tal como em células C2C12 incubadas com uma droga ativadora de AMPK (AICAR) e no músculo esquelético de camundongos transgênicos superexpressando AMPK?2 com o domínio quinase inativo ou AMPK?3 com mutação de ativação crônica (R70Q). Em contraste, a expressão alterada de uma quinase relacionadas com a AMPK, SNF1-AMPK-related kinase (SNARK), recentemente demonstrada por ter sua expressão aumentada em virtude do envelhecimento, exerceu efeitos significativos sobre a expressão do miR- 696, como por exemplo sua redução dependente do knockdown de SNARK em células C2C12. Consistente com estes resultados, a superexpressão de SNARK em células C2C12 resultou no aumento da expressão do miR-696 e redução na expressão do PGC-1?, bem como no consumo de oxigénio. Nossos resultados demonstram que o estresse metabólico aumenta a expressão do miR-696 no músculo esquelético, que por sua vez inibe a sinalização da PGC-1? e a função mitocondrial. Ainda, apesar da AMPK não se apresentar como mediadora da expressão do miR-696, SNARK pode desempenhar um papel neste processo através do mecanismo de sinalização SNARKmiR-696-PGC-1?. / Mitochondrial dysfunction may be a key underlying mechanism for occurrence of metabolic disease and diabetes; thus elucidating how this process occurs is of great value. MicroRNAs (miRs) are known to regulate gene expression in several physiological processes including metabolism, mitochondrial biogenesis, proliferation, differentiation and cell death in multiple tissues including adipose tissue and skeletal muscle. Using \"in silico\" analysis (Sfold2.2) we identified 219 unique microRNAs that potentially bind to the 3\'UTR region of PGC-1?, a gene involved in mitochondrial biogenesis and glucose metabolism. Out of the 219 candidates, there was a high value of hybridization free energy between the microRNA miR-696 and PGC-1? (- 29.8 kcal/mol), suggesting that miR-696 could be involved in the downregulation of PGC-1?, which in turn could cause mitochondrial dysfunction. Consistent with this hypothesis we found that miR-696 expression was increased in the skeletal muscles of two mouse models of diabetes that have impaired mitochondrial function: STZ-induced diabetic mice and chronic high fat fed mice. To understand if miR-696 regulates mitochondrial dysfunction we used C2C12 muscle cells exposed to a high dose of palmitic acid (700 µM) for 24 hours, which caused a decrease in mitochondrial gene expression and in oxygen consumption. Importantly, inhibition of miR-696 using an antisense oligo approach rescued the mitochondrial function by restoration of mitochondrial-related genes and increased oxygen consumption in the palmitic acid-treated C2C12 cells. Interestingly, there was no change in miR-696 levels in models involved with AMPactivated protein kinase such as C2C12 cells incubated with AICAR, skeletal muscle from AMPK?2 dominant-negative transgenic mice, and transgenic mice overexpressing the activating R70Q AMPK mutation. In contrast, altered expression of the AMPK-related kinase, SNF1- AMPK-related kinase (SNARK), recently shown to increase with aging, had significant effects on miR-696 expression. Knockdown of SNARK in C2C12 cells significantly decreased miR-696. Consistent with these findings, SNARK overexpression in C2C12 cells increased miR-696 concomitant with a decrease in PGC-1? expression and decreased oxygen consumption. Our findings demonstrate that metabolic stress increases miR-696 expression in skeletal muscle which in turn inhibits PGC-1? signaling and mitochondrial function. While AMPK does not mediate miR-696 expression, SNARK may play a role in this process through a SNARK-miR- 696-PGC-1? signaling mechanism.

Dans un premier temps, nous avons observé la chronologie des altérations de la biogenèse et de la fonction mitochondriale dans les ventricules droit (VD) et gauche (VG) et le muscle gastrocnémien (GAS) dans un modèle animal d’hypertension artérielle pulmonaire (HTAP). Nous avons constaté une diminution précoce des facteurs impliqués dans la biogénèse mitochondriale du GAS. Plus tard, les mêmes anomalies apparaissaient dans le VD. Au stade décompensé de l’insuffisance cardiaque droite s’ajoutaient une diminution de la protéine PGC-1 , de l’activité de la citrate-synthase et de la respiration mitochondriale. L’expression des ARNm et la respiration mitochondriale du VG n’étaient pas modifiées de façon significative.Dans un deuxième temps, nous avons étudié l’effet de l’entraînement en mode excentrique sur le même modèle d’HTAP. La survie des rats entraînés n’était pas différente de celle des rats sédentaires et la tolérance hémodynamique évaluée par échocardiographie et cathétérisme cardiaque a été bonne. Le bénéfice de l’entraînement s’est traduit par une augmentation de la vitesse maximale de course dans les deux groupes entraînés, malades et témoins. / We assessed the time courses of mitochondrial biogenesis factors and respiration in the right ventricle (RV), gastrocnemius (GAS) and left ventricle (LV) in a model of pulmonary-hypertensive (PH) rats induced by monocrotaline (MT). The expression of the studied genes was decreased early in the MT GAS. At 4 weeks, the MT GAS and MT RV showed decreased mRNA levels whatever the stage of disease, but PGC-1 protein and citrate-synthase activity were significantly reduced only atthe decompensated stage. The functional result was a significant fall in mitochondrial respiration at the decompensated stage in the RV and GAS. The mRNA expression and mitochondrial respiration were not significantly modified in the MT LV. Secondly, we assessed the effects of eccentric exercise training (ECCt) in MT rats with PH. ECCt was initiated 2 weeks after MT injection for 4 weeks. The trained MT rats survival was not different from that of sedentary rats. ECCt was not detrimental on hemodynamic condition estimated by echocardiography and right heart catheterization. Maximal speed significantly increased in trained rats. The mRNA expression of mitochondrial biogenesis factors were not significantly modified in skeletal muscle and in RV.

Le muscle squelettique (MS) est un tissu métabolique important. L'objectif de ma thèse était de caractériser le rôle des corégulateurs de la transcription, PGC-1β (transcriptional coactivator peroxisome proliferator-activated receptor-gammacoactivator 1beta) et TIF2 (Transcriptional Intermediary Factor 2) dans ce tissu. Mon travail a démontré que PGC-1β limite le stress oxydatif est crucial dans le maintien de la structure et de la fonction mitochondriale, via le contrôle de l’expression de gènes impliqués dans les voies de signalisation liées à l’énergie, à la dynamique mitochondriale et à la machinerie d’import mais n'est pas indispensable pour le contenu mitochondrial. Mon travail aussi démontré que TIF2 de la MS a un impact négatif sur la durée de vie des mammifères. De plus, la déplétion de TIF2 conduit à une protection partielle du MS contre les dommages oxydatifs induits par le stress. Ainsi notre travail représente une avancée dans l’établissement futur de traitements contre les troubles liés au stress oxydatif et au vieillissement. / Skeletal muscle (SM) accounting for ~ 40% of total body mass is an important metabolic tissue. The aim of my thesis was to characterize the role of transcriptional coregulators, peroxisome proliferator-activated receptor-gamma coactivator-1β (PGC-1β) and transcriptional intermediary factor 2 (TIF2) in this tissue. My work demonstrated that PGC-1β is crucial to maintain SM mitochondrial structure and function, by controlling expression of genes involved in energy pathways, mitochondrial dynamics and import machinery, but is dispensable for mitochondrial content and fiber type maintenance. Furthermore, it limits oxidative stress. The second part of my work demonstrated that myofiber TIF2 has negative impact onmammalian life span. Moreover, TIF2 ablation leads to partial protection of SM from oxidative stress-induced damage. In conclusion, our work provides a better understanding of SM homeostasis regulation and insights in treatments for disordersrelated to oxidative stress and aging.

A disfunção mitocondrial pode ser um mecanismo chave associado à ocorrência de doenças metabólicas como o diabetes. Neste contexto, é importante obeservar os mecanismos envolvidos nesse processo. MicroRNAs (miRs) são conhecidos por regular a expressão de genes em vários processos fisiológicos, incluindo o metabolismo de glicose e ácidos graxos, biogênese mitocondrial, proliferação, diferenciação e morte celular no músculo esquelético. Usando análise \"in silico\" (Sfold2.2) identificamos 219 microRNAs que, potencialmente, se ligam à região 3 \'UTR do PGC-1?, um gene envolvido na biogênese mitocondrial e no metabolismo de glicose. Dos 219 candidatos, encontramos um alto valor de energia livre de hibridização entre o microRNA miR-696 e PGC-1? (-29,8 kcal / mol), sugerindo que o miR-696 poderia estar envolvido na regulação negativa do PGC-1? resultando em disfunção mitocondrial. Consistente com esta hipótese, observamos que a expressão do miR-696 apresentou-se aumentada nos músculos esqueléticos de dois modelos de camundongos com diabetes: camundongos diabéticos induzidos por STZ e camundongos alimentados com dieta hiperlipídica. Para compreender se o miR-696 regula a disfunção mitocondrial utilizamos células musculares C2C12 expostas a uma alta dose de ácido palmítico (700 µM) durante 24 horas, o que causou uma redução na expressão de genes mitocondriais, bem como no consumo de oxigênio. Vale destacar que a inibição do miR-696 através da transfecção de oligonucleotídeos antisenso (ASO) preveniu, parcialmente, a perda da função mitocondrial de células C2C12 tratadas com ácido palmítico. Curiosamente, não houve nenhuma alteração nos níveis de miR-696 em modelos envolvidos com a proteína AMPK, tal como em células C2C12 incubadas com uma droga ativadora de AMPK (AICAR) e no músculo esquelético de camundongos transgênicos superexpressando AMPK?2 com o domínio quinase inativo ou AMPK?3 com mutação de ativação crônica (R70Q). Em contraste, a expressão alterada de uma quinase relacionadas com a AMPK, SNF1-AMPK-related kinase (SNARK), recentemente demonstrada por ter sua expressão aumentada em virtude do envelhecimento, exerceu efeitos significativos sobre a expressão do miR- 696, como por exemplo sua redução dependente do knockdown de SNARK em células C2C12. Consistente com estes resultados, a superexpressão de SNARK em células C2C12 resultou no aumento da expressão do miR-696 e redução na expressão do PGC-1?, bem como no consumo de oxigénio. Nossos resultados demonstram que o estresse metabólico aumenta a expressão do miR-696 no músculo esquelético, que por sua vez inibe a sinalização da PGC-1? e a função mitocondrial. Ainda, apesar da AMPK não se apresentar como mediadora da expressão do miR-696, SNARK pode desempenhar um papel neste processo através do mecanismo de sinalização SNARKmiR-696-PGC-1?. / Mitochondrial dysfunction may be a key underlying mechanism for occurrence of metabolic disease and diabetes; thus elucidating how this process occurs is of great value. MicroRNAs (miRs) are known to regulate gene expression in several physiological processes including metabolism, mitochondrial biogenesis, proliferation, differentiation and cell death in multiple tissues including adipose tissue and skeletal muscle. Using \"in silico\" analysis (Sfold2.2) we identified 219 unique microRNAs that potentially bind to the 3\'UTR region of PGC-1?, a gene involved in mitochondrial biogenesis and glucose metabolism. Out of the 219 candidates, there was a high value of hybridization free energy between the microRNA miR-696 and PGC-1? (- 29.8 kcal/mol), suggesting that miR-696 could be involved in the downregulation of PGC-1?, which in turn could cause mitochondrial dysfunction. Consistent with this hypothesis we found that miR-696 expression was increased in the skeletal muscles of two mouse models of diabetes that have impaired mitochondrial function: STZ-induced diabetic mice and chronic high fat fed mice. To understand if miR-696 regulates mitochondrial dysfunction we used C2C12 muscle cells exposed to a high dose of palmitic acid (700 µM) for 24 hours, which caused a decrease in mitochondrial gene expression and in oxygen consumption. Importantly, inhibition of miR-696 using an antisense oligo approach rescued the mitochondrial function by restoration of mitochondrial-related genes and increased oxygen consumption in the palmitic acid-treated C2C12 cells. Interestingly, there was no change in miR-696 levels in models involved with AMPactivated protein kinase such as C2C12 cells incubated with AICAR, skeletal muscle from AMPK?2 dominant-negative transgenic mice, and transgenic mice overexpressing the activating R70Q AMPK mutation. In contrast, altered expression of the AMPK-related kinase, SNF1- AMPK-related kinase (SNARK), recently shown to increase with aging, had significant effects on miR-696 expression. Knockdown of SNARK in C2C12 cells significantly decreased miR-696. Consistent with these findings, SNARK overexpression in C2C12 cells increased miR-696 concomitant with a decrease in PGC-1? expression and decreased oxygen consumption. Our findings demonstrate that metabolic stress increases miR-696 expression in skeletal muscle which in turn inhibits PGC-1? signaling and mitochondrial function. While AMPK does not mediate miR-696 expression, SNARK may play a role in this process through a SNARK-miR- 696-PGC-1? signaling mechanism.

Le diabète et/ou l’obésité sont associés à la stéatose hépatique non-alcoolique (SHNA). Cette maladie du foie affecte environ un tiers de la population nord-américaine. Elle peut progresser vers un stade d’inflammation, de stress oxydatif et de fibrose appelé la stéatohépatite pouvant éventuellement entraîner le développement d’un cancer primitif du foie comme le carcinome hépatocellulaire (CHC). Cependant, les mécanismes reliant la diète, les maladies métaboliques et le développement du cancer sont complexes et peu connus. Le coactivateur transcriptionnel PGC-1α est un important régulateur du métabolisme énergétique de la cellule et la perte de ce dernier mène à un métabolisme nutritionnel inefficace, ainsi qu’à des défauts mitochondriaux importants. Fait intéressant, une réduction de PGC-1α est retrouvée chez les patients atteints de la maladie du foie gras non-alcoolique (SHNA) et du carcinome hépatocellulaire (HCC). Nous avons précédemment démontré qu’une réduction de PGC- 1α dans le foie murin en combinaison avec une diète obésogène peut provoquer l’apparition de la stéatohépatite. Cependant, le rôle causal de PGC-1α dans le cancer du foie associé à la diète demeure inconnu. Ensuite, un variant génétique de PGC-1α (SNP rs8192678) modifie un résidu glycine en sérine à la position 482 (PGC-1α G482S) chez l’humain et mène à une perte de stabilité protéique dans des cellules hépatiques humaines. Ce polymorphisme est associé au développement de maladies métaboliques, mais son impact sur le cancer demeure inconnu. Enfin, le gène de PGC-1α (PPARGC1A) est régulé par deux promoteurs (proximal et alternatif) donnant naissance à différents isoformes (PGC-1α1-4) de fonctions inconnues. L’action indépendante de ces variants pourrait fournir des indices quant au paradoxe entourant les recherches sur PGC-1α. Nous posons l’hypothèse principale que la perte d’expression de PGC-1α dans le foie favorise le développement du cancer hépatique en réponse à une diète riche en gras/fructose et à l’agent carcinogène diéthylnitrosamine. Dans cette thèse, nous montrons que la perte de PGC-1α favorise le développement du cancer du foie dans un modèle murin combinant une diète obésogène et un carcinogène hépatique. En effet, PGC-1α est nécessaire au maintien de l’expression du marqueur épithélial E-cadhérine et à la réponse cellulaire (apoptose, yH2AX) face aux dommages hépatiques. Nous montrons également que le variant G481 stabilise PGC-1α au niveau protéique et a un effet protecteur contre le cancer du foie chez la souris. Enfin, à l’aide d’expériences in vivo et in vitro nous montrons que la forme canonique PGC-1α1 et le variant PGC-1α4 exercent des rôles distincts sur la mort des cellules hépatiques en réponse à l’inflammation. En conclusion, cette thèse apporte de nouvelles connaissances sur les fonctions de PGC-1α au sein des complications hépatiques associées aux maladies métaboliques et inflammatoires. / Diabetes and obesity are associated to nonalcoholic fatty liver disease (NAFLD). This pathology affects approximately 30% of the population in North America. It ranges from simple steatosis to a more severe necro-inflammatory form called nonalcoholic steatohepatitis (NASH) that can ultimately lead to cirrhosis and primary liver cancer, such as hepatocellular carcinoma (HCC). However, the relationship between diet, metabolic disorders, and cancer development is poorly understood. PGC-1α is a transcriptional coactivator that regulates cellular energy metabolism. Loss of PGC-1α can lead to inefficient nutrient metabolism and severe mitochondrial defects. Interestingly, patients with NAFLD/NASH and HCC exhibit reduced levels of hepatic PGC-1α. We have previously shown that low hepatic PGC-1α combined with an obesogenic diet leads to hallmarks of NASH in mice. However, whether low hepatic PGC-1α reflects a cause or a consequence of liver cancer remains to be determined. Furthermore, a single nucleotide polymorphism within the PPARGC1A coding sequence (SNP rs8192678) leads to a switch between glycine to serine residue at position 482 (PGC-1α G482S) in humans and is associated with reduced protein stability in human liver cells. This SNP is associated with metabolic disorders, but its impact on liver cancer remains un- known. Lastly, the PGC-1α gene (PPARGC1A) is regulated by two promoters (proximal and alternative) that give rise to different isoforms (PGC-1α1-4) of unknown functions. Independent actions of these isoforms could provide a plausible explanation for the paradox observed in previous studies covering the role of PGC-1α. We proposed the general hypothesis that loss of hepatic PGC-1α promotes diet-associated liver cancer development in mice through increased susceptibility to hepatotoxicity. In this thesis, we show that loss of hepatic PGC-1α promotes diet-associated liver cancer in mice. Indeed, PGC-1α is essential to maintain E-cadherin expression and liver cell response (apoptosis, yH2AX) to damage. We also show that G481 variant stabilizes hepatic PGC-1α protein and protects against liver cancer development in mice. Finally, using in vivo and in vitro experiments we show that canonical PGC-1α1 and the PGC-1α4 variant differentially regulate liver cell apoptosis in response to inflammatory signaling. In conclusion, this thesis sheds new light on the role of PGC-1α in liver complications associated with metabolic disorders and inflammation.

Le déficit héréditaire en Carnitine Palmitoyl Transférase 2 (CPT2), l’un des déficits de l’oxydation mitochondriale des acides gras (OAG) les plus fréquents, est caractérisé dans sa forme adulte par une myopathie métabolique avec des épisodes récurrents de douleurs musculaires, de myoglobinurie et de rhabdomyolyse, habituellement déclenchés par un exercice prolongé ou un jeûne. A l’heure actuelle, il n’y a pas de traitement pharmacologique efficace pour la correction de ce déficit à l’exception de prise en charge nutritionnelle. Mon travail de thèse a porté sur l’étude du potentiel thérapeutique du composé GSK773 un activateur direct de l’AMP-activated Protein Kinase (AMPK), un senseur énergétique de la cellule, dans des myotubes de quatre patients déficitaires en CPT2. En effet, l'AMPK est considérée comme une cible thérapeutique potentielle dans de nombreux troubles métaboliques ou neurodégénératifs courants associés aux dysfonctionnements mitochondriaux. Nous montrons que le composé GSK773 est capable de stimuler les capacités résiduelles de l’OAG et de corriger le flux d’OAG dans des myotubes déficitaires en CPT2 (n=4) après un traitement par 30µM pendant 48h. L’étude par western-blot et par immunofluorescence montre que le GSK773 augmente la quantité de protéine mutante CPT2. L'analyse des intermédiaires C16-acylcarnitines montre que les myotubes déficients en CPT2 présentent, comme prévu, une accumulation de C16-acylcarnitines significativement diminuée après le traitement par le GSK773. De manière intéressante, l'IF et l’xCELLigence, une nouvelle technique basée sur la mesure de l’impédance électrique en temps réel, montrent un processus de différenciation altéré dans les myotubes de patients déficitaires en CPT2 par rapport aux cellules témoins, qui est corrigé par le GSK773. Nous avons également montré que le GSK773 induit une conversion des fibres musculaires vers les fibres de type I lentes/oxydatives, mais aussi une amélioration générale de la qualité du réseau mitochondrial accompagnée d’une biogenèse mitochondriale et une augmentation du niveau de ROS suggérant que le GSK773 agirait sur la plasticité musculaire. D’un point de vue mécanistique, nous avons montré que les effets du GSK773 passent par l’AMPK, PGC-1α, p38 MAPK et les ROS. Ainsi, ces résultats suggèrent que le GSK773 améliore les paramètres métaboliques et structuraux dans les myotubes déficients en CPT2 et que l'AMPK pourrait représenter une cible thérapeutique hautement pertinente pour la correction pharmacologique du déficit en CPT2. / Carnitine Palmitoyl Transferase 2 (CPT2) deficiency is among the most common inherited defects of mitochondrial fatty acid oxidation (FAO). A frequent phenotype is an early adult-onset myopathy characterized by recurrent episodes of muscle pain, myoglobinuria and rhabdomyolysis usually triggered by prolonged exercise or fasting. To date, there is no treatment of this disorder other than dietary management. AMPK is considered as a potential therapeutic target in many common metabolic or neurodegenerative disorders associated to mitochondrial dysfunctions. Thus, we tested the therapeutic potential of the direct AMPK activator GSK773 in myotubes from four CPT2-deficient patients. We show that GSK773 is able to stimulate residual FAO capacities in a dose- and time-dependent manner. Correction of CPT2 defect is achieved after treatment with GSK773 at 30µM for 48h. Western-blots analysis and Immunostaining shows that GSK773 increases the amount of CPT2 mutant protein. Analysis of acylcarnitine intermediates in the culture media shows that CPT2-deficient myotubes exhibit, as expected, an accumulation of C16-acylcarnitines that is significantly decreased after GSK773 treatment. Surprisingly, immunofluorescence and xCELLigence (a real-time monitoring of cell culture technic) show an impaired differentiation process in CPT2-deficient myotubes that is corrected by GSK773. We also show that GSK773 induces a shift in myosin-heavy-chain isoforms toward slow oxidative fiber types, improves the quality of mitochondrial network with an induction of mitochondrial biogenesis and increases ROS levels, suggesting that GSK773 might induce muscle plasticity. Finally, from a mechanistic point of view, siRNAs experiments showed that the effects triggered by GSK773 implicate AMPK, PGC-1α, ROS and p38 MAPK. Altogether these results suggest that GSK773 improves metabolic and structural parameters in CPT2-deficient myotubes and that AMPK might represent a highly relevant therapeutic target for pharmacological correction of inborn CPT2 deficiency.

Skeletal muscle adapts differently to specific modes of exercise, where resistance training results in muscle growth and endurance training induces mitochondrial biogenesis. These are results of molecular events that occur after each exercise session, increasing the expression of specific genes and the rate of both synthesis and breakdown of protein. The rate of protein synthesis is controlled by the mTORC1 signaling pathway, which is potently stimulated by resistance exercise and amino acid, and their combined effect is needed for muscle growth. The essential amino acids (EAA) are responsible for the stimulation of protein synthesis and here leucine has been attributed specific attention, but its particular role among the EAA, and the involvement of the other branched-chain amino acids (BCAA) is unclear. Endurance exercise activates the protein AMPK which, in animal models, has been shown to inhibit mTORC1 signaling and protein synthesis.  Suggesting that concurrent endurance and resistance exercise could restrain muscle growth, but it is unknown if this mechanism is relevant in exercising human muscle. Little is known about the regulation of protein breakdown and although much attention has been given the proteins MuRF-1 and MAFbx which target proteins for degradation, their role requires further investigation. The aim of thesis was to address the mentioned uncertainties by examining how different modes of exercise and amino acids affect mTORC1 signaling, protein synthesis and markers of protein breakdown in human muscle. In study I, the influence of high intensity endurance exercise on subsequent resistance exercised induced mTORC1 signaling was examined. Despite robust activation of AMPK by the endurance exercise there was no inhibition of mTORC1 signaling or protein synthesis during recovery from resistance exercise. Study II utilized a similar set up, but with the difference that resistance exercise was performed with the triceps. The cycling exercise reduced the resistance exercise stimulated mTORC1 signaling immediately after the exercise, but during the recovery period mTORC1 signaling and protein synthesis was similar between trials. Concurrent exercise induced the mRNA expression of MuRF-1 and that of PGC-1α, the master regulator of mitochondrial biogenesis, in both studies, despite that the exercise modes in study II were separated between legs and arms. In study III, the effect of an EAA supplement with or without leucine, in the stimulation of mTORC1 signaling in connection with resistance exercise was examined. Intake of EAA robustly stimulated mTORC1 signaling after exercise, but this was only minor when leucine was excluded from the supplement. In study IV, subjects were supplied with leucine, BCAA, EAA or placebo in a randomized fashion during four sessions of resistance exercise. Leucine alone stimulated mTORC1 signaling after the exercise, but both the amplitude and extent of stimulation was substantially greater with EAA, an effect that was largely mediated by the BCAA as a group. In conclusion, endurance exercise prior to resistance exercise using the leg or arm muscles does not affect mTORC1 signaling or protein synthesis during the three hour recovery period from exercise, supporting compatibility between resistance- and endurance exercise induced signaling. Concurrent exercise increases the expression of the proteolytic marker MuRF-1 compared to resistance exercise only, which could indicate both and increased demand of cellular adaptive remodeling or a more direct detrimental proteolytic effect. Leucine is crucial among the EAA in the stimulation of mTORC1 signaling after exercise, its effect is however potentiated by intake of the remaining EAA. As a supplement a mixture of EAA must be regarded preferable, although the effect is largely mediated by the BCAA as a group.

Maintaining energy organismal homeostasis under changing physiological and environmental conditions is vital, and requires constant adjustments of the energy metabolism. Central to meeting energy demands is the regulation of mitochondrial oxidative capacity. When demands increase, animals can increase mitochondrial content/enzymes, known as mitochondrial biogenesis. Central to mammalian mitochondrial biogenesis is the transcriptional master regulator PPARγ (peroxisome proliferator-activated receptor γ) coactivator-1α (PGC-1α), and the network of DNA-binding proteins it coactivates (e.g. nuclear respiratory factor 1 and 2 [NRF-1, NRF-2], estrogen-related receptor α [ERRα], thyroid receptor α [TRα-1], retinoid X receptor α [RXRα]). However, the mechanisms by which mitochondrial content in lower vertebrates such as fish is controlled are less studied. In my study I investigate underlying mechanisms of the phenomenon that many fish species alter mitochondrial enzyme activities, such as cytochrome c oxidase (COX) in response to low temperatures. In particular, I investigated (i) if the phenomenon of mitochondrial biogenesis during cold-acclimation is related to fish phylogeny, (ii) what role PGC-1α and other transcription factors play in mitochondrial biogenesis in fish, and (iii) if mRNA decay rates are important in the transcriptional control of a multimeric protein like COX. This study shows that mitochondrial biogenesis does not follow a phylogenetic pattern: while distantly related species displayed the same response to low temperatures, closely related species showed opposite responses. In species exhibiting mitochondrial biogenesis, little evidence was found for PGC-1α as a master regulator, whereas NRF-1 is supported to be an important regulator in mitochondrial biogenesis in fish. Further, there was little support for other transcription factors (NRF-2, ERRα, TRα-1, RXRα) to be part of the regulatory network. Lastly, results on the post-transcriptional control mechanism of mRNA decay indicate that this mechanism is important in the regulation of COX under mitochondrial biogenesis: it accounts for up to 30% of the change in subunit transcript levels. In summary, there is no simple temperature-dependent mitochondrial response ubiquitous in fish. Further, the pathways controlling mitochondrial content in fish differ from mammals in the important master regulator PGC-1α, however, NRF-1 is important in regulating cold-induced mitochondrial biogenesis in fish. Lastly, COX subunit mRNA decay rates seem to have a part in controlling COX amounts during mitochondrial biogenesis. / Thesis (Ph.D, Biology) -- Queen's University, 2013-10-21 09:53:59.46

Die Herzinsuffizienz ist eine der häufigsten chronischen Erkrankungen mit progressivem Krankheitsverlauf. Dieser ist verschiedenen Kompensationsmechanismen geschuldet, die zunächst zur Verbesserung, über einen längeren Zeitraum hinweg jedoch zur Verschärfung der Symptomatik führen. Durch erhöhte Konzentrationen inflammatorischer Zytokine im Rahmen dieser Krankheit kommt es unter anderem zu pathologischen Veränderungen in Muskel- als auch Fettgewebe. Das kürzlich in der Literatur als PGC-1α reguliert beschriebene Molekül FNDC5 (Spaltprodukt Irisin) wird als Myokin bezeichnet, welches zwischen Muskel- und Fettgewebe vermittelt. Bis dato ist jedoch ungeklärt, ob und inwiefern es einen Zusammenhang zwischen einer Herzinsuffizienz, erhöhten Konzentrationen proinflammatorischer Zytokine und dem Molekül FNDC5 in der Skelettmuskulatur gibt. Um diesen Fragestellungen nachzugehen, wurde in der vorliegenden Arbeit zum einen durch LAD-Ligatur in Ratten eine Herzinsuffizienz induziert, um die systemischen Verhältnisse dieser Erkrankung im Tiermodell bestmöglich nachzuempfinden. Zum anderen wurden Mäuse mit TNF-α oder Ang II behandelt und C2C12-Myotuben mit TNF-α, Ang II sowie einer Zytokinkombination inkubiert, um den Einfluss der Zytokine bzw. des Hormons auf die FNDC5-Expression zu untersuchen. Anschließend wurden die Expression von FNDC5 auf mRNA- und Proteinebene sowie die Expression der PGC-1α-mRNA in Skelettmuskelbiopsien bestimmt. Es konnte nachgewiesen werden, dass eine Herzinsuffizienz sowie der Einfluss inflammatorischer Zytokine zur signifikanten Expressionsverminderung von FNDC5 führt. Ang II hingegen bewirkte nicht in allen Versuchen eine deutliche Reduktion der FNDC5-Expression. Außerdem wurde gezeigt, dass die Signaltransduktionswege über p38 und p42/44 MAPK nicht für die Wirkung der Zytokinkombination auf die FNDC5-Expression verantwortlich sind.

Right ventricular dysfunction is the most frequent cause of death in patients with pulmonary arterial hypertension. Although abnormal energy substrate use has been implicated in the development of chronic left heart failure, data describing such metabolic remodeling in failing right ventricular tissue remain incomplete. In the present dissertation we sought to characterize metabolic gene expression changes and mitochondrial dysfunction in functional and dysfunctional RV hypertrophy. Two different rat models of RV hypertrophy were studied. The model of right ventricular failure (SU5416/hypoxia) exhibited a significantly decreased gene expression of peroxisome proliferator-activated receptor- coactivator-1α, peroxisome proliferator- activated receptor-α and estrogen-related receptor-α. The expression of multiple peroxisome proliferator-activated receptor- coactivator-1α target genes required for fatty acid oxidation was similarly decreased. Decreased peroxisome proliferator-activated receptor- coactivator-1α expression was also associated with a net loss of mitochondrial protein and oxidative capacity. Reduced mitochondrial number was associated with a downregulation of transcription factor A, mitochondrial, and other genes required for mitochondrial biogenesis. Electron microscopy demonstrated that, in right ventricular failure tissue, mitochondria had abnormal shape and size. Lastly, respirometric analysis demonstrated that mitochondria isolated from right ventricular failure tissue had a significantly reduced ADP- stimulated (state 3) rate for complex I. Conversely, functional right ventricular hypertrophy in the pulmonary artery banding model showed normal expression of peroxisome proliferator-activated receptor- coactivator-1α, whereas the expression of fatty acid oxidation genes was either preserved or unregulated. Moreover, pulmonary artery banding-right ventricular tissue exhibited preserved transcription factor A mitochondrial expression and mitochondrial respiration despite elevated right ventricular pressure-overload. We conclude that right ventricular dysfunction, but not functional right ventricular hypertrophy in rats, demonstrates a gene expression profile compatible with a multilevel impairment of fatty acid metabolism and significant mitochondrial dysfunction, partially independent of chronic pressure-overload.