Introdução: A obesidade é uma doença crônica que aumenta sua prevalência progressivamente no mundo todo. Uma das suas principais complicações é a doença hepática gordurosa não alcoólica (DHGNA), que pode apresentar-se de forma assintomática, com esteatose hepática, esteato-hepatite, cirrose e hepatocarcinoma. Está associada à dislipidemia, hipertensão, diabetes, síndrome metabólica e resistência insulínica. O padrão ouro para seu diagnóstico é a biopsia hepática, considerado método invasivo. Medidas indiretas são usadas para auxiliar na sua detecção, como dados do exame físico, exames de bioquímica e de imagem. Objetivo: Diagnosticar DHGNA usando a ressonância nuclear magnética (RNM) como método não invasivo, correlacionando-a com achados clínicos e laboratoriais. Metodologia: Estudo transversal de 50 crianças e adolescentes seguidas no Ambulatório de Obesidade do Hospital das Clínicas da FMRP-USP. Foram submetidas à questionário sobre histórico pessoal e familiar, à exame físico, exames laboratoriais (lipidograma, transaminases, glicemia e insulina basal) e RNM de abdome para cálculo da de gordura hepática, visceral e subcutânea. Resultados: Diagnosticado esteatose hepática em 14 (28%) dos participantes, sendo 8 com esteatose grave (porcentagem de gordura >18%) e 4 não grave (porcentagem gordura hepática entre 9 e 18%). Houve diferença estatisticamente significante entre a gordura hepática detectada pela RNM e o gênero masculino, triglicérides, TGO, TGP, relação TGO/TGP e acantose nigricans. O cálculo do Homeostasis Model Assessment Insulin Resistance (HOMA-IR) e da síndrome metabólica não apresentaram relação positiva com a porcentagem de gordura hepática. Conclusão: A frequência de esteatose hepática, utilizando a RNM como ferramenta diagnóstica, foi inferior à encontrada na literatura. Dos parâmetros estudados, os melhores preditores de esteatose hepática foram triglicérides, TGO, TGP, relação TGO/TGP, gênero masculino, acantose nigricans e Z escore para IMC elevado / Introduction: Obesity is a chronic disease whose prevalence progressively increasing worldwide. One of its main complications is non-alcoholic fatty liver disease (NAFLD), which may occur in an asymptomatic form, as simple fatty liver, steatohepatites (NASH), cirrhosis and hepatocellular carcinoma. This disease is associated with dyslipidemia, hypertension, diabetes, metabolic syndrome and insulin resistance. The gold standard for diagnosis is a liver biopsy, considered to be an invasive method. Indirect measures are used to aid its diagnosis, such as physical, biochemical and imaging tests. Objective: Diagnosing NAFLD using MRI as non-invasive method and correlating it with clinical and laboratory findings. Methodology: A cross-sectional study of 50 children and adolescents followed at the Obesity clinic of the University Hospital, FMRP-USP. A questionnaire about personal and family history was aplplied and each subject was submitted to physical examination, laboratory tests (lipip profile, transaminases, glycemia and basal insulin) and magnetic resonance imaging (MRI) of the abdomen for calculation of hepatic, visceral and subcutaneous fat. Results: Fatty liver disease was diagnosed in 14 (28%) of the participants, 8 of them with severe steatosis (fat percentage > 18%) and 4 with milder steatosis (fat percentage between 9 and 18%). There was a statistically significant difference between the hepatic fat detected by MRI and male gender, triglycerides, AST, ALT, AST/ALT ratio and acanthosis nigricans. The calculation of the Homeostasis Model Assessment Insulin Resistance (HOMA-IR) and of the metabolic syndrome did not show a positive relationship with the percentage of hepatic fat. Conclusion: The frequency of hepatic steatosis using MRI as a diagnostic tool, was lower than the values reported in the literature. Among the parameters studied, the best predictors of liver steatosis were triglycerides, AST, ALT, AST/ALT ratio, male gender, acanthosis nigricans and Z score for high body mass index

Obesity and insulin resistance are associated with a constellation of features including hypertension, dyslipidaemia, type 2 diabetes, and premature cardiovascular disease, collectively termed the metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) represents the hepatic component of this syndrome, incorporating a spectrum of liver disease with increasing morbidity and mortality, from simple steatosis, to non-alcoholic steatohepatitis (or NASH), fibrosis, cirrhosis and ultimately hepatocellular carcinoma. However, factors influencing this progression are incompletely understood. In this thesis I sought to investigate pathways which promote hepatic inflammation and fibrosis by studying two contrasting dietary models of NAFLD in mice in which the risk of hepatic inflammation, insulin resistance and fibrosis differ; namely the methionine and choline deficient diet (MCDD) which induces steatohepatitis, hepatic insulin resistance, and weight loss, and the choline deficient diet (CDD) which may be protected from insulin resistance, and leads to steatosis without inflammation or weight loss. I investigated the possible molecular mechanisms underlying these differences, and whether they influenced progression to hepatic fibrosis induced by carbon tetrachloride (CCl4).

<p>With the prevalence of obesity and metabolic syndrome rising sharply world-wide, it has become increasingly important to define the molecular mechanisms underlying the pathogenesis and progression of diseases associated with lipid-induced cytotoxicity. Cardiovascular disease, type-2 diabetes mellitus, and nonalchoholic fatty liver disease (NAFLD) have all recently gained recognition as diseases that are exacerbated by lipoapoptosis. In this dissertation, we demonstrate a novel role for caspase-2 as an initiator of lipoapoptosis. Using an unbiased metabolomics approach, we discovered that the activation of caspase-2, the initiator of apoptosis in Xenopus egg extracts, is associated with an accumulation of long-chain fatty acid (LCFA) metabolites. Metabolic treatments that block the buildup of LCFAs potently inhibit caspase-2, while add-back of a saturated LCFA restores caspase activation in the extract setting. Extending these findings to mammalian cells, we show that caspase-2 is engaged and activated in response to treatment with the saturated LCFA, palmitate. Down-regulation of caspase-2 significantly impairs cell death induced by saturated LCFAs, revealing a conserved, critical role for caspase-2 in mediating LCFA-induced lipoapoptosis. </p><p> Since lipoapoptosis has been implicated as a key driver of the progression of NAFLD, we aimed to determine the therapeutic significance of our findings by evaluating the importance of caspase-2 in an in vivo model of this disease. We subjected wild-type and caspase-2 knockout mice to a diet which induces severe liver steatosis and the development nonalcoholic steatohepatitis (NASH), the most advanced stage of NAFLD characterized by liver fibrosis. Interestingly, we observed an increase in caspase-2 protein levels in the livers of wild-type mice fed a NASH-inducing diet. These findings were of particular importance, since caspase-2 expression was also significantly elevated in patients diagnosed with NASH. Most importantly, we demonstrated that caspase-2 knockout mice are protected from apoptosis and fibrosis when fed a NASH-inducing diet, suggesting that caspase-2 is major regulator of hepatocyte lipoapoptosis. Together, these findings reveal a previously unknown role for caspase-2 as an initiator of lipoapoptosis and suggest that caspase-2 may be an attractive therapeutic target for inhibiting pathological lipid-induced apoptosis.</p> / Dissertation

Thesis (PhD (Medicine. Internal Medicine))--Stellenbosch University, 2008. / Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in Western countries, extending from steatosis (FLD) to steatohepatitis (NASH). Differentiation between NASH and nonprogressive NAFLD is difficult on clinical grounds therefore a need exists to identify reliable biomarkers of disease progression. The aims of the study were 1) to describe the disease profile of NAFLD/NASH in South African patients of the Western Cape, 2) to investigate the metabolic derangements associated with this condition, including insulin resistance, lipid abnormalities and liver fibrogenesis, and 3) to assess the possible involvement of candidate genes in relation to the disease phenotype in the patient cohort. A total of 233 patients (73% female) were enrolled in this study, consisting of 69% Cape Coloured, 25% Caucasian, 5% Black and 1% Asian individuals. All subjects were obese or overweight based on the assessment of body mass index (BMI). Screening for NAFLD identified 182 patients (87%) with ultrasonographical evidence of fatty infiltration and/or hepatomegaly. Liver biopsies were performed on patients with persistently abnormal liver functions and/or hepatomegaly. NAFLD was confirmed histologically in 111 patients of whom 36% had NASH and 17% advanced liver fibrosis. None of the Black patients had advanced fibrosis.

Στην παρούσα εργασία, μελετήσαμε την συνεισφορά των μεταβολικών μονοπατιών της HDL και των χυλομικρών/VLDL στην εμφάνιση της παχυσαρκίας, στις διαταραχές του μεταβολισμού της γλυκόζης, στην εναπόθεση των τριγλυκεριδίων στο ήπαρ και στην ανάπτυξη της διατροφικά επαγόμενης μη αλκοολικής λιπώδους νόσου του ήπατος (NAFLD). Έτσι, επιλέξαμε να εστιάσουμε στην μελέτη των απολιποπρωτεϊνών Α-Ι (apoA-I) και Ε (apoE) και του ενζύμου λεκιθινο-χοληστερολική ακυλοτρανσφεράση (LCAT). Η apoA-I αποτελεί το κύριο συστατικό των υψηλής πυκνότητας λιποπρωτεϊνών (HDL) και είναι υπεύθυνη για την σύνθεση τους, η LCAT εστεροποιεί την ελεύθερη χοληστερόλη των λιποπρωτεϊνών του πλάσματος και ευθύνεται για το σχηματισμό των ώριμων σωματιδίων HDL και η apoE συμμετέχει στον καταβολισμό των υπολειμμάτων των χυλομικρών, των πολύ χαμηλής πυκνότητας λιποπρωτεϊνών (VLDL) και των χαμηλής πυκνότητας λιποπρωτεϊνών (LDL) από την κυκλοφορία καθώς και στην de novo βιογένεση της HDL. Προκειμένου να μελετηθεί ο ρόλος αυτών των μορίων στις παραπάνω μεταβολικές διαταραχές, μελετήσαμε πειραματικά μοντέλα ποντικών με έλλειψη στα γονίδια αυτά. Συγκεκριμένα, ομάδες ποντικών με έλλειψη στο γονίδιο που κωδικοποιεί την apoA-I (apoA-I-/-), την LCAT (LCAT-/-), την apoE (apoE-/-) αλλά και μια ομάδα ποντικών που εκφράζουν το πλήρες γονιδίωμα (C57BL/6) τέθηκαν σε δίαιτα πλούσια σε λιπαρά (δίαιτα δυτικού τύπου) για 24 εβδομάδες και πραγματοποιήθηκαν ιστολογικές, βιοχημικές και κινητικές αναλύσεις. Στα apoA-I-/- ποντίκια παρατηρήθηκε αύξηση του σωματικού βάρους, έντονη συσσώρευση τριγλυκεριδίων στο ήπαρ, διαταραγμένη ιστολογική εικόνα του ήπατος και ανάπτυξη διατροφικά επαγόμενης NAFLD όπως, επίσης, παρουσίασαν ανοχή στη γλυκόζη και αντίσταση στην ινσουλίνη. Επιπλέον, η ποσοτικοποίηση του mRNA των γονιδίων FASN, DGAT-1 και PPAR-γ απέκλεισε την de novo σύνθεση των λιπαρών οξέων και των τριγλυκεριδίων σαν πιθανή αιτία της εμφάνισης της νόσου στα apoA-I-/- ποντίκια. Παρόμοια το μεταβολικό προφίλ δεν ανέδειξε σημαντικές διαφορές στην ενεργειακή δαπάνη μεταξύ των apoA-I-/- και των C57BL/6 ποντικών. Επίσης, παρατηρήθηκε ενισχυμένη εντερική απορρόφηση, ταχύτερη κάθαρση των μεταγευματικών τριγλυκεριδίων από την κυκλοφορία και μειωμένη ταχύτητα ηπατικής έκκρισης των πολύ χαμηλής πυκνότητας λιποπρωτεϊνών (VLDL) σε σχέση με την ομάδα ελέγχου. Γονιδιακή μεταφορά της apoA-IMilano μέσω αδενοϊού σε apoA-I-/- ποντίκια που έλαβαν δίαιτα δυτικού τύπου για 12 εβδομάδες, είχε ως αποτέλεσμα την μείωση της συγκέντρωσης των ηπατικών τριγλυκεριδίων και την βελτίωση της ιστολογικής εικόνας και αρχιτεκτονικής του ήπατος. Τα ποντίκια αυτά λόγω της έλλειψης της apoA-I δεν συνθέτουν HDL, επομένως η απουσία της HDL σε συνδυασμό με δίαιτα πλούσια σε λιπαρά οδηγεί στην εμφάνιση παχυσαρκίας, διαταραχών στο μεταβολισμό της γλυκόζης και NAFLD. Για να αξιολογήσουμε τη συνεισφορά της ποιότητας της HDL στην εμφάνιση των παραπάνω διαταραχών, μελετήσαμε LCAT-/- ποντίκια που διαθέτουν ¨ανώριμη¨ δισκοειδή HDL. Όπως και στα ποντίκια που δεν εκφράζουν την apoA-I, έτσι και σε αυτή την ομάδα παρατηρήθηκε σημαντική διατροφικά επαγόμενη εναπόθεση τριγλυκεριδίων στο ήπαρ και διαταραγμένη ιστολογική εικόνα και αρχιτεκτονική του ήπατος. Αντιθέτως στα ποντίκια αυτά παρατηρήθηκε σημαντική αύξηση του σωματικού βάρους σε σχέση με την ομάδα ελέγχου. Επιπλέον, τα LCAT-/- ποντίκια δεν παρουσίασαν διαταραχές στο μεταβολισμό της γλυκόζης ενώ οι κινητικές αναλύσεις έδειξαν ότι η απουσία της LCAT σχετίζεται με αυξημένη εντερική απορρόφηση των διατροφικών λιπιδίων, ταχύτερη κάθαρση των μεταγευματικών τριγλυκεριδίων και μειωμένη ταχύτητα ηπατικής έκκρισης των VLDL σε σχέση με τα C57BL/6 ποντίκια. Γονιδιακή μεταφορά της LCAT μέσω αδενοϊού σε LCAT-/- ποντίκια που έλαβαν δίαιτα δυτικού τύπου για 12 εβδομάδες, είχε ως αποτέλεσμα την σημαντική μείωση της συγκέντρωσης των ηπατικών τριγλυκεριδίων και την βελτίωση της ιστολογικής εικόνας και αρχιτεκτονικής του ήπατος. Τα μέχρι τώρα δεδομένα μας λοιπόν υποδεικνύουν πως το μεταβολικό μονοπάτι της HDL είναι κεντρικός ρυθμιστής διαδικασιών σχετιζόμενων με την εναπόθεση διατροφικών τριγλυκεριδίων στο ήπαρ και την εμφάνιση NAFLD. Επιπλέον, τα αποτελέσματα μας υποστηρίζουν πως η συνύπαρξη μειωμένης και πιθανόν δυσλειτουργικής HDL μαζί με NAFLD σε ασθενείς με μεταβολικό σύνδρομο δεν είναι μια απλή σύμπτωση αλλά υποδηλώνει μία ισχυρή μηχανιστική συσχέτιση ανάμεσα στις δύο αυτές καταστάσεις. Προκειμένου να μελετηθεί ο ρόλος του μεταβολικού μονοπατιού των χυλομικρών, μελετήσαμε ποντίκια με έλλειψη στην apoE τα οποία καταβολίζουν βραδέως τα διατροφικά λιπίδια. Τα apoE-/- ποντίκια αντιστάθηκαν στην παχυσαρκία και στην εμφάνιση της διατροφικά επαγόμενης NAFLD σε σχέση με τα C57BL/6 ποντίκια. Επίσης, δεν παρουσίασαν διαταραχές στο μεταβολισμό της γλυκόζης και οι κινητικές αναλύσεις έδειξαν ότι είχαν βραδύτερη κάθαρση των μεταγευματικών τριγλυκεριδίων από την κυκλοφορία του αίματος. Θέλοντας να ερευνήσουμε και το ρόλο του υποδοχέα της LDL, πραγματοποιήθηκε μια σειρά ανάλογων πειραμάτων σε LDLr-/- ποντίκια που έλαβαν δίαιτα δυτικού τύπου για 24 εβδομάδες. Τα LDLr-/- ποντίκια είχαν σημαντική συσσώρευση τριγλυκεριδίων στο ήπαρ και NAFLD προτείνοντας ότι η ηπατική συσσώρευση τριγλυκεριδίων μέσω της apoE είναι μια διαδικασία ανεξάρτητη από τον LDLr. Τα ευρήματα μας προτείνουν ένα νέο ρόλο κλειδί για την apoE ως ένας περιφερικός συντελεστής στην ομοιόσταση των ηπατικών λιπιδίων και στην ανάπτυξη της διατροφικά επαγόμενης NAFLD. Επιπλέον, δείχνουν ότι οι διαταραχές στο μεταβολικό μονοπάτι των χυλομικρών σχετίζονται άμεσα με την εμφάνιση της NAFLD. Συμπερασματικά, το μεταβολικό σύστημα λιπιδίων και λιποπρωτεϊνών φέρεται να κατέχει κεντρικό ρόλο στην εναπόθεση ηπατικών τριγλυκεριδίων και στην εμφάνιση της NAFLD. / In the present study, we investigated the contribution of HDL and the clylomicron/VLDL pathways in the development of obesity, glucose metabolism and diet-induced non alcoholic fatty liver disease (NAFLD). Thus, we chose to study apolipoproteins A-I (apoA-I) and E (apoE), as well as the enzyme lecithin:cholesterol acyltransferase (LCAT). ApoA-I is the main protein of high density lipoprotein (HDL) and is responsible for it’s synthesis, LCAT esterifies the free cholesterol of plasma lipoproteins and forms mature particles of HDL and apoE participates in the catabolism of chylomicrons, very low density lipoproteins (VLDL) and low density lipoproteins (LDL) and also participates in the de novo biogenesis of HDL. In an attempt to study the role of all these particles in the development of diet-induced NAFLD, apoA-I deficient, LCAT deficient, apoE deficient and control C57BL/6 mice were fed western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5Kcal/g) for 24 weeks and their sensitivity towards NAFLD was assessed by histological and biochemical methods. ApoA-I deficient (apoA-I-/-) mice showed increased body weight, increased diet-induced hepatic triglyceride deposition and disturbed hepatic histology while they exhibited reduced glucose tolerance and insulin sensitivity. Quantification of FASN, DGAT-1, and PPARγ mRNA expression suggested that the increased hepatic triglyceride content of the apoA-I-/- mice was not due to de novo synthesis of triglycerides. Similarly, metabolic profiling did not reveal differences in the energy expenditure between the two mouse groups. However, apoA-I-/- mice exhibited enhanced intestinal absorption of dietary triglycerides, accelerated clearance of postprandial triglycerides, and a reduced rate of hepatic VLDL triglyceride secretion. In agreement with these findings, adenovirus-mediated gene transfer of apoA-IMilano in apoA-I-/- mice fed western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content and an improvement of hepatic histology and architecture. In order to evaluate the contribution of HDL quality in the development of the metabolic disturbances described above, we studied LCAT-/- mice which have immature discoidal HDL circulating in the plasma. Similarly to apoA-I-/- mice, in the LCAT-/- group we observed increased diet-induced hepatic triglyceride deposition and impaired hepatic histology and architecture. In contrast hoewever, these mice gained significantly more body weight, compared to the control group though they did not develop disturbances in their plasma glucose metabolism. Mechanistic analyses indicated that LCAT deficiency was associated with enhanced intestinal absorption of dietary triglycerides, accelerated clearance of postprandial triglycerides, and a reduced rate of hepatic very low density lipoprotein triglyceride secretion. No statistical difference in the average daily food consumption between mouse strains was observed. Adenovirus-mediated gene transfer of LCAT in LCAT-/- mice that were fed western-type diet for 12 weeks resulted in a significant reduction in hepatic triglyceride content and a great improvement of hepatic histology and architecture. Taken together, these data suggested that HDL metabolic pathway is a central modulator of processes associated with diet-induced hepatic lipid deposition and NAFLD development. Furthermore, our results sypport that the the coexistence of reduced and possibly dysfunctional HDL with NAFLD in patients with metabolic syndrome is not a mere coincidence, rather indicates a strong mechanistic link between these two conditions. In order to study the role of the chylomicron metabolic pathway, we employed apoE-deficient mice, which show a very slow catabolism of dietary lipids. Our data indicate that the apoE-/- mice are resistant to obesity and to diet-induced NAFLD compared to control C57BL/6 mice and they don’t reveal disturbances in the glucose metabolism. In an attempt to identify the molecular basis for this phenomenon biochemical and kinetic analyses revealed that apoE-/- mice displayed a significantly delayed post-prandial triglyceride clearance from their plasma. In contrast to apoE-/- mice, LDLr-/- mice fed western-type diet for 24 weeks developed significant accumulation of hepatic triglycerides and NAFLD suggesting that the apoE-mediated hepatic triglyceride accumulation in mice is independent of the LDLr. Our findings suggest a new role of apoE as key peripheral contributor to hepatic lipid homeostasis and the development of diet-induced NAFLD. Furthermore, they show that the disturbances in the metabolic pathway of chylomicron are related, directly, with the development of NAFLD. Overall, our findings reinforce our initial hypothesis that the transport of dietary lipids from the intestine to the liver plays a central role to the deposition of triglycerides in the liver and the development of NAFLD.

Oxidative stress is characterized by the cellular accumulation of reactive oxygen species (ROS). Increased production of ROS, such as the superoxide anion (O2.-), or a deficiency in their clearance by antioxidant defenses, mediates the cellular pathology. Non-alcoholic fatty liver disease (NAFLD) is a broad spectrum liver disorder commonly manifesting in milieu of the metabolic syndrome. Oxidative stress is an important pathogenic mediator in NAFLD, and in its associated morbidities like atherosclerosis. The objective of my research was to investigate the regulation of oxidative stress and the antioxidant actions of natural health products (NHPs) in the context of NAFLD and its associated disorders. The O2.- generating NADPH oxidase contributes to atherogenesis by facilitating macrophage induced vascular injury. In manuscript I, the plant alkaloid berberine effectively abolished NADPH oxidase mediated O2.- production in lipopolysaccharide stimulated macrophages. Real-time PCR analysis and siRNA transfection studies revealed that berberine mediated its effects through down-regulation of the oxidase’s catalytic subunit gp91phox. Berberine also restored the activity of the O2.- clearing enzyme superoxide dismutase (SOD). High fat diet (HFD) fed rodents are a popular model for investigating NAFLD pathogenesis. In manuscript II, folic acid supplementation significantly reduced HFD-induced hepatic oxidative stress and liver injury in mice. Folic acid decreased NF-kB/DNA binding, down-regulated NADPH oxidase gene expression, and inhibited the oxidase. The antioxidant activities of SOD and catalase were restored and the reduced to oxidized glutathione ratio (GSH:GSSG) was re-established with folic acid supplementation. Folic acid’s hepatoprotective antioxidant effects were associated with a marked improvement in liver histology. Homocysteine (Hcy) levels are perturbed in NAFLD, but the etiology is unclear. In manuscript III, HFD fed mice exhibited decreased Hcy levels. Real-time PCR and Western Immunoblotting analysis revealed that Hcy catabolising enzymes cystathionine-b-synthase (CBS) and cystathionine-g-lyase (CSE) were increased in the liver of these animals. The transsulfuration activities of these enzymes were elevated and coincided with enhanced hepatic hydrogen sulfide biosynthesis. Glutathione was maintained despite increased hepatic oxidative stress. Taken together, NHPs such as berberine and folate, and Hcy catabolising enzymes CBS and CSE, might have therapeutic potential for managing oxidative stress in NAFLD and its associated co-morbidities. / October 2015

Oxidative stress, lipid peroxidation, and endotoxaemia with cytokine-mediated injury have been implicated as factors in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The degree of insulin resistance together with co-existing inadequacies of vital antioxidant defence mechanisms may be important determinants of progression to fibrosis in patients with non-alcoholic steatohepatitis (NASH). Current therapies are targeted at improving insulin sensitivity as well as addressing hepatic repair including anti-inflammatory strategies. Anti-oxidants remedies have also been tested but the role of selenoenzymes with antioxidant action, namely thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPX1) have been ignored. The aim of this thesis is to investigate the role of selenium in the pathophysiology of NAFLD both in vitro and in vivo. The in vitro studies used cell lines representing the cell types involved in the disorder; hepatocytes (C3A line) and hepatic stellate cells (LX-2 line). In order to assess the influence of selenium status and selenoenzymes expression on the pathogenesis of NAFLD it was necessary to develop a culture system which allowed good cell viability in selenium free culture medium. This was achieved by the use of an insulin and transferrin (IT)-supplemented medium which importantly was free of any animal serum additions. Using this IT culture medium, selenium addition (as selenite) produced a significant increase in the expression of GPX1 and TR1 in both C3A and LX2 cells. TR1 and GPX1 were expressed at similar levels in both C3A and LX-2 cells. It was also necessary to develop an in-vitro model for fat loading C3A cells to mimic fatty liver pathophysiology. Two models of fat loading were investigated. One model used lactate, pyruvate, octanoate and ammonium (LPON). LPON has been previously used to increase the functionality of C3A cells but it was observed that fat droplets accumulated in these LPON treated cells. Dissection of the agents in the LPON revealed that octanoate was the factor that increased the triglyceride accumulation. Interestingly, octanoate also increased the expression of TR1 and GPX1, suggesting that it could induce oxidative stress leading to the induction of selenoenzymes to afford a protective defence mechanism. In the second model, oleate and/or palmitate were used to fat-load C3A cells. These cells had significantly higher triglyceride content than the LPON-fat-loaded cells. However, oleate and/or palmitate treatments did not increase the expression of either TR1 or GPX1 in C3A cells suggesting perhaps these cells were not under oxidative stress. LPON and oleate/palmitate were also capable of fat loading LX2 cells. Selenium-supplementation of C3A and LX-2 cells efficiently protected (measured by their lactate dehydrogenase retention) them from oxidative damage induced by t-butylhydroperoxide. This suggests that selenium supplementation through its incorporation into selenoenzymes could protect the cells from the oxidative damage. The role of selenium was also investigated in the regulation of α-1 pro-collagen mRNA expression. In LX-2 cells, the expression of α-1 pro-collagen mRNA was unaffected by the selenium status of the cell. Similarly the selenium status of C3A cells had no effect on modifying α-1 pro-collagen mRNA of LX2 cells when co-culture or conditioned medium experiments were performed. These results suggest that LX-2 cells were already largely activated and at a stage unable to be ameliorated by selenium treatment. In contrast, studies on C3A cells revealed that TGF-β1 (common inducer of α-1 pro-collagen mRNA in hepatic stellate cells) dramatically increased the expression of α-1 pro-collagen mRNA in C3A cells to the levels observed in LX-2 cells. More interestingly, selenium supplementation of C3A cells notably decreased α-1 pro-collagen mRNA expression in response to TGF-1. In the in vivo study, plasma selenium in type 2 diabetics (high risk of developing NAFLD) were inversely related to the body mass index and in most patients selenium levels were below that required to maximally express GPX1 in red cells. Furthermore, type 2 diabetics had lower plasma selenium levels compared to the healthy control group. Collectively, this suggests that in the UK population, obesity is a risk factor for both insulin resistance and decreased selenium status leading to sub-optimal antioxidant protection. In conclusion, this study provides evidence that selenium through increasing the expression of selenoenzymes is beneficial in protecting liver cells from oxidative stress. Furthermore, selenium is capable of suppressing α-1 pro-collagen mRNA expression in hepatocytes although not in activated hepatic stellate cells. Taken together these data support the view that suboptimal selenium intake in the UK may be a risk factor in the pathogenesis of NAFLD.

In pharmacotherapeutics, the term "correct dosing" is based on the concept that too high a systemic concentration will lead to drug toxicity, while drug levels that are too low may not produce the intended therapeutic effect. Often, the factors determining the ability of a patient to manage a given dose rely on their capacity to efficiently metabolize and eliminate drugs from the body. The liver plays a crucial role in the processing of many clinically relevant drugs via three stages of hepatic drug management. Drugs must first be taken into hepatocytes by uptake transporters. Drugs are then metabolized by phase I and phase II enzymes to make them more manageable. Finally, metabolites are removed from the hepatocyte by efflux transporters either into the bile for elimination or reintroduction to systemic blood. Alterations in one or more of the hepatic drug management stages increase the potential for adverse drug reactions (ADRs).In the United States, ADRs account for between 3%-12% of admissions to hospitals, and approximately 5% of deaths each year. While less than 20% of these cases are due to genetic polymorphisms, the vast majority of ADRs are due to environmental factors including disease. Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of conditions progressing from steatosis to non-alcoholic steatohepatitis (NASH) and often leading to cirrhosis. Presently, NASH patients represent the greatest population of candidates for liver transplant, illustrating the severity as well as the incidence of this disease. Patients with NAFLD are typically treated for co-existing conditions of the metabolic syndrome (i.e. hyperlipidemina or type II diabetes) and therefore represent a distinct population at risk for adverse drug reactions.The following studies show that experimental NAFLD affects both the signal transduction pathways regulating hepatic drug management genes as well as the hepatic uptake transporter function. Additionally, patient livers diagnosed with progressive stages of NAFLD, display altered CYP activity and efflux transporter expression similar to those previously reported in experimental NAFLD. Given that changes observed in experimental NAFLD result in functional changes in hepatic drug management, similar changes observed in patients with this disease suggest an increased risk for ADRs.

The body encounters an innumerable amount of foreign substances, termed xenobiotics, which it must remove in order to prevent damage to cells and organs. This system of removal is a collection of processes known as ADME (absorption, distribution, metabolism, and excretion). The dynamics of ADME ultimately determine the fate, or pharmacokinetics, of a xenobiotic in the body whether it be an administered pharmaceutical or a potentially harmful toxicant. The major cellular effectors of ADME are the drug metabolizing enzymes (DMEs) and transporters. DMEs function to transform xenobiotics into a metabolite that is more suitable for excretion, whereas drug transporters serve a two-fold function. They may facilitate the uptake of the xenobiotic into the cell so that it can be acted upon by DMEs, or they may function to actively secrete xenobiotics and metabolites from the cell, encouraging their removal from the body. Any perturbations in the expression or function of these critical cellular effectors can result in the diminished therapeutic effect of a pharmaceutical via accelerated removal from the body, or increased toxicity of a pharmaceutical or toxicant due to retention in the body and increased exposure.Perturbations in the ADME processes may result in adverse drug reactions (ADRs) which are an unintended response to a pharmaceutical when administered at the recommended dose. In the last reporting year, the USFDA documented 471,291 serious ADRs causing hospitalization or permanent disabilities, of which 82,724 resulted in death. ADRs can be categorized as two types: dose-related ADRs, and those that are generally unpredictable and mostly occur in susceptible individuals. The major factors that make a person susceptible to ADRs are genetics and disease; however, genetics account for only a small proportion. This dissertation is focused on the contribution of an environmentally-derived component, particularly liver disease, to the occurrence of ADRs. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease of industrialized nations. It represents a spectrum of damage progressing to the severe stage of nonalcoholic steatohepatitis (NASH), and is closely related to obesity and type 2 diabetes. The following studies have determined the effect of NAFLD and NASH on DMEs and transporters, and demonstrated the propensity for NASH to result in serious ADRs.

Différents facteurs sont connus pour affecter les paramètres de qualité du foie gras. Parmi ces paramètres, la fonte est considérée comme étant la plus critique du point de vue des producteurs et des consommateurs. Outre les facteurs déjà connus, le but de ce travail est d’étudier si les activités protéolytiques hépatiques pourraient jouer un rôle dans le déterminisme de la qualité du foie gras. Pour atteindre cet objectif, trois expérimentations ont été menées, dans lesquelles nous avons utilisé des approches biochimiques et protéolytiques ainsi que des mesures de qualité. Dans ces trois expériences nous avons évalué : 1) l’effet du gavage chez deux types génétiques de canards, 2) l’effet de la vitesse de refroidissement et du stockage post mortem et 3) l’effet de la variabilité de taux de fonte sur les activités protéolytiques. Nos résultats montrent que les activités protéolytiques restent constantes ou baissent avec le gavage. De plus, malgré des différences connues quant à leur capacité à développer une stéatose hépatique, les canards de Barbarie et les canards Pékin ont réagis de la même façon en ce qui concerne leurs activités protéolytiques. En ce qui concerne l’effet des différentes vitesses de refroidissement, nous avons montré que plus le refroidissement est lent, plus le foie fond et que ceci est –au moins partiellement- due à des activités protéolytiques plus élevées. Finalement, nous avons montré que les foies à fonte élevée présentés également des activités protéolytiques supérieures à celles des foies à fonte faible. Dans cette thèse, nous avons pu mettre en évidence certaines relations existantes entre la protéolyse hépatique et les qualités du foie gras de canard. Cependant, d’autres études seront nécessaires pour mieux comprendre les mécanismes responsables de ces relations dans le but d’exploiter le potentiel des protéases dans la maîtrise de la qualité du foie gras. / Different factors are known to affect the quality parameters of « foie gras ». Among those parameters, the cooking loss is considered to be the most critical one in the eyes of both producers and consumers. Besides already known factors, the objective of this work is to investigate whether proteolytic activities might play a role in the determinism of « foie gras» cooking losses. To achieve this objective three experiments were conducted in which we used biochemical and proteomic approaches as well as quality analysis. In these three different experiments we evaluated: 1) the effect of overfeeding in two breeds of ducks (Muscovy and Pekin ducks) 2) the effect of chilling rate and post mortem storage and 3) the effect of cooking loss variability on proteolytic activities. Our results show that overfeeding either does not affect proteases activities, either causes their decrease. In addition, despite the known differences regarding their ability to develop hepatic steatosis, in our study Muscovy and Pekin ducks’ proteolytic activities responded similarly to overfeeding. Concerning the effect of chilling rates, we showed that the slower the chilling rate the higher the melting after cooking. This result is - at least partially- due to higher proteolytic activities. Finally we showed that livers with a high melting rate presented higher proteolytic activities than those with a low melting rate. In this thesis, we were able to expose certain relations between hepatic proteolysis and fatty liver qualities. However, more studies are required to better understand the mechanisms underlying these relations in the goal of exploiting the potential of proteases in fatty liver quality control.