Duchenne Muscular Dystrophy (DMD) results from the absence of a functional dystrophin protein. Among its possible therapeutic options is the upregulation of dystrophin’s autosomal analogue, utrophin A. This can be achieved by a pharmacologically induced shift towards a slower, more oxidative skeletal muscle phenotype, which has been shown to confer morphological and functional improvements on models of DMD. Whether these improvements are a result of the utrophin A upregulation or other beneficial adaptations associated with the slow, oxidative phenotype, such as improved autophagy, has not been determined. To understand the importance of utrophin A to the therapeutic value of the slow, oxidative phenotype, we used the utrophin/dystrophin double knockout (dKO) model of DMD. We found the dKO mouse to have a similar skeletal muscle signaling capacity and phenotype to mdx mice. When treated with the adenosine monophosphate activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), both dKO and mdx mice expressed a shift towards a slower, more oxidative phenotype. In the mdx mice, this shift caused improvements in muscle fiber central nucleation, IgM penetration, damage from eccentric contractions, and forelimb grip strength. These morphological and functional benefits were not seen in the AICAR treated dKO mice. This study highlights the importance of utrophin A upregulation to the benefits of the slow, oxidative myogenic program to dystrophic mice. It confirms utrophin A as a therapeutic target in DMD and the slow, oxidative myogenic program as clinically relevant avenue towards treatment of the disease.

L'AMPK est un senseur cellulaire des réserves énergétiques de l’organisme. Les spermatozoïdes, mobilisent beaucoup d’énergie pour leur mobilité et la fécondation de l’ovocyte. L’objectif de ce travail était de caractériser et décrire des éléments clés de la voie de signalisation de l'AMPK, de comprendre leur implication dans les spermatozoïdes de coq et d’étudier comment leurs modulateurs peuvent impacter les fonctions des gamètes conservés in vitro. Nous avons montré une augmentation de la mobilité et de la réaction acrosomique dans les spermatozoïdes exposés à l'AICAR et à la metformine, des activateur de l’AMPK, y compris après avoir été congelés. Ces activateurs ont partiellement restauré les activités des enzymes antioxydantes (SOD, GPx, GR): et diminué les ROS et la LPO dans les spermatozoïdes décongelés. Nous avons établi la présence des CaMKKs (α et β) et de CaMKI dans les spermatozoïdes et leur rôle lié au calcium extracellulaire (via les canaux calcique SOCs) dans la voie de régulation de l'AMPK et dans la mobilité et la réaction acrosomique des spermatozoïdes. En conclusion, ce travail confirme le rôle de différents acteurs de signalisation liés au métabolisme énergétique et aux flux calciques dans les fonctions des spermatozoïdes. / AMPK is a cellular sensor of body energy reserves. Spermatozoa mobilize a lot of energy for their motility and the fertilization of the oocyte. The objective of this work was to characterize and describe key elements of the signaling pathway of AMPK, understand their involvement in chicken spermatozoa and study how their modulators may impact the functions of in vitro preserved gametes. We showed an increase in mobility and acrosome reaction in spermatozoa exposed to AICAR and metformin, activators of AMPK, including after freezing. These activators have partially restored the activities of antioxidant enzymes (SOD, GPx, GR): and decreased ROS and LPO in thawed spermatozoa. We have established the presence of CaMKKs (α and β) and CaMKI in sperm and their role related to extracellular calcium (via calcium channels SOCs) in the control channel of AMPK and in motility and acrosome reaction of spermatozoa. In conclusion, this work confirms the role of different signaling actors related to energy metabolism and calcium fluxes in spermatozoa functions.

L’AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) est un intermédiaire de la voie de biosynthèse des purines. A des concentrations importantes, ce métabolite a un effet cytotoxique sur les cellules cancéreuses aneuploïdes, c’est-à-dire contenant un nombre anormal de chromosome. Or,90% des tumeurs solides sont aneuploïdes. Les mécanismes responsables de cette cytotoxicité doivent donc être mieux étudiés pour une utilisation éventuelle en thérapie anti-cancéreuse.Dans la littérature, l’effet de l’AICAR est expliqué par son rôle mimétique de l’AMP sur l’AMPK.Cependant, certaines données de la littérature et du laboratoire laissent penser que l’inhibition de la croissance par l’AICAR peut impliquer plusieurs types de mécanismes dont certains sont dépendantsde l’AMPK et d’autres indépendants. L’identification des cibles de l’AICAR alternatives à l’AMPK estdonc nécessaire pour une meilleure compréhension de ses effets.Dans ce projet, j’ai pu confirmer la présence d’autres cibles de l’AICAR indépendantes de l’AMPKet responsables de son effet cytotoxique. Grâce à une approche transcriptomique, j’ai montré un effetde l’AICAR sur l’expression et l’activation de LATS1 et LATS2 (large tumor suppressor 1 and 2). Ces protéines kinases fond partie du core enzymatique de la voie HIPPO, dont le rôle en cancérologie est fondamental. Les effecteurs finaux de cette voie sont YAP et TAZ, deux cofacteurs de transcription,aussi régulés par l’AICAR. J’ai pu montrer que la cytotoxicité de l’AICAR est due en partie à l’activation de cette voie. Depuis la découverte récente de la voie HIPPO, de nombreuses études visent à identifier des molécules permettant l’inhibition directe de cette voie. L’AICAR s’avère être une molécule puissante dans le cadre d’une thérapie anticancéreuse ciblant la voie HIPPO. / AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is an intermediate of the purine biosynthesis pathway. At high concentrations, this metabolite has a cytotoxic effect on aneuploid cancer cells that is cells containing an abnormal chromosome number. However, 90% of solid tumorsare aneuploid. The mechanisms responsible for this cytotoxicity should be better studied for possible use in anti-cancer therapy.In the literature, the effect of AICAR is explained by its AMP mimetic role on the AMPK. However,some literature and laboratory data suggest that AICAR growth inhibition may involve several types of mechanisms, some of which are dependent and other independent of AMPK. Therefore, the identification of AMPK alternative targets is necessary for a better understanding the AICAR effects. In this project, I was able to confirm the presence of other AICAR targets independent of AMPK and responsible for its cytotoxic effect. Using a transcriptomic approach, I showed an effect of AICAR on the expression and activation of LATS1 and LATS2 (large tumor suppressor 1 and 2). These proteinkinases form part of the enzymatic nucleus of the HIPPO pathway, whose role in oncology is fundamental. The effectors of this pathway are YAP and TAZ, two transcription cofactors, also regulated by the AICAR. I have been able to show that the cytotoxicity of AICAR is due to the activation of this pathway. Since the recent discovery of the HIPPO pathway, numerous studies aim to identify molecules allowing direct inhibition of this pathway. AICAR has proven to be a potent molecule in anticancer therapy which goal is targeting the HIPPO pathway.

ABSTRACT Metformin is the most common drug used against type 2 diabetes mellitus. However, it was only recently shown, in human and rat hepatocytes, that metformin-like 5-aminoimidazole-4-carboximide ribonucleoside (AICAR), acts via activation of the AMP-activated protein kinase (AMPK), an enzyme that plays a central role in lipid metabolism. Although it is well known that metformin is used in the treatment of type 2 diabetes and results in significant fat loss, no study has investigated the effects of this drug on adipocytes. In this report I studied the effects of metformin on the formation of fat deposits in mouse 3T3-L1 preadipocytes, as well as its effects on the activation of AMPK in these cells. Our results suggested that metformin significantly inhibits the transformation of pre-adipocytes into adipocytes. This is achieved via the inhibition of intracellular lipid accumulation during adipogenesis. In addition to its inhibition of intracellular lipid accumulation, metformin induced a significant increase in the phosphorylation of AMPK. It has been shown that AMPK activation with AICAR results in the inhibition of the nuclear factor-κB (NF-κB) induced gene expression. Since NF-κB is the key nuclear factor used by HIV-1 during the initiation of its gene transcription, I investigated the possibility of inhibiting HIV-1 replication in U1 cells with metformin and AICAR. I observed that AICAR and metformin inhibit HIV-1 replication in U1 cells. This inhibition wasparalleled by the accumulation of NF-κB in the cytoplasm of AICAR and metformin treated cells, and at the same time by a significant decrease in the concentration of this nuclear factor in the nucleus of these cells. However, I failed to observe any phosphorylation of AMPK by metformin and AICAR in U1 cells. In conclusion, metformin inhibits adipogenesis in mouse adipocytes and this inhibition is likely to take place via the activation of AMPK. AICAR and metformin have inhibitory properties against HIV-1 replication. However, this inhibition does not seem to be by the activation of AMPK.

Preeclampsia (PE) remains one of the most enigmatic and pervasive conditions developed during pregnancy and is a leading cause of maternal and fetal morbidity and mortality throughout the world. Afflicting nearly 5-8% of pregnancies in the Unites States, PE is most commonly characterized by an increase in blood pressure and high protein excretion near or after the 20th week of gestation. Unfortunately, few effective treatments are available, and the only "cure" is delivery. While the molecular pathogenesis of PE remains undefined, an interruption in placental blood flow, or placental ischemia, is widely observed as a primary contributor to the syndrome progression. Furthermore, to investigate the role of both pharmacological and non-pharmacological modalities to prevent placental ischemia induced hypertension, we employed a robust model of reduced utero-placental perfusion pressure (RUPP) in the pregnant rat. First, in Chapter IV, exercise initiated during gestation was not effective in the prevention of RUPP-induced hypertension, whereas exercise training prior to and continued through gestation prevented the increase in blood pressure. Though the molecular contributions to this effect are undefined, the effects appear to be independent of angiogenic balance restoration. Finally, in Chapter V, administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) was explored as a novel pharmacological modality to prevent the onset of hypertension and endothelial dysfunction in the RUPP model. As hypothesized, AICAR ameliorated the RUPP-induced hypertension, and the anti-hypertensive effect in the RUPP appears to be dependent on the restoration of angiogenic balance in the maternal plasma. This dissertation includes previously published and unpublished co-authored material.

Essentielle à l'équilibre énergétique de la cellule, la mitochondrie, véritable sentinelle, joue, un rôle majeur dans le destin de la cellule, en modulant les voies de signalisation de mort cellulaire mis en jeu dans l'atrophie musculaire. L'objectif de cette thèse est de proposer des cibles thérapeutiques centrées sur la mitochondrie dans deux modèles murins dont la physiopathologie est caractérisée par une dysfonction mitochondriale associée à une atrophie musculaire : le vieillissement et la dystrophie musculaire de Duchenne (DMD). Pour lutter contre la perte de masse musculaire liée à l'âge, la déficience en myostatine (mstn), associée à un phénotype hypermusculé, est une stratégie thérapeutique prometteuse. Mais, l'altération du métabolisme mitochondrial et oxydatif induite par cette déficience réduit les effets bénéfiques d'une telle stratégie. Nous avons donc testé l'intérêt de l'utilisation de la molécule pharmacologique AICAR, activateur connu de l'AMPK, afin de « booster » la fonction mitochondriale chez la souris âgée KO mstn. Les résultats montrent chez la souris KO mstn, une amélioration du temps d'endurance de course. Au niveau signalétique, le traitement induit des effets bénéfiques mais limités sur la fonction mitochondriale. Les mécanismes restent à préciser mais tendent vers l'hypothèse d'un effet bénéfique de l'AICAR sur le stress du réticulum endoplasmique (RE). Le dysfonctionnement mitochondrial a été également largement impliqué dans la physiopathologie de la DMD. Dans notre seconde étude, ce même traitement à l'AICAR chez le modèle murin de la DMD, la souris mdx atténue le phénotype dystrophique et améliore la fonction contractile du diaphragme. Nous montrons que ces effets bénéfiques sont associés à une induction de mécanisme de survie, l'autophagie, et une limitation des phénomènes d'apoptose induit par la mitochondrie, mettant en évidence une amélioration de l'intégrité mitochondriale par stimulation de leur renouvellement dans des fibres musculaires dystrophiques. Enfin, ce travail a mis en avant pour la première fois la présence à l'état basal de stress du RE chez la mdx, propsant une nouvelle cible thérapeutique. L'impact de ce stress dans la fibre musculaire normal et pathologique est très mal connu. Nos résultats montrent que le stress du RE modifie les liens entre le réticulum sarcoplasmique et la mitochondrie, perturbe l'homéostasie calcique et active les voies de mort cellulaire associées à une dysfonction contractile. Ces résultats ouvrent une perspective de stratégie thérapeutique dans les pathologies musculaire impliquant un stress du RE, comme la DMD. Ce travail de thèse a mis en avant l'importance de développer des thérapies pharmacologiques dans les pathologies musculaires, permettant d'améliorer la fonction à la fois métabolique et de sentinelle de la mitochondrie. / Fundamental for the energetic balance of the cell, mitochondria play a key role for modulation of cell death pathway related to muscular atrophy. Thus, the purpose of this PhD is to find therapeutic strategy focus on mitochondria in two different murine models where the physiopathology is characterized by a mitochondria dysfunction associated with muscle atrophy: Aging process and Duchenne Muscular Dystrophy (DMD).To prevent loss of muscle mass associated with aging, the lack of myostatin, inducing a hypermuscular phenotype, is a promising therapeutic strategy. However, loss of myostatin is associated with a strong reduction of mitochondrial and oxidative metabolism in skeletal muscle, and this strategy need to be potentiated. In this context, we explore if mitochondrial alteration in aged wild-type mice or in aged mstn KO mice are rescued by chronic AMPK-activating treatment, using the synthetic agonist AICAR, considered as “an mimetic of exercise”. Our results show an improvement of aerobic running performance in mstn KO mice. Concerning to signaling pathways, AICAR treatment induces beneficial but limited effects on mitochondrial metabolism. Mechanisms are still under investigation but our results suggest a reduction in ER stress. Moreover, mitochondria dysfunction has been widely implicated in DMD physiopathology. This same treatment of AICAR, in the murine model of DMD, improves the diaphragm histopathology as well as maximal force generating capacity. These beneficial effects were linked with autophagy activation and apoptosis limitation, without inducing muscle fiber atrophy, and promoting the elimination of defective mitochondria.Finally, the last part of this study highlight for the first time, an increase of ER stress at basal level, suggesting a new therapeutic target. Nevertheless, ER stress impact in skeletal muscle fibers is sparsely known. The preliminary results show that ER stress decrease the link between RE and mitochondria, which have an impact on calcium homeostasis and stimulate cell death pathway with a decrease of contractile function.This study highlights the importance to develop pharmacological therapies in muscular pathology, focus on metabolic and sentinel mitochondria function.

Le contrôle de la masse musculaire est sous la dépendance d'un équilibre entre les processus de synthèse et de dégradation. Sur le plan cellulaire, deux voies signalétiques majeures sont impliquées : la voie des facteurs de transcription de la famille FoxO qui contrôle l'expression des gènes impliqués dans les systèmes de dégradation (système ubiquitine-protéasome et autophagie), et la voie IGF-1/Akt/mTORC1 qui représente la voie majeure de la synthèse protéique. Nos travaux mettent en évidence, sur des cellules musculaires le rôle de la protéine kinase AMP-dépendante (AMPK) qui inhibe l'activité de la voie mTOR et régule les systèmes ubiquitine-protéasome et autophagiques de manière FoxO3 dépendante. Une nouvelle cible de l'AMPK a également été identifiée : la protéine Ulk1 qui possède une fonction clé dans l'activation de l'autophagie. Par ailleurs, nous avons montré le rôle centraldu facteur d'initiation à la traduction eIF3f dans l'induction de l'hypertrophie, et dans l'augmentation de l'activité de la voie mTORC1 associée. De plus, nous montrons que la surexpression d'un mutant d'eIF3f résistant à la dégradation est associée à une protection effective contre l'atrophie. / Skeletal muscle mass is depending upon a dynamic balance between anabolic and catabolic processes. At a cellular level, two major signaling pathways are involved: the transcription factors FoxO related pathway, implicated in the control of protein breakdown systems(ubiquitin-proteasome system and autophagy), and the IGF-1/Akt/mTORC1 pathway associated with the canonic pathway of protein synthesis. We show in muscle cells that theAMP-activated protein kinase (AMPK) decreases the mTORC1 pathway activity and simulate subiquitin-proteasome and autophagy systems in a FoxO3-dependant manner. Furthermore,we identify Ulk1 as a new interacting partner of AMPK, which plays a major role in the autophagy induction. Moreover, we demonstrate the key role of the eukaryotic translation initiation factor eIF3f in hypertrophy induction and in the associated increase of the mTORC1activity. In addition, we show that the overexpression of an eIF3f mutant resistant to the degradation is associated with a protection against muscle atrophy.

Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMPK, calcium, and circulating FFAs. Chronic treatment with either AICAR, a chemical activator of AMPK, or increasing circulating FFAs with a high fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high fat diet (HF), AICAR injections (AICAR), or a high fat diet and AICAR injections (HF+AICAR) for six weeks. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on LCAD, cytochrome c, and PGC-α protein, as well as on citrate synthase and β-HAD activity. In contrast, no additive effect was noted in the soleus and in the red quadriceps only β-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high fat diet-induced post transcriptional increase in PGC-α protein and AMPK mediated increase in PGC-α protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.

AMP-activated protein kinase (AMPK), a metabolic regulator, acts in opposition to many of the effects of aging and may provide insights into the development of sarcopenia. However, the effect of aging on AMPK activation is unclear. The purpose of this dissertation was to: 1) clarify the controversy concerning the activation of AMPK in response to endurance-like exercise in aged skeletal muscle; 2) address mechanisms for the age-associated alterations in AMPK activation; and 3) address the known benefits of chronic AICAR treatment in aged skeletal muscle. First, to clarify the effect of age on AMPK activation, young adult (YA) (8 mo.) and old (O) (30 mo.) male Fischer344 x Brown Norway F1 hybrid rats received an in situ bout of endurance-type contractions produced via electrical stimulation of the sciatic nerve (STIM). AMPK activation was attenuated in aging muscle as demonstrated by decreased AMPKα phosphorylation and AMPKα2 protein content and activity in O vs. YA muscle after STIM. In contrast, AMPKα1 content was greater in O vs. YA muscle, and α1 activity increased with STIM in O but not YA muscles. Second, the effect of age on the AMPK heterotrimer composition and nuclear localization was assessed as mechanisms for the altered AMPK activation. The AMPK heterotrimer composition was altered in aging skeletal muscle with lower AMPKγ2 and γ3 content and decreased association of AMPKγ3 with AMPKα1 and α2. Furthermore, activation of AMPK is known to increase translocation of AMPK to the nucleus in YA muscle; however, translocation of phosphorylated AMPK, AMPKα2, and AMPKγ3 were impaired in the aging rat muscle after STIM. Finally, chronic activation of AMPK with 5'-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is known to increase mitochondrial content, activate autophagy, and repress protein synthesis; pathways that are altered with aging. The known benefits of chronic AICAR treatment were assessed in YA (5 mo.) and O (23 mo.) male C57Bl/6 mice. Mice were treadmill tested prior to and after one month of AICAR treatment. In vitro muscle contractions were performed following AICAR treatment. AICAR treatment improved the O mice treadmill endurance and the YA mice rate of fatigue and recovery. Additionally, AICAR increased citrate synthase activity, decreased SQSTM1/p62 protein content , and decreased Myf6 protein content in both the YA and O mice suggesting increased mitochondrial activity, autophagy, and decreased muscle regeneration. Therefore, chronic AICAR treatment may alter metabolic pathways to improve the exercise response in both YA and O mice.

<p><b>Background and aims:</b> Type 2 diabetes mellitus is connected with elevated glucose levels, which cause impaired glucose-stimulated insulin secretion (GSIS) and degeneration of β-cells. Mechanisms for such glucotoxic effects were explored in the present study.</p><p><b>Materials and methods:</b> INS-1E cells were cultured for 5 days in 5.5, 11, 20 or 27 mM glucose in the presence or absence of AMPK-agonist AICAR. GSIS was determined from INS-1E cells and islets obtained from type 2 diabetes and control donors. Human islets and INS-1E cells were functionally characterized (GSIS) and protein profiled (SELDI-TOF MS). Glucose-induced <i>de novo</i> synthesis of fatty acyls (HR-MAS NMR spectroscopy), fatty acid composition (GC-MS), triglyceride content and specific proteins (Western blotting) were determined in INS-1E cells.</p><p><b>Results:</b> Impaired GSIS was observed from INS-1E cells exposed to chronic hyperglycaemia and islets isolated from type 2 diabetics compared to INS-1E cells cultured at normal glucose levels and control islets, respectively. Several glucose-regulated proteins were found when type 2 diabetes and control islets or mitochondria from INS-1E cells cultured at different glucose concentrations were protein profiled. Glucose induced lipid <i>de novo</i> synthesis of both saturated and unsaturated fatty acids in specific proportions. Glucose-induced impairment of function and mass was reverted by inclusion of AICAR, which lowered levels of pro-apoptotic protein CHOP but left triglyceride content unaffected.</p><p><b>Conclusions:</b> Impaired GSIS and increased apoptosis observed in β-cells after prolonged exposure to elevated glucose concentrations involved accumulation of lipid species in specific proportions, AMPK-inactivation, ER-stress activation and complex, coordinated changes in expression patterns of mitochondrial and human islet proteins.</p>