Exposure to toxic bile acids (BA) and retinoic acids (RA) is implicated in toxicities related to excessive oxidative stress. This thesis examined roles and mechanisms of the oxidative stress-responsive nuclear factor (erythroid 2-like) factor 2 (Nrf2) in adaptive cell defense against BA and RA toxicities. Using liver cells and mouse models, many antioxidant proteins known to be Nrf2 target genes, particularly the rate-limiting enzyme for glutathione (GSH) biosynthesis, i.e., glutamate-cysteine ligase subunits (GCLM/GCLC), were induced by BA [lithocholic acid (LCA)] or RA (all-trans, 9-cis and 13-cis) treatment. Evidence for increased Nrf2 transactivation by LCA and all-trans-RA was exemplified in HepG2 by: (1) reduced constitutive and inducible expression of GCLM/GCLC upon Nrf2 silencing via small-interfering RNA; (2) increased inducible expression of GCLM/GCLC genes by Nrf2 overexpression, but overexpression of dominant-negative Nrf2 decreased it; (3) increased nuclear accumulation of Nrf2 as signature event of receptor activation; (4) enhanced Nrf2-dependent antioxidant-response-element (ARE) reporter activity as indicative of increased Nrf2 transactivation; and (5) increased Nrf2 occupancy to AREs of GCLM and GCLC. Additionally, in BA-treated HepG2 cells, we observed concomitant increases of many ATP-binding cassette (ABC) transporters (MRPs 1-5, MDR1 and BCRP) in parallel with increased cellular efflux. Nrf2 silencing in HepG2 cells decreased constitutive and inducible expression of MRP2, MRP3 and ABCG2. However, Nrf2-silenced mouse hepatoma cells, Hepa1c1c7, and Nrf2-/- mice had decreased constitutive and/or inducible expression of Mrps 1-4, suggesting species differences in Nrf2-dependent regulation of hepatic ABC transporters. Protection by Nrf2 against BA and RA toxicities was confirmed by observations that Nrf2 silencing increased cell susceptibility to BA- and RA-induced cell death. Moreover, Nrf2-/- mice suffered more severe liver injury than the wildtype. Increased GSH and efflux activity following increased GCLM/GCLC and ABC transporters, respectively, can mitigate LCA toxicity. Activation of MEK1-ERK1/2 MAPK was shown to primarily mediate Nrf2 transactivation and LCA-induced expression of antioxidant proteins and Nrf2-dependent and -independent ABC transporters. In conclusion, Nrf2 activation by BA and RA led to coordinated induction of antioxidant and ABC proteins, thereby counteracting resultant oxidative cytotoxicity. The potential of targeting Nrf2 in management of BA and RA toxicities merits further investigation.

No description available.

No description available.

A Síndrome de Noonan (SN) é caracterizada por baixa estatura proporcionada de início pós-natal, dismorfismos faciais, cardiopatia congênita e deformidade torácica. A frequência da SN é estimada entre 1:1000 e 1:2500 nascidos vivos, com distribuição semelhante em ambos os sexos. A herança é autossômica dominante com penetrância completa, porém a maioria dos casos é esporádica. Até o momento, mutações em genes da via RAS-MAPK (PTPN11, KRAS, SOS1, RAF1, MEK1, NRAS e SHOC2) foram identificadas em aproximadamente 70% dos pacientes. Uma das principais características fenotípicas da SN é a baixa estatura pós-natal, embora o mecanismo fisiopatológico do déficit de crescimento nesta síndrome ainda não esteja totalmente esclarecido. Estudos que avaliaram o padrão de crescimento linear em crianças com SN foram realizados anteriormente ao conhecimento do diagnóstico molecular dessa síndrome. No presente estudo, avaliamos a frequência de mutação nos genes PTPN11, SOS1, RAF1 e KRAS em 152 pacientes com SN e o padrão de crescimento linear (altura) e ponderal [índice de massa corpórea (IMC)] dos pacientes com mutação identificada. No total, mutações nos genes relacionados foram encontradas em 99 pacientes (65%) do nosso estudo, com predominância do gene PTPN11 (47%), seguido do SOS1 (9%), RAF1 (7%) e KRAS (3%). Foram construídas curvas específicas para SN de Altura e IMC para idade e sexo utilizando o método LMS. Os pacientes com SN apresentaram crescimento pré-natal preservado, porém o comprometimento do crescimento pós-natal foi observado desde o primeiro ano de vida, atingindo uma altura final de -2,5 e -2,2 desvios-padrão da média para população brasileira em homens e mulheres, respectivamente. O prejuízo da altura foi maior nos pacientes com mutação no gene RAF1 em comparação com os genes PTPN11 e SOS1. O IMC dos pacientes com SN apresentou queda de 1 desvio-padrão em relação à média da população brasileira normal. O comprometimento do IMC foi menor nos pacientes carreadores de mutação no RAF1. Pacientes com mutação nos genes PTPN11 e SOS1 apresentaram maior frequência de estenose de valva pulmonar, enquanto a miocardiopatia hipertrófica foi mais frequente nos pacientes com mutação no gene RAF1. A variabilidade fenotípica observada nos pacientes com mutação no PTPN11 não pode ser explicada pelo grau que estas mutações influenciam a atividade tirosina fosfatase da SHP-2 nem pela presença de polimorfismos no gene KRAS. Com a análise dos éxons 3, 8 e 13 do PTPN11, seguido dos éxons 6 e 10 do SOS1 e éxon 7 do RAF1 identificamos 86% dos pacientes carreadores de mutações nos genes relacionados, propondo uma forma mais eficiente de avaliação molecular na SN. Acreditamos que a variabilidade fenotípica presente nessa síndrome esteja diretamente ligada aos diferentes papéis exercidos pelas proteínas que participam da via RAS/MAPK. Entretanto, mais estudos em relação à via RAS/MAPK serão necessários para esclarecer as questões relacionadas ao crescimento e outras características fenotípicas da SN / Noonan Syndrome (NS) is characterized by distinctive facial features, short stature and congenital heart defects. The estimated prevalence is 1:1000 to 1:2500 live births, affecting equally both sexes. It is an autosomal dominant disorder with complete penetrance, but most cases are sporadic. To date, mutations in the RAS/MAPK pathway genes (PTPN11, KRAS, SOS1, RAF1, MEK1, NRAS and SHOC2) were identified in approximately 70% of patients. One of the cardinal signs of NS is proportional postnatal short stature although the physiopathological mechanism of growth impairment remains unclear. The current knowledge about the natural history of growth associated with NS was described before molecular diagnosis era. In this study, we performed PTPN11, SOS1, RAF1, and KRAS mutation analysis in a cohort of 152 NS patients and studied the natural linear (height) and ponderal growth [body mass index (BMI)] of NS patients with related mutations. Mutations in NS-causative genes were found in 99 patients (65%) of our cohort. The most common mutated gene was PTPN11 (47%), followed by SOS1 (9%), RAF1 (7%) and KRAS (3%). Sex-specific percentile curves for height and BMI were constructed using the LMS method. NS patients had birth weight and length within normal ranges but the postnatal growth impairment was observed during the first year of life, reaching a final height of -2.3 and -2.2 standard deviations from the mean for Brazilian healthy men and women, respectively. Postnatal growth impairment was higher in RAF1 mutation patients than in patients with SOS1 and PTPN11 mutations. BMI values in NS patients were lower in comparison with normal Brazilian population. BMI values were higher in patients with RAF1 mutations than in patients with other genotypes. Patients with mutations in PTPN11 and SOS1 genes were more likely to have pulmonary valve stenosis, whereas hypertrophic cardiomyopathy was more common in patients with mutations in the gene RAF1. The intensity of constitutive tyrosine phosphatase activity of SHP-2 due to PTPN11 mutations, as well as the presence of polymorphisms in KRAS gene did not influence the phenotype of NS patients with mutation in PTPN11 gene. Analysis of exons 3, 8 and 13 of PTPN11 gene, followed by exons 6 and 10 of SOS1 gene and exon 7of RAF1 gene identified 86% of patients harboring mutations in related genes, suggesting a more efficient evaluation of NS molecular diagnosis. We believe that the phenotypic variability in this syndrome is directly linked to the different roles played by proteins that participate in RAS/MAPK pathway. However, further studies in RAS/MAPK pathway are needed to clarify issues related to growth and other phenotypic characteristics of SN

Várias condições podem estar relacionadas com a atrofia muscular, tais como inatividade, envelhecimento, septicemia, diabetes, câncer e uso de glicocorticoides. Em tentativa prévia de prevenir tal condição catabólica secundário ao uso de glicocorticoide, através da suplementação de ômega-3 (N-3), observamos um agravamento da atrofia muscular, afetando mais tipos de fibras musculares, usualmente poupadas pelo glicocorticoide, fibras tipo 1 por exemplo. Entretanto, não foi possível determinar quais as propriedades dessa interação. Portanto, o objetivo deste estudo foi de avaliar a ação do Ômega-3 associada a dexametasona e de diferentes glicocorticoides em dose equipotente sobre o peso corporal; área de secção transversa muscular; perfil de ácidos graxos; expressão gênica de fatores de transcrição musculares e atrogenes (Atrogina 1 e MuRF-1); expressão proteica de componentes das vias do IGF-1/Akt/mTOR, Ras/Raf/MEK/ERK e Miostatina/Smad2/3; e expressão de receptores de glicocorticoides na musculatura esquelética de ratos. Metodologia: Ratos Wistar suplementados ou não com ômega-3 (100mg/kg/dia de EPA) por 40 dias receberam dexametasona (DX) subcutânea (2,5 e 1,25mg/kg/dia) nos últimos 10 dias de suplementação. Para estudo dos demais glicocorticoides, ratos sem suplementação receberam deflazacorte (DC), metilprednisolona (MP) em dose/volume equipotente ao de dexametasona (DC 10 e 20mg/kg/dia e MP6,7 e 13,3mg/kg/dia) por 10 dias. Constituindo 10 grupos: CT, N-3, DX1,25, DX2,5, DX1,25+N-3, DX2,5+N-3, MP6, MP13, DC10 e DC20. Através de estudo histológico, imuno-histoquímico, PCR em tempo real e Western blotting, foram avaliados a área transversa dos diferentes tipos de fibras musculares; a expressão de receptor de glicocorticoide na fibra muscular; a expressão gênica dos atrogenes e fatores de transcrição; expressão de proteínas das vias IGF-1, Miostatina e MEK/ERK. Resultados: A administração de N-3 influenciou a atrofia por DX causando maior atrofia em fibras do tipo 1 e 2A, aumento na expressão proteica de FoxO3a total, P-Smad3, LC3-II e gênica (mRNA) de REDD-1, Atrogina-1/MAFbx. De forma isolada o ômega-3 reduziu a expressão de P-FoxO3a, PGC1alfa, a quantidade de ácido araquidônico e a expressão de mRNA do IRS-1 com aumento na expressão de LC3-II. A comparação entre glicocorticoides mostrou que a MP (13mg/kg/dia) acarretou maior impacto no peso corporal e muscular; o DC (10mg/kg/dia) causou menor atrofia em fibras 2B em relação aos demais glicocorticoides. A DX causou maior impacto sobre o Akt total em comparação com os demais glicocorticoides, em P-Akt o grupo DX1,25 teve menor expressão em relação a outros glicocorticoides em dose equipotente. Todos os glicocorticoides afetaram a expressão de P-FOXO3a. Na expressão de ERK1/2 e P-ERK1/2, MP6 foi o grupo com maior prejuízo à fosforilação em relação aos demais em dose equipotente. Já na avaliação da via Miostatina/Smad2/3 os grupos MP 6, MP13 e DC20 mostraram maior expressão de Smad2/3 total e P-Smad3. A expressão gênica de REDD-1 e MYOD foi aumentada nos grupos MP6 e MP13 em relação aos demais grupos; REDD2 no grupo DC20 foi menor em relação ao grupo DX2,5. A expressão de Miostatina foi menor nos grupos DX2,5 e DC20, sendo o DC a droga com menor impacto sobre os atrogenes MuRF-1 e Atrogina-1. DX1,25 e DX2,5 causaram menor expressão de IRS-1 entre os grupos de glicocorticoides. Conclusões: Ômega-3 pode aumentar a atrofia muscular causada por DX em fibras 1 e 2A, possivelmente relacionado com aumento da expressão de FoxO3a, REDD-1 e Atrogina-1, diminuição na expressão de PGC1alfa e P-FoxO3a, nas quantidades de ácido araquidônico com aumento da atividade lisossomal. Comparando diferentes glicocorticoides, a MP tende a produzir maior impacto nos pesos corporal e muscular, o DC é menos prejudicial as fibras do tipo 2B, entretanto, afeta predominantemente fibras do tipo 1, da mesma forma que a DX na dosagem de 1,25mg/kg/dia. A DX tende a afetar mais a expressão de Akt total e fosforilado que os demais glicocorticoides. A MP afeta mais a via Ras/Raf/MEK/ERK e expressão de REDD-1 em relação aos demais glicocorticoides, e o DC e MP mostram maior expressão de Smad2/3 total e fosforilada em relação ao DX após 10 dias de administração / Several conditions may be related to muscle atrophy, such as inactivity, aging, septicemia, diabetes, cancer and use of glucocorticoids. In a previous attempt to prevent such glucocorticoid catabolic condition, through the supplementation of omega-3 (N-3), we observed a worsening of muscular atrophy, affecting more types of muscle fibers, usually spared by glucocorticoid, type 1 fibers for example. However, it was not possible to determine the properties of this interaction. Therefore, the objective of this study was to evaluate the action of omega-3 associated with dexamethasone and different glucocorticoids in equipotent dose on body weight; muscle cross-sectional area; fatty acid profile; gene expression of muscle transcription factors and atrogenes (Atrogin-1 and MuRF-1); protein expression of IGF-1/Akt/mTOR, Ras/Raf/MEK/ERK and Myostatin/Smad2/3 pathways components; and expression of glucocorticoid receptors in the skeletal musculature of rats. Methods: Wistar rats given orally or not with omega-3 (100mg/kg/day of EPA) for 40 days received subcutaneous dexamethasone (DX) (2.5 or 1.25mg/kg/day) during the last 10 days of supplementation. For the other glucocorticoids, rats without supplementation received deflazacorte (DC) or methylprednisolone (MP) in dose/volume equivalent to that of dexamethasone (DC 10 or 20mg/kg/day and MP6.7 or 13.3mg/kg/day) for 10 days. Comprising 10 groups: CT, N-3, DX1.25, DX2.5, DX1.25 + N-3, DX2.5 + N-3, MP6, MP13, DC10 and DC20. Through histological, immunohistochemical, real-time PCR and Western blotting, we evaluated the transverse area of the different muscle fibers; the expression of glucocorticoid receptor; the gene expression of atrogenes and transcription factors; protein expression of the IGF-1, Myostatin and MEK/ERK pathways. Results: N-3 administration influenced DEXA atrophy causing increased atrophy in type 1 and 2A fibers, increased protein expression of total FoxO3a, P-Smad3, LC3-II, and REDD-1 gene (mRNA), Atrogin-1/MAFbx isolated omega-3 reduced the expression of P-FoxO3a, PGC1alpha, the amount of arachidonic acid and the expression of IRS-1 mRNA with increased expression of LC3-II. The comparison between glucocorticoids showed that MP13 had a greater impact on body and muscle weight; the DC10 caused less atrophy in 2B fibers in relation to the other glucocorticoids. DX, caused greater impact on total Akt compared to the other glucocorticoids, in P-Akt the DX1,25 group had lower expression to other equipotent dose glucocorticoids. All glucocorticoids affect the expression of P-FOXO3a. In the of ERK1/2 and P-ERK1/2 protein expression, the MP6 was the group with the greatest damage to the phosphorylation in relation to the others in equipotent dose. In the evaluation of the Myostatin/Smad2/3 pathway MP 6, MP13 and DC20 showed higher expression of total Smad2/3 and P-Smad3. The gene expression of REDD-1 and MYOD was increased in the MP6 and MP13 groups compared to the other groups, REDD2 in the DC20 group was lower in relation to the DX2.5 group. Myostatin expression was lower in the DX2.5 and DC20 groups, with DC being the drug with less impact on atrogenes MuRF-1 and Atrogin-1. DX1.25 and DX2.5 caused lower IRS-1 expression among the glucocorticoid groups. Conclusions: Omega-3 may increase muscle atrophy caused by DX in fibers 1 and 2A, possibly related to increased expression of FoxO3a, REDD-1 and Atrogin-1, decreased expression of PGC1alpha and P-FoxO3a, in the amounts of acid arachidonic with increased lysosomal activity. Comparing different glucocorticoids, MP tends to produce a greater impact on body and muscular weights, DC is less harmful to type 2B fibers, however, it predominantly affects type 1 fibers, in the same way as DX in the dosage of 1.25mg/kg/day. DX tends to affect total and phosphorylated Akt expression more than other glucocorticoids. MP affects more the Ras/Raf/MEK/ERK pathway and REDD-1 expression in relation to the other glucocorticoids, and DC and MP show a higher expression of total and phosphorylated Smad2/3 compared to DX after 10 days of administration

Abstract Cardiovascular diseases are the leading causes of death in the developed countries and their incidence is not expected to decrease in the future. There is a lifetime risk of one in five of developing heart failure, which still has poor prognosis and current treatments only cover part of the pathophysiology behind this syndrome. Pathological processes contributing to heart failure include cardiac hypertrophy and remodeling, which involves neurohumoral activation, reactivation of fetal genes, impaired Ca2+ cycling, increased apoptosis, and increased fibrosis. Intracellular signalling pathways and transcription factors mediating the response to various extracellular stresses have a key role in the regulation of myocardial remodeling and they are investigated in order to develop new approaches for the treatment of heart failure. The aim of this thesis was to elucidate roles of mitogen-activated protein kinases (MAPKs) and transcription factor GATA-4 in the regulation of cardiomyocyte function in cell cultures, and in hearts ex vivo and in vivo. The main findings were that (i) Inhibition of p38α MAPK enhanced function of sarco/endoplasmic reticulum Ca2+ -ATPase and thus cardiac contractility by increasing phosphorylation of protein phosphatase inhibitor-1 and phospholamban, (ii) p38 MAPK isoforms p38α and p38β regulated promoter activity of B-type natriuretic peptide via distinct pathways, (iii) p38α and p38β MAPKs also had different effects on gene expressions related to fibrosis and hypertrophy, and (iv) p38 and ERK1/2 MAPKs mediated stretch-induced activation of GATA-4 by phosphorylation at Ser 105. GATA-4 also seems to be regulated by ubiquitination. This study provides novel data of p38 MAPK and GATA-4 in the regulation of cardiomyocyte function. Inhibition of p38α MAPK could be beneficial in the treatment of heart failure. Also GATA-4 is a potential target for treatment of cardiovascular diseases. / Tiivistelmä Sydän- ja verisuonisairaudet ovat yleisin kuolinsyy länsimaissa, eikä niiden ilmaantuvuus tule vähenemään lähitulevaisuudessa. Elinikäinen riski sairastua sydämen vajaatoimintaan on 20 %, ja sydämen vajaatoiminnan ennuste on edelleen huono. Nykyisillä hoitomuodoilla voidaan puuttua vain osittain sydämen vajaatoiminnan patofysiologisiin mekanismeihin. Sydämen vajaatoiminnan kehittymiseen liittyvät sydänlihaksen liikakasvu ja uudelleenmuovautumisprosessi, johon liittyy neurohumoraalinen aktivaatio, sikiöaikaisten geenien uudelleenilmentyminen, häiriöt solunsisäisessä Ca2+-viestinnässä sekä lisääntynyt ohjelmoitu solukuolema ja sidekudoksen muodostuminen sydämeen. Solunsisäisillä viestinvälitysketjuilla sekä transkriptiotekijöillä, jotka vastaavat solunulkoisten ärsykkeiden välittämisestä solun sisällä, on keskeinen rooli edellämainittujen prosessien säätelyssä. Uusien lähestymistapojen kehittäminen sydämen vajaatoiminnan hoitoon edellyttää myös solunsisäisen viestinvälityksen ja geenien säätelyn mekanismien selvittämistä. Tämän väitöstyön tavoite oli selvittää p38 mitogeeniaktivoituvan proteiinikinaasin (p38 MAPK) ja transkriptiotekijä GATA-4:n merkitystä sydämen vajaatoiminnan patogeneesissä soluviljelymalleissa. Päälöydöksiä olivat: (i) p38α MAPK -isoformin estäminen paransi kalsiumia solulimakalvostoon pumppaavan SERCA2a:n toimintaa ja sydänlihassolun supistumiskykyä lisäämällä fosfolambaanin ja proteiinifosfataasi-inhibiittori-1:n fosforylaatiota. (ii) p38 MAPK isoformit p38α ja p38β säätelivät B-tyypin natriureettisen peptidin geenin promoottorialuetta erillisten reittien kautta. (iii) p38α ja p38β isoformit vaikuttivat myös eri tavoin sydämen sidekudoksen muodostumiseen ja hypertrofiaan liittyvien geenien ilmentymiseen. (iv) p38 ja ERK1/2 välittävät venytyksen aiheuttaman GATA-4:n aktivaation fosforyloimalla seriini-105 fosforylaatiopaikan. Lisäksi GATA-4:n toimintaa säädellään ubiquitinaation avulla. Tämä tutkimus tuo uutta tietoa p38 MAPK:n ja GATA-4:n rooleista sydämen vajaatoiminnan kehittymisessä. p38α-isoformin toiminnan estäminen voisi olla hyödyllinen hoitomuoto sydämen vajaatoiminnassa. Myös GATA-4 on potentiaalinen lääkehoidon kohde sydänsairauksien hoidossa.

La adaptación de la levadura Saccharomyces cerevisiae a condiciones de alta osmolaridad está mediada por la vía de HOG ((high-osmolarity glycerol). La activación de esta vía induce una serie de respuestas que van a permitir la supervivencia celular en respuesta a estrés. La regulación génica constituye una respuesta clave para dicha supervivencia. Se han descrito cinco factores de transcripción regulados por Hog1 en respuesta a estrés osmótico. Sin embargo, éstos no pueden explicar la totalidad de los genes regulados por la MAPK Hog1. En el presente trabajo describimos cómo el complejo transcripcional formado por las proteínas Rtg1 y Rtg3 regula, a través de la quinasa Hog1, la expresión de un conjunto específico de genes. Hog1 fosforila Rtg1 y Rtg3, aunque ninguna de estas fosforilaciones son esenciales para regulación transcripcional en respuesta a estrés. Este trabajo también muestra cómo la deleción de proteínas RTG provoca osmosensibilidad celular, lo que indica que la integridad de la vía de RTG es esencial para la supervivencia celular frente a un estrés osmótico. / In Saccharomyces cerevisiae the adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway, which elicits different cellular responses required for cell survival upon osmostress. Regulation of gene expression is a major adaptative response required for cell survival in response to osmotic stress. At least five transcription factors have been reported to be controlled by the Hog1 MAPK. However, they cannot account for the regulation of all of the genes under the control of the Hog1 MAPK. Here we show that the Rtg1/3 transcriptional complex regulates the expression of specific genes upon osmostress in a Hog1-dependent manner. Hog1 phosphorylates both Rtg1 and Rtg3 proteins. However, none of these phosphorylations are essential for the transcriptional regulation upon osmostress. Here we also show that the deletion of RTG proteins leads to osmosensitivity at high osmolarity, suggesting that the RTG-pathway integrity is essential for cell survival upon stress.

Le diabète de type 2 (DT2) est une maladie métabolique complexe causée par des facteurs génétiques mais aussi environnementaux, tels la sédentarité et le surpoids. La dysfonction de la cellule β pancréatique est maintenant reconnue comme l’élément déterminant dans le développement du DT2. Notre laboratoire s’intéresse à la sécrétion d’insuline par la cellule β en réponse aux nutriments calorigéniques et aux mécanismes qui la contrôle. Alors que la connaissance des mécanismes responsables de l’induction de la sécrétion d’insuline en réponse aux glucose et acides gras est assez avancée, les procédés d’inhibition de la sécrétion dans des contextes normaux ou pathologiques sont moins bien compris. L’objectif de la présente thèse était d’identifier quelques-uns de ces mécanismes de régulation négative de la sécrétion d’insuline dans la cellule β pancréatique, et ce en situation normale ou pathologique en lien avec le DT2. La première hypothèse testée était que l’enzyme mitochondriale hydroxyacyl-CoA déshydrogénase spécifique pour les molécules à chaîne courte (short-chain hydroxyacyl-CoA dehydrogenase, SCHAD) régule la sécrétion d’insuline induite par le glucose (SIIG) par la modulation des concentrations d’acides gras ou leur dérivés tels les acyl-CoA ou acyl-carnitine dans la cellule β. Pour ce faire, nous avons utilisé la technologie des ARN interférants (ARNi) afin de diminuer l’expression de SCHAD dans la lignée cellulaire β pancréatique INS832/13. Nous avons par la suite vérifié chez la souris DIO (diet-induced obesity) si une exposition prolongée à une diète riche en gras activerait certaines voies métaboliques et signalétiques assurant une régulation négative de la sécrétion d’insuline et contribuerait au développement du DT2. Pour ce faire, nous avons mesuré la SIIG, le métabolisme intracellulaire des lipides, la fonction mitochondriale et l’activation de certaines voies signalétiques dans les îlots de Langerhans isolés des souris normales (ND, normal diet) ou nourries à la dière riche en gras (DIO) Nos résultats suggèrent que l’enzyme SCHAD est importante dans l’atténuation de la sécrétion d’insuline induite par le glucose et les acides aminés. En effet, l’oxydation des acides gras par la protéine SCHAD préviendrait l’accumulation d’acyl-CoA ou de leurs dérivés carnitine à chaîne courtes potentialisatrices de la sécrétion d’insuline. De plus, SCHAD régule le métabolisme du glutamate par l’inhibition allostérique de l’enzyme glutamate déshydrogénase (GDH), prévenant ainsi une hyperinsulinémie causée par une sur-activité de GDH. L’étude de la dysfonction de la cellule β dans le modèle de souris DIO a démontré qu’il existe une grande hétérogénéité dans l’obésité et l’hyperglycémie développées suite à la diète riche en gras. L’orginialité de notre étude réside dans la stratification des souris DIO en deux groupes : les faibles et forts répondants à la diète (low diet responders (LDR) et high diet responder (HDR)) sur la base de leur gain de poids corporel. Nous avons mis en lumières divers mécanismes liés au métabolisme des acides gras impliqués dans la diminution de la SIIG. Une diminution du flux à travers le cycle TG/FFA accompagnée d’une augmentation de l’oxydation des acides gras et d’une accumulation intracellulaire de cholestérol contribuent à la diminution de la SIIG chez les souris DIO-HDR. De plus, l’altération de la signalisation par les voies AMPK (AMP-activated protein kinase) et PKC epsilon (protéine kinase C epsilon) pourrait expliquer certaines de ces modifications du métabolisme des îlots DIO et causer le défaut de sécrétion d’insuline. En résumé, nous avons mis en lumière des mécanismes importants pour la régulation négative de la sécrétion d’insuline dans la cellule β pancréatique saine ou en situation pathologique. Ces mécanismes pourraient permettre d’une part de limiter l’amplitude ou la durée de la sécrétion d’insuline suite à un repas chez la cellule saine, et d’autre part de préserver la fonction de la cellule β en retardant l’épuisement de celle-ci en situation pathologique. Certaines de ces voies peuvent expliquer l’altération de la sécrétion d’insuline dans le cadre du DT2 lié à l’obésité. À la lumière de nos recherches, le développement de thérapies ayant pour cible les mécanismes de régulation négative de la sécrétion d’insuline pourrait être bénéfique pour le traitement de patients diabétiques. / Type 2 diabetes (T2D) is a complex metabolic disease caused by genetic as well as environmental factors, such as sedentarity and obesity. Pancreatic β cell dysfunction is now recognized as the key factor in T2D development. Our laboratory is studying the mechanisms of regulation of insulin secretion by the pancreatic β cell in response to nutrients. While the knowledge of the mechanisms responsible for initiation of insulin secretion in response to glucose and fatty acids is quite advanced, the inhibitory processes of insulin secretion in normal or pathological situations are still poorly understood. This doctoral thesis has focused on the identification of some of the mechanisms responsible for negative regulation of insulin secretion in pancreatic β cell. We have addressed this issue under normal situation or pathological conditions related to T2D. We first tested the hypothesis by which a mitochondrial enzyme, short-chain hydroxyacyl-CoA dehydrogenase (SCHAD), negatively regulates glucose-induced insulin secretion (GIIS) by limiting the concentrations of some fatty acids and their derivatives such as acyl-CoA or acyl-carnitine molecules in the β cell. For this purpose, the downregulation of SCHAD by RNA interference (RNAi) was used in the pancreatic β cell line INS832/13. Then, we tested wether a prolonged administration of high-fat diet to mice (diet-induced obesity mouse model, DIO) would modulate intracellular metabolic and molecular pathways responsible for inhibition of insulin secretion. C57BL/6 mice were therefore fed a high-fat diet for 8 weeks followed by insulin secretion, intracellular lipid metabolism, mitochondrial function and intracellular signaling measurements on isolated pancreatic islets of Langerhans of those mice. Our results suggest that SCHAD negatively regulates GIIS and amino acid-induced insulin secretion. We propose that fatty acid oxidation by SCHAD would prevent the accumulation of short-chain acyl-CoAs or acyl-carnitines capable of potentiating insulin secretion. In addition, SCHAD regulates glutamate metabolism by the allosteric inhibition of glutamate dehydrogenase (GDH) preventing the hyperinsulinemia caused by excessive GDH activity. The study of β cell dysfunction in the DIO mouse model stratified LDR and HDR highlighted various fatty acid metabolism pathways involved in the reduction of GIIS. A decrease in the triglycerides/free fatty acid (TG/FFA) cycling associated with an increase in fatty acid oxidation and intracellular accumulation of cholesterol was shown to contribute to the decreased GIIS in DIO-HDR mice. Furthermore, alteration of AMP-activated kinase (AMPK) and protein kinase C epsilon (PKC epsilon) signaling pathways would be responsible for those alterations in metabolic pathways observed in DIO islets and cause decreased insulin secretion. In summary, we have shed light on important pathways negatively regulating insulin secretion in pancreatic β cell. These pathways could either limit the amplitude or duration of insulin secretion after a meal, or help to preserve β-cell function by delaying exhaustion. Some of those signaling pathways could explain the altered insulin secretion observed in T2D obese patients. In light of our research, the development of therapies targeting pathways that negatively regulate insulin secretion may be beneficial for treating diabetic patients.

Les peroxyrédoxines sont des enzymes essentielles de la cellule. Outre leur rôle d’antioxydant, elles sont aussi des régulateurs de la signalisation cellulaire et des suppresseurs de tumeurs. La péroxiredoxine 1 (Prx1) est la plus abondante parmi les six isoformes de peroxyrédoxines humaines. Elle est fréquemment surexprimée dans plusieurs types de cellules cancéreuses, et on a pu associer Prx1 aux processus de carcinogenèse et de métastase, ainsi qu’à la résistance à la radiothérapie ou la chimiothérapie. Ainsi, Prx1 pourrait donc être une cible anticancéreuse intéressante. Au cours de ce travail de thèse, nous avons d’abord évalué l'impact d’une diminution de Prx1 (Prx1 knockdown (Prx1–)) sur la sensibilité cellulaire à des dizaines de médicaments anticancéreux dont la vinblastine, le taxol, la doxorubicine, la daunorubicine, l’actinomycine D, et le 5-fluorouracile, et d’agents connus pour provoquer la production d’espèces réactives de l’oxygène (ROS), dont le peroxyde d'hydrogène, le 2-phényléthyle isothiocyanate, le β-lapachone (β-lap) et la ménadione. Nous avons mis en évidence qu’une diminution de Prx1 augmente significativement la sensibilité des cellules à l'effet cytotoxique de la β-lap et de la ménadione, deux naphtoquinones possédant une activité anti-tumorale.Nous avons étudié les mécanismes responsables de l'augmentation de la cytotoxicité de la β-lap dans un contexte Prx1–. Nous montrons que la toxicité accrue de la β-lap dans des cellules Prx1– est due à une accumulation intracellulaire de ROS. Cet effet est dépendant de l’activité NADPH quinone oxydoréductase (NQO1) et s’accompagne d’une phosphorylation de c-Jun N-terminal kinases (JNK), protein 38 (p38), extracellular signal-regulated kinases (Erk) et des mitogen-activated protein kinases (MAPK), mais aussi d’une diminution des niveaux protéiques de la thiorédoxine 1. En se basant sur le fait que Prx1 est une enzyme antioxydante et un partenaire d'au moins ASK1 et JNK, deux éléments clés de la voie MAPK, nous proposons que la sensibilisation à la β-lap, observée après diminution de Prx1, est provoquée par une action synergique entre l'accumulation de ROS et l'induction de la voie MAPK, conduisant ainsi à l'apoptose.Nous avons ensuite étudié les mécanismes responsables de l'augmentation de la cytotoxicité de la ménadione dans le contexte Prx1–. La sensibilité accrue des cellules à l'effet cytotoxique de la ménadione et également associée à l'accumulation rapide et massive des ROS intracellulaire et à une mort cellulaire ressemblant à la nécrose programmée (necroptosis). L’accumulation de ROS induite par la ménadione et très rapidement détectée dans le cytosol, le noyau, et de façon encore plus importante, dans la matrice mitochondriale. Ce phénomène est en corrélation avec l'oxydation importante des thiorédoxine 2 et peroxiredoxine 3, deux protéines antioxydantes localisées dans la mitochondrie. La diminution de l’expression de Prx1 s’accompagne d’une augmentation des quantités tant de l’ARNm que de la protéine NRH: quinone oxydoréductase 2 (NQO2). Cette augmentation de l'activité de NQO2 est en grande partie responsable de l'accumulation intracellulaire de ROS et de la mort cellulaire après le traitement à la ménadione. Nos données révèlent que l’accumulation de ROS dans les cellules Prx1– provient de la résultante entre l’augmentation de leur production par NQO2 au cours du métabolisme de la ménadione et la diminution de leur élimination par Prx1. Enfin et de façon surprenante, selon la nature des naptoquinones (β-lap ou ménadione), les voies métaboliques qui conduisent à l'accumulation des ROS, ou les voies de signalisation et les mécanismes de mort cellulaire impliqués semblent être distincts. / Peroxiredoxins have multiple cellular functions as major antioxidants, signaling regulators, molecular chaperones and tumor suppressors. Peroxiredoxin 1 (Prx1) is the most abundant among the six isoforms of human peroxiredoxins. It is frequently over-expressed in various cancer cells, which is known associated with carcinogenesis, metastasis and resistance to radiotherapy or chemotherapy. Prx1 could thus be an interesting anticancer target. In this study, we first evaluated the impact of Prx1 knockdown (Prx1–) on cellular sensitivity to dozens of anticancer drugs including vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D, and 5-fluorouracil, and of reactive oxygen species (ROS)-generating agents, including hydrogen peroxide, 2-phenylethyl isothiocyanate, β-lapachone (β-lap) and menadione. We observed that Prx1 knockdown significantly enhanced cancer cell sensitivity to β-lap and menadione, two naphthoquinones with anti-cancer activity.We first investigated the underlying mechanisms responsible for the specifically enhanced cytotoxicity to β-lap in a Prx1 knockdown context. Prx1 knockdown markedly potentiated β-lap-induced cytotoxicity through ROS accumulation. This effect was largely NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent and associated with the phosphorylation of c-Jun N-terminal kinases (JNK), protein 38 (p38) and extracellular signal-regulated kinases (Erk) proteins in mitogen-activated protein kinase (MAPK) pathways, and a decrease in thioredoxin 1 protein levels. Based on the fact that Prx1 is a major ROS scavenger and a partner of apoptosis signaling kinase 1 (ASK1) and JNK, two key components of MAPK pathways, we propose that Prx1 knockdown-induced sensitization to β-lap is achieved through the combined action of ROS accumulation and MAPK pathway activation, leading to cell apoptosis.We then investigated the underlying mechanisms responsible for the specifically enhanced cytotoxicity to menadione in Prx1– cells. Enhanced sensitivity to menadione was associated with a rapid and significant intracellular ROS accumulation and necroptotic-like cell death. Menadione-induced ROS accumulation occurred immediately in the cytosol, the nucleus, and even more noticeably in the mitochondrial matrix, correlated with significant oxidation of both mitochondria-localized thioredoxin 2 and peroxiredoxin 3. Prx1 knockdown significantly up-regulated mRNA and protein levels of NRH: quinone oxidoreductase 2 (NQO2). Increased activity of NQO2 was largely responsible for menadione-induced ROS accumulation and consequent cell death. Our data indicate that massive ROS accumulation results from the combined effect of increased ROS generation by higher NQO2 activity during menadione metabolism, and diminished Prx1 scavenging activity. Finally and noteworthy, the metabolic pathways that lead to ROS accumulation, downstream signaling pathways and cell death mechanisms appear to be distinct for β-lap and menadione.

Le diabète de type 2 (DT2) est une maladie métabolique complexe causée par des facteurs génétiques mais aussi environnementaux, tels la sédentarité et le surpoids. La dysfonction de la cellule β pancréatique est maintenant reconnue comme l’élément déterminant dans le développement du DT2. Notre laboratoire s’intéresse à la sécrétion d’insuline par la cellule β en réponse aux nutriments calorigéniques et aux mécanismes qui la contrôle. Alors que la connaissance des mécanismes responsables de l’induction de la sécrétion d’insuline en réponse aux glucose et acides gras est assez avancée, les procédés d’inhibition de la sécrétion dans des contextes normaux ou pathologiques sont moins bien compris. L’objectif de la présente thèse était d’identifier quelques-uns de ces mécanismes de régulation négative de la sécrétion d’insuline dans la cellule β pancréatique, et ce en situation normale ou pathologique en lien avec le DT2. La première hypothèse testée était que l’enzyme mitochondriale hydroxyacyl-CoA déshydrogénase spécifique pour les molécules à chaîne courte (short-chain hydroxyacyl-CoA dehydrogenase, SCHAD) régule la sécrétion d’insuline induite par le glucose (SIIG) par la modulation des concentrations d’acides gras ou leur dérivés tels les acyl-CoA ou acyl-carnitine dans la cellule β. Pour ce faire, nous avons utilisé la technologie des ARN interférants (ARNi) afin de diminuer l’expression de SCHAD dans la lignée cellulaire β pancréatique INS832/13. Nous avons par la suite vérifié chez la souris DIO (diet-induced obesity) si une exposition prolongée à une diète riche en gras activerait certaines voies métaboliques et signalétiques assurant une régulation négative de la sécrétion d’insuline et contribuerait au développement du DT2. Pour ce faire, nous avons mesuré la SIIG, le métabolisme intracellulaire des lipides, la fonction mitochondriale et l’activation de certaines voies signalétiques dans les îlots de Langerhans isolés des souris normales (ND, normal diet) ou nourries à la dière riche en gras (DIO) Nos résultats suggèrent que l’enzyme SCHAD est importante dans l’atténuation de la sécrétion d’insuline induite par le glucose et les acides aminés. En effet, l’oxydation des acides gras par la protéine SCHAD préviendrait l’accumulation d’acyl-CoA ou de leurs dérivés carnitine à chaîne courtes potentialisatrices de la sécrétion d’insuline. De plus, SCHAD régule le métabolisme du glutamate par l’inhibition allostérique de l’enzyme glutamate déshydrogénase (GDH), prévenant ainsi une hyperinsulinémie causée par une sur-activité de GDH. L’étude de la dysfonction de la cellule β dans le modèle de souris DIO a démontré qu’il existe une grande hétérogénéité dans l’obésité et l’hyperglycémie développées suite à la diète riche en gras. L’orginialité de notre étude réside dans la stratification des souris DIO en deux groupes : les faibles et forts répondants à la diète (low diet responders (LDR) et high diet responder (HDR)) sur la base de leur gain de poids corporel. Nous avons mis en lumières divers mécanismes liés au métabolisme des acides gras impliqués dans la diminution de la SIIG. Une diminution du flux à travers le cycle TG/FFA accompagnée d’une augmentation de l’oxydation des acides gras et d’une accumulation intracellulaire de cholestérol contribuent à la diminution de la SIIG chez les souris DIO-HDR. De plus, l’altération de la signalisation par les voies AMPK (AMP-activated protein kinase) et PKC epsilon (protéine kinase C epsilon) pourrait expliquer certaines de ces modifications du métabolisme des îlots DIO et causer le défaut de sécrétion d’insuline. En résumé, nous avons mis en lumière des mécanismes importants pour la régulation négative de la sécrétion d’insuline dans la cellule β pancréatique saine ou en situation pathologique. Ces mécanismes pourraient permettre d’une part de limiter l’amplitude ou la durée de la sécrétion d’insuline suite à un repas chez la cellule saine, et d’autre part de préserver la fonction de la cellule β en retardant l’épuisement de celle-ci en situation pathologique. Certaines de ces voies peuvent expliquer l’altération de la sécrétion d’insuline dans le cadre du DT2 lié à l’obésité. À la lumière de nos recherches, le développement de thérapies ayant pour cible les mécanismes de régulation négative de la sécrétion d’insuline pourrait être bénéfique pour le traitement de patients diabétiques. / Type 2 diabetes (T2D) is a complex metabolic disease caused by genetic as well as environmental factors, such as sedentarity and obesity. Pancreatic β cell dysfunction is now recognized as the key factor in T2D development. Our laboratory is studying the mechanisms of regulation of insulin secretion by the pancreatic β cell in response to nutrients. While the knowledge of the mechanisms responsible for initiation of insulin secretion in response to glucose and fatty acids is quite advanced, the inhibitory processes of insulin secretion in normal or pathological situations are still poorly understood. This doctoral thesis has focused on the identification of some of the mechanisms responsible for negative regulation of insulin secretion in pancreatic β cell. We have addressed this issue under normal situation or pathological conditions related to T2D. We first tested the hypothesis by which a mitochondrial enzyme, short-chain hydroxyacyl-CoA dehydrogenase (SCHAD), negatively regulates glucose-induced insulin secretion (GIIS) by limiting the concentrations of some fatty acids and their derivatives such as acyl-CoA or acyl-carnitine molecules in the β cell. For this purpose, the downregulation of SCHAD by RNA interference (RNAi) was used in the pancreatic β cell line INS832/13. Then, we tested wether a prolonged administration of high-fat diet to mice (diet-induced obesity mouse model, DIO) would modulate intracellular metabolic and molecular pathways responsible for inhibition of insulin secretion. C57BL/6 mice were therefore fed a high-fat diet for 8 weeks followed by insulin secretion, intracellular lipid metabolism, mitochondrial function and intracellular signaling measurements on isolated pancreatic islets of Langerhans of those mice. Our results suggest that SCHAD negatively regulates GIIS and amino acid-induced insulin secretion. We propose that fatty acid oxidation by SCHAD would prevent the accumulation of short-chain acyl-CoAs or acyl-carnitines capable of potentiating insulin secretion. In addition, SCHAD regulates glutamate metabolism by the allosteric inhibition of glutamate dehydrogenase (GDH) preventing the hyperinsulinemia caused by excessive GDH activity. The study of β cell dysfunction in the DIO mouse model stratified LDR and HDR highlighted various fatty acid metabolism pathways involved in the reduction of GIIS. A decrease in the triglycerides/free fatty acid (TG/FFA) cycling associated with an increase in fatty acid oxidation and intracellular accumulation of cholesterol was shown to contribute to the decreased GIIS in DIO-HDR mice. Furthermore, alteration of AMP-activated kinase (AMPK) and protein kinase C epsilon (PKC epsilon) signaling pathways would be responsible for those alterations in metabolic pathways observed in DIO islets and cause decreased insulin secretion. In summary, we have shed light on important pathways negatively regulating insulin secretion in pancreatic β cell. These pathways could either limit the amplitude or duration of insulin secretion after a meal, or help to preserve β-cell function by delaying exhaustion. Some of those signaling pathways could explain the altered insulin secretion observed in T2D obese patients. In light of our research, the development of therapies targeting pathways that negatively regulate insulin secretion may be beneficial for treating diabetic patients.