Increasing age is the single biggest independent risk factor for cardiovascular disease, which is in turn the leading cause of morbidity and mortality worldwide. Ageing is associated with hypertension and metabolic changes which all increase the risk of the development of cardiovascular disease. In young, healthy individuals, perivascular adipose tissue (PVAT) secretes factors that can influence vascular contractility, exerting a net anti-contractile effect against numerous vasoconstrictors including the thromboxane A2 mimetic U46619 and α1-adrenoceptor phenylephrine. Whilst it is known that dysfunction in PVAT can contribute to obesity-related hypertension, little is known whether similar dysfunction occurs with ageing. In young Wistar rats, wire myography and pharmacological studies showed that the anti-contractile effect of PVAT in the presence of U46619 is dependent on both PVAT-derived nitric oxide and prostaglandins, whereas the anti-contractile effect in the presence of phenylephrine is nitric oxide independent. This finding was supported by Western blot experiments that showed increased phosphorylation of endothelial nitric oxide synthase (eNOS) in PVAT following U46619 incubation, but not phenylephrine. In the Wistar rat model of ageing used, wire myograph studies revealed that the PVAT anti-contractile effect in the presence of phenylephrine is preserved at 24 months of age, but in in the presence of U46619 is lost. Furthermore PVAT from aged animals had a deleterious effect on endothelial function, suggesting changes in its secreted factors. These changes are accompanied by alterations in the expression and activation of key enzymes in the nitric oxide synthesis pathway within the PVAT as measured by Western blot, as well as alterations in cardiometabolic phenotype including hypertension, hyperglycaemia and insulin resistance. Taken together these findings suggest that previously unidentified age-related PVAT dysfunction may contribute to age-related hypertension and thus may provide a potential therapeutic target for future study.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a common location for breast cancer cell metastasis, and progression of tumor in bone can lead to bone loss and affect human health. Osteocytes have important roles in bone homeostasis and osteogenesis, and their interaction with metastasized cancer cells are known to affect progression of metastasized tumor. However, the potential role of metabolic signaling and actin- cytoskeleton-associated moesin in the interaction of osteocytes and tumor cells remain poorly understood. In this study, we first examined the roles of metabolic signaling, specifically global AMPK modulators and mitochondria-specific AMPK inhibitor (Mito-AIP), as well as mechanical force in beta catenin signaling through interaction between osteocytes and precursor osteoblasts as well as osteocytes and breast cancer cells. We also evaluated the role of metabolic signaling in Rho GTPases including RhoA, Rac1 and Cdc42. We observed that AMPK activator (A769662) and Mito-AMPK stimulated beta catenin translocation to the nucleus, indicating the activation of Wnt signaling, while Mito-AIP did not significantly affect beta catenin activation in osteoblasts. We also observed that osteocyte conditioned medium (CM) treated with Mito-AIP substantially increased beta catenin signaling in osteoblasts, while decreasing beta catenin signaling in breast cancer cells. CM of osteocytes treated with fluid flow increased beta catenin signaling in breast cancer cells. A769662 and Mito-AIP also decreased the activities of RhoA, Rac1, and Cdc42 in cancer cells which are known to regulate cancer cell migration. Additionally, we evaluated the roles of intracellular and extracellular moesin (MSN) protein in well-established oncogenic signaling proteins, such as FAK, Src, and RhoA as well beta catenin signaling. Constitutively active MSN (MSN+) significantly increased FAK and Src activities in cancer cells, but decreased the activity of RhoA. Surprisingly, CM of mesenchymal stem cells treated with MSN+ decreased the activities of FAK, Src, and RhoA, suggesting the inhibitory role of extracellular MSN in tumor-promoting signaling. Our results suggest the distinct role of AMPK signaling, specifically at mitochondria of osteocytes, in the activities of beta-catenin signaling in osteoblasts and breast cancer cells and the distinct role of intracellular and extracellular MSN in these two types of cell.

This study was to determine (1) if genetic selection for high (HWS) or low (LWS) body weight in chickens has altered the hypothalamic AMP-activated protein kinase (AMPK) system and (2) if this alteration contributes to the dissimilar feeding response to various appetite modulators between HWS and LWS lines. Compared to HWS, LWS chickens had higher levels of AMPK α and acetyl-CoA carboxylase (ACC) phosphorylation, which was caused by upregulation of the upstream factor calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK β). There was greater mRNA expression of carnitine palmitoyltransferase I (CPT1), leptin receptor (LEPR) and neuropeptide Y (NPY) and less mRNA expression of ACC α, fatty acid synthase (FAS), fat mass and obesity associated gene (FTO), pro-opiomelanocortin (POMC) and orexin in LWS than HWS chickens. At 5 days of age, intracerebroventricular (ICV) injection of AICAR, 5-amino- 4-imidazolecarboxamide riboside, caused a quadratic dose-dependent decrease in food intake in LWS but not HWS chicks. Compound C, (6-(4-(2-piperidin-1-yl-ethoxy)-phenyl))-3-pyridin-4-yl-pyrazolo(1,5-a)-pyrimidine, caused a quadratic dose-dependent increase in food intake in HWS but not LWS chicks. The anorexigenic effect of AICAR in LWS chicks and orexigenic effect of Compound C in HWS chicks resulted from either activation or inhibition of other kinase pathways separate from AMPK. There is a lower threshold for the anorexigenic effect of ghrelin in LWS than HWS chicks, which was associated with differential hypothalamic AMPK signaling. ICV injection of ghrelin inhibited corticotrophin-releasing hormone (CRH), 20-hydroxysteroid dehydrogenase (20HSD), glucocorticoid receptor (GR), CPT1 and FTO expression in LWS but not HWS chicks. Additionally, the hypothalamic mRNA level of ghrelin was significantly higher in LWS than HWS chicks, which may also contribute to the differential threshold response to ghrelin in these two lines. Obestatin caused a linear dose-dependent increase in food intake in HWS but not LWS chicks. The orexigenic effect of obestatin in HWS chicks was not associated with altered AMPK. Obestatin inhibited LEPR and FTO expression in HWS but not LWS chicks. Thus, selection for body weight may alter the hypothalamic response to ghrelin by the AMPK pathway, CRH pathway, CPT1 and FTO, and to obestatin by LEPR and FTO. / Ph. D.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13598号 / 論医博第2308号 / 新制||医||1072(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 浅野 雅秀, 教授 山中 伸弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

La tétraploïdie – cellule contenant le double du génome d’une cellule diploïde – est considérée comme un état métastable, à l’origine de l’aneuploïdie des cancers. Présentes dans les lésions précancéreuses, les cellules tétraploïdes sont associées à la progression tumorale. Etant donné le rôle clé de la tétraploïdie au cours de l’oncogenèse, le développement d’agents pharmacologiques éliminant spécifiquement les cellules tétraploïdes pourrait permettre de prévenir et limiter l’évolution vers un état cancéreux. Afin d'identifier des composés délétères pour les cellules tétraploïdes, nous avons développé une méthode de criblage basée sur la vidéomicroscopie à fluorescence automatisée. La chimiothèque de l'Institute of Chemistry and Cell Biology (ICCB) a été criblée, et nous avons mis en évidence plusieurs hits aux effets cytostatiques et/ou cytotoxiques préférentiels pour les cellules tétraploïdes. En raison de ses propriétés chimiopréventives, le resvératrol est le premier composé dont nous avons choisi de poursuivre la caractérisation. Sa sélectivité pour les cellules tétraploïdes a été confirmée sur différents types cellulaires, stables ou en cours de polyploïdisation. L’étude du mécanisme d’action anti-tétraploïde du resvératrol a permis d’identifier le senseur de la charge énergétique 5’-adenosine monophosphate-activated kinase (AMPK), comme une cible moléculaire responsable de la mort sélective des cellules tétraploïdes. Une série d’agents pharmacologiques activant directement ou indirectement AMPK – parmi lesquels l’aspirine et son métabolite le salicylate, dont l’action chimiopréventive a été établie par plusieurs études épidémiologiques et essais cliniques – a montré une toxicité préférentielle pour les cellules tétraploïdes. De la même manière que le resvératrol, ces agents éliminent les cellules tétraploïdes stables et limitent la polyploïdisation. Finalement, l’effet anti-tétraploïde du resvératrol et de l’aspirine a été évalué in vivo. L’administration orale de ces deux composés aux doses décrites comme chimiopréventives, réduit le développement des cellules épithéliales tétraploïdes et aneuploïdes des cryptes intestinales des souris ApcMin/+, le modèle murin de la polypose adénomateuse familiale. Collectivement les résultats de cette étude suggèrent que l’action chimiopréventive du resvératrol et de l’aspirine est associée à l’élimination des cellules tétraploïdes précurseur de tumeurs. / Tetraploidy – cells that contain twice the normal amount of chromosomes – is a metastable state leading to aneuploidy in cancer. Tetraploid cells have been observed in precancerous lesions and constitute a step toward tumor progression. Given the importance of tetraploidization for oncogenesis, developing drugs that selectively target tetraploid cells should prevent cancer.To discover compounds toxic to tetraploid cells, we developed an assay-system based on automatic fluorescence videomicroscopy. We screened the Institute of Chemistry and Cell Biology (ICCB) chemical library and identified several hits exerting a selective cytostatic and/or cytotoxic effect on tetraploid cells. Due to its well known chemopreventive properties, resveratrol was the first compound we further characterized. Its selectivity for tetraploid cells was confirmed on various stable or polyploidizing cancer cell lines, as well as primary epithelial cells. The mechanism accounting for the preferential killing of tetraploid cells involves the 5’-adenosine monophosphate-activated kinase (AMPK) signaling pathway. A series of additional agents that stimulate AMPK – including aspirin and salicylate whose chemopreventive action have been established by several epidemiological studies and clinical trials – display a selective toxicity toward tetraploid cells. Similar to resveratrol, these drugs eliminate stable tetraploid cells and reduce polyploidization. Finally, we validated the anti-tetraploid effect of resveratrol and aspirin in vivo. Oral treatment with either of these two compounds at chemopreventive doses, repressed the accumulation of tetraploid and subsequently aneuploid intestinal epithelial cells from the crypts of the ApcMin/+ mouse model of familial adenomatous polyposis.Collectively, our results suggest that the chemopreventive action of resveratrol and aspirin involves the elimination of tetraploid cancer cell precursors.

A l’état basal, les cellules souches musculaires sont quiescentes. Après blessure, ces cellules s’activent, s’amplifient et se différencient afin de réparer les myofibres lésées. Cependant, une petite population de ces cellules myogéniques activées ne va pas entrer dans la voie de la myogenèse, mais va retourner en quiescence par un phénomène appelé auto-renouvellement. Cette étape est cruciale afin de maintenir une réserve de cellules souches musculaires tout au long de la vie. Mais, les mécanismes cellulaires et moléculaires régulant ce phénomène sont peu décrits dans la littérature. La régénération musculaire est composée d’une série d’évènements complexes et bien orchestrés selon une cinétique précise. Le challenge de son étude est donc de pouvoir distinguer les évènements les uns des autres, tout en sachant qu’ils sont interconnectés. Bien que les cellules souches musculaires aient un fort potentiel de régénération, elles ont besoin d’interagir avec d’autres cellules au cours de la régénération, notamment avec les macrophages qui ont un rôle prépondérant dans ce processus. Après une blessure, les monocytes circulants sont recrutés sur le site de lésion et se différencient en macrophages inflammatoires. Ensuite, ces macrophages changent leur statut inflammatoire et acquièrent un profil anti-inflammatoire. Plusieurs études in vitro ont suggéré un rôle pour le métabolisme et son régulateur principal, la kinase activée par l’AMP (AMPK), dans la résolution de l’inflammation et dans le devenir des cellules souches adultes. Ainsi, j’ai étudié l’influence extrinsèque (via les macrophages) et intrinsèque du métabolisme sur le devenir des cellules souches musculaires au cours de la régénération. Pour cela, j’ai utilisé divers modèles déficients pour l’AMPK1 dans le macrophage, dans la cellule souche musculaire et dans la myofibre qui m’ont permis d’établir des cultures primaires de macrophages et de cellules musculaires. Dans un premier temps, grâce à ces outils, nous avons pu démontrer le rôle primordial de l’AMPK dans la résolution de l’inflammation au cours de la régénération musculaire et dans l’acquisition des fonctions anti-inflammatoires des macrophages. Dans ce contexte, l’activation de l’AMPK est dépendante de la kinase CAMKK et régule la phagocytose, principal phénomène cellulaire permettant le changement de statut inflammatoire des macrophages. Ce travail a été publié en 2013 dans le journal Cell Metabolism. Ensuite, j’ai mis en évidence un lien entre le métabolisme et le devenir des cellules souches musculaires. La suppression de l’AMPK dans les cellules souches musculaires augmente leur auto-renouvellement. Cette modification du devenir des cellules souches est due à un changement de métabolisme similaire à l’effet Warburg observé dans les cellules souches cancéreuses, qui s’effectue via la modulation de l’activité de l’enzyme Lactate Déshydrogénase, enzyme clé de la glycolyse. En conclusion, j’ai pu mettre en évidence deux nouveaux rôles de l’AMPK dans le devenir des cellules souches musculaires, établissant un lien de causalité entre métabolisme, inflammation et devenir des cellules souches. / During skeletal muscle regeneration, muscle stem cells activate and recapitulate the myogenic program to repair the damaged myofibers. A subset of these cells does not enter into the myogenesis program but self-renews to return into quiescence for further needs. Control of muscle stem cell fate choice is crucial to maintain homeostasis but molecular and cellular mechanisms controlling this step are poorly understood. A difficulty of understanding muscle stem cell self-renewal is that skeletal muscle regeneration is a coordinated and non-synchronized process. Various and dissociated molecular and cellular mechanisms regulate muscle stem cell fate. Indeed, skeletal muscle regeneration requires the interaction between myogenic cells and other cell types, among which the macrophages. Macrophages infiltrate the muscle and adopt distinct and sequential phenotypes. They act on the sequential phases of muscle regeneration and resolving the inflammation by skewing their inflammatory profile to an anti-inflammatory state. Some in vitro studies suggested a role for the metabolism and the AMP-activated protein Kinase (AMPK), the master metabolic regulator of cells, in both inflammation and stem cell fate. Thus, I investigated the role of metabolism on muscle stem cell fate within the muscle stem cells (cell autonomous regulations) and through the action of macrophages (non-cell autonomous regulations) during skeletal muscle regeneration. To analyze muscle stem cell fate, I used in vitro (macrophages and muscle stem cell primary cultures), ex vivo (isolated myofibers) and in vivo (using specific mice model deleted specifically for AMPK1 in the myeloid lineage, in muscle stem cells or in myofibers) experiments. First, I highlighted that macrophagic AMPK1is required for the resolution of inflammation during skeletal muscle regeneration and for the trophic functions of macrophages on muscle stem cell fate. Moreover, CAMKK-AMPK1 activation regulates phagocytosis, which is the main cellular mechanism inducing macrophage skewing. This work was published in 2013 in Cell Metabolism. Second, I demonstrated that depletion of myogenic AMPK1 tailors muscle stem cell metabolism in a LKB1 independent manner, orients their fate to the self-renewal by promoting metabolic switch from an oxidative to a glycolytic metabolism pathway, through the over activation of a new molecular target, which is a key enzyme for glycolysis: the Lactate Dehydrogenase. To conclude, during my thesis, I established two new crucial roles of AMPK1 in muscle stem cell fate choice, linking for the first time metabolism, inflammation and fate choice.

O ácido palmitoleico (POA) é descrito como uma lipocina, capaz de melhorar a responsividade à insulina, estimular a oxidação de lipídios e reduzir a inflamação, efeitos essenciais para o controle da doença do fígado gorduroso não alcoólico (NAFLD). Assim, investigamos se o POA pode proteger contra os efeitos deletérios de uma dieta rica em gordura saturada (HF-L) ou gordura trans (HF-T), verificando a participação de PPARα e PPARγ. Para tanto utilizamos camundongos selvagens alimentados com uma dieta padrão (SD), ou animais knockout total de PPARα (PPARαKO) ou com deleção de PPARγ em células mielóides (PPARγKOLyzCre+) alimentados com HF. As dietas foram administradas por 12 semanas, e a partir da 10ª semana os animais foram suplementados com ácido oleico ou POA (300mg/kg). Utilizamos também macrófagos intraperitoneais extraídos de animais WT, PPARαKO e PPARγKOLyzCre+ alimentados com SD com LPS ou LPS + POA (600uM). A HF promoveu resistência à insulina, esteatose e inflamação exacerbada no fígado dos animais, independente do genótipo. Embora não tenha reduzido o acúmulo ectópico de lipídios no fígado, ou modulado a expressão de fatores lipogênicos, POA melhorou a resposta à insulina periférica e hepática. No fígado, POA não modulou fatores da cascata de insulina, ou a produção de adiponectina, porém, estimulou a ativação de AMPK e aumentou os níveis FGF-21, de forma dependente de PPARα. POA reduziu a inflamação hepática, por diminuir TLR4, NFκB e fatores do inflamassoma, mas principalmente, por inibir a polarização de macrófagos do fígado para o fenótipo M1, um efeito que ocorreu mesmo em animais PPARγKOLyzCre+, apesar da maior expressão gênica e proteica de PPARγ observada em macrófagos com POA, in vivo e in vitro. Nossos dados indicam que o POA promove efeitos benéficos contra intolerância à glicose e resistência à insulina hepática e periférica induzida por dieta rica em gordura, por ativação de AMPK, FGF21 e PPARα. E, por reduzir a polarização M1 de macrófagos, independente de PPARs, este ácido graxo 16:1n-7 é capaz de reduzir a inflamação no fígado induzida por dieta hiperlipídica. / Palmitoleic acid (POA) is described as a lipokine, capable of improving insulin responsiveness, stimulating lipid oxidation and reducing inflammation, essential for the control of non-alcoholic fatty liver disease (NAFLD). Thus, we investigated whether POA can protect against the deleterious effects of a diet rich in saturated fat (HF-L) or trans fat (HF-T), verifying the role of PPARα and PPARγ. We used wild-type mice fed a standard diet (SD), or whole-body PPARα knockout (PPARαKO) mice or mice with myeloid cells selective-delected PPARγ (PPARγKOLyzCre+) fed a HF. The diets were administered for 12 weeks, and from the 10th week the animals were supplemented with oleic acid or POA (300mg/kg). Intraperitoneal macrophages extracted from WT, PPARαKO and PPARγKOLyzCre+ mice fed SD and stimulated with LPS or LPS+POA (600μM), were also analyzed. HF promoted insulin resistance, steatosis and exacerbated inflammation in the liver of mice, regardless of genotype. Although it did not reduce the lipids ectopic accumulation in the liver, or modulated the lipogenic factors expression, POA improved the peripheral and hepatic insulin response. In the liver, POA did not modulate factors of the insulin cascade, or adiponectin production, however, stimulated AMPK activation and increased FGF-21 levels, in a PPARα-dependent manner. POA reduced liver inflammation by decreasing TLR4, NFκB and inflammatory factors, but mainly by inhibiting the macrophage polarization of the liver to the M1 phenotype, an effect that occurred even in PPARγKOLyzCre+ mice, despite the increased gene and protein expression of PPARγ observed in macrophages with POA, in vivo and in vitro. Our data indicate that the POA promotes beneficial effects against glucose intolerance and hepatic and peripheral insulin resistance induced by a high fat diet through the activation of AMPK, FGF21 and PPARα. And, by reducing the M1 polarization of macrophages, independent of PPARs, this 16: 1n-7 fatty acid is able to reduce inflammation in the liver induced by high fat diet.

Durant la régénération du muscle suite à une lésion, les cellules souches musculaires, aussi appelées les cellules satellites, quittent leur état de quiescence et s’activent. Elles pourront soit emprunter la voie de la myogenèse afin de former de nouvelles fibres musculaires, soit retourner à leur état de quiescence pour reformer la réserve de cellules souches mobilisable en cas de lésion ultérieure. La régulation du devenir de la cellule souche est modulée par de nombreuses voies de signalisation telles que la voie Wnt, la voie Notch ou la voie des TGFb. Cependant, rares sont les données concernant l’implication du métabolisme sur le devenir de la cellule souche. Pourtant il a été démontré que l’activation des cellules satellites est étroitement liée avec le métabolisme cellulaire, dont l’un des principaux acteurs est la protéine kinase AMPK. Ce complexe hétérotrimérique, composé de trois sous-unités a, b et g est responsable de l’équilibre entre consommation énergétique et production d’énergie au sein de la cellule. Grâce à la modulation de mTORC1, il a également été prouvé que l’AMPKa1 était responsable de la croissance cellulaire et de la prolifération des précurseurs myogéniques. A l’aide de différents modèles murins, de lignées primaires et de cellules satellites en sortie de tri, nous avons déterminé le rôle que pouvait jouer chacun des isoformes, AMPKa1 et AMPKa2 au sein de la cellule souche, sur le déroulement de la myogenèse adulte post- lésionnelle ainsi que sur l’homéostasie du muscle régénéré. Dans un premier temps nous avons démontré que la voie de signalisation AMPKa1-LDH permettait de réguler l’autorenouvellement des cellules satellites grâce au contrôle du métabolisme. En effet, au moment de l’entrée de la cellule dans la voie de la différenciation, la voie de l’AMPKa1 induit une diminution de l’activité de la LDH, permettant aux cellules d’adopter un métabolisme de phosphorylation oxydative répondant à leurs besoins énergétiques. Dans un second temps, nous avons démontré que l’isoforme AMPKa2, exprimé uniquement après l’entrée de la cellule dans la voie de la myogenèse, était responsable d’une modulation de la régénération musculaire et que son absence induisait un défaut de différenciation et un retard de maturation des fibres néoformées. Nos travaux nous ont ainsi permis de confirmer la place centrale de la protéine kinase AMPK dans la modulation via le métabolisme du devenir de la cellule souche musculaire dans un contexte de régénération du tissu musculaire squelettique dans un modèle murin / During muscle regeneration following injury, muscle stem cells, also called satellite cells,leave their quiescent state and activate. MuSCs are capable of both differentiating torepair muscle tissue after an injury and self-renewing to replenish the pool of stem cells.The regulation of their fate is modulated by several signaling pathways such as Wnt,Notch or TGFb pathway. However, there are few data concerning the involvement ofmetabolism in the fate of satellite cells. Yet it has been shown that the activation ofsatellite cells is closely related to cellular metabolism, which one of the main players isAMPK protein kinase. This heterotrimeric complex, composed of three subunits a, b andg, is responsible for the balance between energy consumption and energy productionwithin the cell. With the modulation of mTORC1, AMPKa1 has also been shown to be responsible for cell growth and proliferation of myogenic precursors. Using different mouse models, primary lines and sorted satellite cells, we determined the role that each isoform, AMPKa1 and AMPKa2, could play within the cell, on myogenesis and on the homeostasis of the regenerated muscle. First, we demonstrated that AMPKa1-LDH signaling pathway regulates the satellite cells self-renewal by controlling metabolism. Indeed, at the time of cell fate choice between commitment into terminal differentiation versus self-renewal, the AMPKa1 pathway induces a decrease in LDH activity, allowing cells to adopt an oxidative phosphorylation metabolism responding to their energy needs. In a second time, we demonstrated that the AMPKa2 isoform, expressed during myogenesis only after the induction of muscle cell differentiation, was responsible for a modulation of the muscular regeneration and that its absence induced a lack of differentiation and a delay in maturation of the new formed myofibers. Our work allowed us to confirm the central role of AMPK protein kinase in the regulation, by the modulation of metabolism, of muscle stem cell fate in a context of skeletal muscle regeneration in a mouse model

Le muscle strié squelettique régénère ad integrum après une lésion aigüe stérile grâce aux cellules satellites qui sont les cellules souches du muscle strié squelettique. L'inflammation, et notamment les macrophages, joue un rôle important durant ce processus. En effet, après une lésion, les monocytes sanguins infiltrent le tissu et deviennent des macrophages avec un phénotype pro-inflammatoire associé à la lésion. Ces macrophages phagocytent les débris cellulaires et promeuvent la prolifération des cellules souches musculaires. Ensuite, les macrophages changent leur phénotype vers un phénotype anti-inflammatoire associé à la restauration du tissu. Ils promeuvent la différenciation, puis la fusion des cellules souches musculaires et la croissance des myofibres. Cette séquence de phénotypes inflammatoires est essentielle pour une régénération musculaire efficace. Le laboratoire a montré que ce changement de phénotype est dépendant d'un senseur énergétique majeur de la cellule qui contrôle le métabolisme cellulaire, l'AMP kinase (AMPK)al. Par ailleurs, les glucocorticoïdes sont utilisés depuis des décennies pour leurs effets anti-inflammatoires sur l'inflammation. Leur action est médiée par le Récepteur aux Glucocorticoïdes qui induit ou réprime l'expression de gènes par interaction directe ou indirecte à l'ADN. Comme l'AMPKal et les glucocorticoïdes induisent des effets anti-inflammatoires similaires sur les macrophages, nous avons posé l'hypothèse que ces 2 voies de signalisation pourraient être interconnectées dans les macrophages afin de permettre leur changement de phénotype et la régénération musculaire. Les données issues d'un modèle in vitro de lésion musculaire utilisant des macrophages dérivés de la moelle osseuse de souris ont montré que : i) les glucocorticoïdes induisaient la phosphorylation de l'AMPKal ; ii) l'AMPKal était requise pour l'acquisition fonctionnelle du statut anti-inflammatoire des macrophages induit par les glucocorticoïdes puisque des macrophages déficients pour l'AMPKal ne modifiaient pas leur phénotype et ne stimulaient pas la myogenèse. Les expériences in vivo utilisant des souris LysMCre/+;AMPKalfl/fl dans lesquelles l'AMPKal est invalidée uniquement dans les cellules myéloïdes ont montré que l'AMPKal dans les macrophages régulait les effets bénéfiques des glucocorticoïdes au cours de la régénération du muscle strié squelettique. En effet, en absence d'AMPKal dans les macrophages, les glucocorticoïdes induisaient un retard de régénération et une modification de la maturation des fibres attestée par une modification de l'expression des isoformes des chaînes lourdes de myosines. En conclusion, ces données montrent que l'AMPKal est requise pour le changement de phénotype des macrophages induit par les glucocorticoïdes et une régénération musculaire efficace / Skeletal muscle regenerates ad integrum after a sterile acute injury thanks to satellite cells (muscle stem cells). Inflammation, and notably macrophages, plays important roles during this process. Just after injury, monocytes infiltrate the tissue from the blood and convert into pro-inflammatory damaged associated macrophages. These macrophages phagocyte muscle debris and promote the proliferation of muscle stem cells. Then, macrophages switch their phenotype toward an anti-inflammatory restorative profile and promote muscle stem differentiation, fusion and myofiber growth. This sequence of macrophage profile is essential for an efficient skeletal muscle regeneration. The lab has shown that this phenotype switch is dependent of AMP kinase (AMPK)a1, a major energetic sensor in the cell controlling cellular metabolism. Besides, glucocorticoids have been used for decades for their anti-inflammatory effects on inflammation. Their actions are mediated by the Glucocorticoid Receptor which induces or represses gene expression by direct or indirect DNA-binding. As AMPKa1 and glucocorticoids induce similar anti-inflammatory effects on macrophages, we hypothesized that these 2 pathways could be interconnected in macrophages to allow the resolution of inflammation and muscle repair. Data from an in vitro model of skeletal muscle injury using bone marrow derived macrophages showed that: i) glucocorticoids induce AMPK phosphorylation; ii) AMPKa1 is required for the functional acquisition of the anti-inflammatory phenotype induced by glucocorticoids. Indeed, AMPKa1-deficient macrophages did not switch their phenotype and did not sustain myogenesis. In vivo experiments using LysMCre/+;AMPKa1fl/fl mice in which AMPKa1 is depleted only in myeloid cells, showed that macrophagic AMPK drove the beneficial effects of glucocorticoids during skeletal muscle regeneration. Inversely, in absence of AMPK in macrophages, glucocorticoids induced a delayed muscle regeneration and a modification in myofiber maturation, assessed by the alteration of myosin heavy chain expression. Altogether, these data show that glucocorticoids need AMPKa1 in macrophages for the resolution of inflammation and an efficient skeletal muscle regeneration