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Background and Purpose –The mammalian target of rapamycin (mTOR) pathway has been implicated in cellular responses to hypoxia and inflammation. Cerebral palsy (CP) is a neurodevelopmental disorder often linked to hypoxic and inflammatory injury to the brain, however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR inhibition would prevent neuronal death and diminish inflammation in a mouse model of CP. Methods – Post-natal day 6 mouse pups were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing injury to several brain areas. Mice received rapamycin (5mg/kg) following HIL, and then daily for 3 subsequent days. The phospho-activation of the mTOR effector mTOR effector proteins S6, S6K and 4EBP as well as upstream negative regulators, TSC1 and Redd1, were assessed as an in vivo measure of the mTOR signaling cascade. Expression of hypoxia inducible factor 1 (HIF-1 alpha) was assayed as an indicator of hypoxia-mediated cellular injury. Neuronal cell death was defined with Fluoro-Jade C (FJC) and cleaved-caspase 3 (CC3), a marker of apoptosis. Autophagy was measured using Beclin-1 and LC3II expression. Lastly, neuroninflammation following HIL was evaluated by examining Iba-1 labeled microglia number and morphology, as well as P-STAT3 expression. Results – Neuronal death, HIF-1alpha expression, and numerous Iba-1 labeled microglia were evident at 24 and 48 hours following HIL. Basal mTOR signaling was unchanged by HIL. Coincident with persistent mTOR signaling, a decreased in Redd1 expression but not TSC1 was observed in HIL. Increased P-STAT3 expression was observed at 24 and 48 hours post-HIL. Rapamycin treatment following HIL significantly reduced neuronal death, decreased HIF-1 alpha and P-STAT3 expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. Increase in neuronal death was observed with concomitant administration of rapamycin and chloroquine, an autophagy inhibitor. Administration of a S6K inhibitor, PF-4708671, following HIL also decreased FJC staining further supporting an mTOR-dependent effect of HIL. Conclusions – mTOR inhibition prevented neuronal cell death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in CP. / Biomedical Neuroscience

The mechanistic target of rapamycin (mTOR) is a key serine/threonine protein kinase that functions in complexes mTORC1 and mTORC2. mTORC1, originally discovered due to its sensitivity towards the mTOR inhibitor rapamycin, responds to extracellular growth factor signaling, WNT signaling, and nutrient abundance via glucose and amino acid-triggered signaling. Downstream effectors of mTORC1 include autophagy, mitochondrial metabolic function, protein synthesis, and ribosome biogenesis. mTORC2, initially discovered as a rapamycin-insensitive complex of mTOR, responds to insulin, growth factor signaling, and inflammatory signaling such as tumor necrosis factor-alpha, with its downstream effectors being Akt, a key serine/threonine kinase that functions in cell division and is frequently dysregulated in many types of cancer, the NFkB pathway, and cytoskeletal reorganization and protein synthesis. Much research has been devoted to mTORC1 signaling, with mTORC2 receiving significantly less attention, despite both complexes’ regulation of key cellular activities and response to rapamycin, as well as to other rapamycin-derived drugs (rapalogs). We have targeted both mTORC1 and mTORC2 for hepatocyte-specific deletion during the gestational period of mice, with the goal of describing mTORC1 and mTORC2 signaling and its perturbation in the adult mouse hepatocyte. Our model has shown that deletion of RAPTOR, the regulatory associated protein of mTOR, and RICTOR, the rapamycin insensitive component of mTOR, in mTORC1 and mTORC2 respectively, leads to widespread effects on the hepatocyte transcriptome. We have found that a subset of genes responds both to Raptor and Rictor knockout, and an analysis of these genes indicates their function in key disorders of the liver, such as non-alcoholic fatty liver disease and hepatocellular carcinoma. Bioinformatic analysis following hepatocyte RNA sequencing of mTORC1 and mTORC2 knockout mice has revealed an unexpected upregulation of genes known to be regulated by these respective complexes. We have also found that cross talk exists between both complexes, in which the knockout of one yields the activation of the other. We have additionally found translationally relevant enrichments following Ingenuity Pathway Analysis (IPA) of RNA sequencing data. These results provide a key mechanistic discovery of mTOR signaling activity, and allow for a better understanding of the potential physiological effects of mTOR inhibition in human patients.

Duchene muscular dystrophy (DMD) is a lethal X-linked disease that afflicts approximately 1 in 3500 newborn males. Boys with DMD will become progressively weaker causing wheelchair dependence by their early teens and death by their mid to late twenties. Currently there is no cure for DMD, the exact mechanism of disease action remains elusive, and treatments to improve quality of life are limited. Two areas of DMD research that could begin to fill this void and provide simple, cost effective therapy aimed to improve quality of life are neutriceutical and exercise therapies. We hypothesized that leucine, a branched chain amino acid (BCAA) with anabolic properties, given to sedentary and exercised x-linked dystrophic mice (mdx) over 4 weeks would improve skeletal muscle function and decrease markers of skeletal muscle degradation. In sedentary mdx mice, leucine improved tetanic extensor digitorum longus (EDL) stress (p < 0.05), gastrocnemius mammalian target or rapamycin (mTOR) phosphorylation (p < 0.05), while decreasing the rate of real-time calpain activity in flexor digitorum brevis (FDB) fibers (p < 0.05) compared to sedentary mice given no leucine. In exercised mdx mice, leucine improved total running distance over the 4 week testing period by 40% (p < 0.02) and increased EDL stress at every frequency recorded (p < 0.05). Our data lead us to the conclusion that the BCAA leucine can increase EDL muscle stress in dystrophic animals, and that the effects of leucine treatment are enhanced when leucine supplementation is combined with exercise. Leucine supplementation should be explored further and in higher order species of muscular dystrophy to determine if its use could provide clinical improvements in DMD patients. / Ph. D.

Improving our understanding of milk protein production regulation and AA transport is important for successfully formulating diets for AA and improving N efficiency. The objectives were to study protein synthesis regulation and AA transport using in vitro and in vivo models. In the first experiment, the objective was to evaluate the ability of five distinct AA profiles and balancing Lys to Met ratio to 3:1 to stimulate protein translation. No single AA profile uniquely stimulated phosphorylation of translational machinery related proteins suggesting identification of a single optimal AA profile as unlikely. In the second experiment, an in vitro method using three different AA isotopes was developed to trace AA movement. The method assesses bi-directional transport of multiple AA simultaneously enabling evaluation of unidirectional uptake kinetics. This method was used to evaluate AA concentrations representing 16, 100, 186, and 271% of cow plasma AA concentrations. Amino acid uptake was not saturable within the in vivo range for eleven AA. Arginine, Val, and Pro exhibited saturation with the Michaelis-Menten km being 95, 49, and 65% of in vivo concentrations. Results suggest that AA transport is generally non-saturable and that high bi-directional transport exists which enables a mechanism for mitigating AA shortages. In experiment 3, the objective was to evaluate milk protein production and regulation from infusing Met, Lys, and His (MKH) or Ile and Leu (IL). The two EAA groups independently and additively increased milk protein yield. This finding contradicts the single limiting AA theory that a single nutrient will limit milk protein yield. Changes in udder AA extraction and blood flow from supplemental EAA reveal flexible delivery mechanisms. The phosphorylation state of proteins associated with the mTOR pathway was impacted by both EAA treatments. Changes in the udder proteome suggest negative feedback on mTOR pathway activation when milk protein yield was increased by the EAA groups separately but when supplemented together, negative feedback was lessened. Results indicate that multiple EAA can stimulate milk protein production, the ability of AA transport to match intracellular needs, and that the single limiting AA theory or existence of a unique optimal AA profile is likely irrelevant in dairy cows. / Doctor of Philosophy / Post absorptive metabolism of dietary protein and conversion to milk protein is not well described in dairy nutrition models resulting in poor predictions of response to changing protein supply. This partially constrains diet formulation with respect to successfully balancing diets for protein and amino acids or for improving N efficiency. The efficiency of absorbed AA into milk protein varies and udder AA uptake may contribute to this varying efficiency through transport regulation in an attempt to maintain intracellular AA homeostasis. Amino acid transport was assessed when AA supplies were varied below and above in vivo supplies. High bi-directional AA transport was saturating uptake for Arg, Pro, and Val within the normal in vivo range for lactating dairy cows. The high AA exchange suggest strong ability to manage changes in AA supply to meet needs for milk protein translation. When intracellular AA supply declines, efflux of the limiting AA out of the cell declines which results in greater uptake of the limiting AA by the cell. The theory that milk protein yield is limited by a single most limiting amino acid (e.g., the barrel and stave analogy) and that a single optimal EAA profile exists predominates in the field of animal nutrition, but implementation of this theory has not greatly improved N efficiency or been adopted widely. We observed that various AA profiles can equally stimulate milk protein translational machinery in mammary epithelial cells and that balancing Lys to Met ratio to 3 to 1 only had a minor effect. Multiple EAA can regulate milk protein production through signaling to synthesis machinery and delivery of AA to the udder. Supplementation of two iv groups of EAA, 1) methionine, lysine, and histidine and 2) isoleucine and leucine, independently and additively increased milk protein yield in dairy cows. These increases were achieved by changes in blood flow in the udder, AA uptake, and nutrient signaling related to protein translation regulation. Hence, results of this dissertation tend to not support the idea of a single limiting amino acid or a unique optimal profile of AA for milk protein production in dairy cattle.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25181号 / 医博第5067号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 武藤 学, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25199号 / 医博第5085号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 藤田 恭之, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La transplantation d'organe est le meilleur traitement en cas de défaillance terminale d'un organe vital. Cependant, la survie sur le long terme est limitée par la perte inexorable de la fonction des greffons. Cette dernière est attribuée à l'inflammation microvasculaire (1MV) causée par la réponse anticorps contre les alloantigènes (rejet humoral chronique (RHC)). En analysant une cohorte de 129 transplantés rénaux présentant de l'1MV sur une biopsie de greffon, nous avons trouvé que, dans la moitié des cas, les lésions n'étaient pas médiées par les anticorps. Chez ces patients, des études génétiques ont révélé une prévalence plus élevée de « mismatches » entre les molécules HLA de classe 1 (HLA-1) du donneur et les « Killer-cell immunoglobulin-receptors » (K1R) inhibiteurs des NK du receveur. Nous avons émis l'hypothèse que la nature allogénique de l'endothélium du greffon pouvait créer un « pseudo-missing-self ». De ce fait, les NK du receveur, exposés à des stimuli inflammatoires, ne reçoivent plus les signaux inhibiteurs transmis par le HLA-1 de la part des cellules endothéliales du donneur. Dans un modèle de co-culture de cellules endothéliales et de NK humains, nous avons démontré que l'absence d'un ligand HLA-1 du soi sur la cellule endothéliale peut activer les NK. Cette activation dépend de la voie mTOR dans les NK, qui peut être bloquée par la rapamycine, un inhibiteur de mTORC1 disponible en clinique. Enfin, nous avons confirmé l'existence de rejets NK induit par le « missing-self » et leur sensibilité à la rapamycine dans un modèle murin de transplantation cardiaque. Notre travail identifie un nouveau type de rejet chronique, exclusivement médié par l'immunité innée, les NK, ayant le même impact délétère sur la survie des greffons que le RHC. Cependant, alors qu'il n'y a pas de traitement disponible pour le RHC, les inhibiteurs de mTOR préviennent efficacement le développement de lésions dans un modèle murin de rejet vasculaire chronique induit par le « missing self » / Organ transplantation is the best treatment for terminal organ failure. However, long-term outcome of organ transplantation remains limited by inexorable loss of graft function, which the prevalent dogma links to the microvascular inflammation (MVI) triggered by the recipient's antibody response against alloantigens (antibody-mediated chronic rejection, AMR). Analysing a cohort of 129 renal transplant patients with MVI on graft biopsy, we found that, in half of the cases, histological lesions were not mediated by antibodies. In these patients, genetic studies revealed a higher prevalence of mismatches between donor HLA-I and inhibitory Killer-cell immunoglobulin-receptors (KIR) of recipient's NK cells. We hypothesized that the allogeneic nature of graft endothelium could create a "pseudo-missing self" situation, thereby the recipient's NK cells exposed to inflammatory stimuli would not receive HLA I-mediated inhibitory signals from donor endothelial cells. In co-culture experiments with human NK cells and endothelial cells, we demonstrated that the lack of self HLA-I on endothelial cells can activate NK. This activation triggers mTOR pathway in NK, which can be blocked by rapamycin, a commercially available inhibitor of mTORC1. Finally, we confirmed the existence of missing self-induced rejection and its sensitivity to mTOR inhibition in a murine heart transplantation model. Our work identifies a new type of chronic rejection, exclusively mediated by innate NK cells, with the same detrimental impact on graft survival as AMR. However, while no therapy is available for AMR, mTOR inhibitors efficiently prevent the development of lesions in murine models of NK cell-mediated chronic vascular rejection

Recentemente suspeitas de que mecanismos epigenéticos poderiam estar relacionados à fisiopatologia da depressão foram levantadas. Estudos recentes indicam que as alterações na transcrição gênica, induzidas por estresse ou por drogas antidepressivas, parecem envolver mecanismos epigenéticos. Nesse sentido, resultados preliminares de nosso grupo de pesquisa indicaram pioneiramente inibição global da metilação de DNA através da administração sistêmica do agente inibidor da DNA metiltransferase (DNMTs), 5-aza-2-deoxicitidina (5-azaD), induz efeito tipo-antidepressivo, dose-dependente, no modelo animal do nado forçado em ratos(Sales et al., 2011). O córtex pré-frontal medial ventral (CPFMv) é uma estrutura límbica intimamente relacionada com a neurobiologia da depressão. Evidências recentes indicam que o efeito tipo-antidepressivo aparece associado a aumento dos níveis da neurotrofina BDNF (brain derived neurotrophic factor) e de seu receptor TrkB no CPFMv, sendo a sinalização intracelular mediada pela ativação da proteína m-TOR. Contudo, não há evidências de que esses mecanismos moleculares estariam envolvidos nos efeitos induzidos pelos inibidores da metilação do DNA. Sabe-se, no entanto, que tanto o BDNF quanto TrkB têm sua expressão regulada por metilação do DNA. Diante disso, o objetivo presente trabalho será investigar a participação da via BDNF-TrkB-mTOR do CPFMv no efeito antidepressivo induzido por inibidores da metilação de DNA. Para tanto, ratos tratados com inibidores da metilação de DNA (5-azaD ou RG-108), em dois momentos diferentes (imediatamente após o PT e 23horas após o PT) foram submetidos ao teste do nado forçado (FST). Outro grupo de animais recebeu uma injeção intra-CPMv de k252a ou Rapamicina, 40 minutos antes do teste e uma injeção de BDNF intra-CPFMv, 30 minutos antes do teste. Em outro experimento, grupos independentes de animais submetidos ao nado forçado foram tratados sistemicamente com RG-108 e receberam injeção intra-CPFMv de K252a (antagonista de Trk) ou de rapamicina (inibidor da m-Tor), a fim de investigar se o efeito dessas drogas depende da via BDNF-TrkB-mTOR no CPFMv. Um grupo independente foi tratado com RG108 e CPFM desses animais foi dissecado para posterior análise da expressão de BDNF, TrkB e m-TOR, bem como da metilação de DNA. O tratamento com RG108 e 5azaD sistêmico reduziu o tempo de imobilidade dos animais submetidos ao nado forçado nos dois tempo de administração. A administração intra-CPFMv de BDNF promoveu efeito antidepressivo no FST, e esse efeito foi bloqueado pela administração de k252a ou Rapamicina no CPFMv. No mesmo sentido, o efeito antidepressivo do RG108 sistêmico foi bloqueado pela administração intra-CPFMv de k252a ou Rapamicina. Entretanto, a medida dos níveis de metilação global no CPFMv não apresentou alteração como tratamento com RG108, e também não mostrou alteração nos níveis de BDNF presente no CPF. O tratamento com RG108 não alterou a expressão, bem como a ativação de TRkB e mTOR. Concluímos que os inibidores da metilação do DNA apresentam agudamente efeito tipo antidepressivo rápido, que necessita da funcionalidade integral da via BDNF-TRkB-mTOR. Entretanto, esse efeito parece não alterar a síntese e expressão das proteínas envolvidas nessa via no que diz respeito ao CPFmv. / Recent studies indicate that changes in gene transcription induced by stress or antidepressant drugs appear to involve epigenetic mechanisms. Accordingly, results of our research group pioneered indicated global inhibition of DNA methylation through systemic administration of an inhibitor of DNA methyltransferase (DNMTs), 5-aza-2-deoxycytidine (5-AzaD), induces antidepressant-like effect dose-dependent in the animal model of forced swimming in rats (Sales et al., 2011). The ventral medial prefrontal (vmPFC) cortex is a limbic structure closely related to the neurobiology of depression. Recent evidence indicates that the antidepressant-like effect appears associated with increased levels BDNF (Brain derived neurotrophic factor) and its receptor TrkB in vmPFC, and intracellular signaling mediated by activation of protein mTOR. However, there is no evidence that these molecular mechanisms are involved in the effects induced by inhibitors of DNA methylation. It is known, however, both as BDNF and TrkB expression is regulated by DNA methylation. Thus, the goal of this work is to investigate the role of BDNF-TRkB pathway mTOR-vmPFC in the antidepressant effect induced by inhibitors of DNA methylation. To this end, rats treated with inhibitors of DNA methylation (5-Azad or RG-108), at two different times (immediately after 23hours after the PT and PT) were subjected to the forced swim test (FST). Another group received an intra-vmPFC injection of K252a or Rapamycin 40 minutes before the test, and an injection intra-vmPFC of BDNF 30 minutes before the test. In another experiment, separate groups undergoing the forced swim were treated systemically with RG-108 and received intra-vmPFC of K252a (Trk antagonist) or injection of rapamycin (m-Tor inhibitors) in order to investigate the effect these drugs depends on BDNF-TrkB-mTOR pathway in vmPFC. A separate group was treated with RG108 and mPFC these animals were dissected for analysis of the expression of BDNF and TrkB m-TOR, as well as DNA methylation. The systemic treatment whit 5azaD and RG108 reduced the immobility time of rats subjected to FST administration in both time. The intra-vmPFC BDNF administration promoted antidepressant effect in the FST, and this effect was blocked by the administration of K252a or Rapamycin in vmPFC. Similarly, the antidepressant effect of systemic RG108 was blocked by intra-vmPFC of K252a or Rapamycin administration. However, the measurement of the levels of global methylation in CPFMv did not change as treatment with RG108, and also showed no change in the levels of BDNF present in the CPF. Treatment with RG108 did not alter the expression and activation of TrkB and mTOR. We conclude that inhibitors of DNA methylation present acutely antidepressant-like effect, it needs the full functionality of the BDNF-TrkB-mTOR pathway. However, this effect seems not to alter the synthesis and expression of proteins involved in this pathway at vmPFC.

A arginina é um aminoácido condicionalmente essencial que participa de inúmeras reações metabólicas no organismo como, por exemplo, o ciclo da uréia, a síntese de creatina e a geração de óxido nítrico (NO). Além dessas funções a arginina é associada, com a sensibilidade à insulina, a secreção de GH e mais recentemente com a síntese protéica muscular. O objetivo deste trabalho foi investigar o efeito da suplementação via oral crônica de L-arginina sobre a síntese protéica muscular, pela via da mTOR, a fim de contribuir com as novas discussões científicas acerca deste aminoácido de ampla atuação. Métodos: Foram utilizados ratos wistar machos adultos com cerca de 200g de peso corporal divididos em quatro grupos de quatorze animais denominados na seguinte forma: Arginina Treinado (AT), Arginina Sedentário (AS), Dieta-Controle Treinado (CT) e Dieta-Controle Sedentário (CS). Ambas as dietas foram elaboradas com base das recomendações da AIN-93, sendo que a dieta enriquecida com arginina recebeu acréscimo de 2% deste aminoácido e a dieta controle recebeu um mix de aminoácidos não essenciais para garantir que ambas fossem isonitrogenadas e isocalóricas e as proporções de aminoácidos presente nas rações foi conferida por aminograma. O treinamento dos animais consistiu em exercício anaeróbio com sessões que eram compostas de 4 séries de 10 saltos com um minuto de descanso entre estas em tanque de água. Os saltos eram desempenhados com carga de 50% do peso corporal acoplado ao tórax dos animais na freqüência de 5 dias por semana por 6 semanas. A evolução da massa corporal dos animais bem como o consumo de ração foram avaliadas três vezes por semana e estimada uma média semanal. Foram realizados testes de tolerância oral à glicose (OGTT) e tolerância à insulina (ITT) no início e ao final do experimento em todos os animais para avaliar alterações na sensibilidade à insulina. Após 72hs da última sessão de treinamento os animais foram anestesiados para infusão de insulina, coleta dos músculos gastrocnêmio e plantar, fígado, sangue e eutanasiados conforme protocolo aprovado pelo CEA-USP. As análises bioquímicas foram determinações séricas de insulina, GH, IGF-1 e a proteína transportadora de IGF-1 (IGFBP-3), glicose plasmática, uréia e creatinina séricas, IGF-1 muscular e hepático por kits comerciais de tecnologia multiplex Luminex e aminograma sérico por cromatografia. As análises moleculares foram realizadas para as proteínas chaves envolvidas na via de síntese protéica muscular em sua forma total e fosforilada, sendo estas: IRS-1, Akt, mTOR, 4E-BP1 e p70S6K determinadas por método de western blotting. Resultados: Não foram encontradas diferenças estatisticamente significativas nos parâmetros avaliados com exceção da creatinina que se mostrou mais elevada nos grupos suplementados com arginina. A suplementação de arginina, nas concentrações administradas, bem como o exercício de alta intensidade pelo período determinado não foram capazes de alterar a expressão das proteínas envolvidas na regulação de síntese protéica muscular de ratos nem a sensibilidade celular à insulina. Conclusão: não houve aumento da síntese protéica muscular com a suplementação de arginina, nestas condições experimentais. / The arginine is an amino acid conditionally essential that participates in innumerous metabolic reactions in the body like, for instance, the urea cycle, the synthesis of creatine and production of nitric oxide (NO). Besides those functions the arginine is associated, with the insulin sensitivity, GH secretion and most recently with muscle protein synthesis. The aim of this work was to investigate the effect of L-arginine chronic oral supplementation on the muscle protein synthesis, through mTOR pathway, in order to contribute with new scientific discussions about this broad action amino acid. Methods: Wistar male adult rats were used with about 200g body weight distribute into four groups of fourteen animals named this way: Trained Arginine (TA), sedentary Arginine (SA), Trained Diet-Control (TC) and Sedentary Diet-control (SC). Both diets were elaborated based on the AIN-93 recommendations, considering that the enriched diet with arginine was added 2% of this amino acid and the control diet received a mix of non-essential amino acid in order to ensure that both were isonitrogenous and isocaloric and the proportions of present amino acids in the rations have been checked through aminogram. The animals training consisted of anaerobic exercise with sections composed by four jump series, with one minute rest among these in a PVC cube water. The jumps were performed with a load of 50% of their body weight attached in the animal\'s trunk, five days a week over six weeks. The animals\' body weight evolution as well as the food intake were evaluated three times a week in order to figure a weekly average. Oral glucose test tolerance (OGTT) and insulin test tolerance (ITT) have been done in the beginning and in the end of the experiment in all animals to evaluate insulin sensitive changes. The animals were anesthetized to insulin infusion, gastrocnemic and plantaris muscles, liver and blood collects 72 hrs after the last training section and afterwards sacrificed according to CEA-USP approved protocol. The biochemical analysis were blood determination of insulin, GH, IGF-1 and its binding protein 3 (IGFBP-3), glucose, urea and creatinine, and muscle and liver IGF-1 through commercial kits of multiplex Luminex technology and seric aminogram through chromatography. The molecular analysis were performed for the key proteins of the muscle protein synthesis pathway in its total and phosphorylated form: IRS-1, Akt-1, mTOR, 4E-BP1 and p70S6K determined by western blotting method. Results: Significant statistical differences were not found to all evaluated biomarkers in this experiment except for creatinine which was more elevated in groups supplemented with arginine. The arginine supplementation, in these given doses, as well as the high-intense exercise, failed in stimulate both the expression of the proteins involved in the muscle protein synthesis regulation and the insulin sensitivity in the rats in this condition. Conclusion: There hasn\'t been any increase in the muscle protein synthesis with arginine supplementation, in these experimental conditions.