Diante da ausência de dados prévios, foi pretendido estabelecer uma correlação entre as possíveis modificações metabólicas e moleculares na resistência insulínica e inflamação em modelos animais de obesidade, induzida pela dieta, recebendo a suplementação de ômega 3/ALA. Além disto, pretendeu-se obter dados que permitam uma melhor elucidação dos mecanismos envolvidos na resistência insulínica neste modelo e um possível efeito preventivo da administração de ALA sobre este processo, podendo, desta forma, auxiliar no desenvolvimento de novas terapias. O objetivo do estudo foi demonstrar que a suplementação ALA reduz a resistência à insulina e a inflamação em modelo animal de obesidade. Foram divididos 40 camundongos machos (C57/BL6) em 4 grupos: controle (C), controle + ômega 3/ALA (CW), obesos (O) e obesas + ômega 3/ALA (OW). Por um período de oito semanas, os grupos O e OW receberam uma dieta hiperlipídica com 60% de lipídeos, enquanto o C e CW receberam ração padrão. Depois, os grupos CW e OW receberam suplementação de 10% de ômega 3/ALA liofilizado, extraído de semente de linhaça, diariamente, por mais 8 semanas. Observando os resultados, todos os grupos de animais tiveram o mesmo ganho de peso, assim como consumo alimentar, eficiência enérgica e eficiência alimentar. O uso do ALA diminuiu o peso da gordura subcutânea no grupo OW comparado ao O e manteve os valores semelhantes entre os outros grupos, no entanto, as comparações do peso do fígado, gordura epididimal, pâncreas e músculo gastrocnemio, foram semelhantes entre todos os grupos. Observou-se uma diminuição na resistência insulínica nos animais OW comparado ao O pelo teste de IPGTT, sendo a área sob a curva de glicose similar entre C e CW. Além disso, os níveis totais de gordura no fígado foram significativamente menores no CW e OW, em comparação com C e O, estes resultados são reforçados pela análise dos tecidos em avaliação histopatológica. Os níveis séricos de glicemia e insulina, ao final do estudo, mostraram uma redução importante em OW comparado a CW, porém, não foi observado diferenças entre os animais obesos suplementados ou não com ômega-3/ALA. Entretanto, quanto à avaliação de resistência insulínica pelo cálculo do HOMA IR, este mostrou-se menor comprando-se OW com O. Os níveis séricos de colesterol total e HDL foram maiores no grupo CW comparados ao C, sendo que os níveis de colesterol total foram menores, também, no grupo OW comparados ao O. No entanto, os valores de triglicérides séricos foram semelhantes entre todos os grupos, assim como valores de triglicérides hepáticos. Colesterol total hepático teve um aumento significativo entre OW e O. As dosagens séricas de IL1, IL6 e MCP1 mostraram uma redução importante em O comparadas com OW. Já quanto a IL17 e TNF, ambas foram equivalentes, nas comparações entre diferentes grupos. A avaliação da ativação do estresse do retículo endoplasmático mostrou que a proteína BIP teve um aumento importante tanto na comparação entre C e CW e também entre O e OW. A também chaperona HSP70 mostrou aumento significativo em ambas as comparações entre grupos, tanto em C e CW quanto em O e OW. GRP94 e IRE1 tiveram resultados semelhantes, sem diferenças entre os grupos, assim como a DAPK1. CHOP teve diminuição importante comparando-se C e CW, e O e OW. Em contrapartida, XBP1 teve diminuição importante na comparação entre os grupos C e CW. Por fim, a suplementação de ômega 3/ALA mostrou ser eficaz na prevenção de esteatose hepática, redução de resistência insulínica, diminuição do processo inflamatório, e redução da ativação do estresse do retículo endoplasmático em tecido hepático. / Given the absence of previous data, it is intended to establish a possible correlation between metabolic and molecular changes in insulin resistance and inflammation in animals receiving supplementation of -linoleic acid (ALA) found in flaxseeds oil. Furthermore, we intend to obtain data for an elucidation of the mechanisms involved in insulin resistance and preventive effect of administration of ALA on this process and may thus aid the development of new therapies. The aim of study was prove the ALA supplementation reduces insulin resistance and inflammation in an obesity animal model. 40 male mice (C57/BL6) were divided into 4 groups: control (C), control + omega 3/ALA (CW), obese (O) + omega 3/ALA and obese (OW). For a period of eight weeks, the groups O and OW received a high-fat diet with 60% fat, while the C and CW received regular chow. Then, the CW and OW groups were supplemented with 10% omega 3/ALA lyophilized, extracted from flaxseed daily, for another 8 weeks. Analyzing the results, all groups of animals had the same weight gain, and food consumption, food efficiency and energetic efficiency. The use of ALA decreased subcutaneous fat weight in group OW compared to O and remained similar values between the other groups, however, comparisons of the size of liver tissue, epididymal fat, pancreas and gastrocnemius muscle were similar among all groups. There was a decrease in insulin resistance in animals OW compared to O by GTT test, with an area under the curve of glucose similar between C and CW. Moreover, the total liver fat levels were significantly lower in CW and OW compared to C and O, these results are reinforced by histopathological tissues analysis. Serum glucose and insulin levels, at the end of the study, showed a significant reduction in OW compared to CW, however, no differences were observed between the obese animals supplemented or not with omega 3/ALA. Meanwhile, about the valuation of insulin resistance calculating by HOMA IR, this was lower in OW compared with O. Serum total cholesterol and HDL levels were higher in CW compared to the group C, and total cholesterol levels were also lower in the OW group compared to O. However, the serum levels of triglyceride were similar among all groups as well as hepatic triglycerides values. Hepatic total cholesterol increased significantly between OW and O. Serum levels of IL1, IL6 and MCP1 showed a significant reduction in O compared to OW. As for IL17 and TNF were similar for both comparisons between different groups. The evaluation of endoplasmic reticulum stress activation showed the BIP protein had a significant increase in the comparison between CW and C and also between OW and O. The also chaperone HSP70 showed a significant increase in both comparisons between groups, both in C and CW as for O and OW. GRP94 and IRE1 had similar results, with no differences between groups, as well as DAPK1. CHOP had significant decrease comparing CW and C, and O and OW. In contrast, XBP1 had significant reduction in the comparison between groups C and CW. Finally, omega 3/ALA supplementation showed to be effective in preventing hepatic esteatose, the reduction of insulin resistance, inflammation decrease, and reduction of activation of endoplasmic reticulum stress in liver tissue.

Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.

Les maladies inflammatoires chroniques de l'intestin (MICI) se caractérisent par une inflammation sévère de l'intestin grêle et du côlon et comprennent la maladie de Crohn (MC) et la rectocolite hémorragique (RCH). Les MICI sont des maladies complexes faisant intervenir des facteurs génétiques : certains senseurs bactériens, l'autophagie et le stress du réticulum endoplasmique. Un défaut de barrière de l'épithélium digestif est également fortement impliqué dans la physiopathologie du processus inflammatoire. La fonction barrière de l'épithélium digestif est assurée par plusieurs types cellulaires, synthétisant entre autres, des peptides antimicrobiens (PAM) et des mucines. Dans les MICI, une augmentation de la perméabilité intestinale et une perte de muco-sécrétion ont été décrites. Les protéases jouent un rôle fondamental dans la digestion du bol alimentaire mais également dans le maintien de l'homéostasie intestinale en activant ou dégradant divers motifs moléculaires, ou in induisant des signaux spécifiques aux cellules par l'activation de quatre récepteurs : les PARs (Protease-Activated Receptor). Dans les MICI, un excès d'activité protéolytique de type trypsine est observé. L'origine de cette activité est théoriquement attribuée aux cellules immunitaires, à une surproduction pancréatique ou au microbiote, mais les cellules épithéliales intestinales semblent également être une source majeure de protéases. L'objectif de mon projet de thèse visait à étudier l'impact des principales voies impliquées dans les MICI sur l'homéostasie des protéases épithéliales et le rôle de celles-ci dans la déstabilisation de la fonction de barrière. Nos résultats ont confirmé un excès de protéases à sérine dans les cellules épithéliales de patients atteint de MC ou de RCH. In vitro, sur des monocouches de cellules Caco-2, l'induction de l'autophagie diminuait la libération apicale de protéase de type trypsine, alors que le senseur bactériens NOD2 n'avait aucun effet. A l'inverse, une stimulation du Stress du réticulum endoplasmique (SRE) par la Thapsigargin, induisait une libération accrue de protéases actives de type trypsine au pôle apical des cellules. [...] / Crohn's disease (CD) and Ulcerative colitis (UC) are two forms of Inflammatory Bowel Disease (IBD), a chronic inflammatory pathology affecting the digestive tract. Patients suffer from relapsing flares, diarrhea, abdominal pain and bleeding. Although the molecular mechanisms of IBD are poorly understood, recent data suggest that IBD occurs in genetically predisposed individuals developing an abnormal immune response to intestinal microbes after, being exposed to specific environmental triggers. Genetic studies have reported more than 170 polymorphisms susceptible to be involved in IBD pathogenesis. The strongest associations have highlighted three main pathways altered in IBD including bacterial sensing (NOD2, CD), autophagy (ATG16L1 and IRGM, CD) and endoplasmic reticulum stress (ER-Stress) (XBP1, UC). The role of intestinal barrier function is also strongly implicated in IBD pathogenesis, and is modulated by factors present in the lumen derived from microbiota, food or at a molecular level, by factors such as proteases. In IBD pathophysiology, the inflammatory process is characterized by impaired intestinal biology including disruption of tight junctions and leaky gut, decreased amount of Paneth and Goblet cells, and translocation of luminal antigens triggering inflammation. Previous studies have demonstrated an increased level of active serine proteases in the stools and tissues of IBD patients, supposing that proteases originate from infiltrated immune cells, pancreatic secretion or microbiota. However, our team has reported that intestinal epithelial cells are a major source of serine proteases, in particular trypsin-like enzymes, are released by a stressed epithelium in pathogenic context such as irritable bowel syndrome. In this project, we aimed at better understanding whether the three main pathways involved in IBD (Nod2, autophagy, ER-stress) could be linked to an epithelial release of trypsin and reciprocally, if epithelial trypsin is able to induce or modulate these three IBD pathways. We confirmed that trypsin-like activity was significantly higher in biopsies from UC and CD patients compared to healthy controls. In Caco-2 monolayers cultured in transwells, secreted trypsin-like proteolytic activity remained stable upon NOD2 stimulation but decreased under autophagy induction. Thapsigargin (Tg) stimulation a well-known ER-stress inducer, enhanced the apical release of trypsin-like activity in Caco2 cells. [...]

Le tissue adipeux (TA) est un organe complexe specialisé dans le stockage et la libération d'énergie sous forme de lipides. Cet organe adipeux est essentiel pour le maintien de l'homéostasie énergétique. Les adipocytes sont les cellules prototypiques du TA. Elles se forment durant la différenciation de précurseurs, un processus appelé adipogenèse. L'adipogenèse est intimement associée à la synthèse des acides gras et de triglycérides lors de la lipogenèse. Néanmoins, divers facteurs peuvent perturber l'adipogénèse et la lipogenèse, contribuant au dysfonctionnement du TA et au développement des maladies métaboliques. Le but de cette thèse a été d'étudier la lipogenèse dans le contexte du stress du réticulum endoplasmiques (SRE), de la restriction calorique et du vieillissement. Dans le projet A, nous avons montré que l'exposition d'adipocytes à un SRE aigu inhibe l'expression des gènes liés à la lipogenèse et empêche l'incorporation du glucose dans les lipides. En plus, l'exposition des préadipocytes à un SRE chronique, détériore à la fois la lipogenèse et l'adipogenèse. Par contre, pour les adipocytes, un SRE chronique mais modéré n'a pas d'effet évident sur la lipogenèse. Ces effets du SRE pourraient contribuer à la détérioration de la fonction de TA vue dans l'obésité. La capacité du TA à stocker des lipides diminue avec l'âge, probablement à cause de l'accumulation de cellules sénescentes ou un SRE plus élevé. Dans le projet B, nous avons étudié la capacité lipogénique du TA humain en relation à la sénescence et aux marqueurs du SRE au sein d'une cohorte de femmes obèses jeunes ou âgées. Tandis que l'expression des principaux marqueurs de la sénescence était augmentée dans le TA des femmes âgées, l'expression génique des enzymes de lipogenèse et des chaperonnes était diminuée dans le TA des personnes âgées. Ces résultats étaient partiellement retrouvés dans les adipocytes différenciés in vitro des mêmes individus ce qui suggère une moins bonne capacité à faire face au SRE lors du vieillissement. Le régime à très basses calories (VLCD) est souvent prescrit en première intention pour une rapide perte de poids. L'amélioration de la sensibilité à l'insuline se voit dès 2 jours de VLCD. Néanmoins, on ne sait quasiment rien des modifications métaboliques du TA survenant durant les premiers jours. Dans le projet C, nous avons donc comparé les réponses métaboliques et inflammatoires du TA sous-cutané précocément (2 jours) et plus tardivement (28 jours) lors d'un VLCD. A 2 jours de régime, l'expression des gènes lipolytiques était augmentée, alors que l'expression des gènes lipogéniques était diminuées. Les marqueurs d'inflammation n'étaient pas changés dans le TA. Néanmoins, les changements d'expression dans le TA lors de la phase précoce du régime ne pouvait pas expliquer l'effet de ce régime court à l'amélioration de la sensibilité à l'insuline. Dans la phase tardive, l'expression des gènes impliqués dans la lipogenèse et la ß-oxydation était largement réduite, tandis que l'expression des marqueurs inflammatoires était augmentée. Nous avons donc montré que les réponses métaboliques et inflammatoires du TA sous-cutané à 2 jours et 28 jours de VLCD sont différentes. Dans le projet D, nous avons comparé et défini les effets de la restriction calorique modérée sur la physiologie des préadipocytes et des adipocytes différenciés in vitro chez des jeunes obèses ou des personnes âgées obèses. De façon surprenante, on n'a observé aucun effet de l'intervention sur le métabolisme des préadipocytes dans les deux groupes. Par contre, un effet bénéfique de l'intervention sur le métabolisme adipocytaire n'a été observé que chez les personnes âgées. Nos données montrent donc qu'une restriction calorique modérée peut avoir un effet positif sur le métabolisme adipocytaire des séniors. Pour conclure, cette thèse montre que la lipogenèse dans le TA humain peut être inhibée par le SRE, la restriction calorique sévère et le vieillissement. / Adipose tissue (AT) is a complex organ specialised in safe storage and release of energy as lipids. The adipose organ is therefore essential for the maintenance of energy homeostasis. The prototypical cells of AT are adipocytes, emerging from the precursors in a process called adipogenesis. Adipogenesis itself is tightly connected with lipogenesis, i.e. with the synthesis of fatty acids and triglycerides. Various stimuli can disturb adipocyte differentiation and lipogenesis and thus contribute to AT dysfunction and development of associated metabolic diseases. This thesis was focused on the investigation of lipogenesis in the context of endoplasmic reticulum stress (ERS), calorie restriction and aging. In Project A, we showed that exposition of adipocytes to high acute ERS inhibits expression of lipogenic genes and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to ERS impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in adipocytes. These effects of ERS could therefore contribute to the worsening of AT function seen in obesity. The capacity of AT to store lipids decreases in aging, possibly due to the accumulation of senescence cells or higher ERS. In Project B, we investigated lipogenic capacity of human AT in relation to senescence and markers of ERS. AT and adipose cells from young and elderly women were investigated. While mRNA expression of major senescent markers was increased in AT from the elderly compared to young individuals, mRNA expression of lipogenic enzymes and chaperones was decreased in AT from elderly individuals. These results were also partly observed in vitro in differentiated adipocytes from AT of the same individuals suggesting the reduced capability to cope with ERS in aging. Very-low calorie diet (VLCD) is first line lifestyle intervention to achieve rapid weight loss. The improvement of whole body insulin sensitivity can be seen as soon as after 2 days of VLCD. However, little is known about AT metabolic changes in those early days. Thus, in Project C, we compared metabolic and inflammation-related characteristics of subcutaneous AT in the early (2 days) and later (28 days) phase of a VLCD. In the early phase of VLCD, the expression of lipolytic genes was increased, whereas the expression of lipogenic genes was suppressed. The inflammatory markers remained unchanged in AT. The changes in AT gene expression in the early phase of VLCD could not explain the effect of short calorie restriction on the improvement of insulin sensitivity. At the later phase, expression of genes involved in lipogenesis and ß-oxidation was markedly suppressed, whereas the expression of inflammatory markers was increased. Thus, we found that the early and later phases of VLCD differ with respect to metabolic and inflammatory responses in subcutaneous AT. In Project D, we compared and defined the effects of moderate calorie restriction on preadipocytes and in vitro differentiated adipocytes in two groups of obese men: juniors and seniors. We did not observe any effect of the intervention on metabolism of preadipocytes in either group. However, we observed an intervention-driven improvement in adipocyte metabolism selectively in the group of seniors. Therefore, our data suggest that moderate calorie restriction could initiate positive changes in metabolism of adipocytes in seniors. In conclusion, this thesis brought several pieces of evidence that lipogenesis in human AT can be inhibited by ER stress, severe caloric restriction and aging.

La fibrose hépatique est la conséquence de toutes les maladies chroniques du foie et se caractérise par un dépôt excessif de matrice extracellulaire synthétisée par les myofibroblastes. Les myofibroblastes portaux (MFP), l'une des sous populations de myofibroblastes, jouent un rôle majeur dans la progression de la fibrose et sont pro-angiogéniques. Des études ont montré un rôle important du stress du réticulum endoplasmique (RE) dans la fibrose du foie. Nos objectifs étaient de déterminer si un stress du RE survient dans les MFP lors de la fibrose et affecte les fonctions de ces cellules, et d'étudier l'effet du TUDCA, une molécule chaperonne utilisée en clinique dans les maladies biliaires, sur le stress du RE. Le phénotype de MFP activés in vivo, isolés à partir de foie de rats fibreux après cholestase, a été comparé à celui de MFP contrôles que nous avons préalablement bien caractérisés. Nos résultats montrent que les MFP activés in vivo subissent un stress du RE se traduisant par l'activation de la voie PERK. Ce stress du RE n'a pas d'effet sur la différenciation myofibroblastique, diminue les capacités de prolifération et de migration des MFP mais augmente leur pouvoir angiogénique. En revanche, le TUDCA n'a aucun effet sur les paramètres étudiés. Les MFP subissent donc un stress du RE lors de leur activation myofibroblastique qui stimule leur propriété pro-angiogénique et pourrait ainsi favoriser la progression de la fibrose. Cependant le stress du RE inhibe également leurs fonctions de prolifération et de migration ce qui pourrait induire une boucle de contrôle négative limitant leur expansion. / Hepatic fibrosis is the consequence of all chronic liver diseases and is characterized by an abnormal extra cellular matrix deposition by myofibroblasts. Portal myofibroblasts (PMF), a subpopulation of hepatic myofibroblasts, play a major role in fibrosis progression and angiogenesis. Accumulating evidences indicate an important role of endoplasmic reticulum (ER) stress in hepatic fibrosis. The aims of this study were to determine whether an ER stress occured in PMF during fibrosis and affected the functions of these cells, and to study the effect of the molecular chaperone TUDCA used in biliary diseases, on ER stress. The phenotype of in vivo activated-PMF obtained from rat fibrotic liver after cholestasis was compared with the phenotype of control PMF that we previously characterized. Our results showed that in vivo activated-PMF underwent ER stress with PERK pathway activation. This ER stress had no effect on myofibroblastic differentiation but reduced PMF proliferation and migration and increased PMF angiogenesis capacity. TUDCA had no effect on these parameters. In conclusion, PMF display ER stress during their activation. ER stress stimulates their pro-angiogenic proprieties and thereby may promote fibrosis progression. However, ER stress also inhibits their proliferation and migration functions, and thereby could provide a negative control loop to restrict their expansion.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 3, 2010). Includes bibliographical references.

Activation of the Unfolded Protein Response (UPR) following stress in the Endoplasmic Reticulum (ER) is an important mechanism by which obesity results in insulin resistance and type II diabetes. We uncovered a role for the adaptor protein Nck in modulating the UPR. In this study, we report that obese Nck1-/- mice, which show lower levels of UPR in liver and adipose tissue, present improved insulin signalling in these tissues. We established that the effect of Nck1 is cell autonomous by showing that HepG2 cells treated with Nck1 siRNA have reduced ER stress-induced UPR and Insulin Receptor Substrate-1 (IRS-1) serine phosphorylation. In these cells, we observed that the IRS-1 levels and activation of signalling components downstream of the insulin receptor were increased. This correlates with enhanced cell survival to stress and insulin stimulated glycogen synthesis. Overall, we demonstrated that Nck1 participates in ER-stress-induced insulin resistance and regulation of IRS-1-dependent signalling.

Pancreatic beta-cells are responsible for secreting insulin into the circulation to maintain whole body glucose homeostasis. While pancreatic beta-cells have a large capacity to secrete insulin, their function progressively deteriorates during the pathogenesis of type 2 diabetes as a result of both genetic predisposition and environmental factors. Obesity is the largest risk factor for developing type 2 diabetes and is associated with various conditions that can impair normal beta-cell function, including excess free fatty acids, inflammation and insulin resistance. Accumulating evidence in the literature suggests that endoplasmic reticulum (ER) stress contributes to the molecular mechanism of pancreatic beta-cell failure during the progression of type 2 diabetes. In this thesis, I have examined the role of the ER stress sensor ATF6-alpha and also the ER-resident chaperone GRP78 in pancreatic beta-cell homeostasis and function. Work presented in Chapter 2 examined the function of naturally occurring ATF6-alpha protein variants associated with type 2 diabetes. I also examined the role of endogenous ATF6-alpha in pancreatic beta-cells, which is described in Chapter 3. Results from these analyses suggest that the ATF6-alpha gene is not a type 2 diabetes susceptibility gene; however, ATF6-alpha protein expression is important to beta-cell function and survival. Finally, ER stress markers have been detected in pancreatic beta-cells and insulin sensitive tissues (such as adipose and liver), which promote beta-cell dysfunction and insulin resistance, respectively. In Chapter 4, I examined the contribution of ER stress in beta-cell dysfunction specifically by generating transgenic mice over-expressing GRP78. The mice were subsequently challenged by high fat diet to determine their susceptibility to developing symptoms of type 2 diabetes. Indeed increased chaperone capacity in pancreatic beta-cells protected against obesity-induced glucose intolerance and insulin resistance. Overall, these data support the hypothesis that ER stress contributes to beta-cell dysfunction in type 2 diabetes progression.

Maintainence of appropriate redox homeostasis is crucial for processes such as protein folding in the endoplasmic reticulum (ER) and to minimise genesis of oxidative stress. Previous studies have indicated a possible link between ER stress and production of reactive oxygen species (ROS) although the cellular mechanisms involved were not fully elucidated. To investigate the cellular mechanisms involved in tolerance to oxidative stress and ER stress, genome-wide screens were performed to identify mutants sensitive to chronic ER stress induced by dithiothreitol and tunicamycin. These screens identified the Cu,Zn superoxide dismutase (SOD1) and genes involved in NADPH generation (RPE1, TKL1) as important for chronic ER stress tolerance. Superoxide anion has been identified as one of the ROS generated during ER stress. The ER oxidoreductase Ero1p, previously implicated in ROS production in vitro, did not appear to be a source of superoxide when the protein was over-expressed. It was also found that cellular NADP(H) levels affected induction of the unfolded protein response (UPR), since cells lacking TKL1 or RPE1 exhibited decreased UPR induction. These data indicate an important role for superoxide dismutase and cellular NADP(H) in survival of cells during ER stress. Subsequent analysis determined that NADPH generation was also required for adaptation to H2O2. Mutants affected in NADPH production were chronically sensitive to H2O2 but resistant to an acute dose. These mutants over-accumulated reduced glutathione (GSH) but maintained normal cellular redox homeostasis. This over- production of GSH was not regulated at the transcriptional level of GSH1 encoding ??- glutamyl cysteine synthetase. These data raise the important question as to how cells maintain cellular glutathione redox balance. To better understand how cells respond to perturbations in glutathione redox homeostasis, cells deleted for GLR1, encoding GSSG reductase, were exposed to extracellular oxidised glutathione (GSSG) and intracellular GSH and GSSG were monitored over time. Intriguingly cells lacking GLR1 showed increased levels of GSH accumulation upon GSSG treatment in a manner independent of GSH synthesis. It was subsequently found that the cytosolic thioredoxin-thioredoxin reductase system contributes to the reduction of GSSG in vivo.

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Department of Cell Biology. Title from pdf file main screen (viewed on October 24, 2009). Includes bibliographical references.