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.

Orientadores: Licio Augusto Velloso, Marcio Alberto Torsoni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T11:00:58Z (GMT). No. of bitstreams: 1 Oliveira_MaristelaCesquinide_D.pdf: 2056313 bytes, checksum: fe162225f6402886dd228faa021438b9 (MD5) Previous issue date: 2006 / Resumo: O aumento da prevalência da obesidade e diabete tipo 2 nas sociedades modernas está fortemente associado às doenças cardiovasculares, hipertensão, infarto e aterosclerose. Estas patologias têm sido relacionadas com ingestão calórica excessiva e diminuição do gasto energético. Muitos fatores regulam a ingestão alimentar, incluindo hormônios como insulina e leptina, alimentos e comportamento. Estes fatores são integrados no hipotálamo que media um sistema de sinalização celular para regular o comportamento alimentar e o balanço energético da célula. A proteína quinase ativada por AMP (AMPK) tem sido proposta como capaz de mediar as adaptações celulares às variações nutricionais ambientais. O resultado da ativação da AMPK é a inibição de vias biossintetizantes que consomem energia, tais como as vias de síntese de ácidos graxos e proteínas, e a ativação de vias catabólicas, como a via de oxidação de ácidos graxos e a glicólise. No hipotálamo, a AMPK desempenha o papel de mediar os efeitos hormonais na ingestão alimentar e no balanço energético. Outras moléculas, como a acetil-CoA carboxilase (ACC) e o seu produto, o malonil-CoA têm sido propostas como possíveis intermediários na sinalização hipotalâmica que monitora o estado energético do corpo. No presente trabalho, nós administramos citrato, um ativador alostérico da ACC, no hipotálamo de ratos a fim de investigarmos o efeito deste composto na atividade hipotalâmica e hepática da AMPK. O resultado desta modulação no metabolismo energético, metabolismo de glicose e comportamento alimentar foi comparado aos ratos tratados com salina. Nós observamos que o tratamento com citrato inibiu a fosforilação da AMPK hipotalâmica seguido de inibição da fosforilação da ACC hipotalâmica, indicando menor atividade da AMPK neste tecido. A inibição da AMPK promoveu diminuição da ingestão alimentar, perda de peso corpóreo e aumento da expressão dos neuropeptídeos anorexigênicos POMC e CRH. No grupo de ratos tratados com citrato, a captação de glicose foi maior do que nos ratos que receberam solução salina, como mostrada pelo teste tolerância à glicose (GTT) e pelo clamp hiperinsulinêmico-euglicêmico. Consistente com estes resultados, no fígado, os ratos tratados com citrato mostraram maior fosforilação das proteínas da cascata de sinalização da insulina, reduzidos níveis de fosforilação da AMPK e ACC e expressão aumentada da PEPCK e G6Pase, se comparado aos animais controle. O efeito central do citrato na melhora da sinalização da insulina nos tecidos periféricos, na ativação da produção de glicose pelo fígado e na inibição da AMPK hepática foi bloqueado por antagonista ?-adrenérgico. De acordo com estes resultados, nós podemos sugerir que a modulação da AMPK por intermediários metabólicos possa ser um mecanismo importante de controle da homeostase energética e consequentemente do diabetes e obesidade / Abstract: The increased prevalence of obesity and type II diabetes in modern societies is largely linked to cardiovascular disease, hypertension, stroke and atherosclerosis. These pathologies have been associated to excessive caloric intake and decreased energy expenditure. Many factors regulate food intake, including hormones such as insulin and leptin, fuels and behavior. These factors are integrated in the hypothalamus that mediates a signaling system to regulate food behavior and cellular energy balance. The AMPactivated protein quinase (AMPK) has been proposed to be capable of mediating the cellular adaptations to nutritional environmental variation. The result of AMPK activation is the inhibition of nergy-consuming biosynthetic pathways, such as fatty acid and protein synthesis, and activation of ATP-catabolic pathways, such as fatty acid oxidation and glycolysis. In the hypothalamus, AMPK mediates hormonal effects on food intake and energy balance. Other molecules, such as acetyl-CoA carboxylase (ACC) and its product malonyl-CoA have been proposed as possible intermediaries in the hypothalamic signaling pathway that monitors energy status. In the present work we administrated citrate, an allosteric activator of ACC, in the hypothalamus to investigate its effect on hypothalamic and hepatic AMPK activity and the result of this modulation in the energetic cellular metabolism, such as glucose metabolism and food behavior. Results were compared to saline-treated rats. Here we show that citrate treatment inhibited hypothalamic AMPK phosphorylation followed by an inhibition of hypothalamic ACC phosphorylation, indicating lower AMPK activity in this tissue. The AMPK inhibition promoted decrease in food intake, loss of body weight and increased expression of anorexigenic neuropeptides (POMC and CRH). In the citrate group, the glucose uptake was higher than in animals receiving saline according to GTT and hyperinsulinemic-euglycemic clamp. Consistent with these results, in the liver, citrate-treated rats showed also higher phosphorylation of insulin signaling proteins than control rats, reduced AMPK and ACC phosphorylation and increased PEPCK and G6Pase expression. Supported by these results, we suggest that AMPK modulation by citrate can be an important mechanism to understand deregulated glucose metabolism involved in diabetes and obesity. The central effect of citrate in the improvement of peripheral insulin signaling, in the activation of liver glucose production, and in the inhibition of hepatic AMPK was blocked by the ?-adrenergic antagonist. According to these results, we suggest that AMPK modulation by metabolic intermediaries can be an important mechanism controlling the energetic homeostase, diabetes and obesity / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Prevalence of obesity and associated diseases like type 2 diabetes has increased rapidly during last years. These diseases closely relate to each other. Obesity leads to insulin resistence, which directly precedes type 2 diabetes. Metformin is the most prescribed medicament for type 2 diabetic patients and insulin resistant people. It improves glucose tolerance and insulin resistance. Enzyme AMP-activated protein kinase (AMPK) is strogly involved in metformin action. The latest studies using transgenic models lacking AMPK suggest, that notable part of mechanisms involved in metformin action is independent on AMPK. n-3 polyunsaturated fatty acids (n-3 PUFA), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are abundant in sea fish, have beneficial effects on metabolism. These fatty acids lower plasma lipids and exert cardioprotective effects. n-3 PUFA also prevent development of insulin resistence and type 2 diabetes in rodents. The aim of this thesis was to characterise acute effects of metformin on glucose homeostasis, impact of short term diet intervention with diet rich in n-3 PUFA on metformin action and the role of insulin stimulated signalling pathways and AMPK. Results suggest that early effect of metformin is dose dependent and that single dose of metformin...

The dystrophin-associated protein complex (DAPC) provides a mechanical link between the intracellular cytoskeleton and extracellular matrix, serving as a mechanosensor and signal transducer across the sarcolemma. Pharmacological stimulation of AMP-activated protein kinase (AMPK) induces the expression of DAPC components in skeletal muscle, whereas physiological reductions in AMPK are associated with DAPC dysfunction. We sought to determine whether AMPK was necessary for the maintenance of DAPC expression in skeletal muscle. Fast glycolytic extensor digitorum longus (EDL) and slow oxidative soleus (SOL) muscles from wild-type (WT) mice, as well as from littermates deficient in both isoforms of the AMPK-β subunit in skeletal muscle (MKO) were analyzed. DAPC mRNA levels, as well as protein expression and localization were similar between genotypes, with the exception of nNOS, which displayed a compensatory sarcolemmal enrichment in MKO muscles. The content of transcriptional and post-transcriptional regulators of the DAPC, such as PGC-1α and KSRP, were also not affected by the loss of AMPK. However, MyoD and myogenin expression was significantly diminished in MKO muscles, which is consistent with previous reports of myopathy in these animals. Furthermore, we observed decrements in extrasynaptic utrophin expression selectively in MKO SOL muscles, despite an adaptive accumulation of PGC-1α at the sarcolemmal compartment. Collectively the evidence indicates that AMPK is sufficient, but not essential for the maintenance of DAPC expression in skeletal muscle. However, AMPK is required for preserving extrasynaptic utrophin levels in slow, oxidative muscles, which underscores the role of AMPK in the gene expression of this disease modifying protein. / Thesis / Master of Science (MSc) / The dystrophin-associated protein complex (DAPC) connects the interior and exterior of muscle cells. Activation of AMP-activated protein kinase (AMPK) increases the expression of the DAPC in skeletal muscle. We sought to determine whether AMPK was necessary for DAPC expression in skeletal muscle. Fast and slow muscles from normal mice, as well as from those deficient in skeletal muscle AMPK (MKO) were analyzed. We found DAPC levels and localization were similar between both groups, with the exception of nNOS, which was enriched at the muscle membrane in MKO muscles. Regulators of the DAPC were also not affected by the loss of AMPK. However, genes important for the production of muscle were significantly diminished in MKO muscles. Furthermore, we observed decrements in utrophin at the muscle membrane selectively in slow MKO muscles. Our work indicates that AMPK is not essential for the DAPC expression in skeletal muscle, however it is required for preserving utrophin levels in slow, oxidative muscles.

Yes / Recent clinical trials of the hypoglycaemic sodium-glucose co-transporter-2 (SGLT2) inhibitors, which inhibit renal glucose reabsorption, have reported beneficial cardiovascular outcomes. Whether SGLT2 inhibitors directly affect cardiovascular tissues, however, remains unclear. We have previously reported that the SGLT2 inhibitor canagliflozin activates AMP-activated protein kinase (AMPK) in immortalised cell lines and murine hepatocytes. As AMPK has anti-inflammatory actions in vascular cells, we examined whether SGLT2 inhibitors attenuated inflammatory signalling in cultured human endothelial cells. Incubation with clinically-relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin activated AMPK and inhibited IL-1β-stimulated adhesion of pro-monocytic U937 cells and secretion of IL-6 and monocyte chemoattractant protein-1 (MCP-1). Inhibition of MCP-1 secretion was attenuated by expression of dominant-negative AMPK and was mimicked by the direct AMPK activator, A769662. Stimulation of cells with either canagliflozin or A769662 had no effect on IL-1β-stimulated cell surface levels of adhesion molecules or nuclear factor-κB signalling. Despite these identical effects of canagliflozin and A769662, IL-1β-stimulated IL-6/MCP-1 mRNA was inhibited by canagliflozin, but not A769662, whereas IL-1β-stimulated c-jun N-terminal kinase phosphorylation was inhibited by A769662, but not canagliflozin. These data indicate that clinically-relevant canagliflozin concentrations directly inhibit endothelial pro-inflammatory chemokine/cytokine secretion by AMPK-dependent and -independent mechanisms without affecting early IL-1β signalling. / Project Grant (PG/13/82/30483 to IPS and TMP) and PhD studentships (FS/16/55/32731 and FS/14/61/31284 to DB and AS) from the British Heart Foundation and an equipment grant (BDA11/0004309 to IPS and TMP) from Diabetes UK. OJK was supported by a Scholarship from the Iraqi Ministry of Higher Education and Scientific Research. TAA was supported by a Libyan Ministry of Education PhD Studentship.

Yes / Aims/hypothesis Hypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG). Methods To test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios. Results AMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure. Conclusions/interpretation Taken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes. / Diabetes UK (RD Lawrence Fellowship to CB; 13/0004647); the Medical Research Council (MR/N012763/1) to KLJE, ADR and CB; and a Mary Kinross Charitable Trust PhD studentship to CB, ADR and RW to support PGWP. Additional support for this work came from awards from the British Society for Neuroendocrinology (to CB and KLJE), the Society for Endocrinology (CB), Tenovus Scotland (CB) and the University of Exeter Medical School (CB and KLJE). AR was also supported by a Royal Society Industry Fellowship.

Les mutations germinales activatrices de la voie RAS sont responsables de maladies rares regroupées sous le nom de RASopathies : le Syndrome de Noonan, le Syndrome de Noonan avec de Multiples Lentigines, la Neurofibromatose de type 1, le Syndrome de Malformations Capillaires et Malformations Artério-Veinseuses, le Syndrome Cardio-Facio-Cutané, le Syndrome de Legius et le Syndrome de Costello. Cette thèse s’intéresse au syndrome de Costello causé par une mutation hétérozygote de novo du gène HRAS. Ce syndrome est révélé dans les premiers mois de la vie et se caractérise par un retard de croissance postnatal, des traits du visage épais, un déficit intellectuel, des anomalies cutanées, ainsi qu’une prédisposition à développer des tumeurs. De plus, les patients atteints du syndrome de Costello développent une cardiomyopathie hypertrophique, de l’hypertension, une hypotonie et une myopathie d'origine moléculaire inconnue. En lien avec une association de malade et le service de génétique du CHU de Bordeaux, nous avons mené une exploration des anomalies protéomiques dans les tissus d’une souris modèle du syndrome de Costello ainsi que dans des fibroblastes de patients et des cellules modèles exprimant les formes mutées de HRASG12S et HRASG12A. Cette analyse globale et sans a priori a révélé des altérations au niveau du métabolisme énergétique et plus particulièrement de la composition des mitochondries. Le déficit fonctionnel des mitochondries, centrale énergétique du corps humain, a été caractérisé par des approches de biochimie, de bioénergétique et de biologie cellulaire. De plus, l’analyse des données ‘omiques’ a permis de suggérer une nouvelle hypothèse dans la physiopathologie du syndrome de Costello. Cette hypothèse considère l’implication d’un micro-ARN, le miR-221* dans l’inhibition du métabolisme oxydatif. Les analyses génétiques réalisées sur les cellules de patients et les cellules modèles ont démontré l’inhibition de l’expression de la protéine AMPK, un régulateur majeur du métabolisme mitochondrial, par le miR-221* sous le contrôle de HRASG12S et HRASG12A. Ces découvertes ont permis d’élaborer une stratégie thérapeutique visant à réduire la cardiomyopathie dans le syndrome de Costello. Les analyses précliniques effectuées sur les modèles cellulaires et le modèle murin ont permis d’évaluer l’efficacité d’une stimulation pharmacologique du métabolisme mitochondrial. Cette thèse révèle donc l’implication des mitochondries dans le syndrome de Costello et l’analyse moléculaire réalisée propose une série de données ‘Omiques’ qui permettront de progresser dans la compréhension de cette maladie rare. / Germline activating mutations of the RAS pathway are responsible for rare diseases grouped under the name of RASopathies: Noonan Syndrome, Noonan Syndrome with multiple Lentigines, Type 1-neurofibromatosis, Capillaries malformations and arteriovenous malformations syndrome, Cardio-Facio-Cutaneous Syndrome, Legius Syndrome and Costello Syndrome. This Ph.D thesis focuses on Costello syndrome that is caused by a heterozygous de novo mutation of the HRAS gene. This syndrome is revealed in the first months of life and is characterized by postnatal growth retardation, thick facial features, intellectual deficit, skin abnormalities, and a predisposition to developing tumors. In addition, patients with Costello syndrome develop hypertrophic cardiomyopathy, hypertension, hypotonia and myopathy of unknown molecular origin. In connection with a patients association and the genetics department of Bordeaux University Hospital, we conducted an exploration of proteomic abnormalities in the tissues of a mouse model of the Costello syndrome as well as in patients’ fibroblasts and cell models expressing mutated forms of HRASG12S and HRASG12A. This global and unbiased analysis revealed alterations in energy metabolism and more particularly in the composition of mitochondria. The functional deficiency of mitochondria, energy plants of the human body, has been characterized by biochemistry, bioenergetics and cell biology approaches. In addition, the 'omic' analysis of Costello syndrome suggested a new pathophysiology hypothesis that considered the involvement of a microRNA, miR-221* in the alteration of oxidative metabolism. Functional genetic analyzes performed on patient cells and cell models demonstrated the inhibition of the expression of the major mitochondrial metabolism regulator AMPK protein by miR-221* under the control of HRASG12S and HRASG12A. These findings led to the development of a preclinical therapeutic strategy to reduce cardiomyopathy in Costello syndrome. Preclinical investigations performed on the cellular models and the murine model made it possible to evaluate the efficacy of a pharmacological stimulation of mitochondrial metabolism. This thesis thus reveals the involvement of mitochondria in Costello syndrome and the molecular analysis carried out makes available a series of 'Omics' data that will allow progress in the understanding of this rare disease.

O acúmulo de lipídeos associado à obesidade, resistência à insulina (RI) e diabetes mellitus tipo 2 (DM2) pode levar ao desenvolvimento de danos renais, e diversos mecanismos podem estar envolvidos neste processo, dentre os quais: 1) redução na atividade da proteína ativada por AMP (AMPK); 2) hiperativação do sistema renina angiotensina (SRA) e consequente aumento na produção de angiotensina II (Ang II). O treinamento físico aeróbio (TFA) promove melhora metabólica significativa, no entanto, pouco se sabe sobre os mecanismos celulares induzidos pelo TFA contra o desenvolvimento de danos renais associados com doenças metabólicas. Sendo assim, o objetivo deste estudo foi avaliar o potencial do TFA para a prevenção de danos renais induzidos por dieta de cafeteria, e a participação do SRA e da proteína AMPK nessa resposta. Para isso, camundongos machos adultos C57BL6/J foram separados em grupos (n=13/grupo) sedentários (SED) alimentados com dieta normocalórica (NO) ou de cafeteria (CAF) (SED-NO e SED-CAF, respectivamente) e treinados (TF) alimentados com dieta NO ou CAF (TF-NO e TF-CAF, respectivamente). O TFA foi realizado a 60% da capacidade máxima, simultaneamente com as dietas durante 8 semanas. A dieta de cafeteria causou maior adiposidade, intolerância à glicose e RI no grupo SED-CAF, enquanto o TFA preveniu esses prejuízos no grupo TF-CAF. Os animais SED-CAF apresentaram 88% de aumento no ritmo de filtração glomerular (RFG), maior deposição lipídica renal e redução do espaço de Bowman comparado ao SED-NO, as quais foram prevenidas no grupo TF-CAF. Não houve alteração no conteúdo de colágeno IV e fibronectina, entretanto o TNF-alfa aumentou em ambos os grupos alimentados com dieta de cafeteria. Houve aumento de 27% da expressão proteica da p-AMPK no grupo TF-CAF, sem diferenças na expressão de t-ACC, p-ACC, PGC1-alfa e SIRT-1. A expressão gênica do SREBP-1 não diferiu entre os grupos, porém a expressão do SREBP-2 aumentou nos grupos SED-CAF e TF-CAF comparado aos grupos SED-NO e TF-NO. No soro, apenas a atividade da ECA2 aumentou nos grupos TF-NO e TF-CAF comparados aos sedentários. No rim, a atividade da ECA aumentou 46% no grupo SED-CAF comparado ao SED-NO, e o TFA foi capaz de prevenir esse aumento. No entanto, a Ang II renal aumentou nos grupos SED-CAF, TF-NO e TF-CAF comparados ao grupo SED-NO. Não houve diferença nos componentes do SRA ECA2/Ang 1-7/Mas renal. Em conclusão, o TFA preveniu os danos renais causados pela dieta de cafeteria, tais como acúmulo de lipídeos nos rins, aumento do RFG e redução do espaço de Bowman, e essa resposta está associada, pelo menos em parte, com a maior ativação da AMPK independente da contribuição do SRA / Lipid accumulation observed in the obesity, insulin resistance (IR) and Diabetes Mellitus type 2 (DM2) may lead to the development of renal damage, and several mechanisms may be involved in this process, such as: 1) reduction in the AMP-activated protein (AMPK) activity; 2) hyperactivation of the renin angiotensin system (RAS) and consequent increase in the production of Angiotensin II (Ang II). Aerobic exercise training (AET) promotes significant metabolic improvement, however, little is known about the cellular mechanisms induced by AET against the development of kidney damage associated with metabolic diseases. Thus, the present study aimed to evaluate the potential of AET to prevent kidney damage induced by cafeteria diet, and the participation of RAS and AMPK protein in this response. Adult male C57BL6/J mice were separated into sedentary (SED) groups fed a normocaloric (NO) or cafeteria (CAF) (SED-NO and SED-CAF, respectively) and trained (TF) fed a NO or CAF diet (TF-NO and TF-CAF, respectively). The AET was performed at 60% of the maximum capacity simultaneously with the diets during 8 weeks. The cafeteria diet induced adiposity increase, glucose intolerance and IR, while AET prevented these changes. Animals SED-CAF increased 88% of glomerular filtration rate (GFR), increased renal lipid deposition and reduced Bowman\'s space compared to SED-NO, which were prevented by AET in the TF-CAF group. There was no change in the collagen IV and fibronectin, however TNF-alpha increased in both cafeteria diet fed groups. There was a 27% increase in the protein p-AMPK expression in the TF-CAF group, with no changes in t-ACC, p-ACC, PGC1-alpha and SIRT-1 expression. The SREBP-1 gene expression did not change among groups, but SREBP-2 gene expression increased in the SED-CAF and TF-CAF groups compared to the SED-NO and TF-NO groups. In the serum, only the activity of ACE 2 increased in TF-NO and TF-CAF groups compared to sedentary groups. In the kidney, ACE activity increased 46% in the SED-CAF group compared to SED-NO, nevertheless the AET was able to prevent this increase. Renal Ang II concentration increased in SED-CAF, TF-NO and TF-CAF groups compared to the SED-NO. No differences were observed in the components of renal RAS ACE2/Ang 1-7/Mas. In conclusion, AET prevented the renal damage caused by cafeteria diet, such as lipid accumulation, increased GFR and reduced Bowman space, and these responses are associated, at least in part, with greater activation of the AMPK protein independent of the RAS contribution

O acúmulo de lipídeos associado à obesidade, resistência à insulina (RI) e diabetes mellitus tipo 2 (DM2) pode levar ao desenvolvimento de danos renais, e diversos mecanismos podem estar envolvidos neste processo, dentre os quais: 1) redução na atividade da proteína ativada por AMP (AMPK); 2) hiperativação do sistema renina angiotensina (SRA) e consequente aumento na produção de angiotensina II (Ang II). O treinamento físico aeróbio (TFA) promove melhora metabólica significativa, no entanto, pouco se sabe sobre os mecanismos celulares induzidos pelo TFA contra o desenvolvimento de danos renais associados com doenças metabólicas. Sendo assim, o objetivo deste estudo foi avaliar o potencial do TFA para a prevenção de danos renais induzidos por dieta de cafeteria, e a participação do SRA e da proteína AMPK nessa resposta. Para isso, camundongos machos adultos C57BL6/J foram separados em grupos (n=13/grupo) sedentários (SED) alimentados com dieta normocalórica (NO) ou de cafeteria (CAF) (SED-NO e SED-CAF, respectivamente) e treinados (TF) alimentados com dieta NO ou CAF (TF-NO e TF-CAF, respectivamente). O TFA foi realizado a 60% da capacidade máxima, simultaneamente com as dietas durante 8 semanas. A dieta de cafeteria causou maior adiposidade, intolerância à glicose e RI no grupo SED-CAF, enquanto o TFA preveniu esses prejuízos no grupo TF-CAF. Os animais SED-CAF apresentaram 88% de aumento no ritmo de filtração glomerular (RFG), maior deposição lipídica renal e redução do espaço de Bowman comparado ao SED-NO, as quais foram prevenidas no grupo TF-CAF. Não houve alteração no conteúdo de colágeno IV e fibronectina, entretanto o TNF-alfa aumentou em ambos os grupos alimentados com dieta de cafeteria. Houve aumento de 27% da expressão proteica da p-AMPK no grupo TF-CAF, sem diferenças na expressão de t-ACC, p-ACC, PGC1-alfa e SIRT-1. A expressão gênica do SREBP-1 não diferiu entre os grupos, porém a expressão do SREBP-2 aumentou nos grupos SED-CAF e TF-CAF comparado aos grupos SED-NO e TF-NO. No soro, apenas a atividade da ECA2 aumentou nos grupos TF-NO e TF-CAF comparados aos sedentários. No rim, a atividade da ECA aumentou 46% no grupo SED-CAF comparado ao SED-NO, e o TFA foi capaz de prevenir esse aumento. No entanto, a Ang II renal aumentou nos grupos SED-CAF, TF-NO e TF-CAF comparados ao grupo SED-NO. Não houve diferença nos componentes do SRA ECA2/Ang 1-7/Mas renal. Em conclusão, o TFA preveniu os danos renais causados pela dieta de cafeteria, tais como acúmulo de lipídeos nos rins, aumento do RFG e redução do espaço de Bowman, e essa resposta está associada, pelo menos em parte, com a maior ativação da AMPK independente da contribuição do SRA / Lipid accumulation observed in the obesity, insulin resistance (IR) and Diabetes Mellitus type 2 (DM2) may lead to the development of renal damage, and several mechanisms may be involved in this process, such as: 1) reduction in the AMP-activated protein (AMPK) activity; 2) hyperactivation of the renin angiotensin system (RAS) and consequent increase in the production of Angiotensin II (Ang II). Aerobic exercise training (AET) promotes significant metabolic improvement, however, little is known about the cellular mechanisms induced by AET against the development of kidney damage associated with metabolic diseases. Thus, the present study aimed to evaluate the potential of AET to prevent kidney damage induced by cafeteria diet, and the participation of RAS and AMPK protein in this response. Adult male C57BL6/J mice were separated into sedentary (SED) groups fed a normocaloric (NO) or cafeteria (CAF) (SED-NO and SED-CAF, respectively) and trained (TF) fed a NO or CAF diet (TF-NO and TF-CAF, respectively). The AET was performed at 60% of the maximum capacity simultaneously with the diets during 8 weeks. The cafeteria diet induced adiposity increase, glucose intolerance and IR, while AET prevented these changes. Animals SED-CAF increased 88% of glomerular filtration rate (GFR), increased renal lipid deposition and reduced Bowman\'s space compared to SED-NO, which were prevented by AET in the TF-CAF group. There was no change in the collagen IV and fibronectin, however TNF-alpha increased in both cafeteria diet fed groups. There was a 27% increase in the protein p-AMPK expression in the TF-CAF group, with no changes in t-ACC, p-ACC, PGC1-alpha and SIRT-1 expression. The SREBP-1 gene expression did not change among groups, but SREBP-2 gene expression increased in the SED-CAF and TF-CAF groups compared to the SED-NO and TF-NO groups. In the serum, only the activity of ACE 2 increased in TF-NO and TF-CAF groups compared to sedentary groups. In the kidney, ACE activity increased 46% in the SED-CAF group compared to SED-NO, nevertheless the AET was able to prevent this increase. Renal Ang II concentration increased in SED-CAF, TF-NO and TF-CAF groups compared to the SED-NO. No differences were observed in the components of renal RAS ACE2/Ang 1-7/Mas. In conclusion, AET prevented the renal damage caused by cafeteria diet, such as lipid accumulation, increased GFR and reduced Bowman space, and these responses are associated, at least in part, with greater activation of the AMPK protein independent of the RAS contribution

La protéine kinase activée par AMP (AMPK) est un senseur ubiquitaire du statut énergétique de la cellule eucaryote. Elle est exprimée sous la forme d'un complexe hétérotrimèrique comprenant les sous unités catalytique (α) et régulatrices (β et γ). Ce large complexe protéique (130kDa), fonctionne comme un hub central de la signalisation cellulaire, régulateur du métabolisme énergétique et au-delà. La (dé)régulation de l'AMPK est impliquée dans de nombreuses pathologies et l'AMPK apparait comme une cible de choix pour développer de nouveaux médicaments contre le diabète de type 2. Une fois activée, l'AMPK va restaurer l'homéostasie énergétique en diminuant le métabolisme demandeur d'énergie (anabolisme) et en stimulant le métabolisme produisant le l'énergie (catabolisme). In vivo, l'AMPK est activée par des mécanismes multiples et complexes permettant la fine régulation de son activité lors de différentes situations de stress métaboliques. Premièrement, l'activité de l'AMPK est modulée de manière systémique par phosphorylation et déphosphorylation de la sous unité α (par des kinases et phosphatases en amont respectivement). De plus, l'attachement d'AMP et d'ADP à la sous unité γ augmente la phosphorylation de l'AMPK. Deuxièmement, l'AMPK est activée de manière allostérique par l'AMP qui se lie à sous unité γ lors de chutes du ratio ATP/AMP. Tous ces mécanismes requièrent une communication entre les sous unités α et γ, mais un modèle consensus complet de l'activation de l'AMPK est toujours manquant. Se basant sur différentes études structurales, d'autres et nous-mêmes avons proposé un changement de conformation induit par AMP au sein de l'hétérotrimère AMPK. Afin de mieux élucider ce mécanisme, nous avons tiré profit de ces changements conformationels pour imaginer et créer un hétérotrimère d'AMPK permettant de suivre directement et en temps réel l'état de conformation de l'AMPK par FRET. Une limite importante lors du développement de complexes multiprotéiques est l'augmentation exponentielle de la quantité de travail liée à la modification et la combinaison de nombreux gènes hétérologues lors du remaniement de ces complexes protéiques et de leurs productions. Nous avons utilisé la technologie ACEMBL, qui exploite des techniques de recombinaisons homologues, pour faciliter la révision rapide et itérative de la production et de l'analyse fonctionnelle, après ingénierie, de complexes multi protéiques. Le senseur fluorescent génétiquement codé ainsi crée, et nommé AMPfret, a la propriété de rapporter les changements de conformation induits par les nucléotides ayant lieu au sein de l'AMPK. De plus, les changements de signal FRET corrèlent avec l'activation allostérique de l'AMPK. Le senseur répond à de faible concentrations en AMP (micromolaire) et a démontré la capacité exclusive qu'a l'ATP, et non l'ATP-Mg, à concurrencer l'AMP. De plus, son utilisation a permis une meilleure compréhension du rôle des sites CBS lors de l'activation allostérique. AMPfret peut aussi être considérer comme un outil de choix pour le criblage de molécules ciblant l'AMPK, et pour le monitoring de l'état énergétique intracellulaire. / AMP-activated protein kinase (AMPK) is a ubiquitous sensor of cellular energy and nutrient status in eukaryotic cells. It is expressed as heterotrimeric complexes comprising catalytic (α) and regulatory (β and γ) subunits. This large protein complex (130kDa), conserved from yeast to plants and mammals, functions as a central signaling hub and master regulator of energy metabolism and beyond. (Dys)regulation of AMPK signaling has been implicated in various pathologies. In particular, AMPK emerged as a suitable target to develop novel drugs for type II diabetes. Once activated AMPK will attempt to restore the energy homeostasis by down-regulating energy demanding pathways (anabolism) and up-regulating the energy producing ones (catabolism). AMPK is activated in vivo by multiple, complex mechanisms allowing fine tuning of AMPK activity in different situations of metabolic stress. First, AMPK activity is systemically modulated via activating phosphorylation at the α-subunit (by upstream kinases) and inactivating dephosphorylation (by upstream phosphatases). In addition, AMP and ADP binding to the γ-subunit increase AMPK phosphorylation. Second, AMPK is allosterically activated by AMP binding to the γ-subunit when the ATP/AMP ratio is falling. All these mechanisms require close communication between the γ- and α subunits, but a complete consensus model for AMPK activation is still lacking. We and others have proposed an AMP-induced conformational switch within the full-length heterotrimeric AMPK complex based on different, complementary structural studies. To further elucidate this mechanism, we have profited from these structural rearrangements to imagine and engineer an AMPK complex that allows a direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). A definite bottleneck in engineering multiprotein complexes is the exponential increase in work-load if several heterologous genes need to be altered, engineered and combined for revised protein complex production experiments. We used the ACEMBL technology which harnesses site-specific and homologous recombination techniques in tandem to facilitate rapid, iterative revision of multi-protein complex expressions after engineering and functional analysis of multiprotein complex. The resulting genetically encoded fluorescent biosensor, named AMPfret, can report conformational changes within the AMPK heterotrimer induced by nucleotide binding and the monitored FRET correlates with AMPK allosteric activation. The sensor responds to low micromolar concentrations of AMP, shows the exclusive ability of ATP, but not Mg-ATP, to compete with AMP, and allows insight into the role of CBS domains for allosteric AMPK activation. It may also be a tool of choice for AMPK targeted drug screening, and reporting the intracellular energy state.