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.

Le diabète de type 2 (T2D) est un trouble métabolique de l’homéostasie du glucose. Il est caractérisé par une hyperglycémie chronique qui résulte en partie d’une production excessive de glucose par le foie conséquence au développement d’une résistance à l’insuline. Le T2D est une pathologie multifactorielle à la fois génétique et environnementale. Récemment des études d’associations de gènes (GWAS) dans différentes cohortes ont mis en évidence une forte corrélation entre le locus CDKN2A et le risque de développement du T2D en se basant sur certains paramètres métaboliques tel que la glycémie à jeun. Le locus CDKN2A code pour des protéines régulatrices du cycle cellulaire dont la protéine p16INK4a. p16INK4a est largement décrite dans la littérature pour son rôle suppresseur de tumeurs et comme marqueur de sénescence, cependant son rôle dans le contrôle de l’homéostasie hépatique du glucose n’a jamais été rapporté. Afin de déterminer le rôle de p16INK4a dans le métabolisme hépatique du glucose, nous avons utilisé in vivo des souris sauvages (p16+/+) et déficientes pour p16INK4a (p16-/-) et in vitro des hépatocytes primaires ainsi que la lignée AML12. Nous avons montrés qu’après un jeune, les souris p16-/- présentent une hypoglycémie moins prononcée qui se traduit par une expression hépatique plus élevée de gènes de la néoglucogenèse tels que PEPCK, G6Pase et PGC1a. De plus, les hépatocytes primaires de souris p16-/- présentent une meilleur réponse au glucagon que ceux des p16+/+. Enfin, nous avons montrés que la diminution d’expression de p16INK4a par siRNA dans les AML12 suffit à induire l’expression des gènes de la néoglucogenèse et potentialise la réponse de ces cellules à différents stimuli gluconéogenique. L’effet observé dépend de l’activation de la voie PKA-CREB-PGC1A. L’ensemble de ces données montrent pour la première fois que p16INK4a pourrait jouer un rôle un cours du développement du T2D. / P16INK4a is a tumor suppressor protein well described as a cell cycle regulator. p16INK4a blocks cyclin D/ cyclin dependent kinase (CDK) 4 activity by binding to the catalytic subunit of CDK4, preventing retinoblastoma protein phosphorylation and subsequently the release of the E2F1 transcription factor. As a consequence; the transcription of genes required for progression to the S phase is restrained. Recently, genome-wide association studies (GWAS) associated the CDKN2A locus, encoding, amongst other genes, p16INK4A, with an increased risk of type 2 diabetes (T2D) development. However, the pathophysiological link between p16INK4a and hepatic glucose homeostasis remains unknown. In this context, we investigated the role of p16INK4a in hepatic glucose metabolism in vivo using p16+/+ and p16-/- mice and in vitro using primary hepatocytes and the AML12 hepatocyte cell line.p16-/- mice exhibited a higher response to fasting as shown by an increased hepatic gluconeogenic gene expression including phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-biphosphatase (F1,6P) and glucose-6-phosphatase (G6Pase). p16-/- mice displayed an enhanced hepatic gluconeogenic activity in vivo upon administration of pyruvate, a gluconeogenic substrate. Consistent with this, in vitro data show that p16-/- primary hepatocytes display an enhanced gluconeogenic response to glucagon. In addition, knock down of p16INK4a by siRNA in AML12 cells increased gluconeogenic gene expression. These effects were associated with an increased activity of the PKA-CREB signaling pathway which leads to increased PPARg coactivator 1 (PGC1)α expression, a key transcriptional co-activator that regulates genes involved in energy metabolism. These findings describe a new function for p16INK4a as an actor in the hepatic adaptation to metabolic stress and suggest that p16INK4a could play a role during T2D development .

OBJECTIVE. To identify clinical, socio-demographic, dietary and biological markers to be used in a non-invasive and cost-effective clinical tool for screening for non-alcoholic fatty liver disease (NAFLD) in obese adolescents. METHODS. We conducted a cross-sectional analysis using baseline data from 77 obese adolescents enrolled in a drug trial for the Glaser Pediatric Research Network, between October 2003 and August 2007. NAFLD was defined as the presence of fatty liver infiltration assessed by computed tomography. Receiver operation characteristic (ROC) analyses were performed to identify variables with the highest area under the curve (AUC) for NAFLD. Serum biomarkers were dichotomized using sensitivity analyses to identify the best cutoff point for NAFLD. Multiple logistic regression models were created to predict prevalent NAFLD. RESULTS. Serum triglycerides was identified as the best biomarker for NAFLD (AUC 0.790; pseudo R2 0.235). Additional adjustment for sex, age and Tanner stage improved the AUC to 0.846 and the pseudo R2 to 0.290. We then explored adding a simple biochemical marker for predicting NAFLD (HOMA-B, ALT or glutamate) and found that HOMA-B led to greater improvement in AUC, ALT to a greater improvement in sensitivity and glutamate to a greater improvement in the pseudo R2. Thus, all three factors individually improved overall model performance to some degree and inclusion of all three led to an AUC=0.907 and pseudo R2=0.433. Our second objective was to develop a more complex exploratory model starting with the inclusion of important clinical predictors (triglycerides, sex, age, Tanner stage, SBP, BMI, waist circumference); this yielded an AUC of 0.871 and pseudo R2 of 0.342. Further adjustment for HOMA-B, ALT and glutamate gave an AUC=0.913 and pseudo R2=0.497. CONCLUSION. Simple clinical and biochemical factors may be used to screen for prevalent NAFLD. Our simplest clinically relevant model using triglycerides, age, sex and Tanner stage provided a reasonable screening tool for NAFLD in obese adolescents. A second more complex model that warrants further testing includes triglycerides, sex, age, Tanner stage, SPB, BMI, waist circumference, HOMA-B, ALT and glutamate. In this study, this model was more accurate for detecting undiagnosed cases of NAFLD in this pediatric population.

The non-alcoholic fatty liver disease (NAFLD) is the most common liver pathology in developed countries with an estimated prevalence of 20 to 30% in the American population. A typically benign and asymptomatic pathology, NAFLD is characterized by hepatic steatosis and abnormal levels of hepatic enzymes stemming from an increase in circulating free fatty acids originating from white adipose tissue lipolysis, an increased de novo lipogenesis, reduced fatty acid oxidation and decreased hepatic triglycerides secretion, all within an insulin resistance context. NAFLD has the potential to progress to the non-alcoholic steatohepatitis (NASH), a condition marked by inflammation, advanced oxidative stress and fibrosis. NASH is expected to be the leading cause of liver transplant by 2020 due to its complications (i.e.: cirrhosis, hepatocellular carcinoma and liver failure). Various xenobiotics such as pesticides have been shown to promote the apparition and development of NAFLD. Of interest to this study is the neonicotinoid imidacloprid, more contemporarily known for its suspected role in the colony collapse disorder of various anthophilae species. Imidacloprid has been shown to induce hepatic oxidative stress in rats, a significant factor in the development of NAFLD and its progression to NASH. Lifestyle modifications, namely physical exercise, is a current treatment which has been proven beneficial to prevent and treat NAFLD by reducing hepatic steatosis, oxidative stress and improving insulin sensitivity. The role of any neonicotinoid on the development of NAFLD has yet to be examine and few have looked at the role of exercise in the treatment of NAFLD brought about by pesticide contamination.

研究背景及實驗目的: 非酒精性脂肪性肝炎(NASH)使得肥胖和2 型糖尿病變得複雜，肝臟炎症的持續產生是其主要的發病機理。CXCL10 是一種促進炎症的細胞因數，其在肥胖和2 型糖尿病中的表達顯著升高。CXCL10 以及其受體CXCR3 是否在NASH 的發生發展中起作用尚不清楚。在本研究中，我們探索了CXCL10 以及其受體CXCR3 在脂肪性肝炎中的功能， 並評估了CXCL10 在NASH 中的臨床價值。 / 實驗方法：CXCL10 基因敲除鼠，CXCR3 敲除鼠以及野生型C57BL/6 小鼠給予蛋氨酸膽鹼缺乏食(MCD)4 周或者8 周。CXCL10 的信號通路以及下游靶點通過細胞因數分析，cDNA array, 蛋白DNA 結合實驗，自噬溶酶體系統分析進行檢測。為了闡明CXCL10 抑制對NASH 的預防治療作用，我們給MCD 餵養的小鼠注射抗CXCL10 抗體。用不同濃度的CXCL10 抗體以及CXCR3 抑制劑NIBR2130 幹預MCD 培養的肝細胞株AML-12。臨床研究中，我們收集了147個非酒精性脂肪肝患者以及73 個健康對照的血清，用酶聯免疫吸附試驗檢測血清中CXCL10 的水準。 / 結果：野生型小鼠給予MCD 餵養後，CXCL10 以及CXCR3 的表達升高，並出現脂肪性肝炎的表現。然而，MCD 飼養的CXCL10 以及CXCR3 基因敲除鼠中，脂肪性肝炎明顯減輕。CXCL10 通過促炎細胞因數的產生以及NK-κB 信號通路促進MCD 飼養的小鼠NASH 的發生。CXCL10 通過促進脂質合成的基因SREBP-1c, ChREBP 和 SCD-1 引起脂肪變性，並通過CYP2E1 以及 C/EBPβ 的上調引起氧化應激。值得注意的是，自噬的損傷在CXCL10 以及CXCR3 導致的脂肪性肝炎的進展中起重要作用。 MCD 飼養的野生型小鼠中p62 以及LC3-II 表達明顯高於CXCL10 以及CXCR3 基因敲除鼠。通過抗CXCL10 抗體中和CXCL10 可以減輕MCD 食引起的小鼠脂肪性肝炎以及MCD 培養液引起的AML-12 細胞損傷。高選擇性的CXCR3 抑制劑NIBR2130 也可以抑制MCD 引起的肝細胞損傷。我們進一步研究了CXCL10 的臨床應用價值，發現NASH 患者血清以及肝臟中CXCL10 的水準明顯升高。更重要的是，血液中CXCL10 的水準與肝小葉炎症程度有關，是NASH 的獨立危險因素。 / 結論：我們的研究首次發現CXCL10 以及其受體CXCR3 通過促進炎症，脂質聚集，氧化應激以及自噬缺乏在NASH 的發病中起重要作用。抑制CXCL10 或者CXCR3 為NASH 患者的治療提供了新的方法。CXCL10 可作為NASH 患者非侵入性診斷的標誌物。 / Background and aims: Non-alcoholic steatoheaptitis (NASH) complicates obesity and type 2 diabetes, while recruitment and perpetuation of liver inflammation is central to its pathogenesis. Expression of C-X-C motif chemokine 10 (CXCL10), a proinflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 and its receptor CXCR3 play a role in NASH is unknown. In this study, we investigated the functional significance of CXCL10 and its receptor CXCR3 in steatoheaptitis. Moreover, the clinical impact of CXCL10 in NASH was examined. / Methods: Gene-deleted CXCL10 (CXCL10-/-), CXCL10 receptor CXCR3 (CXCR3-/-) and C57BL/6 wildtype (WT) mice were fed methionine and choline-deficient (MCD) diet for 4 or 8 weeks. Cytokine profiling assay, cDNA array, protein-DNA binding activity assay and autophagosome-lysosome system analysis of CXCL10 signaling and downstream targets were performed. In other experiments, we injected neutralizing anti-CXCL10 monoclonal antibodies (mAb) into MCD diet-fed WT mice, while AML-12 cells were cultured in MCD medium in the presence of anti-CXCL10 mAb or CXCR3 inhibitor (NIBR2130) for 24 hours. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 controls. Circulating CXCL10 levels were determined by enzyme-linked immunosorbent assay. / Results: MCD-fed WT mice developed steatohepatitis with higher hepatic CXCL10 and CXCR3 expression. CXCL10-/- and CXCR3-/- mice were refractory to MCDinduced steatohepatitis. In WT mice with steatohepatitis, but not in CXCL10-/- mice, CXCL10 was associated with the induction of pro-inflammatory chemokines and cytokines, as well as activation of nuclear factor-κB (NF-κB) signaling. CXCL10 expression was linked to steatosis through lipogenic factors, including liver X receptors and its downstream targets (SREBP-1c, ChREBP and SCD-1), and also to oxidative stress (up-regulation of CYP2E1 and C/EBPβ). In particular, autophagy deficiency was involved in CXCL10- and CXCR3-induced steatohepatitis as indicated by p62 and LC3-I/II protein accumulation in MCD-fed WT mice than in CXCL10-/- and CXCR3-/- mice. Moreover, the impaired autophagic function was related to the reduction of lysosomal function in CXCL10- or CXCR3-induced NASH. Blockade of CXCL10 by anti-CXCL10 mAb protected against MCD-induced steatohepatitis in vivo and against MCD-mediated injury to AML-12 cells in vitro. The highly selective CXCR3 antagonist NIBR2130 also inhibited MCD-induced injury in AML-12 hepatocytes. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. / Conclusions: We demonstrate for the first time that CXCL10 and its receptor CXCR3 plays a pivotal role in the pathogenesis of NASH by promoting inflammation, fatty acid accumulation, oxidative stress and autophagy deficiency. Blockade of CXCL10 or CXCR3 is a potential novel approach for NASH intervention. CXCL10 is a noninvasive biomarker for NASH patients. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Xiang. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 145-167). / Abstracts also in Chinese.

Obesity can lead to a range of health problems including type 2 diabetes (T2DM), cardiovascular disease and non-alcoholic fatty liver disease (NAFLD), and causes an estimated 2.8 million deaths annually (2016). It is a growing epidemic affecting over 600 million people worldwide (in 2014), with 26.8% of the adult population in England alone being obese, an increase of 10% in the last decade, and 62.9% overweight or obese. This trend is predicted to continue, and is attributed to an increasingly sedentary lifestyle, coupled with a high calorie “western diet”, which is estimated to cost >£25billion/year in the UK (2015), which is predicted to rise to £49.9 billion by 2050. It is clear that both sex and genetics affect the extent to which individuals exposed to a high fat diet develop adiposity and its associated morbidities, although the mechanisms underlying these differences are not well understood. Here we explore this aetiology, focusing on poly(A)-binding protein 4 (PABP4), an RNA-binding protein in which polymorphisms associated with altered cholesterol levels and cardiovascular disease risk were identified in human GWAS studies. To this end, I take advantage of an unpublished Pabp4 knock-out mouse, maintained on either normal (ND) or high fat diet (HFD), to explore the role of PABP4 in determining the response to high fat diet. PABP4 is a poorly characterised member of the PABP family, which are multifunctional central regulators of global and mRNA-specific translation, and stability. In cell lines, PABP4 is predominantly cytoplasmic, consistent with such functions. However, analogously to PABP1, the prototypical PABP family member, PABP4 is relocalised to stress granules or the nucleus in response to specific cellular stresses and/or viral infections, suggesting a role in altering gene expression programs in responses to changing cellular conditions. Whilst the expression pattern of PABP4 within tissues has not been previously characterised, western blotting of adult mouse tissues revealed that PABP4 is highly expressed in tissues relevant to obesity, T2DM and NAFLD, such as adipose, pancreas, liver and muscle, consistent with the idea that it may play a role in regulating gene expression programs in response to HFD. Immunohistochemistry of tissue sections provided additional insight, revealing a distinct cellular distribution of PABP4 in some tissues, when compared to the well characterised PABP1. Birth weight and post-birth growth can affect adult metabolism. In particular, low birth weight and catch-up growth, characterised by preferentially putting down adipose over lean mass, increases the risk of metabolic conditions in adulthood, such as obesity, T2DM and cardiovascular disease. Therefore, Pabp4-/- and wildtype mice were weighed at birth and daily until weaning. Interestingly this revealed that Pabp4-/- mice have a reduced weight at birth that is exacerbated to weaning (21days (P21)) (5.7% and 18.3% reductions respectively). This analysis also uncovered a reduced survival to weaning, with both male and female Pabp4-/- mice being present at sub-Mendelian ratios by P21 (p=0.0056). Whilst most death occurred neonatally, Pabp4-/- mice showed an increased rate of attrition until weaning, preceded in some cases by an arrest of weight gain. Weight gain was also tracked from 4 weeks to 12 weeks of age on normal diet showing that Pabp4-/- mice had reduced weight into adulthood (12% reduction at 12wks). Analysis of weight gain by sex uncovered a sexually dimorphic effect of Pabp4-deficiency, with female, but not male, Pabp4-/- mice remaining reduced in weight compared to wildtype after 8 weeks on ND (13.4% reduction in female weight). Body composition analysis showed that fat mass was equivalent to wildtype at 12 weeks of age in both sexes but that female Pabp4-/- mice had a 14.3% reduction in lean mass. Neither the catch-up growth in males nor the reduced lean mass in females was sufficient to result in a change in glucose homeostasis. As the risk of developing metabolic disorders in adult life is a consequence of both genetic and environmental factors, such as diet, Pabp4-/- were placed on a HFD at 4 weeks of age for 8 weeks. HFD models the ‘western’ diet, and has been shown to induce obesity, insulin resistance and glucose intolerance in wildtype mice. Whereas Pabp4-/- mice were only distinguishable from wild-type in terms of female lean mass on normal diet, pronounced sexually dimorphic differences were observed in HFD fed mice. Male Pabp4-/- mice appeared to be partially protected from the negative effects of an 8 week HFD regimen, with a 44% decrease in adipose mass gain compared to wildtype despite equal lean mass. Pabp4-/- male mice also had significantly reduced ectopic lipid stores, with an 81% decrease in hepatic triglyceride concentration compared to wildtype, meaning that NAFLD has not developed. Furthermore, Pabp4- /- male mice did not develop hyperinsulinemia on HFD and retained insulin sensitisation (assessed via glucose tolerance test (GTT) and insulin tolerance test (ITT)), although they displayed wildtype-like elevated plasma glucose concentrations (compared to ND). Western blotting had detected high PABP4 levels in the pancreas, indicating a possible pancreatic origin of these alterations. However, immunofluorescence revealed that PABP4 was confined to the exocrine portion of the pancreas, and was undetectable in the insulin producing pancreatic beta cells, suggesting this phenotype may not be beta cell in origin. This is consistent with the fact that the Pabp4-/- male mice retained an appropriate glucose-induced burst of insulin secretion, and therefore insulin production appears unimpaired. Thus, the primary defect may reside in the exocrine pancreas, which aids digestion, or in other key metabolism related tissues (e.g. muscle, liver, adipose and brain), or a combination thereof. In HFD fed wildtype mice, insulin resistance is caused by increased adiposity and ectopic lipid depots, which blunt insulin stimulated signalling cascades, meaning that the normal responses to insulin (e.g. cellular up take of glucose in muscle and arrested glucose production in liver, to decrease plasma glucose concentrations), are impaired. Therefore, the absence of insulin resistance in HFD fed Pabp4-/- male mice may be a consequence of the reduced increase in adipose mass and ectopic lipid deposits detected in these mice, and their consequent lack of inhibition on insulin signalling pathways. The reduced adiposity was not a result of reduced food intake or dietary fat absorption as male Pabp4-/- mice did not eat less nor exhibit apparent steatorrhea (fatty stools). These results highlight that the Pabp4-/- male mice appear to have an alteration in energy use/storage, and the investigation of this will form the basis of future work. When fed HFD, female Pabp4-/- mice revealed a divergent phenotype to that of wildtype female mice and Pabp4-/- male mice. HFD fed Pabp4-/- female mice showed no difference to HFD-fed wildtype mice in terms of weight, but still exhibited the reduction in lean mass seen on ND, but now with a 22.8% increase in volume of adipose tissue. Together, this means that HFD fed Pabp4-/- females have a higher body fat percentage (32.6% compared to 25.9 % for wildtype females). In contrast to the males, there was no difference in terms of hepatic triglycerides in HFD fed Pabp4-/- female mice and they showed greater hyperglycaemia than wildtype (GTT), although like males they retained insulin sensitisation (ITT). These potentially conflicting results in terms of insulin sensitivity and plasma glucose concentrations may result from the alterations in body composition, which can confound results when lean mass is altered and total body weight is used for calculating doses for GTT/ITT. Interestingly, adiponectin, an adipokine normally found in inverse proportion to adipose mass, was increased in plasma from HFD fed Pabp4-/- female mice (21% increase from HFD fed wildtype mice). Whilst surprising given the increase fat mass of Pabp4-/- females, the insulin sensitising properties of adiponectin may help to explain the retained insulin sensitivity detected in the female Pabp4-/- mice. / The finding that HFD revealed metabolic differences in the Pabp4-/- mice lead to the question of whether Pabp4-/- mice have issues adapting to other situations which require modulation of energy storage and glucose homeostasis. One such event is pregnancy, when maternal regulation of insulin resistance is tightly modulated throughout gestation. We therefore characterised the maternal Pabp4-/- environment in late pregnancy (E18.5), when insulin sensitivity decreases to 40-60% lower than pre-pregnancy which results reduced maternal glucose uptake, freeing the glucose up for the rapidly developing foetus. Pregnant Pabp4-/- mice had elevated plasma insulin concentration post fasting (63.7% increase), however glucose homeostasis was wildtype-like, both in terms of plasma glucose and insulin concentrations, throughout a GTT. However, plasma glucose and insulin concentrations in E18.5 Pabp4-/- foetuses were significantly decreased (9% and 44.3% respectively). Pabp4-/- foetuses also had reduced foetal and placental weight/length parameters. This establishes that the differences in weight observed at birth were present by late gestation and secondly, that the reductions in both foetal glucose and insulin concentrations which may contribute to or underlie the reduced growth. It also suggests that the differences seen in adulthood on HFD may be a consequence of metabolic differences present during pregnancy. Taken together, these data support the hypothesis that PABP4 plays a key role in the regulation of mRNAs which are important in growth, post-natal survival and metabolic adaption to high fat diet.