Este estudo teve como objetivo investigar incialmente o efeito do pHf na qualidade da carne, nas concentrações de HSP27 e HSP70, bem como no perfil proteômico da carne (Longissimus thoracis) de bovinos cruzados - 1/2 (Simental Sul Africano x Nelore) de forma a contribuir para a predição de biomarcadores que poderão ser agregados em programas de melhoramento genético. Foram avaliados 415 bovinos (180 fêmeas e 235 machos imunocastrados) terminados em confinamento. As carcaças foram classificadas de acordo com pHf, mensurados 48 horas após o abate, em dois grupos: normal (pHf ≤ 5,80) e intermediário (pHf = 5,81 - 6,19). Os parâmetros físico-químicos pH, cor (L*, a* e b*), perda de água por cocção (PAC, %) e força de cisalhamento (FC, kg), foram mensurados para avaliar a qualidade da carne em dois tempos de maturação (1 e 14 dias). Para a quantificação de HSP27 e HSP70 foram selecionadas 38 amostras em função dos grupos de pHf, dentro de cada tempo de maturação, totalizando 76 amostras. Para a realização das análises proteômicas foram selecionadas três amostras de cada grupo de pHf nos dois tempos de maturação, totalizando doze amostras. Observou-se interação (p<0,05) entre os grupos de pHf e os tempos de maturação para a cor e FC. A concentração de HSP27 foi maior (p<0,05) em pHf intermediário e com 1 de maturação, para a HSP70 só foi possível observar maior concentração (p<0,05) aos 14 dias de maturação, as correlações indicam que as HSPs modulam a qualidade da carne. Foram identificadas 13 proteínas diferencialmente expressas (p<0,05) envolvidas com funções metabólicas (triosefosfato isomerase - TPI1; fosfoglicerato mutase 2 - PGAM2; malato desidrogenase - MDH1; piruvato quinase - PKM2; e adenalato quinase isoenzima 1 - AK1), estrutural (troponina I tipo 2 - TTNI2; tropomiosina cadeia beta - TPM2; troponina C tipo 2 - TNNC2; e cofilina 2 - CFL2) e defesa em resposta ao estresse (proteína deglicase DJ-1 - PARK7; αβ-cristalina - CRYAB; proteína do choque térmico 27 - HSPB1; e peroxirredoxina 1 - PRDX1). As correlações demonstraram que essas proteínas estão envolvidas na determinação do pHf e dos demais atributos da qualidade da carne. A bioinformática permitiu observar que essas proteínas estão envolvidas nas três categorias de ontologia gênica, sendo 18 termos de componentes celulares, 1 termo de função molecular e 61 termos dos processos biológicos, foram identificadas também 6 vias metabólicas. A proteína MDH1, esteve associada a mecanismos de produção de energia, confirmando o potencial desta como um biomarcador para a determinação do pHf assim como para a cor da carne, enquanto que a TPI1 foi relacionada somente no estabelecimento do pHf. Outras proteínas como a PKM2, com função metabólica, e a PARK7, com função protetora, estiveram associadas aos atributos maciez e cor da carne, respectivamente. Os resultados obtidos são de grande relevância no âmbito das ciências da carne e do melhoramento genético animal, por permitir a identificação de possíveis biomarcadores que poderão ser utilizados na seleção animal. / The objective of this study was to investigate the effect of pHf on meat quality, on the concentrations of HSP27 and HSP70, as well as on the proteomic profile in beef (Longissimus thoracis) of crossbreed cattle - 1/2 (South African Simmental x Nellore) - in order to explore the contribute to the prediction of biomarkers that can be added to the process of animal selection in breeding programs. Were evaluated 415 cattle (180 females and 235 immunocastrated males) feedlot-finished. Carcasses were classified according to pHu, measured 48 hours after slaughter, in two groups: normal (pHu ≤ 5.80) and intermediate (pHu = 5.81 - 6.19). The physical-chemical parameters pH, color values (L *, a * and b *), cooking loss (CL,%) and shear force (SF, kg) were measured to evaluate the meat quality in two aging times (1 and 14 days). For quantification of HSP27 and HSP70, 38 samples were selected according to the pHu groups, and aging time, totaling 76 samples. To perform the proteomic analysis were selected three samples from each group and aging time, totaling twelve samples. Color and SF meat quality attributes showed interaction (p<0.05) between pHu groups and aging times. The concentration of HSP27 was higher (p<0.05) at intermediate pHu and at 1 day of aging. It was only possible to observe a higher concentration (p<0.05) for HSP70 at 14 days of aging. Correlations analysis indicate that HSPs modulate the quality of meat. Thirteen differentially expressed proteins (p<0.05) were identified as involved with metabolic (triosephosphate isomerase - TPI, phosphoglycerate mutase 2 - PGAM2; malate dehydrogenase, cytoplasmic - MDH1; pyruvate kinase - PKM2; and adenylate kinase isoenzyme 1- AK1), structural (troponin I type 2 - TTNI2; tropomyosin beta chain - TPM2; troponin C type 2 - TNNC2; and cofilin 2 - CFL2) and defense in response to stress functions (protein deglycase DJ-1- PARK7; αβ-cristallin - CRYAB; heat shock protein 27 - HSPB1; and peroxiredoxin 1 - PRDX1). Correlations analysis showed that these proteins are involved in the determination of pHu and other attributes of meat quality. Using bioinformatics it was possible to observe that these proteins are involved in three gene ontology categories, which were 18 cellular components terms, 1 molecular function term and 61 biological processes terms. Other six metabolic pathways have also been identified. The MDH1 protein was associated with energy production mechanisms, confirming its potential as a biomarker for the determination of pHu as well as meat color, while TPI was related to the establishment of pHu only. Other proteins such as PKM2, with metabolic function, and PARK7, with protective function, were associated with the attributes of meat tenderness and color, respectively. The obtained results are of great relevance in the field of meat science and animal breeding genetic, it allows the identification of biomarkers that can be used in animal selection.

A oligopeptidase neurolina (E.C.3.4.24.16; nln ) foi identificado pela primeira vez em membranas sinápticas de cérebro de ratos como sendo capaz de participar no metabolismo de peptídeos bioativos, como neurotensina e bradicinina. Recentemente, foi sugerido que a ausência de Nln pode melhorar a sensibilidade a insulina. Aqui, nós mostrado que camundongos knockout para Nln (KO) são mais tolrerantes à glicose, sensíveis à insulina e apresentam maior gliconeogênese. Os animais KO apresentou um aumento na expressão de mRNA de vários genes relacionados com a gliconeogênese no fígado. A semiquantificação de peptídeos intracelulares revelou um aumento em peptídeos intracelulares específicos no gastrocnêmio e tecido adiposo epididimal, que estão envolvidos com o aumento da tolerância a glicose e maior sensibilidade à insulina nos animais KO. Esses resultados sugerem fortemente a nova possibilidade de que Nln é uma enzima chave no metabolismo energético e pode ser um novo alvo terapêutico para melhorar a captação de glicose e sensibilidade a insulina. / The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln knockout mice (KO) have increased glucose tolerance, insulin sensitivity and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semi-quantitative peptidomic analysis suggests increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity.

Embora seja bem estabelecido que a exposição a baixas temperaturas resulte em ativação simpática e hiperglicemia, os mecanismos moleculares envolvidos na regulação neural da produção hepática de glicose ainda não são bem conhecidos. Portanto, o objetivo do presente estudo foi investigar o papel da inervação simpática na regulação de genes e enzimas chaves da neoglicogênese hepática, em roedores expostos agudamente ao frio. Para isso, foram utilizados dois modelos experimentais de desnervação simpática: a simpatectomia química em camundongos induzida pelo tratamento com 6-hidroxidopamina (100 mg.kg-1.dia-1; 1º, 2º e 7º dia de vida pós-natal; i.p). e a desnervação local dos nervos hepáticos por fenol (95%) em ratos. A exposição de camundongos ao frio (4ºC), durante 1, 3 e/ou 6h, induziu hiperglicemia, hipotermia, depleção do conteúdo de glicogênio hepático e ativação da neoglicogênese, estimada pela elevada expressão gênica e atividade das enzimas glicose-6-fosfatase e fosfoenolpiruvato carboxiquinase (PEPCK). Em paralelo, verificou-se que a exposição ao frio induziu um drástico aumento na expressão do RNAm do Nr4a1 e do PGC1-?, dois genes-alvos de CREB que participam do complexo de ativação transcricional dos genes que codificam as enzimas neoglicogênicas estudadas. A simpatectomia não afetou a depleção dos estoques de glicogênio hepático induzida pelo frio, mas reduziu o aumento do conteúdo hepático de noradrenalina e AMPc e acentuou a hipotermia, bem como preveniu a hiperglicemia, sendo este efeito associado ao bloqueio da expressão gênica e atividade da glicose-6-fosfatase e da PEPCK e do RNAm do Nr4a1, em todos os tempos estudados. Resultados bastante semelhantes foram observados em ratos submetidos à lesão dos nervos hepáticos com fenol e expostos ao frio durante 24h. Tanto a adrenalectomia como a adrenodemedulação (remoção da medula da adrenal) não alteraram o efeito estimulatório do frio na atividade e expressão gênica das enzimas neoglicogênicas de camundongos. Porém, em ratos expostos ao frio, a adrenodemedulação bloqueou aIII hiperglicemia e reduziu parcialmente a hiperexpressão dos genes da glicose-6-fosfatase e PEPCK. Estes resultados mostram a importância fisiológica da inervação simpática do fígado de roedores na estimulação do programa gênico da neoglicogênese, durante o estresse térmico agudo. / Although it is well established that exposure to low temperatures leads to sympathetic activation and hyperglycemia, the molecular mechanisms related to the neural regulation of hepatic glucose production remain elusive. Therefore, the goal of the present study was to investigate the role of sympathetic innervation in the regulation of genes and key enzymes of hepatic gluconeogenesis in liver of rodents exposed to acute cold. For this, two experimental models of sympathetic denervation were used: the chemical sympathectomy in mice induced by 6-hydroxydopamine treatment (100 mg.kg-1.day-1; 1º, 2º and 7ºdays of neonatal life; i.p.) and local denervation of hepatic nerves by phenol (95%) in rats. Exposure of mice to cold (4° C) for 1, 3 and / or 6 h induced hyperglycemia, hypothermia, depletion of hepatic glycogen content and activation of gluconeogenesis, estimated by high gene expression and activity of the enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK). In parallel, it was found that exposure to cold induced a dramatic increase in Nr4a1 and PGC1-? mRNA expression, two CREB target genes that participate in the transcriptional activation complex of the genes encoding the gluconeogenesis enzymes studied. The sympathectomy did not affect the depletion of cold-induced hepatic glycogen stores but reduced the hepatic content of noradrenaline and cAMP and increased hypothermia, as well as prevented hyperglycemia, and this effect was associated with the blockade of gene expression and enzymatic activity of glucose-6-phosphatase and PEPCK and Nr4a1 mRNA levels, at all time intervals investigated. Similar results were observed in rats submitted to hepatic nerve damage with phenol and exposed to cold for 24h. Either adrenalectomy or adrenodemedullation (surgical removal of the adrenal medulla) did not alter the stimulatory effect of cold on the activity and gene expression of the gluconeogenesis enzymes of mice. However, in cold-exposed rats, adrenodemedullation blocked hyperglycemia and partially reduced overexpression of the glucose-6-phosphatase and PEPCK mRNA.V These results show the physiological role of the hepatic sympathetic innervation in the transcriptional program associated with gluconeogenesis, during acute thermal stress.

Au cours de ce travail, nous avons recherché l’effet du nitrate et de l’acétate d’uranyle sur des tubules proximaux isolés de reins de souris et de reins humains métabolisant l’un de ses substrats physiologiques, le lactate. Notre étude montre que le nitrate et l’acétate d’uranyle diminuent la gluconéogenèse et le niveau intracellulaire d’ATP de manière dose-dépendante pour des concentrations de l’ordre du millimolaire. Après l’incubation dans un milieu Krebs dépourvu d’ions phosphate en présence de L-[1-13C]-, ou L-[2-13C]-, ou L-[3-13C]lactate, la consommation du substrat et la formation de produits ont été mesurées par des méthodes enzymatiques et par spectroscopie RMN du 13C. En combinant les résultats obtenus par chacune de ces méthodes à l’aide d’un modèle mathématique du métabolisme du lactate, il a pu être montré qu’à la concentration de 3 mM, le nitrate d’uranyle exerce un effet inhibiteur sur les flux enzymatiques de la lactate deshydrogénase (LDH) et de 3 enzymes clés de la gluconéogenèse ; chez la souris et l’homme, les flux sont inhibés respectivement de 14% et 20% (LDH), de 32% et 27% (pyruvate carboxylase), de 36% et 35% (phosphoénolpyruvate carboxykinase) et de 45% et 39% (glucose-6-phosphatase). De plus, une diminution de la quantité de glutathion est observée chez la souris (-36%) et chez l’homme (-12%). Contrairement à ce que l’on attendait, car les ions phosphate pourraient faciliter l’entrée de l’uranium dans la cellule, dans les tubules proximaux murins, l’ajout d’ions phosphate au milieu d’incubation ne semble pas accroître l’inhibition de la gluconéogenèse rénale par le nitrate d’uranyle 3 mM / As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate and acetate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate and acetate reduced gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-13C]-, ou L-[2-13C]-, ou L-[3-13C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were reduced by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These effects were associated with a 12% and 36 % decrease in the cellular content of glutathione in human and mouse tubules, respectively. Contrary to what expected because phosphate was supposed to facilitate the entry of uranium, the addition of phosphate in the medium did not enhance the inhibitory effect of 3 mM uranyl nitrate on gluconeogenesis in isolated mouse tubules

Les carcinomes hépatocellulaires (CHC) mutés CTNNB1 ont des caractéristiques phénotypiques propres en termes de polarité et de métabolisme (absence de stéatose). Nous avons émis l’hypothèse que ce phénotype pouvait être secondaire à l’activation du gène suppresseur de tumeurs LKB1 qui code une Ser/Thr kinase multitâches.Nous avons tout d’abord montré qu’il existait effectivement un dialogue complexe entre les voies Wnt/β-Caténine et LKB1 dans le foie. Les mutations de CTNNB1 sont en effet capables d’induire l’expression protéique de LKB1 dans des lignées hépatomateuses humaines, et les CHC mutés CTNNB1 présentent une expression protéique accrue de LKB1 et une signature transcriptionnelle d’activation de LKB1. De plus, dans deux modèles murins d’invalidation hépatospécifique de Lkb1, LKB1 est apparu comme requis pour l’activation complète du programme transcriptionnel de β-Caténine mais de façon dépendante du stade de développement et du contexte nutritionnel. Enfin, la signalisation LKB1 est apparue comme nécessaire à la survie des hépatocytes activés pour β-Caténine dans deux modèles murins différents.Nous avons aussi caractérisé les rôles métaboliques de LKB1 dans le foie. L’invalidation hépatospécifique de Lkb1 induisait une augmentation progressive de la masse grasse corporelle avec utilisation préférentielle des glucides comme substrat énergétique. Il existait une activation de la néoglucogenèse hépatique avec hyperglycémie et une lipogenèse accrue avec accumulation hépatocytaire de lipides. Enfin, nous avons mis en évidence une activation paradoxale de la signalisation AKT dans les hépatocytes, même à jeun, et une dépendance énergétique aux acides aminés. Enfin, nous avons identifié une nouvelle isoforme protéique de LKB1 délétée de son domaine N-Terminal et d’une partie de son domaine kinase. D’expression tissulaire préférentiellement musculaire et myocardique, cette isoforme catalytiquement inactive se comportait comme dominant positif sur l’activation de l’AMPK par la forme conventionnelle mais comme dominant négatif dans l’activité polarisation induite par LKB1. Enfin, elle était capable d’induire, en l’absence de la forme conventionnelle, la prolifération cellulaire et la tumorigenèse chez la souris nude. Elle pourrait exercer des rôles métaboliques particuliers dans les tissus fortement oxydatifs et des rôles oncogéniques dans certains contextes. / CTNNB1-Mutated hepatocellular carcinomas (HCC) share a specific polarity and metabolic phenotype without steatosis. We hypothesized that such phenotype could imply the tumor suppressor gene LKB1 that encodes for a multi-Task Ser/Thr kinase.We first demonstrated that a complex crosstalk indeed exists in the liver between LKB1 and the Wnt/β-Catenin pathway. LKB1 proteic expression was controlled by mutant β-Catenin in hepatomatous cell line and CTNNB1-Mutated HCCs had an enhanced LKB1 proteic expression as well a transcriptomic signature of LKB1 activation. In two mouse model of liver-Specific invalidation of Lkb1, we showed that LKB1 was required for full activation of the β-Catenin transcriptomic program, but it depended on the developmental stage and nutritional context. At least, LKB1 appeared to be required for the survival of β-Catenin activated liver cells in two other mouse models.Then, we wanted to caracterize the metabolic roles of LKB1 in the liver. Liver-Specific invalidation of Lkb1 progressively raised the body fat mass and we observed that carbohydrates were preferred as whole-Body energetic fuel. In the liver, gluconeogenesis and lipogenesis were enhanced, resulting in mild hyperglycemia and lipid accumulation in the hepatocytes. At least, we identified an aberrant activation of the AKT signaling in the liver, even during fasting, and an energetic dependence towards amino acids.At least, we identified a novel LKB1 proteic isoform that is deleted of its N-Terminal domain and part of its kinase domain. Highly expressed in the muscle and in the heart, this catalytically inactive isoform however acted as a positive dominant towards AMPK activation by full length LKB1 but as a negative dominant towards LKB1-Induced cell polarization. This isoform is also able to enhance cell proliferation and to induce tumors in a xenograft model, even when expressed alone. It could play specific metabolic roles in oxidative tissues and could be oncogenic in some contexts.

Le champignon filamenteux Podospora anserina constitue un système modèle pour l’étude de plusieurs processus biologiques, notamment le vieillissement. Une relation de causalité entre le fonctionnement de la chaine respiratoire et la longévité a été établie pour la première fois chez P. anserina et cette relation de causalité semble conservée. Le lien entre fonctionnement de la chaîne respiratoire et longévité pourrait se faire en partie par l’induction d’une régulation rétrograde en réponse à un dysfonctionnement mitochondrial. Les études des régulations rétrogrades mitochondriales et chloroplastiques montrent qu’en réponse à un dysfonctionnement de ces organites, il y a une reprogrammation de l’expression génique qui participe à la plasticité adaptative des espèces en réponse à l’environnement. Un gène cible d’une régulation rétrograde mitochondriale a été identifié chez P. anserina: le gène aox. L’oxydase alternative appartient à une voie respiratoire alternative en conditions de dysfonctionnement de la voie des cytochromes. Deux facteurs de transcription acteurs de cette régulation rétrograde mitochondriale ont été identifiés par une approche génétique. Ces protéines, RSE2 et RSE3, appartiennent à la famille des protéines Zn2Cys6 et sont les régulateurs majeurs du gène aox. Outre le gène aox, ces deux facteurs de transcription corégulent les deux enzymes de la néoglucogenèse FBP (Fructose-1,6-biphosphatase) et PCK (Phosphoénolpyruvate carboxykinase). Des allèles gain de fonction des gènes rse2 et rse3 ont été isolés et le crible pratiquement saturé. L’analyse de ces mutations ainsi que des approches de mutagenèse dirigée, ont permis de proposer l’existence de régions régulatrices dans les séquences protéiques RSE2 et RSE3. Une approche transcriptomique a permis d’identifier de nouvelles cibles de RSE2 et RSE3 activées lors d’un dysfonctionnement mitochondrial : le gène fhb (codant une flavohémoglobine) et le gène Pa_6_4030 (codant une α/β hydrolase). Une approche métabolomique a permis une meilleure compréhension de la reprogrammation métabolique en réponse à un dysfonctionnement de la chaîne respiratoire et du rôle de RSE2/RSE3 dans cette reprogrammation.RSE2 et RSE3 sont donc des acteurs d’une régulation rétrograde mitochondriale responsables de l’activation de voies respiratoires alternatives mais aussi d’une reprogrammation du métabolisme de P. anserina qui permettrait de maintenir une homéostasie cellulaire en condition de dysfonctionnement mitochondrial. / The filamentous fungus Podospora anserina is a model system for the study of many biological processes, including aging. A causal relationship between the respiratory chain efficiency and longevity has been established for the first time in P. anserina. This causal relationship seems to be conserved between species. The link between the respiratory chain efficiency and longevity could be in part explained by the induction of retrograde regulation in response to mitochondrial dysfunction. Studies of mitochondrial and chloroplast retrograde regulation show that in response to a dysfunction of these organelles, there is a reprogramming of gene expression involved in the adaptive plasticity of organisms in response to the environment.A target gene of mitochondrial retrograde regulation was identified in P. anserina: the aox gene. The alternative oxidase belongs to the respiratory alternative pathway induced when the mitochondrial electron transport chain is impaired. Two transcription factors involved in mitochondrial retrograde regulation were identified by a genetic approach. These proteins, RSE2 and RSE3 belong to the family of Zn2Cys6 proteins and are major regulators of aox. Besides the aox gene, these two transcription factors are responsible for the coregulation of two gluconeogenic enzymes: FBP (fructose-1,6-bisphosphatase) and PCK (phosphoenolpyruvate carboxykinase). By a genetic approach, several gain-of-function mutations were isolated in RSE2 and RSE3 and our screen was likely to be saturated. Analysis of these mutations, together with other mutagenesis approaches allowed us to propose the existence of regulatory regions in RSE2 and RSE3 sequences.Microarray transcriptional profiling was conducted on rse2 and rse3 gain of function mutants. This identified new targets of RSE2 and RSE3 activated during mitochondrial dysfunction: FHB (a gene encoding a flavohemoglobin) and Pa_6_4030 (a gene encoding an α / β hydrolase). A metabolomic approach was also used and it gave new insight to the metabolic reprogramming in response to a malfunction of the respiratory chain and the role of RSE2/RSE3 in this reprogramming.Thus we showed that RSE2 and RSE3 play a role in mitochondrial retrograde regulation that is responsible for the activation of the respiratory alternative pathway, and also for the activation of metabolic reprogramming of P. anserina that would maintain cellular homeostasis under conditions of mitochondrial dysfunction.

The incidences of obesity and type 2 diabetes are reaching epidemic proportions worldwide. A cardinal feature of these conditions is resistance to the effects of the hormone insulin and a resulting hepatic overproduction of glucose. Insulin resistance is also implicated in a range of liver diseases including non-alcoholic fatty liver disease (NAFLD) and hepatitis C infection. Insulin is released after a meal and acts on liver, skeletal muscle and adipose tissue to reduce blood glucose concentration. In the liver, insulin inhibits the production and release of glucose into the circulation and stimulates its storage as glycogen. Glucagon, on the other hand, is present in the fasting state and causes breakdown of hepatic glycogen along with production of new glucose. This glucose is released from hepatocytes into the circulation. For the studies in this thesis, functional assays to measure various aspects of hepatic glucose metabolism in vitro were developed. This included measuring glucose output into culture medium, hepatocyte uptake of radiolabelled glucose and incorporation into glycogen, and total cellular glycogen content. These assays were used to investigate glucose metabolism in primary rat hepatocytes and FaO rat hepatoma cells. Both cell types responded to physiological concentrations of insulin, showing decreased glucose output and increased glycogen synthesis. Glucagon increased glucose output and reduced glycogen synthesis in primary cells but had no effect on FaO cells. Factors that have been identified that may inhibit or potentiate insulin action were investigated. Increased body iron stores have been linked with insulin resistance. De-ironing patients improves insulin sensitivity, suggesting a causal relationship between iron and insulin resistance. Hepatocytes store the majority of the body’s excess iron. This project investigated the effects of increasing hepatocyte iron stores, through addition of ferric ammonium citrate (FAC), or depleting iron stores by chelation with dipyridyl. Small increases or decreases of iron in primary cells had negative effects on cell viability, resulting in significantly reduced glucose output and glycogen synthesis. Dipyridyl treatment had similar effects on FaO cells as on primary cells but FAC treatment increased FaO glucose output, although significant iron loading was not achieved. With concentrations of FAC and dipyridyl low enough to not significantly influence cell viability, insulin sensitivity was not affected. Adiponectin is an insulin sensitiser and appears to exert this effect primarily through the liver. Adiponectin can also reduce hepatic glucose output (HGO) independent of insulin. It is believed adiponectin mediates its effects in liver, skeletal muscle and adipose tissue through activation of AMP-activated protein kinase (AMPK). In muscle, p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a downstream component of adiponectin signalling. In this study, recombinant human adiponectin was produced and collected in culture medium which was then concentrated. Despite the presence of both high molecular weight (HMW) and low molecular weight (LMW) adiponectin multimers, the concentrated medium had no effect on HGO in the presence or absence of insulin. Concentrated adiponectin medium did not affect AMPK or p38 MAPK phosphorylation in hepatocytes or other cell types previously shown to respond to adiponectin. However, commercially-sourced purified recombinant adiponectin also failed to elicit any observable responses. AICAR and metformin are pharmacological activators of AMPK and were used to treat primary rat hepatocytes and FaO cells. These treatments reduced HGO independent of insulin in both cell types. In primary cells, these reductions were partially inhibited with Compound C, an AMPK inhibitor, suggesting that both AICAR and metformin action is at least partly AMPK dependent. In FaO cells, Compound C only inhibited the AICAR-mediated reduction of glucose output, indicating that metformin may act independently of AMPK in these cells. Compound C significantly inhibited AICAR and metformin-mediated increases in AMPK phosphorylation in primary hepatocytes and FaO cells. There was a trend towards inhibition of AICAR-mediated p38 MAPK phosphorylation with Compound C treatment, suggesting that p38 MAPK may lie downstream of AMPK in hepatocytes. Adenoviral expression of constitutively active (CA) and dominant negative (DN) AMPK in primary rat hepatocytes was used to further study the role of AMPK in hepatic glucose metabolism. Despite significant expression of CA AMPK, phosphorylation of downstream acetyl-CoA carboxylase (ACC) was not affected nor was HGO. CA AMPK did, however, increase phosphorylation of p38 MAPK. DN AMPK completely inhibited AICAR-mediated AMPK phosphorylation and partially inhibited phosphorylation of ACC. In addition, AICAR-mediated phosphorylation of p38 MAPK was inhibited by DN AMPK. Taken together, these results suggest that p38 MAPK is downstream of AMPK in hepatocytes. The implication that p38 MAPK is involved in hepatic AMPK signalling is a novel finding. A greater understanding of this pathway in the liver may identify novel therapeutic targets, leading to improved treatment strategies for metabolic disorders linked to obesity and type 2 diabetes.

Made available in DSpace on 2015-07-13T12:10:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-11-07. Added 1 bitstream(s) on 2015-07-13T12:25:27Z : No. of bitstreams: 1 000836373.pdf: 1063290 bytes, checksum: 78b75c8d70712a2dbd5548af14f2bdef (MD5) / O fipronil é um inseticida utilizado para o controle de infestações parasitárias em animais e plantas. O seu mecanismo de ação ocorre por meio da inibição dos receptores do ácido γ-aminobutírico e dos canais de Cl-. Em mamíferos, além do sistema nervoso central, outros órgãos podem ser afetados pelo tratamento com fipronil, dentre eles o fígado. O presente estudo objetivou caracterizar os efeitos do fipronil sobre o metabolismo hepático, por meio da técnica de perfusão isolada de fígado de rato. Foram avaliados parâmetros associados ao metabolismo de carboidratos no período pós-prandial e no jejum e também o metabolismo de aminoácidos no jejum. O fipronil foi infundido no fígado na faixa de concentrações de 10 a 50 μM. O consumo de oxigênio foi monitorado por polarografia e nas amostras do perfusado efluente foram quantificadas as produções de glicose, lactato, piruvato, ureia e amônia. Em animais alimentados, o fipronil inibiu o consumo de oxigênio. Houve aumento da liberação de glicose e lactato provenientes do catabolismo do glicogênio, indicando estímulo da glicogenólise e glicólise. No fígado de animais em jejum, o fipronil inibiu a produção de glicose e de ureia a partir da L-alanina, enquanto a produção de amônia e de lactato foi aumentada. Esses resultados revelam que o fipronil inibe os processos biossintéticos e estimula os processos compensatórios de geração de ATP (glicogenólise e glicólise). A inibição do consumo de oxigênio corrobora que uma ação inibitória sobre a atividade respiratória mitocondrial e, portanto, uma interferência sobre o metabolismo energético seja o mecanismo de ação citotóxica do fipronil / Fipronil is an insecticide used to control parasitic infestations in animals and plants. Its mechanism of action is through inhibition of receptor γ-aminobutyric acid and the Cl- channel. In mammals, besides the central nervous system, other organs may be affected by treatment with fipronil, including the liver. The present study aimed to characterize the effects of fipronil on hepatic metabolism, through the technique of isolated perfused rat liver. Parameters related to carbohydrate metabolism in the postprandial and fasting period and also the aminoacids metabolism during fasting were evaluated. Fipronil was infused in the perfused livers in the concentration range of 10-50 μM. Oxygen consumption was monitored by polarography and in samples of effluent perfusate the production of glucose, lactate, pyruvate, urea and ammonia was quantified. In fed animals, fipronil inhibited the oxygen consumption significantly. There was increased release of glucose and lactate from the catabolism of glycogen, indicating stimulation of glycogenolysis and glycolysis. In the liver of fasted animals, fipronil inhibited the production of glucose and urea from exogenous L-alanine, whereas the production of ammonia and lactate was increased. These results indicate that the fipronil inhibits biosynthetic processes (gluconeogenesis and ureagenesis) and stimulates compensatory processes of ATP formation (glycolysis and glycogenolysis). Inhibition of oxygen consumption corroborates the suggestion that an inhibitory effect on mitochondrial respiratory activity and therefore an interference with energy metabolism is the possible mechanism of cytotoxic action of fipronil

Orientador: Fábio Ermínio Mingatto / Banca:Karina Sayuri Utsunomiya / Banca: Gisele Fabrino Machado / Resumo: O fipronil é um inseticida utilizado para o controle de infestações parasitárias em animais e plantas. O seu mecanismo de ação ocorre por meio da inibição dos receptores do ácido γ-aminobutírico e dos canais de Cl-. Em mamíferos, além do sistema nervoso central, outros órgãos podem ser afetados pelo tratamento com fipronil, dentre eles o fígado. O presente estudo objetivou caracterizar os efeitos do fipronil sobre o metabolismo hepático, por meio da técnica de perfusão isolada de fígado de rato. Foram avaliados parâmetros associados ao metabolismo de carboidratos no período pós-prandial e no jejum e também o metabolismo de aminoácidos no jejum. O fipronil foi infundido no fígado na faixa de concentrações de 10 a 50 μM. O consumo de oxigênio foi monitorado por polarografia e nas amostras do perfusado efluente foram quantificadas as produções de glicose, lactato, piruvato, ureia e amônia. Em animais alimentados, o fipronil inibiu o consumo de oxigênio. Houve aumento da liberação de glicose e lactato provenientes do catabolismo do glicogênio, indicando estímulo da glicogenólise e glicólise. No fígado de animais em jejum, o fipronil inibiu a produção de glicose e de ureia a partir da L-alanina, enquanto a produção de amônia e de lactato foi aumentada. Esses resultados revelam que o fipronil inibe os processos biossintéticos e estimula os processos compensatórios de geração de ATP (glicogenólise e glicólise). A inibição do consumo de oxigênio corrobora que uma ação inibitória sobre a atividade respiratória mitocondrial e, portanto, uma interferência sobre o metabolismo energético seja o mecanismo de ação citotóxica do fipronil / Abstract: Fipronil is an insecticide used to control parasitic infestations in animals and plants. Its mechanism of action is through inhibition of receptor γ-aminobutyric acid and the Cl- channel. In mammals, besides the central nervous system, other organs may be affected by treatment with fipronil, including the liver. The present study aimed to characterize the effects of fipronil on hepatic metabolism, through the technique of isolated perfused rat liver. Parameters related to carbohydrate metabolism in the postprandial and fasting period and also the aminoacids metabolism during fasting were evaluated. Fipronil was infused in the perfused livers in the concentration range of 10-50 μM. Oxygen consumption was monitored by polarography and in samples of effluent perfusate the production of glucose, lactate, pyruvate, urea and ammonia was quantified. In fed animals, fipronil inhibited the oxygen consumption significantly. There was increased release of glucose and lactate from the catabolism of glycogen, indicating stimulation of glycogenolysis and glycolysis. In the liver of fasted animals, fipronil inhibited the production of glucose and urea from exogenous L-alanine, whereas the production of ammonia and lactate was increased. These results indicate that the fipronil inhibits biosynthetic processes (gluconeogenesis and ureagenesis) and stimulates compensatory processes of ATP formation (glycolysis and glycogenolysis). Inhibition of oxygen consumption corroborates the suggestion that an inhibitory effect on mitochondrial respiratory activity and therefore an interference with energy metabolism is the possible mechanism of cytotoxic action of fipronil / Mestre

Este estudo teve como objetivo investigar incialmente o efeito do pHf na qualidade da carne, nas concentrações de HSP27 e HSP70, bem como no perfil proteômico da carne (Longissimus thoracis) de bovinos cruzados - 1/2 (Simental Sul Africano x Nelore) de forma a contribuir para a predição de biomarcadores que poderão ser agregados em programas de melhoramento genético. Foram avaliados 415 bovinos (180 fêmeas e 235 machos imunocastrados) terminados em confinamento. As carcaças foram classificadas de acordo com pHf, mensurados 48 horas após o abate, em dois grupos: normal (pHf ≤ 5,80) e intermediário (pHf = 5,81 - 6,19). Os parâmetros físico-químicos pH, cor (L*, a* e b*), perda de água por cocção (PAC, %) e força de cisalhamento (FC, kg), foram mensurados para avaliar a qualidade da carne em dois tempos de maturação (1 e 14 dias). Para a quantificação de HSP27 e HSP70 foram selecionadas 38 amostras em função dos grupos de pHf, dentro de cada tempo de maturação, totalizando 76 amostras. Para a realização das análises proteômicas foram selecionadas três amostras de cada grupo de pHf nos dois tempos de maturação, totalizando doze amostras. Observou-se interação (p<0,05) entre os grupos de pHf e os tempos de maturação para a cor e FC. A concentração de HSP27 foi maior (p<0,05) em pHf intermediário e com 1 de maturação, para a HSP70 só foi possível observar maior concentração (p<0,05) aos 14 dias de maturação, as correlações indicam que as HSPs modulam a qualidade da carne. Foram identificadas 13 proteínas diferencialmente expressas (p<0,05) envolvidas com funções metabólicas (triosefosfato isomerase - TPI1; fosfoglicerato mutase 2 - PGAM2; malato desidrogenase - MDH1; piruvato quinase - PKM2; e adenalato quinase isoenzima 1 - AK1), estrutural (troponina I tipo 2 - TTNI2; tropomiosina cadeia beta - TPM2; troponina C tipo 2 - TNNC2; e cofilina 2 - CFL2) e defesa em resposta ao estresse (proteína deglicase DJ-1 - PARK7; αβ-cristalina - CRYAB; proteína do choque térmico 27 - HSPB1; e peroxirredoxina 1 - PRDX1). As correlações demonstraram que essas proteínas estão envolvidas na determinação do pHf e dos demais atributos da qualidade da carne. A bioinformática permitiu observar que essas proteínas estão envolvidas nas três categorias de ontologia gênica, sendo 18 termos de componentes celulares, 1 termo de função molecular e 61 termos dos processos biológicos, foram identificadas também 6 vias metabólicas. A proteína MDH1, esteve associada a mecanismos de produção de energia, confirmando o potencial desta como um biomarcador para a determinação do pHf assim como para a cor da carne, enquanto que a TPI1 foi relacionada somente no estabelecimento do pHf. Outras proteínas como a PKM2, com função metabólica, e a PARK7, com função protetora, estiveram associadas aos atributos maciez e cor da carne, respectivamente. Os resultados obtidos são de grande relevância no âmbito das ciências da carne e do melhoramento genético animal, por permitir a identificação de possíveis biomarcadores que poderão ser utilizados na seleção animal. / The objective of this study was to investigate the effect of pHf on meat quality, on the concentrations of HSP27 and HSP70, as well as on the proteomic profile in beef (Longissimus thoracis) of crossbreed cattle - 1/2 (South African Simmental x Nellore) - in order to explore the contribute to the prediction of biomarkers that can be added to the process of animal selection in breeding programs. Were evaluated 415 cattle (180 females and 235 immunocastrated males) feedlot-finished. Carcasses were classified according to pHu, measured 48 hours after slaughter, in two groups: normal (pHu ≤ 5.80) and intermediate (pHu = 5.81 - 6.19). The physical-chemical parameters pH, color values (L *, a * and b *), cooking loss (CL,%) and shear force (SF, kg) were measured to evaluate the meat quality in two aging times (1 and 14 days). For quantification of HSP27 and HSP70, 38 samples were selected according to the pHu groups, and aging time, totaling 76 samples. To perform the proteomic analysis were selected three samples from each group and aging time, totaling twelve samples. Color and SF meat quality attributes showed interaction (p<0.05) between pHu groups and aging times. The concentration of HSP27 was higher (p<0.05) at intermediate pHu and at 1 day of aging. It was only possible to observe a higher concentration (p<0.05) for HSP70 at 14 days of aging. Correlations analysis indicate that HSPs modulate the quality of meat. Thirteen differentially expressed proteins (p<0.05) were identified as involved with metabolic (triosephosphate isomerase - TPI, phosphoglycerate mutase 2 - PGAM2; malate dehydrogenase, cytoplasmic - MDH1; pyruvate kinase - PKM2; and adenylate kinase isoenzyme 1- AK1), structural (troponin I type 2 - TTNI2; tropomyosin beta chain - TPM2; troponin C type 2 - TNNC2; and cofilin 2 - CFL2) and defense in response to stress functions (protein deglycase DJ-1- PARK7; αβ-cristallin - CRYAB; heat shock protein 27 - HSPB1; and peroxiredoxin 1 - PRDX1). Correlations analysis showed that these proteins are involved in the determination of pHu and other attributes of meat quality. Using bioinformatics it was possible to observe that these proteins are involved in three gene ontology categories, which were 18 cellular components terms, 1 molecular function term and 61 biological processes terms. Other six metabolic pathways have also been identified. The MDH1 protein was associated with energy production mechanisms, confirming its potential as a biomarker for the determination of pHu as well as meat color, while TPI was related to the establishment of pHu only. Other proteins such as PKM2, with metabolic function, and PARK7, with protective function, were associated with the attributes of meat tenderness and color, respectively. The obtained results are of great relevance in the field of meat science and animal breeding genetic, it allows the identification of biomarkers that can be used in animal selection.