Indiana University-Purdue University Indianapolis (IUPUI) / Study has shown that plasma membrane (PM) cholesterol and cortical filamentous actin (F-actin) influence skeletal muscle glucose transport. Of fundamental and clinical interest is whether diabetogenic insults promote membrane/cytoskeletal dysfunction amendable for therapy. As exposure to excess fatty acid (FA)s induce glucose intolerance by mechanisms imperfectly understood, we tested if PM cholesterol/F-actin changes could contribute to FA-induced glucose transporter GLUT4 dysregulation in skeletal muscle. High-fat fed, insulin-resistant animals displayed elevated levels of skeletal muscle PM cholesterol and a loss in cortical F-actin, compared to normal-chow fed animals. Consistent with a PM cholesterol component of glucose intolerance, human skeletal muscle biopsies revealed an inverse correlation between PM cholesterol and whole-body glucose disposal. Mechanistically, exposure of L6 myotubes to the saturated FA palmitate induced an increase in PM cholesterol that destabilized actin filaments and decreased insulin-stimulated PM GLUT4 and glucose transport, which could be reversed with cholesterol lowering. Next, study tested if the lipid-lowering action of the antidiabetic AMP-activated protein kinase (AMPK) had a beneficial influence on PM cholesterol balance. Consistent with AMPK inhibition of 3-hydroxy-3-methylglutaryl CoA reductase, a rate-limiting enzyme of cholesterol synthesis, we found that AMPK activation promoted a significant reduction in PM cholesterol and amplified basal and insulin-stimulated GLUT4 translocation. A similar loss of PM cholesterol induced by β-cyclodextrin caused an analogous enhancement of GLUT4 regulation. Interestingly, PM cholesterol replenishment abrogated the AMPK effect on insulin, but not basal, regulation of GLUT4 translocation. Conversely, AMPK knockdown prevented the enhancement of both basal and insulin-stimulated GLUT4 translocation. As a whole these studies show PM cholesterol accrual and cortical F-actin loss uniformly in skeletal muscle from glucose-intolerant mice, swine, and humans. In vivo and in vitro dissection demonstrated this membrane/cytoskeletal derangement induces insulin resistance and is promoted by excess FAs. Parallel studies unveiled that the action of AMPK entailed lowering PM cholesterol that enhanced the regulation of GLUT4/glucose transport by insulin. In conclusion, these data are consistent with PM cholesterol regulation being an unappreciated aspect of AMPK signaling that benefits insulin-stimulated GLUT4 translocation during states of nutrient excess promoting PM cholesterol accrual.

O indivíduo com diabetes mellitus tipo 2 apresenta um risco de 2 a 4 vezes maior de desenvolver doença cardiovascular (DCV) quando comparado ao não-diabético, sendo que este aumento do risco para o desenvolvimento da DCV também é observado quando na intolerância à glicose (IG) que ocorre em fases mais precoces da história natural do diabetes. Atribui-se ser a presença da síndrome metabólica (SM), que ocorre na maioria dos pacientes com DM2 e IG, um fator importante para o desenvolvimento da DCV nestes indivíduos. Dos componentes da SM, inúmeros estudos destacam a dislipidemia como um dos principais fatores para este risco. A dislipidemia comumente encontrada na IG é caracterizada por hipertrigliceridemia, baixo HDL-C e presença de LDL pequena e densa. Entretanto, como a elevação dos níveis séricos do LDL-C associada ao surgimento de aterosclerose prematura em indivíduos não diabéticos na maioria das vezes não é observada em pacientes com IG, questiona-se se outras alterações do metabolismo lipídico, tais como alterações da cinética do colesterol ou a transferência de lípides das lipoproteínas para a HDL, poderiam estar relacionadas ao maior risco cardiovascular nestes pacientes. Estudo prévio, utilizando uma nanoemulsão lipídica artificial de LDL, verificou uma remoção mais rápida do colesterol na forma livre em pacientes normolipidêmicos com doença arterial coronária (DAC) quando comparada com controles. No presente estudo, utilizou-se a nanoemulsão lipídica artificial para avaliar se esses dois processos envolvidos no metabolismo da LDL e da HDL estão alterados em pacientes com intolerância à glicose que os predispõem à DAC, relacionando estes resultados com fatores de risco cardiovasculares, tais como a resistência à insulina, a obesidade e a dislipidemia. Para tanto, foram estudados 14 pacientes com IG e 15 controles, sem manifestação clínica de DCV, que não utilizam antidiabéticos orais e hipolipemiantes, comparados com controles pareados para idade, sexo, raça, IMC, tabagismo, consumo de álcool, prática de atividade física e doenças associadas. Para o estudo cinético, a nanoemulsão marcada foi injetada endovenosamente e amostras de sangue coletadas ao longo de 24h para a determinação da radioatividade, das curvas de decaimento plasmático e da taxa fracional de remoção (TFR) dos lípides marcados a partir de um modelo de análise compartimental. Foi medida a taxa de esterificação do 3Hcolesterol livre da nanoemulsão no plasma e avaliada a transferência in vitro de lípides da nanoemulsão para a fração HDL. A resistência à insulina foi estimada pelo modelo matemático de homeostase glicêmica (HOMA) e a adiposidade abdominal por tomografia computadorizada de abdômen. A concentração plasmática de colesterol total, LDL-C, HDL-C, triglicérides e de apolipoproteínas não diferiu entre os grupos. O perfil antropométrico relacionado ao peso, IMC e circunferência abdominal foi semelhante entre os grupos. O grupo IG apresentou maior concentração de insulina de jejum (p=0,01), menor sensibilidade à insulina (p<0,01) e maior índice de resistência à insulina (p<0,01). A TFR 14C-EC foi similar nos dois grupos, porém a TFR 3H-CL foi mais rápida no grupo IG comparado com controle (p=0,04). A porcentagem de esterificação do 3H-colesterol da nanoemulsão bem como a transferência de lípides da nanoemulsão para a fração HDL foram semelhantes entre os grupos. A remoção mais rápida do 3H-colesterol livre mostra que ocorreu uma dissociação das partículas de colesterol da nanoemulsão lipídica nos pacientes com intolerância à glicose. Essa dissociação do colesterol pode refletir alterações no metabolismo intravascular da lipoproteína LDL, as quais podem favorecer a aterogênese nesses pacientes / Individuals with diabetes mellitus type 2 are 2 to 4 times more susceptible to cardiovascular disease (CVD) than non-diabetic individuals. This increased risk is also observed for glucose intolerance (GI) which appears in the initial stages of diabetes. The presence of the metabolic syndrome (MS), present in most DM2 and GI patients, is also an important factor contributing to the development of CVD in these individuals. Various MS component studies emphasize dyslipidemia as one of the main contributors for this risk factor. The dyslipidemia commonly associated to GI is characterized by hypertriglyciridemia, low HDL-C and the presence of a small and dense LDL. However, since associated LDL-C levels with the development of premature atherosclerosis in non diabetic individuals is for the most part not observed in GI patients, it is questioned whether other lipid metabolism alterations such as cholesterol kinetics or the lipid transfer to HDL could be related to a greater CVD risk in these individuals. A previous study using an artificial LDL nanoemulsion showed a faster removal rate of the free cholesterol in normolipidemic with coronary artery disease (CAD) patients when compared to control individuals. In this study an artificial lipid nanoemulsion was used to evaluate both these processes involved in the metabolism of LDL and HDL which are both altered in patients with GI that expose them to CAD, and relating the results to CVD factors such as insulin resistance, obesity and dyslipidemia. 14 GI and 15 control individuals participated in this study. All without manifestations of CVD, none using any oral antidiabetic medication or hypolipimeants, paired for age, sex, race, BMI, smoking, alcoholic consumption, physical activity and comorbidities. For the kinetic study, a labeled nanoemulsion was interveneously injected and blood samples collected at determined intervals over a 24 hour period to determine the radiactive plasma decay curves and fractional clearance rate (FCR) of the labeled nanoemulsion lipids through a compartmental analysis model. Plasma esterification rate of the 3H-free cholesterol of the nanoemulsion was measured as was the in vitro transfer from the nanoemulsion to HDL fraction. Insulin resistance was obtained by the glycemic homeostasis mathematical model (HOMA) and abdominal adipose by a computerized tomography of the abdomen. No differences were observed for total cholesterol plasmatic concentrations, LDL-C, HDL-C, triglycerides or apolipoproteins between the two groups. The anthropometric profile related to weight, BMI and abdominal circumference was similar for both groups. The GI group presented higher fasting insulin concentration (p=0.01), less insulin sensitivity (p=0.01) and a greater insulin resistance (p=0.01). The TFR 14C-CE was similar in both groups, although the TFR 3H-CL was faster in the GI group compared to the control group (p=0.04). The esterification percentage of the nanoemulsions 3H-colesterol, as well as the lipid transfer from the nanoemulsion to HDL fraction were similar for both groups. The faster 3H-free cholesterol removal shows that a dissociation of the cholesterol particles of the lipidic nanoemulsion occurred in those patients with GI. This dissociation could possibly reflect alterations in the intravascular LDL lipoprotein metabolism which in turn, may favor atherogenesis in these patients

O câncer é considerado uma das principais causas de morte no mundo. Para o tratamento dessa doença, frequentemente são utilizadas estratégias farmacológicas baseadas na intervenção quimioterápica, no qual a doxorubicina (DOX) é largamente utilizada. Visto que, o músculo esquelético possui importante papel na captação de glicose, o objetivo do presente trabalho foi investigar o efeito da DOX na intolerância à glicose. Para isso foram utilizados ratos Wistar, os quais receberam uma dose única de DOX ou salina intraperitoneal (15mg/kg). Avaliamos a expressão de proteínas envolvidas na sensibilidade à insulina e captação de glicose. Os ensaios captação de glicose foram realizados em cultura de miócitos, no qual foi utilizado o agonista de AMPK. O tratamento com DOX causou resistência à insulina e hiperglicemia. No músculo EDL e em miócitos houve menor expressão de GLUT-4 e de AMPk. Em conclusão, o tratamento com DOX causou intolerância à glicose e redução da expressão de AMPk e GLUT-4. A utilização do agonista de AMPk foi capaz de recuperar à intolerância à glicose. / The cancer is considered a major cause of death worldwide. For the treatment of this disease, with frequency are used pharmacological strategies based in chemotherapeutic intervention, in which doxorubicin (DOX) is widely used. Since the skeletal muscle plays an important role in glucose uptake, the aim of this study was to investigate the effect of DOX in glucose intolerance. For this Wistar rats which received a single dose of DOX or saline intraperitoneally (15mg / kg). We evaluated the expression of proteins involved in insulin sensitivity and glucose uptake. The glucose uptake assays were performed on culture myocytes, which was used in the agonist of AMPK. The treatment with DOX caused insulin resistance and hyperglycemia. In the EDL muscle myocytes and there was less expression of GLUT4 and AMPK. In conclusion, treatment with DOX caused impaired glucose tolerance and reduction of expression of AMPK and GLUT-4. The use of AMPK agonist was able to recover glucose intolerance.

Physical activity guidelines encouraging activity among healthy pregnant women have been issued by the Centers for Disease Control and Prevention, yet Latina women remain more sedentary than non-Latina white women. Latina women are also at higher risk for gestational diabetes mellitus and, among Latina women, Puerto Rican women have the highest rates of low birth weight and preterm-related infant death. This dissertation utilized data from the Latina GDM study, a prospective cohort study of 1,231 Latina women recruited early in pregnancy and followed through delivery. Participants were interviewed in early and mid pregnancy for assessment of sociodemographics, acculturation, medical, and behavioral factors, in addition to administration of the Kaiser Physical Activity Survey for assessment of physical activity and sedentary behaviors. Birth outcomes were abstracted from medical records following delivery. In the first chapter, we assessed the prevalence of three health behaviors (meeting physical activity guidelines, meeting fruit/vegetable consumption guidelines, and cigarette smoking) in early and mid pregnancy and identified multiple factors associated with meeting health behavior guidelines in pregnancy. In the second chapter, we examined participation in sedentary behaviors, such as time spent TV watching, sitting at work, and low levels of sports and exercise, in pre, early and mid pregnancy in relation to maternal glucose intolerance and gestational diabetes mellitus. In the final chapter, we analyzed four types of physical activity (sports/exercise, household/caregiving, occupational, and active transportation) as well as total activity in relation to risk of preterm birth and small-for-gestational age. Findings represent the first study of physical activity and maternal/fetal outcomes conducted exclusively among Latina women, a group largely understudied in epidemiologic research. Results will guide culturally specific intervention programs in this high risk population.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

L’existence d’une relation entre les troubles cardio-métaboliques composant le syndrome métabolique et une hyperactivité sympathique est bien admise dans la littérature sans que la relation causale entre ces deux entités soit clairement définie. Nos travaux sur un modèle murin d’hyperactivité sympathique constitutive (délétion partielle et/ou complète du transporteur de recapture de la noradrénaline) ont permis de mettre en évidence le rôle de celle-ci dans le développement de troubles glucidiques : 1) Une augmentation de l’activité sympathique est un facteur suffisant pour le développement de troubles glucidiques précoces associant une intolérance au glucose à une hyperinsulinémie basale sans hyperglycémie. 2) Ces désordres seraient dus à un retard de sécrétion d’insuline en réponse au glucose, probablement consécutif à une sous-expression du transporteur GLUT2. Ces résultats montrent que l’hyperactivité sympathique chronique pourrait constituer un facteur pronostic permettant le diagnostic précoce de patients à risque de développer des troubles de l’homéostasie glucidique et ouvre des perspectives dans le traitement du diabète de type 2. / Several studies have established an association between cardiometabolic disorders composing the metabolic syndrome and sympathetic hyperactivity. The causal relationship is however not clearly defined. Our work on a murine model of constitutive sympathetic hyperactivity (partial and / or complete deletion of the norepinephrine reuptake transporter) highlights its role in the development of carbohydrate disorders: 1) An increase in the sympathetic activity is a sufficient factor for early carbohydrate disorders associating glucose intolerance with basal hyperinsulinemia without hyperglycemia. 2) These disorder are thought to be due to a delay in insulin secretion in response to glucose stimulation, probably consecutive to a decreased expression of the GLUT2 transporter. These results show that chronic sympathetic hyperactivity may constitute a prognostic factor allowing the early diagnosis of patients at risk to develop glucose homeostasis disorders and opens perspectives in the treatment of type 2 diabetes mellitus.

O indivíduo com diabetes mellitus tipo 2 apresenta um risco de 2 a 4 vezes maior de desenvolver doença cardiovascular (DCV) quando comparado ao não-diabético, sendo que este aumento do risco para o desenvolvimento da DCV também é observado quando na intolerância à glicose (IG) que ocorre em fases mais precoces da história natural do diabetes. Atribui-se ser a presença da síndrome metabólica (SM), que ocorre na maioria dos pacientes com DM2 e IG, um fator importante para o desenvolvimento da DCV nestes indivíduos. Dos componentes da SM, inúmeros estudos destacam a dislipidemia como um dos principais fatores para este risco. A dislipidemia comumente encontrada na IG é caracterizada por hipertrigliceridemia, baixo HDL-C e presença de LDL pequena e densa. Entretanto, como a elevação dos níveis séricos do LDL-C associada ao surgimento de aterosclerose prematura em indivíduos não diabéticos na maioria das vezes não é observada em pacientes com IG, questiona-se se outras alterações do metabolismo lipídico, tais como alterações da cinética do colesterol ou a transferência de lípides das lipoproteínas para a HDL, poderiam estar relacionadas ao maior risco cardiovascular nestes pacientes. Estudo prévio, utilizando uma nanoemulsão lipídica artificial de LDL, verificou uma remoção mais rápida do colesterol na forma livre em pacientes normolipidêmicos com doença arterial coronária (DAC) quando comparada com controles. No presente estudo, utilizou-se a nanoemulsão lipídica artificial para avaliar se esses dois processos envolvidos no metabolismo da LDL e da HDL estão alterados em pacientes com intolerância à glicose que os predispõem à DAC, relacionando estes resultados com fatores de risco cardiovasculares, tais como a resistência à insulina, a obesidade e a dislipidemia. Para tanto, foram estudados 14 pacientes com IG e 15 controles, sem manifestação clínica de DCV, que não utilizam antidiabéticos orais e hipolipemiantes, comparados com controles pareados para idade, sexo, raça, IMC, tabagismo, consumo de álcool, prática de atividade física e doenças associadas. Para o estudo cinético, a nanoemulsão marcada foi injetada endovenosamente e amostras de sangue coletadas ao longo de 24h para a determinação da radioatividade, das curvas de decaimento plasmático e da taxa fracional de remoção (TFR) dos lípides marcados a partir de um modelo de análise compartimental. Foi medida a taxa de esterificação do 3Hcolesterol livre da nanoemulsão no plasma e avaliada a transferência in vitro de lípides da nanoemulsão para a fração HDL. A resistência à insulina foi estimada pelo modelo matemático de homeostase glicêmica (HOMA) e a adiposidade abdominal por tomografia computadorizada de abdômen. A concentração plasmática de colesterol total, LDL-C, HDL-C, triglicérides e de apolipoproteínas não diferiu entre os grupos. O perfil antropométrico relacionado ao peso, IMC e circunferência abdominal foi semelhante entre os grupos. O grupo IG apresentou maior concentração de insulina de jejum (p=0,01), menor sensibilidade à insulina (p<0,01) e maior índice de resistência à insulina (p<0,01). A TFR 14C-EC foi similar nos dois grupos, porém a TFR 3H-CL foi mais rápida no grupo IG comparado com controle (p=0,04). A porcentagem de esterificação do 3H-colesterol da nanoemulsão bem como a transferência de lípides da nanoemulsão para a fração HDL foram semelhantes entre os grupos. A remoção mais rápida do 3H-colesterol livre mostra que ocorreu uma dissociação das partículas de colesterol da nanoemulsão lipídica nos pacientes com intolerância à glicose. Essa dissociação do colesterol pode refletir alterações no metabolismo intravascular da lipoproteína LDL, as quais podem favorecer a aterogênese nesses pacientes / Individuals with diabetes mellitus type 2 are 2 to 4 times more susceptible to cardiovascular disease (CVD) than non-diabetic individuals. This increased risk is also observed for glucose intolerance (GI) which appears in the initial stages of diabetes. The presence of the metabolic syndrome (MS), present in most DM2 and GI patients, is also an important factor contributing to the development of CVD in these individuals. Various MS component studies emphasize dyslipidemia as one of the main contributors for this risk factor. The dyslipidemia commonly associated to GI is characterized by hypertriglyciridemia, low HDL-C and the presence of a small and dense LDL. However, since associated LDL-C levels with the development of premature atherosclerosis in non diabetic individuals is for the most part not observed in GI patients, it is questioned whether other lipid metabolism alterations such as cholesterol kinetics or the lipid transfer to HDL could be related to a greater CVD risk in these individuals. A previous study using an artificial LDL nanoemulsion showed a faster removal rate of the free cholesterol in normolipidemic with coronary artery disease (CAD) patients when compared to control individuals. In this study an artificial lipid nanoemulsion was used to evaluate both these processes involved in the metabolism of LDL and HDL which are both altered in patients with GI that expose them to CAD, and relating the results to CVD factors such as insulin resistance, obesity and dyslipidemia. 14 GI and 15 control individuals participated in this study. All without manifestations of CVD, none using any oral antidiabetic medication or hypolipimeants, paired for age, sex, race, BMI, smoking, alcoholic consumption, physical activity and comorbidities. For the kinetic study, a labeled nanoemulsion was interveneously injected and blood samples collected at determined intervals over a 24 hour period to determine the radiactive plasma decay curves and fractional clearance rate (FCR) of the labeled nanoemulsion lipids through a compartmental analysis model. Plasma esterification rate of the 3H-free cholesterol of the nanoemulsion was measured as was the in vitro transfer from the nanoemulsion to HDL fraction. Insulin resistance was obtained by the glycemic homeostasis mathematical model (HOMA) and abdominal adipose by a computerized tomography of the abdomen. No differences were observed for total cholesterol plasmatic concentrations, LDL-C, HDL-C, triglycerides or apolipoproteins between the two groups. The anthropometric profile related to weight, BMI and abdominal circumference was similar for both groups. The GI group presented higher fasting insulin concentration (p=0.01), less insulin sensitivity (p=0.01) and a greater insulin resistance (p=0.01). The TFR 14C-CE was similar in both groups, although the TFR 3H-CL was faster in the GI group compared to the control group (p=0.04). The esterification percentage of the nanoemulsions 3H-colesterol, as well as the lipid transfer from the nanoemulsion to HDL fraction were similar for both groups. The faster 3H-free cholesterol removal shows that a dissociation of the cholesterol particles of the lipidic nanoemulsion occurred in those patients with GI. This dissociation could possibly reflect alterations in the intravascular LDL lipoprotein metabolism which in turn, may favor atherogenesis in these patients

O câncer é considerado uma das principais causas de morte no mundo. Para o tratamento dessa doença, frequentemente são utilizadas estratégias farmacológicas baseadas na intervenção quimioterápica, no qual a doxorubicina (DOX) é largamente utilizada. Visto que, o músculo esquelético possui importante papel na captação de glicose, o objetivo do presente trabalho foi investigar o efeito da DOX na intolerância à glicose. Para isso foram utilizados ratos Wistar, os quais receberam uma dose única de DOX ou salina intraperitoneal (15mg/kg). Avaliamos a expressão de proteínas envolvidas na sensibilidade à insulina e captação de glicose. Os ensaios captação de glicose foram realizados em cultura de miócitos, no qual foi utilizado o agonista de AMPK. O tratamento com DOX causou resistência à insulina e hiperglicemia. No músculo EDL e em miócitos houve menor expressão de GLUT-4 e de AMPk. Em conclusão, o tratamento com DOX causou intolerância à glicose e redução da expressão de AMPk e GLUT-4. A utilização do agonista de AMPk foi capaz de recuperar à intolerância à glicose. / The cancer is considered a major cause of death worldwide. For the treatment of this disease, with frequency are used pharmacological strategies based in chemotherapeutic intervention, in which doxorubicin (DOX) is widely used. Since the skeletal muscle plays an important role in glucose uptake, the aim of this study was to investigate the effect of DOX in glucose intolerance. For this Wistar rats which received a single dose of DOX or saline intraperitoneally (15mg / kg). We evaluated the expression of proteins involved in insulin sensitivity and glucose uptake. The glucose uptake assays were performed on culture myocytes, which was used in the agonist of AMPK. The treatment with DOX caused insulin resistance and hyperglycemia. In the EDL muscle myocytes and there was less expression of GLUT4 and AMPK. In conclusion, treatment with DOX caused impaired glucose tolerance and reduction of expression of AMPK and GLUT-4. The use of AMPK agonist was able to recover glucose intolerance.

Les maladies métaboliques ont pris une ampleur considérable dans le monde ces dernières décennies, telle que certains parlent à ce jour de pandémie. Le diabète de type 2 est l’une de celles qui progressent avec la plus importante prévalence. L'un des facteurs à l’origine du développement de cette physiopathologie est l’insulino-résistance. Il s'agit d'une altération de la réponse à l’insuline des tissus cibles tels que le muscle squelettique, le tissu adipeux et le foie, induisant une dérégulation de l'homéostasie du glucose. Les tissus sensibles deviennent incapables, entre autres, d'absorber adéquatement le glucose sanguin conduisant ainsi à l’établissement d’une hyperglycémie chronique. Les travaux présentés dans cette thèse ont pour objectif de caractériser le rôle de la protéine Growth-arrest specific 6 (GAS6) dans la dérégulation de l’équilibre glycémique et le développement de la résistance à l’insuline. GAS6 est une protéine γ-carboxylée sécrétée qui agit comme ligand pour la famille des récepteurs tyrosines kinases TAM comprenant : TYRO3, AXL et MERTK. GAS6 et ses récepteurs jouent un rôle essentiel dans le système immunitaire, la progression tumorale et les métastases cancéreuses. Cependant, des études récentes menées chez l’humain ont montré que les niveaux circulants de GAS6 ou des variations dans le gène GAS6 sont associés à l’hyperglycémie, la résistance à l'insuline et le risque de développer le diabète de type 2. Cependant, le mécanisme par lequel GAS6 influence ces désordres métaboliques reste méconnu. Dans une première étude, nous avons évalué, pour la première fois dans une cohorte de femmes canadiennes, la corrélation éventuelle entre les niveaux circulants de GAS6 et des facteurs de risque liés au diabète. Cette cohorte nommée MONET (Montréal and Ottawa New Emerging Team) est constituée de 126 femmes post-ménopausées, en surpoids ou obèses. Ces femmes ne sont pas diabétiques, mais présentent un risque plus élevé de développer la maladie à cause de leur poids et de leur statut sédentaire. Nous avons constaté que les femmes ayant des taux élevés de GAS6 dans le sang ont une tolérance au glucose significativement plus faible que celles avec des niveaux plus faibles de GAS6. Par ailleurs, certains paramètres de dysfonctionnements hépatiques (AST, ALT) et des marqueurs d’inflammation (IL-6) concordent positivement avec des taux élevés de GAS6. Nos résultats suggèrent que GAS6 pourrait être un biomarqueur de l’intolérance au glucose chez des patientes obèses et qu’il pourrait être associé à l’inflammation et à certains problèmes hépatiques, qui sont des facteurs impliqués dans le développement du diabète. Dans une seconde étude, à l’aide de modèles murins modifiés génétiquement, nous avons pu démontrer que la délétion du gène Gas6 est suffisante pour améliorer la sensibilité à l’insuline et la tolérance au glucose, sans affecter la sécrétion d’insuline. Par ailleurs, les souris déficientes pour GAS6 sont protégées contre la résistance à l'insuline induite par un régime alimentaire riche en graisses et en sucres. À l’inverse, l'augmentation in vivo des taux circulants de GAS6 est suffisante pour réduire la sensibilité à l'insuline. L'analyse de l'expression génique des récepteurs TAM dans les tissus sensibles à l’insuline a révélé qu’Axl est fortement exprimé dans le muscle squelettique. Dans une lignée de cellules musculaires, nous avons démontré que la voie de signalisation de GAS6-AXL affecte la réponse à l'insuline en inhibant la phosphorylation du récepteur de l'insuline (RI) et de son effecteur en aval AKT. Mécaniquement, AXL s'hétérodimérise avec le RI et GAS6 reprogramme les voies de signalisation en aval du RI dans les cellules musculaires. Il en résulte une activation accrue de la voie des Rab, notamment Rab7 induisant une internalisation de RI. Ensemble, ces résultats décrivent le mécanisme cellulaire par lequel GAS6 et AXL influencent la sensibilité à l'insuline. Finalement, nos derniers résultats soulignent un autre mécanisme d’action de GAS6 sur le métabolisme des cellules musculaires. Nous avons démontré, par protéomique, que GAS6 augmente significativement les niveaux protéiques de plusieurs enzymes impliquées dans la glycolyse et la production de lactate. Le profil métabolique des cellules musculaires traitées avec GAS6 démontre une augmentation du niveau de la glycolyse anaérobique et de la production de lactate. Par ailleurs, nos résultats suggèrent que le lactate lui-même induit une inhibition de la phosphorylation du RI en réponse à l’insuline. Ainsi, GAS6, en reprogrammant les voies métaboliques et l’utilisation du glucose des cellules musculaires, favoriserait la production de lactate induisant une diminution de la sensibilité à l'insuline. / Metabolic diseases have taken on a considerable scale in the world in recent decades, such that some speak of a pandemic. Type 2 diabetes is one of those diseases that progress with the highest prevalence. One of the factors behind the development of this pathophysiology is insulin resistance. It is an alteration of the insulin response of targeted tissues such as skeletal muscle, adipose tissue and liver, inducing dysregulation of glucose homeostasis. Sensitive tissues become incapable, among other things, of adequately absorbing blood glucose, thus leading to the establishment of chronic hyperglycemia. The work presented in this thesis focuses on characterizing the role of Growth-arrest specific protein 6 (GAS6) in the dysregulation of glycemic balance and the development of insulin resistance. GAS6 is a secreted γ-carboxylated protein that acts as a ligand for the TAM family of receptor tyrosine kinases including: TYRO3, AXL and MERTK. GAS6 and its receptors play an essential role in the immune system, tumor progression and cancer metastasis. However, recent studies in humans have shown that circulating GAS6 levels or variations in GAS6 gene are associated with hyperglycemia, insulin resistance and the risk of developing type 2 diabetes. However, the mechanism by which GAS6 influences these metabolic disorders remains unknown. In a first study, carried out for the first time in a cohort of Canadian women, we evaluated the potential correlation between circulating GAS6 levels and risk factors linked to diabetes. This cohort, named MONET (Montreal and Ottawa New Emerging Team), is composed of 126 post-menopausal, overweight or obese women. These women are not diabetic but have high risks of developing the disease because of their weight and sedentary status. We found that women with high levels of GAS6 in the blood have significantly lower glucose tolerance than those with lower levels of GAS6. In addition, certain liver dysfunction parameters (AST, ALT) and inflammation markers (IL-6) positively correlated with high levels of GAS6. Our results suggest that GAS6 could be a biomarker of glucose intolerance in obese patients and be associated with inflammation and certain liver problems, which are factors involved in the development of diabetes.  In a second study, using genetically modified mouse models, we were able to demonstrate that deletion of the Gas6 gene was sufficient to improve insulin sensitivity and glucose tolerance, without affecting insulin secretion. Furthermore, GAS6-deficient mice were protected against insulin resistance induced by a diet high in fats and sugars. Conversely, in vivo, increase of GAS6 circulating levels is sufficient to reduce insulin sensitivity. Analysis of TAM receptors gene expression in insulin-responsive tissues revealed that Axl is highly expressed in skeletal muscle. In a muscle cell line, we demonstrated that the GAS6-AXL signaling pathway affects the insulin response by inhibiting the phosphorylation of the insulin receptor (IR) and its downstream effector AKT. Mechanistically, AXL heterodimerizes with IR and GAS6 reprograms signaling pathways downstream of IR in muscle cells. This results in an increased activation of the Rab pathway, in particular Rab7, inducing an internalization of IR. Together, these results describe the cellular mechanism by which GAS6 and AXL influence insulin sensitivity. Finally, our latest results highlight another mechanism of action of GAS6 on muscle cell metabolism. We demonstrated by proteomics that GAS6 significantly increases the protein levels of several enzymes involved in glycolysis and lactate production. The metabolic profile of muscle cells treated with GAS6 demonstrates an increase in the level of anaerobic glycolysis and lactate production. Furthermore, our results suggest that lactate itself induces an inhibition of IR phosphorylation in response to insulin. Thus, GAS6, by reprogramming the metabolic pathways and the use of glucose in muscle cells, would promote lactate production inducing a decrease in insulin sensitivity.

De nombreuses études indiquent que la prématurité, qui représente 8 % des naissances, estassociée à des indices précoces de dysfonction vasculaire, d’élévation de la pression sanguineet de survenue de diabète de type 2. Les enfants nés prématurément sont plus sujets auxblessures oxydatives de par l’immaturité de leurs défenses antioxydantes et de leur expositionà des situations pro-oxydantes (exposition à l’air ambiant, à un supplément d’oxygène, ou àune exposition aux infections). Cependant, les conséquences à long terme des blessuresoxydatives induites par une exposition à l’oxygène en période périnatale restent méconnues.Le but de ce doctorat a été de mettre en évidence certains mécanismes pouvant relier lesdommages de la prématurité induits par l’oxygène, et le risque à long terme de développer desmaladies cardiovasculaires et métaboliques dans le concept global d’une programmationdéveloppementale de l’hypertension et des pathologies reliées au syndrome métabolique. Des ratons Sprague-Dawley (SD) ont été exposés à 80 % O2 (O2) vs air ambiant (AA) du 3èmeau 10ème jour de vie. Concernant les paramètres cardiovasculaires, nous avons mesuré aucours de la croissance, la pression sanguine à la queue (de la 4ème semaine à la 15ème semaine)et à l’âge adulte : la réactivité vasculaire à l’angiotensine II (AngII) et au carbachol (ex vivo,carotides) avec ou sans le tempol; la production d’oxyde nitrique (NO) en présence ou non Larginineet de L-sépiaptérine (aorte, immunohistochimie) ainsi que l’expression de la nitricoxyde synthase endothéliale (eNOS) (aorte, immunohistochimie et western blot); le stressoxydant vasculaire (aorte, chemiluminescence) par la mesure de la production d’anionssuperoxide en présence ou non des inhibiteurs de la nicotinamide-adenine-dinucleotide-phosphate (NADPH oxydase) et de la nitric oxyde synthase endotheliale (eNOS), l’apocynine,et N-nitro-L-arginine methyl ester (L-NAME) respectivement, ainsi que le stress oxydantcirculant par la mesure des niveaux plasmatiques de malondialdéhyde (MDA, HPLC); ladensité microvasculaire a été évaluée au niveau du muscle tibial antérieur,immunohistochimie); la vitesse d’onde pulsée (VOP) (entre la valve aortique et juste avant labifurcation ilio-fémorale) a été mesurée par ultrason; le nombre de néphrons a été compté pardigestion acide. L’ontogenèse de la plupart de ces mécanismes a été regardée à l’âge de 4semaines.Concernant les paramètres métaboliques, le poids a été mesuré au cours de la croissance. Àl’âge adulte, la composition corporelle et la tolérance au glucose ont été évaluées. À l’âge de 4 semaines, aucune différence n’a été observée dans la pression sanguine, laréactivité vasculaire et le stress oxydant, mais chez les rats O2 vs AA, la densitémicrovasculaire est moindre, et des changements histologiques suggèrent la présence d’unerigidité artérielle augmentée.À l’âge adulte chez les rats O2 vs AA (n = 6-8 /groupe) : i) les pressions sanguines systoliqueset diastoliques sont augmentées; ii) la réactivité vasculaire à l’AngII est augmentée et celle aucarbachol est diminuée, le tempol prévient ces dysfonctions; iii) la production de NO est plusfaible au niveau basal et après stimulation par le carbachol, mais est restaurée après la préincubationavec L-arginine et L-sépiaptérine; iv) l’expression d’eNOS est diminuée parimmunohistochimie et augmentée par western blot; v) les niveaux d’anions superoxide, auniveau basal et en réponse à l’AngII, sont augmentés et sont induits par la NADPH oxydase etle non-couplage d’eNOS; vi) les niveaux plasmatiques de MDA sont augmentés; vii) Ladensité microvasculaire est moindre; viii) la VOP est augmentée; ix) le nombre de néphrons par rein est réduit; x) le poids est plus faible au cours de la croissance et un catch up estobservé à l’âge adulte; la composition corporelle n’est pas différente entre les groupes; xi) latolérance au glucose est diminuée. Ces résultats supportent l’hypothèse d’une programmation développementale des maladiescardiovasculaires et métaboliques à l’âge adulte à la suite d’un stress hyperoxique néonatal. / Many studies showed that prematurity, which represents 8 % of birth, is associated with earlyindices of vascular dysfunction, increased blood pressure and Type 2 diabetes. Prematuritybabies are more susceptible to oxidative injury, consequence of the immaturity of theirantioxidant defences, and exposure to pro-oxidant situations (oxygen supplementation,infection). However, the long-term consequences of oxidative injury induced by oxygenexposure in the neonatal period are unknown.The aim of these PhD studies was to unravel some mechanisms that might underlie thedamage induced by oxygen and the long-term risk of developing vascular and metabolicdiseases in the overall concept of developmental programming of hypertension and metabolicsyndrome-related diseases. Sprague-Dawley pups were kept with their mother in 80 % O2 (O2) or room air (RA) from day3 to 10 of life. Cardiovascular parameters, tail blood pressure was measured between 4 and15 weeks of life. In adulthood : vascular reactivity (ex vivo carotid rings) to angiotensine II(AngII) and carbachol with and without tempol was studied; studies of nitric oxide (NO)production with and without L-arginine and L-sépiaptérine (aorta, immunohistochemistry)and endothelial nitric oxide synthase expression (eNOS; aorta, immunohistochemistry,western blot) were performed; vascular oxidative stress (aorta, using chemiluminescence) bymeasuring superoxide anion production with and without inhibitors of nicotinamide-adeninedinucleotide-phosphate (NADPH oxydase) and nitric oxyde synthase endotheliale (eNOS),apocynin and N-nitro-L-arginine methyl ester (L-NAME) respectively, and circulating oxidative stress by measuring the plasma levels of malondialdéhyde (MDA, HPLC) wereevaluated; microvascular density was assessed on tibialis anterior muscle sections; pulse wavevelocity (PWV) was measured by ultrasound, between aortic valve and ilio-femoralbifurcation; nephrons were counted after hydrochloric acid digestion. The main observationswere also evaluated at 4 weeks of age. Metabolic parameters: body weight has beenmeasured during the growth. In adulthood, body composition, glucose tolerance wereevaluated. A 4 weeks of age, no difference was observed regarding blood pressure, vascular reactivity,and oxidative stress indices, but in rats O2 vs. RA (n = 6-8 /group), microvascular rarefactionand histological changes suggesting enhanced vascular stiffness were present.To adulthood, rats O2 vs. RA (n = 6-8/group) : i) systolic and diastolic blood pressures areincreased; ii) vascular reactivity to Ang II is increased and to carbachol is decreased, thesedysfunction were totally abolished by co-incubation of the vessel rings with tempol; iii) NOproductionis decreased in basal condition and after carbachol stimulation, but is restored afterpre-incubation of aorta sections with L- arginine and L-sépiaptérine; iv) eNOS expression isdecreased by immunohistochemistry but increased by western blot; v) vascular superoxideanion levels are increased in basal condition, after AngII stimulation and this is mediated byNADPH oxydase and eNOS uncoupling; vi) the plasma levels of MDA are increased; vii)microvascular density is decreased; viii) PWV is increased; ix) nephron count per kidney isdecreased; x) body weight is less during growth, but a catch up is observed in adulthood,body composition is similar; xi) the glucose tolerance is decreased in adults. These results support the hypothesis of developmental programming of vascular andmetabolic diseases in adulthood, after exposure to hyperoxic stress in the neonatal period.