Le diabète de type 2 (DT2) est caractérisé par une résistance des tissus périphériques à l’action de l’insuline et par une insuffisance de la sécrétion d’insuline par les cellules β du pancréas. Différents facteurs tels que le stress du réticulum endoplasmique (RE) et l’immunité innée affectent la fonction de la cellule β-pancréatique. Toutefois, leur implication dans la régulation de la transcription du gène de l’insuline demeure imprécise. Le but de cette thèse était d’identifier et de caractériser le rôle du stress du RE et de l’immunité innée dans la régulation de la transcription du gène de l’insuline. Les cellules β-pancréatiques ont un RE très développé, conséquence de leur fonction spécialisée de biosynthèse et de sécrétion d’insuline. Cette particularité les rend très susceptible au stress du RE qui se met en place lors de l’accumulation de protéines mal repliées dans la lumière du RE. Nous avons montré qu’ATF6 (de l’anglais, activating transcription factor 6), un facteur de transcription impliqué dans la réponse au stress du RE, lie directement la boîte A5 de la région promotrice du gène de l’insuline dans les îlots de Langerhans isolés de rat. Nous avons également montré que la surexpression de la forme active d’ATF6α, mais pas ATF6β, réprime l’activité du promoteur de l’insuline. Toutefois, la mutation ou l’absence de la boîte A5 ne préviennent pas l’inhibition de l’activité promotrice du gène de l’insuline par ATF6. Ces résultats montrent qu’ATF6 se lie directement au promoteur du gène de l’insuline, mais que cette liaison ne semble pas contribuer à son activité répressive. Il a été suggéré que le microbiome intestinal joue un rôle dans le développement du DT2. Les patients diabétiques présentent des concentrations plasmatiques élevées de lipopolysaccharides (LPS) qui affectent la fonction de la cellule β-pancréatique. Nous avons montré que l’exposition aux LPS entraîne une réduction de la transcription du gène de l’insuline dans les îlots de Langerhans de rats, de souris et humains. Cette répression du gène de l’insuline par les LPS est associée à une diminution des niveaux d’ARNms de gènes clés de la cellule β-pancréatique, soit PDX-1 (de l’anglais, pancreatic duodenal homeobox 1) et MafA (de l’anglais, mammalian homologue of avian MafA/L-Maf). En utilisant un modèle de souris déficientes pour le récepteur TLR4 (de l’anglais, Toll-like receptor), nous avons montré que les effets délétères des LPS sur l’expression du gène de l’insuline sollicitent le récepteur de TLR4. Nous avons également montré que l’inhibition de la voie NF-kB entraîne une restauration des niveaux messagers de l’insuline en réponse à une exposition aux LPS dans les îlots de Langerhans de rat. Ainsi, nos résultats montrent que les LPS inhibent le gène de l’insuline dans les cellules β-pancréatiques via un mécanisme moléculaire dépendant du récepteur TLR4 et de la voie NF-kB. Ces observations suggèrent ainsi un rôle pour le microbiome intestinal dans la fonction de la cellule β du pancréas. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la répression du gène de l'insuline en réponse aux divers changements survenant de façon précoce dans l’évolution du diabète de type 2 et d'identifier des cibles thérapeutiques potentielles qui permettraient de prévenir ou ralentir la détérioration de l'homéostasie glycémique au cours de cette maladie, qui affecte plus de deux millions de Canadiens. / Type 2 diabetes is characterized by insulin resistance and impaired insulin secretion from the pancreatic β-cell. Endoplasmic reticulum (ER) stress and innate immunity have both been reported to alter pancreatic β-cell function. However, it is not clear whether these factors can affect the transcription of the insulin gene. The aim of this thesis was to assess the role of ER stress and innate immunity in the regulation of the insulin gene. Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. In a first study, using several approaches we showed that ATF6 (activating transcription factor 6), a protein implicated in the ER stress response, directly binds to the A5/Core of the insulin gene promoter in isolated rat islets. We also showed that overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibits the activity of an insulin promoter-reporter construct. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity. In recent years, the gut microbiota was proposed has an environmental factor increasing the risk of type 2 diabetes. Subjects with diabetes have higher circulating levels of lipopolysaccharides (LPS) than non-diabetic patients. Recent observations suggest that the signalling cascade activated by LPS binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. We showed that exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of two key transcription factors of the insulin gene, PDX-1 (pancreatic duodenal homeobox 1) and MafA (mammalian homologue of avian MafA/L-Maf). LPS repression of insulin, PDX-1 and MafA expression was not observed in islets from TLR4-deficient mice and was completely prevented in rat islets by inhibition of the NF-kB signalling pathway. These results demonstrate that LPS inhibits β-cell gene expression in a TLR4-dependent manner and via NF-kB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function. Our findings provide a better understanding of the molecular mechanisms underlying insulin gene repression in type 2 diabetes, and suggest potential therapeutic targets that might prevent or delay the decline of β-cell function in the course of type 2 diabetes, which affects more than two million Canadians.

Produtos de glicação avançada, carbamilação e estresse oxidativo contribuem como fatores de risco não tradicionais para a aterosclerose na doença renal crônica (DRC), em parte, por prejudicarem o metabolismo lipídico e por representarem um mecanismo de injúria memorizado ao longo do desenvolvimento da doença renal. A albumina sérica, isolada de animais com DRC, reduz a remoção de colesterol mediado por apoA-I e subfrações de HDL, prejudicando o fluxo de colesterol de macrófagos arteriais ao fígado por meio do transporte reverso de colesterol. Objetivo: Avaliou-se a influência do tratamento com N-acetilcisteína (NAC) em ratos com DRC sobre a concentração plasmática de produtos de oxidação e glicação avançada e o reflexo sobre os efeitos da albumina sérica sobre o efluxo de colesterol e o estresse de retículo endoplasmático em macrófagos. Métodos: Ratos Wistar com 2 meses de idade, pesando aproximadamente 200-250g foram submetidos à nefrectomia 5/6 e mantidos por 60 dias (grupo DRC) com ou sem tratamento com N-acetilcisteína na água (600mg/L), após o 7° dia de indução da DRC (grupo DRC + NAC). Animais controles foram falso-operados (grupo C) e um subgrupo submetido ao tratamento com NAC (C + NAC). No início e no final do estudo foram determinadas as concentrações plasmáticas de glicose, colesterol (CT), triglicérides (TG), ureia, creatinina e na urina, excreção urinária de proteína de 24 h. AGE totais, pentosidina, TBARS (marcador de peroxidação lipídica) e pressão arterial sistólica (PAS) foram determinados no final do estudo. A albumina sérica foi isolada por cromatografia rápida para separação de proteínas e purificada por extração alcoólica. Macrófagos J774 foram incubados por 18 h com as albuminas dos diferentes grupos experimentais para determinação do conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) e de marcadores de estresse de retículo endoplasmático (chaperonas Grp 78, Grp94 e proteína dissulfeto isomerase, PDI) por imunolbot e efluxo de colesterol, mediado por apo A-I e HDL2. Para isto, as células foram previamente enriquecidas com LDL-acetilada e 14C-colesterol. Macrófagos foram também incubados isoladamente com concentrações crescentes de NAC para avaliação do conteúdo dos receptores de HDL. Resultados: Ao final do estudo, o peso corporal foi 10% menor no grupo DRC em comparação ao C (p=0,006). Esta alteração foi prevenida pelo tratamento com NAC. A PAS (mmHg) foi maior no grupo DRC (130 ± 3) em comparação ao grupo DRC+NAC (109±3; p=0,0004). Ureia, creatinina, CT, TG (mg/dL), proteinúria (mg/24 h), AGE total, pentosidina (unidades arbitrárias de fluorescência) e TBARS (nmol/mL) foram maiores nos grupos DRC em comparação ao grupo C (122 ± 8 vs. 41 ± 0,9 ; 0,9 ± 0,07 vs. 0,4 ± 0,03; 151 ± 6 vs. 76 ± 2,7; 83 ± 4 vs. 51,5 ± 3; 46 ± 2,5 vs. 14 ± 0,9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6,6 ± 0,5 vs. 2 ± 0,2, respectivamente) (p < 0,0001) e nos grupos DRC+NAC em comparação ao grupo C+NAC (91,4 ± 5 vs. 40 ± 0,9 ; 0,6 ± 0,02 vs. 0,3 ± 0,02; 126 ± 7,5 vs. 76 ± 2,6; 73 ± 6 vs. 68 ± 4; 51 ± 3,5 vs. 18,4 ± 1,5; 24720 ± 1114 vs. 20040 ± 700; 10080 ± 748 vs. 5050 ± 267; 4,5 ± 0,5 vs. 1,8 ± 0,2, respectivamente) (p < 0,0001). No grupo DRC + NAC, PAS, CT, ureia, creatinina, AGE total, pentosidina e TBARS foram, respectivamente, 17% (p=0,0004), 17% (p=0,02), 25% (p=0,02), 33% (p=0,06), 24% (p < 0,0001), 40% (p=0,0008), 28% (p=0,009) menores do que no grupo DRC. A glicemia foi maior nos grupos C + NAC (107+-4,6) e DRC + NAC (107+-2,6) em comparação ao C (96+-1,8) e DRC (98+-1,6), respectivamente. Macrófagos tratados com albumina-DRC apresentaram maior conteúdo de PDI (5 vezes; p=0,02 e 7 vezes p=0,02) e Grp94 (66 %; p =0,02 e 20 %; p=0,02) quando comparados aos tratados com albumina-C ou albumina-DRC + NAC, respectivamente. O conteúdo do receptor ABCA-1 foi menor 87% e 70% (p < 0,01) nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação com albumina-C. O conteúdo de ABCG-1 foi, respectivamente, 4 e 7,5 vezes maior nos macrófagos tratados com albumina-C+NAC e albumina-DRC+NAC em comparação as respectivas situações sem tratamento. O efluxo de colesterol mediado por apo A-I foi 59 % e 70 % (p < 0,0001) menor nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação a albumina-C. O efluxo de colesterol mediado pela HDL2 foi 52 % maior nos macrófagos tratados com albumina-C+NAC em comparação as células tratadas com albumina-C. Não houve diferença no conteúdo do receptor ABCA-1 nos macrófagos tratados com concentrações crescentes NAC por 8 h. No entanto, após 18 h, o ABCA-1 diminuiu 50 %, 69 % e 72 % nos macrófagos tratados respectivamente com 10 mM, 20 mM e 30 mM de NAC isoladamente em comparação aos macrófagos controles. O conteúdo de ABCG-1 nos macrófagos tratados com NAC, em 8 h e 18 h não sofreu alteração. Conclusão: A N-acetilcisteína reduz produtos de oxidação e glicação avançada no plasma de animais com DRC e previne o estresse de RE em macrófagos, induzido pela albumina isolada destes animais. Apesar de diminuir o conteúdo de ABCA-1 e o efluxo de colesterol mediado por apo A-I, a NAC aumenta o conteúdo de ABCG-1. Desta forma, a NAC pode contribuir para atenuar os efeitos deletérios da albumina modificada na DRC sobre o acúmulo lipídico em macrófagos, contribuindo para a prevenção da aterosclerose / Advanced glycation, carbamylation and oxidative stress c contribute to atherosclerosis in chronic kidney disease (CKD) as nontraditional risk factors. They impair lipid metabolism and promote a long last injury during the development of CKD. Serum albumin isolated from CKD-animals reduces cholesterol efflux mediated by apoa A-I and HDl subfractions, impairing the cholesterol flux from arterial wall macrophage to the the liver by the reverse cholesterol transport (RCT).Objective: In the present study it was analyzed the influence of N-acetylcysteine treatment in CKD-rats in plasma concentration of lipid peroxides and advanced glycation end products and the effect of serum albumin in macrophage cholesterol efflux and endoplasmic reticulum stress development. Methods: Two months male Wistar weighting 200-250g were submitted to a 5/6 nephrectomized maintained for 60 days (CKD group) treated or not with N-acetylcysteine in water (600 mg/L), after the seventh day of CKD induction (CKD+NAC group). Sham animals were false-operated (SHAM group) and a subgroup was treated with NAC (SHAM+NAC group). In the basal and final periods it was determined plasma concentration of glucose, total cholesterol (TC), triglycerides (TG), urea, creatinine and 24h-urinary protein excretion (UPE). Total AGE, pentosidine, thiobarbituric reactive substances (TBARS) levels and systolic blood pressure (SBP) were measured at the final period only. Serum albumin was isolated by fast protein liquid chromatography and purified by alcoholic extraction. J774 macrophage were incubated for 18 h with albumin isolated from the experimental groups in order to determine the content of HDL receptors and markers of endoplasmic reticulum stress (Grp78, Grp94 and protein dissulfide isomerase, PDI) by immunioblot and cholesterol efflux mediated by apo A-I and HDL2. For this, cells were previously overloaded with acetylated LDL and 14C-cholesterol. Macrophage were also incubated with different concentrations of NAC alone in order to measure HDL-receptors and cholesterole efflux. Results: In the end of the protocol, body weight was 10% lower in CKD group in comparison to SHAM group (p=0.006). This change was preserved by treatment with NAC. SBP (mmHg) was higher in CKD group (130±3) in comparison to CKD+NAC (109±3; p=0.0004). Urea, creatinine, TC, TG (mg/dL), UPE (mg/24 h), total AGE, pentosidine (arbitrary units of fluorescence) and TBARS (nmol/mL) were higher in CKD group in comparison to SHAM (122±8 vs. 41 ± 0.9; 0.9 ± 0.07 vs. 0.4 ± 0.03; 151 ± 6 vs. 76±2.7; 83 ± 4 vs. 51.5 ± 3; 46 ± 2.5 vs. 14 ± 0.9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6.6 ± 0.5 vs. 2 ± 0.2, respectively) (p < 0.0001) and in CKD+NAC in comparison to C+NAC (91.4±5 vs. 40±0.9 ; 0.6±0.02 vs. 0.3 ± 0.02; 126±7.5 vs. 76 ± 2.6; 73±6 vs. 68±4; 51 ± 3.5 vs. 18.4±1.5; 24720 ± 1114 vs. 20040±700; 10080±748 vs. 5050 ± 267; 4.5±0.5 vs. 1.8±0.2, respectively) (p < 0.0001). In CKD+NAC group, SBP, TC, urea, creatinine, total AGE, pentosidine and TBARS were, respectively, 17 % (p=0.0004), 17 % (p=0.02), 25 % (p=0.02), 33 % (p=0.06), 24 % (p<0.0001), 40 % (p=0.0008), 28 % (p=0.009) lower than CKD group. Glycemia was higher in SHAM+NAC (107+-4.6) and CKD+NAC (107+-2.6) in comparison to SHAM (96+-1.8) and CKD group (98+-1.6), respectively. Macrophages treat with CKD-albumin presented higher content of PDI (5 times; p=0.02 e 7 times p=0.02) and Grp94 (66 %; p=0.02 e 20 %; p=0.02) when compared to SHAM-albumin and CKD+NAC-albumin- treated cells, respectively. ABCA-1 protein content was 87 % and 70 % (p < 0.01) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively compared with SHAM-albumin. ABCG-1 protein level was respectively 4 and 7.5 times higher in macrophages treated with SHAM+NAC-albumin and CKD+NAC-albumin in comparison to their respective controls without treatment. The cholesterol efflux mediated by apo A-I was 59 % and 70 % (p < 0.0001) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively, when compared to SHAM-albumin. The HDL2-mediated cholesterol efflux was 52 % higher in macrophages treated with SHAM+NAC-albumin compared to macrophages treated with SHAM-albumin. No difference was observed in the ABCA-1 protein level in macrophages treated with crescent concentrations of NAC alone for 8 h. Nonetheless, after 18 h, ABCA-1 was 50 %, 69 % and 72 % reduced in macrophages treated, respectively, with 10 mM, 20 mM and 30 mM NAC in comparison to control cells. ABCG-1 content in macrophages treated with NAC for 8 h and 18 h was not changed. Conclusion: NAC reduces plasma lipid peroxidation and AGE in CKD animals and prevents the endoplasmic reticulum stress induced by CKD-albumin in macrophages. Despite diminishing ABCA-1 and apo A-I-mediated cholesterol efflux, NAC increases ABCG-1. Then, NAC may contribute to attenuate the deleterious effects of the in vivo modified albumin on lipid accumulation in macrophages helping to prevent atherosclerosis in CKD

O remodelamento vascular é um determinante fundamental do lúmen em doenças vasculares, porém os mecanismos envolvidos não estão completamente elucidados. Nós investigamos o papel da chaperona redox residente do retículo endoplasmático Dissulfeto Isomerase Proteica (PDI) e sua fração localizada na superfície celular (peri/epicelular=pecPDI) no calibre e arquitetura vascular durante reparação à lesão. Em artérias ilíacas de coelho submetidas à lesão in vivo, houve importante aumento do mRNA e expressão proteica (~25x aumento 14 dias pós-lesão vs. controle) da PDI. O silenciamento da PDI por siRNA (cultura de órgãos) acentuou o estresse do retículo e apoptose, diferentemente da inibição da pecPDI com anticorpo neutralizante (PDI Ab). Bloqueio in vivo da pecPDI por aplicação de gel perivascular contendo PDI Ab no 12° dia após lesão, com análise após 48 h, promoveu ca.25% redução no calibre vascular analisado por arteriografia e diminuição similar na área total do vaso detectada por tomografia de coerência óptica. Neste processo, não ocorreu alteração no tamanho da neoíntima, indicando assim, que PDI Ab acentuou remodelamento constrictivo. Neutralização da pecPDI promoveu importantes alterações na arquitetura da matriz de colágeno e citoesqueleto, resultando em fibras com orientação invertida e desorganizadas. Diminuição na produção de espécies reativas de oxigênio e óxidos de nitrogênio também ocorreu. Análise de propriedades viscoelásticas nas artérias indicou redução na ductilidade vascular, evidenciada pela menor distância para ruptura. As alterações subcelulares no citoesqueleto observadas in vivo após PDI Ab foram recapituladas em um modelo de estiramento cíclico em células musculares lisas vasculares, com importante redução na formação das fibras de estresse. Em modelo de migração randômica de células musculares lisas, a exposição a PDI Ab reduziu a resiliência de regulação da polaridade. Embora a neutralização da pecPDI não tenha afetado a atividade global de RhoA, ela promoveu alterações no padrão de marcação em resposta ao estiramento, na redistribuição de RhoA na superfície celular e na associação com regiões contendo caveolina. Além disso, em aterosclerose nativa em humanos, a expressão da PDI correlacionou-se inversamente com remodelamento constrictivo. Dessa forma, PDI é fortemente expressa após a lesão e sua fração peri/epicelular remodela a arquitetura da matriz e citoesqueleto, promovendo um efeito anti-remodelamento constrictivo / Whole-vessel remodeling is a critical lumen caliber determinant in vascular disease, but underlying mechanisms are poorly understood. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase(PDI) and cell-surface PDI(peri/epicellular=pecPDI) pool in vascular caliber and architecture during vascular repair after injury(AI). After rabbit iliac artery balloon injury, there was marked increase in PDI mRNA and protein (25-fold vs. basal at day 14AI), with increase in both intracellular and pecPDI. Silencing PDI by siRNA (organ culture) induced ER stress augmentation and apoptosis, contrarily to pecPDI neutralization with PDI-antibody(PDI Ab). PecPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted ca.25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. PecPDI neutralization promoted marked changes in collagen and cytoskeleton architecture, with inverted fiber orientation and disorganization. Decreased ROS and nitrogen oxide production also occurred. Viscoelastic artery properties assessment showed decreased ductility, evidenced by decreased distance to rupture. Subcellular cytoskeletal disruption by PDI Ab was recapitulated in vascular smooth muscle cell stretch model, with marked decrease in stress fiber buildup. Also, PDI Ab incubation promoted decreased regulation resilience of vascular smooth muscle migration properties. While pecPDI neutralization did not affect global RhoA activity, there was altered RhoA redistribution to the cell surface and association with caveolin-containing clusters, which mislocalized after stretch. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. Thus, strongly-expressed PDI after injury reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect

Le diabète de type 2 (DT2) est caractérisé par une résistance des tissus périphériques à l’action de l’insuline et par une insuffisance de la sécrétion d’insuline par les cellules β du pancréas. Différents facteurs tels que le stress du réticulum endoplasmique (RE) et l’immunité innée affectent la fonction de la cellule β-pancréatique. Toutefois, leur implication dans la régulation de la transcription du gène de l’insuline demeure imprécise. Le but de cette thèse était d’identifier et de caractériser le rôle du stress du RE et de l’immunité innée dans la régulation de la transcription du gène de l’insuline. Les cellules β-pancréatiques ont un RE très développé, conséquence de leur fonction spécialisée de biosynthèse et de sécrétion d’insuline. Cette particularité les rend très susceptible au stress du RE qui se met en place lors de l’accumulation de protéines mal repliées dans la lumière du RE. Nous avons montré qu’ATF6 (de l’anglais, activating transcription factor 6), un facteur de transcription impliqué dans la réponse au stress du RE, lie directement la boîte A5 de la région promotrice du gène de l’insuline dans les îlots de Langerhans isolés de rat. Nous avons également montré que la surexpression de la forme active d’ATF6α, mais pas ATF6β, réprime l’activité du promoteur de l’insuline. Toutefois, la mutation ou l’absence de la boîte A5 ne préviennent pas l’inhibition de l’activité promotrice du gène de l’insuline par ATF6. Ces résultats montrent qu’ATF6 se lie directement au promoteur du gène de l’insuline, mais que cette liaison ne semble pas contribuer à son activité répressive. Il a été suggéré que le microbiome intestinal joue un rôle dans le développement du DT2. Les patients diabétiques présentent des concentrations plasmatiques élevées de lipopolysaccharides (LPS) qui affectent la fonction de la cellule β-pancréatique. Nous avons montré que l’exposition aux LPS entraîne une réduction de la transcription du gène de l’insuline dans les îlots de Langerhans de rats, de souris et humains. Cette répression du gène de l’insuline par les LPS est associée à une diminution des niveaux d’ARNms de gènes clés de la cellule β-pancréatique, soit PDX-1 (de l’anglais, pancreatic duodenal homeobox 1) et MafA (de l’anglais, mammalian homologue of avian MafA/L-Maf). En utilisant un modèle de souris déficientes pour le récepteur TLR4 (de l’anglais, Toll-like receptor), nous avons montré que les effets délétères des LPS sur l’expression du gène de l’insuline sollicitent le récepteur de TLR4. Nous avons également montré que l’inhibition de la voie NF-kB entraîne une restauration des niveaux messagers de l’insuline en réponse à une exposition aux LPS dans les îlots de Langerhans de rat. Ainsi, nos résultats montrent que les LPS inhibent le gène de l’insuline dans les cellules β-pancréatiques via un mécanisme moléculaire dépendant du récepteur TLR4 et de la voie NF-kB. Ces observations suggèrent ainsi un rôle pour le microbiome intestinal dans la fonction de la cellule β du pancréas. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la répression du gène de l'insuline en réponse aux divers changements survenant de façon précoce dans l’évolution du diabète de type 2 et d'identifier des cibles thérapeutiques potentielles qui permettraient de prévenir ou ralentir la détérioration de l'homéostasie glycémique au cours de cette maladie, qui affecte plus de deux millions de Canadiens. / Type 2 diabetes is characterized by insulin resistance and impaired insulin secretion from the pancreatic β-cell. Endoplasmic reticulum (ER) stress and innate immunity have both been reported to alter pancreatic β-cell function. However, it is not clear whether these factors can affect the transcription of the insulin gene. The aim of this thesis was to assess the role of ER stress and innate immunity in the regulation of the insulin gene. Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. In a first study, using several approaches we showed that ATF6 (activating transcription factor 6), a protein implicated in the ER stress response, directly binds to the A5/Core of the insulin gene promoter in isolated rat islets. We also showed that overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibits the activity of an insulin promoter-reporter construct. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity. In recent years, the gut microbiota was proposed has an environmental factor increasing the risk of type 2 diabetes. Subjects with diabetes have higher circulating levels of lipopolysaccharides (LPS) than non-diabetic patients. Recent observations suggest that the signalling cascade activated by LPS binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. We showed that exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of two key transcription factors of the insulin gene, PDX-1 (pancreatic duodenal homeobox 1) and MafA (mammalian homologue of avian MafA/L-Maf). LPS repression of insulin, PDX-1 and MafA expression was not observed in islets from TLR4-deficient mice and was completely prevented in rat islets by inhibition of the NF-kB signalling pathway. These results demonstrate that LPS inhibits β-cell gene expression in a TLR4-dependent manner and via NF-kB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function. Our findings provide a better understanding of the molecular mechanisms underlying insulin gene repression in type 2 diabetes, and suggest potential therapeutic targets that might prevent or delay the decline of β-cell function in the course of type 2 diabetes, which affects more than two million Canadians.

Produtos de glicação avançada, carbamilação e estresse oxidativo contribuem como fatores de risco não tradicionais para a aterosclerose na doença renal crônica (DRC), em parte, por prejudicarem o metabolismo lipídico e por representarem um mecanismo de injúria memorizado ao longo do desenvolvimento da doença renal. A albumina sérica, isolada de animais com DRC, reduz a remoção de colesterol mediado por apoA-I e subfrações de HDL, prejudicando o fluxo de colesterol de macrófagos arteriais ao fígado por meio do transporte reverso de colesterol. Objetivo: Avaliou-se a influência do tratamento com N-acetilcisteína (NAC) em ratos com DRC sobre a concentração plasmática de produtos de oxidação e glicação avançada e o reflexo sobre os efeitos da albumina sérica sobre o efluxo de colesterol e o estresse de retículo endoplasmático em macrófagos. Métodos: Ratos Wistar com 2 meses de idade, pesando aproximadamente 200-250g foram submetidos à nefrectomia 5/6 e mantidos por 60 dias (grupo DRC) com ou sem tratamento com N-acetilcisteína na água (600mg/L), após o 7° dia de indução da DRC (grupo DRC + NAC). Animais controles foram falso-operados (grupo C) e um subgrupo submetido ao tratamento com NAC (C + NAC). No início e no final do estudo foram determinadas as concentrações plasmáticas de glicose, colesterol (CT), triglicérides (TG), ureia, creatinina e na urina, excreção urinária de proteína de 24 h. AGE totais, pentosidina, TBARS (marcador de peroxidação lipídica) e pressão arterial sistólica (PAS) foram determinados no final do estudo. A albumina sérica foi isolada por cromatografia rápida para separação de proteínas e purificada por extração alcoólica. Macrófagos J774 foram incubados por 18 h com as albuminas dos diferentes grupos experimentais para determinação do conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) e de marcadores de estresse de retículo endoplasmático (chaperonas Grp 78, Grp94 e proteína dissulfeto isomerase, PDI) por imunolbot e efluxo de colesterol, mediado por apo A-I e HDL2. Para isto, as células foram previamente enriquecidas com LDL-acetilada e 14C-colesterol. Macrófagos foram também incubados isoladamente com concentrações crescentes de NAC para avaliação do conteúdo dos receptores de HDL. Resultados: Ao final do estudo, o peso corporal foi 10% menor no grupo DRC em comparação ao C (p=0,006). Esta alteração foi prevenida pelo tratamento com NAC. A PAS (mmHg) foi maior no grupo DRC (130 ± 3) em comparação ao grupo DRC+NAC (109±3; p=0,0004). Ureia, creatinina, CT, TG (mg/dL), proteinúria (mg/24 h), AGE total, pentosidina (unidades arbitrárias de fluorescência) e TBARS (nmol/mL) foram maiores nos grupos DRC em comparação ao grupo C (122 ± 8 vs. 41 ± 0,9 ; 0,9 ± 0,07 vs. 0,4 ± 0,03; 151 ± 6 vs. 76 ± 2,7; 83 ± 4 vs. 51,5 ± 3; 46 ± 2,5 vs. 14 ± 0,9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6,6 ± 0,5 vs. 2 ± 0,2, respectivamente) (p < 0,0001) e nos grupos DRC+NAC em comparação ao grupo C+NAC (91,4 ± 5 vs. 40 ± 0,9 ; 0,6 ± 0,02 vs. 0,3 ± 0,02; 126 ± 7,5 vs. 76 ± 2,6; 73 ± 6 vs. 68 ± 4; 51 ± 3,5 vs. 18,4 ± 1,5; 24720 ± 1114 vs. 20040 ± 700; 10080 ± 748 vs. 5050 ± 267; 4,5 ± 0,5 vs. 1,8 ± 0,2, respectivamente) (p < 0,0001). No grupo DRC + NAC, PAS, CT, ureia, creatinina, AGE total, pentosidina e TBARS foram, respectivamente, 17% (p=0,0004), 17% (p=0,02), 25% (p=0,02), 33% (p=0,06), 24% (p < 0,0001), 40% (p=0,0008), 28% (p=0,009) menores do que no grupo DRC. A glicemia foi maior nos grupos C + NAC (107+-4,6) e DRC + NAC (107+-2,6) em comparação ao C (96+-1,8) e DRC (98+-1,6), respectivamente. Macrófagos tratados com albumina-DRC apresentaram maior conteúdo de PDI (5 vezes; p=0,02 e 7 vezes p=0,02) e Grp94 (66 %; p =0,02 e 20 %; p=0,02) quando comparados aos tratados com albumina-C ou albumina-DRC + NAC, respectivamente. O conteúdo do receptor ABCA-1 foi menor 87% e 70% (p < 0,01) nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação com albumina-C. O conteúdo de ABCG-1 foi, respectivamente, 4 e 7,5 vezes maior nos macrófagos tratados com albumina-C+NAC e albumina-DRC+NAC em comparação as respectivas situações sem tratamento. O efluxo de colesterol mediado por apo A-I foi 59 % e 70 % (p < 0,0001) menor nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação a albumina-C. O efluxo de colesterol mediado pela HDL2 foi 52 % maior nos macrófagos tratados com albumina-C+NAC em comparação as células tratadas com albumina-C. Não houve diferença no conteúdo do receptor ABCA-1 nos macrófagos tratados com concentrações crescentes NAC por 8 h. No entanto, após 18 h, o ABCA-1 diminuiu 50 %, 69 % e 72 % nos macrófagos tratados respectivamente com 10 mM, 20 mM e 30 mM de NAC isoladamente em comparação aos macrófagos controles. O conteúdo de ABCG-1 nos macrófagos tratados com NAC, em 8 h e 18 h não sofreu alteração. Conclusão: A N-acetilcisteína reduz produtos de oxidação e glicação avançada no plasma de animais com DRC e previne o estresse de RE em macrófagos, induzido pela albumina isolada destes animais. Apesar de diminuir o conteúdo de ABCA-1 e o efluxo de colesterol mediado por apo A-I, a NAC aumenta o conteúdo de ABCG-1. Desta forma, a NAC pode contribuir para atenuar os efeitos deletérios da albumina modificada na DRC sobre o acúmulo lipídico em macrófagos, contribuindo para a prevenção da aterosclerose / Advanced glycation, carbamylation and oxidative stress c contribute to atherosclerosis in chronic kidney disease (CKD) as nontraditional risk factors. They impair lipid metabolism and promote a long last injury during the development of CKD. Serum albumin isolated from CKD-animals reduces cholesterol efflux mediated by apoa A-I and HDl subfractions, impairing the cholesterol flux from arterial wall macrophage to the the liver by the reverse cholesterol transport (RCT).Objective: In the present study it was analyzed the influence of N-acetylcysteine treatment in CKD-rats in plasma concentration of lipid peroxides and advanced glycation end products and the effect of serum albumin in macrophage cholesterol efflux and endoplasmic reticulum stress development. Methods: Two months male Wistar weighting 200-250g were submitted to a 5/6 nephrectomized maintained for 60 days (CKD group) treated or not with N-acetylcysteine in water (600 mg/L), after the seventh day of CKD induction (CKD+NAC group). Sham animals were false-operated (SHAM group) and a subgroup was treated with NAC (SHAM+NAC group). In the basal and final periods it was determined plasma concentration of glucose, total cholesterol (TC), triglycerides (TG), urea, creatinine and 24h-urinary protein excretion (UPE). Total AGE, pentosidine, thiobarbituric reactive substances (TBARS) levels and systolic blood pressure (SBP) were measured at the final period only. Serum albumin was isolated by fast protein liquid chromatography and purified by alcoholic extraction. J774 macrophage were incubated for 18 h with albumin isolated from the experimental groups in order to determine the content of HDL receptors and markers of endoplasmic reticulum stress (Grp78, Grp94 and protein dissulfide isomerase, PDI) by immunioblot and cholesterol efflux mediated by apo A-I and HDL2. For this, cells were previously overloaded with acetylated LDL and 14C-cholesterol. Macrophage were also incubated with different concentrations of NAC alone in order to measure HDL-receptors and cholesterole efflux. Results: In the end of the protocol, body weight was 10% lower in CKD group in comparison to SHAM group (p=0.006). This change was preserved by treatment with NAC. SBP (mmHg) was higher in CKD group (130±3) in comparison to CKD+NAC (109±3; p=0.0004). Urea, creatinine, TC, TG (mg/dL), UPE (mg/24 h), total AGE, pentosidine (arbitrary units of fluorescence) and TBARS (nmol/mL) were higher in CKD group in comparison to SHAM (122±8 vs. 41 ± 0.9; 0.9 ± 0.07 vs. 0.4 ± 0.03; 151 ± 6 vs. 76±2.7; 83 ± 4 vs. 51.5 ± 3; 46 ± 2.5 vs. 14 ± 0.9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6.6 ± 0.5 vs. 2 ± 0.2, respectively) (p < 0.0001) and in CKD+NAC in comparison to C+NAC (91.4±5 vs. 40±0.9 ; 0.6±0.02 vs. 0.3 ± 0.02; 126±7.5 vs. 76 ± 2.6; 73±6 vs. 68±4; 51 ± 3.5 vs. 18.4±1.5; 24720 ± 1114 vs. 20040±700; 10080±748 vs. 5050 ± 267; 4.5±0.5 vs. 1.8±0.2, respectively) (p < 0.0001). In CKD+NAC group, SBP, TC, urea, creatinine, total AGE, pentosidine and TBARS were, respectively, 17 % (p=0.0004), 17 % (p=0.02), 25 % (p=0.02), 33 % (p=0.06), 24 % (p<0.0001), 40 % (p=0.0008), 28 % (p=0.009) lower than CKD group. Glycemia was higher in SHAM+NAC (107+-4.6) and CKD+NAC (107+-2.6) in comparison to SHAM (96+-1.8) and CKD group (98+-1.6), respectively. Macrophages treat with CKD-albumin presented higher content of PDI (5 times; p=0.02 e 7 times p=0.02) and Grp94 (66 %; p=0.02 e 20 %; p=0.02) when compared to SHAM-albumin and CKD+NAC-albumin- treated cells, respectively. ABCA-1 protein content was 87 % and 70 % (p < 0.01) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively compared with SHAM-albumin. ABCG-1 protein level was respectively 4 and 7.5 times higher in macrophages treated with SHAM+NAC-albumin and CKD+NAC-albumin in comparison to their respective controls without treatment. The cholesterol efflux mediated by apo A-I was 59 % and 70 % (p < 0.0001) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively, when compared to SHAM-albumin. The HDL2-mediated cholesterol efflux was 52 % higher in macrophages treated with SHAM+NAC-albumin compared to macrophages treated with SHAM-albumin. No difference was observed in the ABCA-1 protein level in macrophages treated with crescent concentrations of NAC alone for 8 h. Nonetheless, after 18 h, ABCA-1 was 50 %, 69 % and 72 % reduced in macrophages treated, respectively, with 10 mM, 20 mM and 30 mM NAC in comparison to control cells. ABCG-1 content in macrophages treated with NAC for 8 h and 18 h was not changed. Conclusion: NAC reduces plasma lipid peroxidation and AGE in CKD animals and prevents the endoplasmic reticulum stress induced by CKD-albumin in macrophages. Despite diminishing ABCA-1 and apo A-I-mediated cholesterol efflux, NAC increases ABCG-1. Then, NAC may contribute to attenuate the deleterious effects of the in vivo modified albumin on lipid accumulation in macrophages helping to prevent atherosclerosis in CKD

Parmi les tumeurs cérébrales primaires, les gliomes sont les tumeurs les plus fréquemment rencontrées. Les glioblastomes (GBM) représentent 60 à 70% de ces tumeurs et malgré de récents progrès dans leur traitement, leur pronostic reste sombre. Les gliomes malins sont caractérisés par une prolifération cellulaire importante, un taux élevé de néo-angiogenèse et une migration diffuse des cellules tumorales gliales dans le parenchyme cérébral, ce qui rend impossible une résection chirurgicale complète. De plus, les cellules gliales tumorales migrantes opposent une résistance particulière aux traitements chimiothérapiques de type pro-apoptotique, causant une récidive quasi inévitable de ce type de tumeur. La compréhension des aspects moléculaires à la base de la prolifération, de la migration et de la chimiorésitance des cellules gliales tumorales est donc essentielle pour élaborer des approches ciblées capables d’entraver ces processus. La littérature mentionne plusieurs stratégies qui permettraient, en théorie, de court-circuiter la résistance à l’apoptose des cellules tumorales gliales migrantes. Il s’agirait entre autres :<p>- de réduire le taux d’activation des voies de signalisation contrôlées par PI3K /Akt /mTOR et NFkappaB, qui diminuerait le taux de croissance des gliomes malins, ainsi que le taux de migration des cellules tumorales isolées dans le parenchyme cérébral;<p>- de réduire le taux de migration des cellules tumorales gliales afin de restaurer un certain degré de sensibilité à des agents chimiothérapiques pro-apoptotiques;<p>- d’endiguer l’export des agents chimiothérapiques par les pompes à efflux surexprimées dans les gliomes <p>- d'induire d’autres processus de mort cellulaire que l’apoptose, car les cellules tumorales gliales migrantes sont plus sensibles à d’autres formes de mort cellulaire.<p>Ces besoins de nouvelles stratégies thérapeutiques ont motivé ce travail qui se focalisera sur le potentiel antitumoral des ligands du R-sigma1 dans les glioblastomes. Ainsi, nous montrerons que les ligands des Rs-sigma sont capables de produire certains des effets visés dans les stratégies ci-dessus, dont la réduction de la prolifération et de la migration des cellules cancéreuses avec une certaine potentialisation des chimiothérapies. Ces propriétés ouvrent de nouvelles perspectives en thérapie anticancéreuse pour cette famille de ligands, dont plusieurs membres sont déjà utilisés depuis de nombreuses années comme antipsychotique. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

O remodelamento vascular é um determinante fundamental do lúmen em doenças vasculares, porém os mecanismos envolvidos não estão completamente elucidados. Nós investigamos o papel da chaperona redox residente do retículo endoplasmático Dissulfeto Isomerase Proteica (PDI) e sua fração localizada na superfície celular (peri/epicelular=pecPDI) no calibre e arquitetura vascular durante reparação à lesão. Em artérias ilíacas de coelho submetidas à lesão in vivo, houve importante aumento do mRNA e expressão proteica (~25x aumento 14 dias pós-lesão vs. controle) da PDI. O silenciamento da PDI por siRNA (cultura de órgãos) acentuou o estresse do retículo e apoptose, diferentemente da inibição da pecPDI com anticorpo neutralizante (PDI Ab). Bloqueio in vivo da pecPDI por aplicação de gel perivascular contendo PDI Ab no 12° dia após lesão, com análise após 48 h, promoveu ca.25% redução no calibre vascular analisado por arteriografia e diminuição similar na área total do vaso detectada por tomografia de coerência óptica. Neste processo, não ocorreu alteração no tamanho da neoíntima, indicando assim, que PDI Ab acentuou remodelamento constrictivo. Neutralização da pecPDI promoveu importantes alterações na arquitetura da matriz de colágeno e citoesqueleto, resultando em fibras com orientação invertida e desorganizadas. Diminuição na produção de espécies reativas de oxigênio e óxidos de nitrogênio também ocorreu. Análise de propriedades viscoelásticas nas artérias indicou redução na ductilidade vascular, evidenciada pela menor distância para ruptura. As alterações subcelulares no citoesqueleto observadas in vivo após PDI Ab foram recapituladas em um modelo de estiramento cíclico em células musculares lisas vasculares, com importante redução na formação das fibras de estresse. Em modelo de migração randômica de células musculares lisas, a exposição a PDI Ab reduziu a resiliência de regulação da polaridade. Embora a neutralização da pecPDI não tenha afetado a atividade global de RhoA, ela promoveu alterações no padrão de marcação em resposta ao estiramento, na redistribuição de RhoA na superfície celular e na associação com regiões contendo caveolina. Além disso, em aterosclerose nativa em humanos, a expressão da PDI correlacionou-se inversamente com remodelamento constrictivo. Dessa forma, PDI é fortemente expressa após a lesão e sua fração peri/epicelular remodela a arquitetura da matriz e citoesqueleto, promovendo um efeito anti-remodelamento constrictivo / Whole-vessel remodeling is a critical lumen caliber determinant in vascular disease, but underlying mechanisms are poorly understood. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase(PDI) and cell-surface PDI(peri/epicellular=pecPDI) pool in vascular caliber and architecture during vascular repair after injury(AI). After rabbit iliac artery balloon injury, there was marked increase in PDI mRNA and protein (25-fold vs. basal at day 14AI), with increase in both intracellular and pecPDI. Silencing PDI by siRNA (organ culture) induced ER stress augmentation and apoptosis, contrarily to pecPDI neutralization with PDI-antibody(PDI Ab). PecPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted ca.25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. PecPDI neutralization promoted marked changes in collagen and cytoskeleton architecture, with inverted fiber orientation and disorganization. Decreased ROS and nitrogen oxide production also occurred. Viscoelastic artery properties assessment showed decreased ductility, evidenced by decreased distance to rupture. Subcellular cytoskeletal disruption by PDI Ab was recapitulated in vascular smooth muscle cell stretch model, with marked decrease in stress fiber buildup. Also, PDI Ab incubation promoted decreased regulation resilience of vascular smooth muscle migration properties. While pecPDI neutralization did not affect global RhoA activity, there was altered RhoA redistribution to the cell surface and association with caveolin-containing clusters, which mislocalized after stretch. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. Thus, strongly-expressed PDI after injury reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect