Le tissu musculaire squelettique contribue considérablement au maintien de l’homéostasie du glucose chez l’humain. Que ce soit en situation postprandiale ou lors d’un travail musculaire, le muscle squelettique capte de grandes quantités de glucose sanguin à des fins d’entreposage ou de production d’énergie. Les voies de signalisation cellulaire impliquées dans la régulation du transport du glucose à l’intérieur de la cellule musculaire sont nombreuses et complexes. En présence de désordres physiologiques et/ou métaboliques, de nombreux médiateurs chimiques et enzymatiques peuvent interagir avec les différentes protéines de ces voies de signalisation et entraîner des perturbations importantes au niveau de l’homéostasie du glucose. Notre première étude nous a permis de confirmer l’implication de la protéine AMPK dans le transport du glucose induit par la contraction musculaire. L’AMPK est un senseur énergétique important activé dans le muscle squelettique au cours d’un effort physique. Toutefois, son rôle dans le transport du glucose induit par la contraction musculaire demeure controversé. Grâce à un modèle murin d’invalidation génétique de l’AMPK spécifique au tissu musculaire et à l’élaboration d’un protocole de contraction ex vivo approprié, nous avons établi l’importance de l’AMPK dans la régulation du transport du glucose. Notre seconde étude nous a permis de démontrer que l’incubation ex vivo prolongée du muscle épitrochléen modifie l’expression du transporteur de glucose GLUT1. Nous avons également observé l’induction de la protéine iNOS et la production du NO. Parallèlement, nous avons mesuré une augmentation de l’expression de GLUT1 à la suite d’une exposition au NO dans un modèle de cellules musculaires ainsi qu’une augmentation du transport basal du glucose. L’ensemble de nos résultats nous permet de consolider le lien causal entre la production du NO et la modulation de l’expression de GLUT1 et potentiellement, le développement de perturbations au niveau du métabolisme du glucose musculaire.

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Parmi la population nord-américaine, les cas de diabète de type 2 et d’obésité ont atteint des niveaux alarmants. Leurs conséquences sur la santé publique, la vie économique et l’avenir des populations sont majeures. Mon projet de maîtrise avait pour but de caractériser les dérèglements métaboliques et vasculaires suscités chez le rat soumis à une alimentation riche en gras saturés et en sucre raffinés, et à examiner les effets d’un antioxydant (tempol). Nos résultats indiquent qu’un traitement avec tempol améliore la sensibilité à l’insuline mesurée lors de clamp euglycémique hyperinsulinémique, réduit le gain de poids, augmente l’expression endothéliale de eNOS (estimée par microscopie confocale) et diminue la quantité de protéines nitrotyrosinées dans l’aorte. Il améliore aussi le transport du glucose dans le muscle squelettique et la réactivité vasculaire in vitro. Ces résultats suggèrent non seulement une association étroite entre la diète, les maladies cardiovasculaires, le stress oxydatif et eNOS, mais démontrent aussi l’efficacité du tempol à améliorer la sensibilité à l’insuline et la fonction endothéliale dans ce modèle animal. / In the Western hemisphere, the incidence of type 2 diabetes and obesity have been growing at an alarming rate. Their consequences on public health, economic situation and population’s future are major. My research project at the Master degree was designed to characterize metabolic and vascular dysfunctions elicited in a rat model fed a high fat and high sucrose diet, and to examine in this animal model the effect of a chronic treatment with the antioxidant, tempol. Our results indicate that treatment with tempol significantly improves insulin sensitivity measured during a euglycemic hyperinsulinemic clamp, reduces weight gain, increases endothelium eNOS protein expression (confocal microscopy) and reduces nitrotyrosine formation in aortas. An improvement in insulin-mediated glucose transport activity in skeletal muscles and in vascular reactivity were also noted. These results not only suggest the presence of a close link between diet, cardiovascular diseases, oxidative stress and eNOS, but also indicate the ability of treatment with tempol to significantly improve insulin sensitivity and endothelial functions in this rat model.

La voie de signalisation mTORCl/S6Kl est impliquée dans le développement de la résistance à l'insuline associée à l'obésité en exerçant une boucle de rétro-contrôle négative sur la voie de signalisation PI3K-Akt. Tandis que l'utilisation à court terme de la rapamycine, l'inhibiteur pharmacologique de la voie mTORCl, permet d'inhiber le rétro-contrôle négatif, les effets de l'inhibition chronique de la voie sur le métabolisme ne sont pas connus. L'objectif principal des études présentées dans cette thèse était d'investiguer les effets métaboliques de l'inhibition chronique de la voie de signalisation mTORCl/S6Kl in vitro et in vivo. Dans la première étude, nous avons montré que l'inhibition chronique de mTORCl/S6Kl découple l'activation d'Akt de la PI3K ce qui cause une résistance à l'insuline dans les cellules 3T3-L1. Dans une deuxième étude, nous avons découvert que l'utilisation chronique de la rapamycine in vivo cause une intolérance au glucose et une résistance à l'insuline de par une augmentation de la gluconéogénèse hépatique en plus d'affecter négativement le métabolisme des lipides. Dans une troisième étude, nous avons démontré que l'inhibition chronique de mTORCl/S6K1 dans les cellules hépatique FAO découple l'activation d'Akt de la PI3K ce qui augmente la production de glucose hépatique. Finalement, dans une quatrième étude, nous avons déterminé que l'inhibition chronique de mTORCl/S6K1 dans les cellules L6 ne permet pas de restaurer le transport du glucose stimulé par l'insuline en présence d'un excès de nutriments. L'ensemble de nos études démontre le rôle important joué par la voie de signalisation mTORCl/S6K1 sur le contrôle du métabolisme du glucose et des lipides et limite l'inhibition chronique de mTOR comme cible thérapeutique pour le traitement du diabète de type 2 et de l'obésité.

Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D). To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.

O fungo filamentoso Trichoderma reesei é caracteristicamente reconhecido pela produção de celulases e hemicelulases, que lhe permitem utilizar uma ampla variedade de polissacarídeos como fonte de carbono. Neste trabalho, descrevemos a caracterização de dois genes de T. reesei, Trhxt1 e Trhxt2, que codificam proteínas com alta similaridade a transportadores de glicose de vários microorganismos. Os dois genes foram identificados em um banco de dados de ESTs de T. reesei. A análise computacional de Trhxt1 e Trhxt2 indica que ambos fazem parte da major facilitator superfamily (MFS), apresentando, tipicamente, 12 segmentos transmembrânicos. A expressão de Trhxt1 ocorre apenas em baixos níveis de glicose(≈ 100 µmol 1-1), enquanto a de Trhxt2 parece ocorrer de forma constitutiva, independentemente da fonte de carbono. Em baixas concentrações de oxigênio, a expressão de Trhxt1 é induzida e a de Trhxt2, reprimida. O sistema de transporte em T. reesei apresenta um componente de afinidade muito alta por glicose (Km ≈ 20 µmol 1-1) semelhante ao de outros fungos filamentosos. Dados sobre o transporte de glicose em uma cepa mutante ΔTrhxt1 indicam que o gene Trhxt1 está envolvido com o transporte em baixos níveis de glicose (≤ 100 µmol 1-1) que correspondem, provavelmente, aos valores encontrados no solo, o habitat natural de T. reesei.. Interessantemente, a indução do sistema de celulases de T. reesei por celulose está retardada no mutante ΔTrhxt1, o que sugere a importância do transporte de glicose na expressão dos genes das celulases. Finalmente, além de descrever os primeiros genes de transportadores de glicose em T. reesei, esperamos que este trabalho possa contribuir para o preenchimento de uma lacuna em relação ao transporte de açúcares em fungos filamentosos em geral. / The filamentous fungus Trichoderma reesei is a natural soil inhabitant capable of metabolizing a vast number of polysaccharide substrates. In this work, we describe two genes of T. reesei, named Trhxt1 and Trhxt2, which code for proteins with significant similarities to glucose transporters from other fungi. These genes were identified in an EST database of T. reesei. Sequence analysis of TrHXT1 and TrHXT2 revealed 12 putative transmembrane domains and several other characteristic motifs found in members of the major facilitator superfamily (MFS). Trhxt1 is transcriptionally induced only by low levels of glucose(≈ 100 µmol 1-1), while Trhxt2 expressionis independent of both glucose concentration and carbon source. We also show that Trhxt1 expression is enhanced when cells are exposured to low oxygen levels; in contrast, Trhxt2 expression seems to be repressed at these conditions. Glucose transport in T. reesei is apparently mediated by a multicomponent uptake system, in which the high-affiníty component has a Km of approximately 20 µmol 1-1. This low Km value is similar to the values reported for glucose uptake by other filamentous fungi. Kinetics of glucose transport in a T. reesei ΔTrhxt1 strain suggests that Trhxt1 is involved in glucose uptake in conditions of low glucose (≤ 100 µmol 1-1), which are most probably found in the soil, a low-nutrient environment. Interestingly, índuction ofthe T. reesei cellulase system by cellulose ís significantly delayed in the ΔTrhxt1 mutant, suggesting that glucose transport may be important to the mechanisms of expression of the cellulase genes. Finally, we hope that this work may be helpful to provide a better understanding of sugar uptake in filamentous fungi, for which there is little information available.

O fungo filamentoso Trichoderma reesei é caracteristicamente reconhecido pela produção de celulases e hemicelulases, que lhe permitem utilizar uma ampla variedade de polissacarídeos como fonte de carbono. Neste trabalho, descrevemos a caracterização de dois genes de T. reesei, Trhxt1 e Trhxt2, que codificam proteínas com alta similaridade a transportadores de glicose de vários microorganismos. Os dois genes foram identificados em um banco de dados de ESTs de T. reesei. A análise computacional de Trhxt1 e Trhxt2 indica que ambos fazem parte da major facilitator superfamily (MFS), apresentando, tipicamente, 12 segmentos transmembrânicos. A expressão de Trhxt1 ocorre apenas em baixos níveis de glicose(≈ 100 µmol 1-1), enquanto a de Trhxt2 parece ocorrer de forma constitutiva, independentemente da fonte de carbono. Em baixas concentrações de oxigênio, a expressão de Trhxt1 é induzida e a de Trhxt2, reprimida. O sistema de transporte em T. reesei apresenta um componente de afinidade muito alta por glicose (Km ≈ 20 µmol 1-1) semelhante ao de outros fungos filamentosos. Dados sobre o transporte de glicose em uma cepa mutante ΔTrhxt1 indicam que o gene Trhxt1 está envolvido com o transporte em baixos níveis de glicose (≤ 100 µmol 1-1) que correspondem, provavelmente, aos valores encontrados no solo, o habitat natural de T. reesei.. Interessantemente, a indução do sistema de celulases de T. reesei por celulose está retardada no mutante ΔTrhxt1, o que sugere a importância do transporte de glicose na expressão dos genes das celulases. Finalmente, além de descrever os primeiros genes de transportadores de glicose em T. reesei, esperamos que este trabalho possa contribuir para o preenchimento de uma lacuna em relação ao transporte de açúcares em fungos filamentosos em geral. / The filamentous fungus Trichoderma reesei is a natural soil inhabitant capable of metabolizing a vast number of polysaccharide substrates. In this work, we describe two genes of T. reesei, named Trhxt1 and Trhxt2, which code for proteins with significant similarities to glucose transporters from other fungi. These genes were identified in an EST database of T. reesei. Sequence analysis of TrHXT1 and TrHXT2 revealed 12 putative transmembrane domains and several other characteristic motifs found in members of the major facilitator superfamily (MFS). Trhxt1 is transcriptionally induced only by low levels of glucose(≈ 100 µmol 1-1), while Trhxt2 expressionis independent of both glucose concentration and carbon source. We also show that Trhxt1 expression is enhanced when cells are exposured to low oxygen levels; in contrast, Trhxt2 expression seems to be repressed at these conditions. Glucose transport in T. reesei is apparently mediated by a multicomponent uptake system, in which the high-affiníty component has a Km of approximately 20 µmol 1-1. This low Km value is similar to the values reported for glucose uptake by other filamentous fungi. Kinetics of glucose transport in a T. reesei ΔTrhxt1 strain suggests that Trhxt1 is involved in glucose uptake in conditions of low glucose (≤ 100 µmol 1-1), which are most probably found in the soil, a low-nutrient environment. Interestingly, índuction ofthe T. reesei cellulase system by cellulose ís significantly delayed in the ΔTrhxt1 mutant, suggesting that glucose transport may be important to the mechanisms of expression of the cellulase genes. Finally, we hope that this work may be helpful to provide a better understanding of sugar uptake in filamentous fungi, for which there is little information available.

Hydrodynamic analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. Some detergents (e.g. octylglucoside) maintain the multimeric complex while other detergents (e.g. CHAPS and dodecylmaltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer. Quantitatively deglycosylated GLUT1 displays aberrant electrophoretic mobility, but each protein band contains full-length GLUT1 and the less mobile species, when treated with additional detergent and reductant, converts to the more mobile species. Preliminary structural analysis suggests that denaturing detergent- and thiol chemistry-related changes of α-helical content may mirror mobility shifts. Limited proteolysis of membrane-resident GLUT1 (± ligands) releases membrane-spanning α-helical domains suggesting that (i) some bilayer-resident helices are highly solvent exposed; (ii) membrane-spanning domains 1, 2, & 4 and 7, 8, & 10 are destabilized upon ligand binding; and (iii) helix packing compares well with high-resolution structures of prokaryotic transporters from the same superfamily. Results are consistent with a central, hydrophilic, translocation pathway comprised of amphipathic, membrane-spanning domains that alter associations upon ligand/substrate binding. We have resolved technical difficulties (heterogeneity, lipid/detergent removal, glycosylation, small molecule contamination) associated with GLUT1 analysis by mass spectrometry; and we map global conformational changes between sugar uptake and sugar efflux.