Дисертації з теми "Non-Alcoholic fatty liver diseases"

Non-alcoholic fatty liver disease is the commonest cause of chronic liver disease in developed countries. The accurate assessment of steatosis is central to the diagnosis of non-alcoholic fatty liver disease. I compared the assessment of steatosis by histology, biochemical triglyceride assays, digital image analysis, with and without Oil Red-O staining, in mouse livers and human liver biopsies. In each case Oil Red-O digital image analysis was the most reliable technique for quantitating steatosis. I then investigated a potential, non-invasive technique for distinguishing steatosis from steatohepatitis as only the latter causes progressive liver disease. The liver contains fluorophores which can be detected by autofluorescence spectroscopy. The fluorophore levels vary depending on the levels of oxidative stress and fibrosis within the liver. Mouse and human livers, were assessed to measure the fluorescence intensity at different wavelengths and this was compared with the histology. The probe was able to accurately identify biopsies which had inflammation and fibrosis with a high degree of sensitivity and specificity. Autophagocytosis has recently been suggested to play a role in fat metabolism. Using liver differentiated HUH7 cells grown in normal or oleate containing media with or without Rapamycin (an autophagocytosis activator) the role of autophagocytosis was investigated. This involved examining steatosis by Oil Red-O digital image analysis, biochemical triglyceride assays, electron microscopy and confocal immunofluorescence. I concluded that activating autophagocytosis decreased the levels of steatosis within the cells. This work has shown Oil Red-O digital image analysis is the most accurate way of assessing steatosis within the liver, that autofluorescence spectroscopy has the ability to distinguish, in real-time, isolated steatosis from steatohepatitis and that autophagocytosis has a role in fat metabolism within the liver which may be exploited therapeutically.

Non Alcoholic fatty Liver disease (NAFLD) is the commonest chronic liver disease worldwide and is a spectrum which includes simple fatty liver (simple steatosis), non-alcoholic steatohepatitis (NASH) and cirrhosis. Simple steatosis is a benign condition but NASH may progress to liver fibrosis and cirrhosis. Why only some develop progressive disease is not known and maybe dependant on the pathophysiology.

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum that spans simple steatosis, through steatohepatitis (NASH) to fibrosis and ultimately cirrhosis. NAFLD is characterised by substantial inter-patient variation in rate of progression and disease outcome: whilst up to 25% of the general population are at risk of progressive disease, only a minority experience associated liver-related morbidity. Inter-patient genetic variation and environment determine severity and progression of NAFLD. This thesis reports a series of studies examining the association of genetic variations in two genes patatin-like phospholipase domain-containing 3 (PNPLA3, rs738409 c.444 C > G, p.I148M) and transmembrane 6 superfamily member 2, (TM6SF2, rs58542926 c.449 C > T, p.E167K) with severity of NAFLD and risk of NAFLD-associated hepatocellular carcinoma (HCC). Addressing first the role of PNPLA3, I demonstrate that the rs738409 variant is associated with steatosis, steatohepatitis and fibrosis in the largest histologically characterised NAFLD cohort of European-Caucasian descent (n=1,005) studied to date. Subsequently, adopting a case-control analyses in a cohort of 100 consecutive Northern European Caucasian patients with NAFLD-associated HCC arising and a cohort of patients with histologically characterised NAFLD, I demonstrate that carriage of the rs738409 minor (G) allele is significantly associated with increased risk of developing NAFLD-associated HCC, independent of potential confounding factors including gender, age at diagnosis, presence of advanced fibrosis/cirrhosis, T2DM and BMI. During my studies, a genome-wide association study identified a SNP in TM6SF2 as a modifier of hepatic triglyceride accumulation measured by MR Spectroscopy. It was therefore pertinent to determine whether this variant also affected risk of steatohepatitis or fibrosis in NAFLD. Using the aforementioned cohorts, I demonstrate for the first time that, in addition to its association with steatosis, the rs58542926 SNP is significantly associated with stage of fibrosis in NAFLD. In contrast to PNPLA3 however, no association with NAFLD-HCC was found. In conclusion, the current thesis confirms the association of PNPLA3 with NAFLD severity and provides new evidence of its association with HCC risk. In addition, itdemonstrates for the first time that TM6SF2 is associated with NAFLD-fibrosis severity. These studies provide important new insights into NAFLD pathogenesis and mandate further functional study.

Background: NAFLD is one of the most common causes of liver disease worldwide. It is a spectrum of disease characterized by macrovesicular steatosis of the liver that ranges from simple fatty liver (steatosis), to non-alcoholic steatohepatitis (NASH). NASH may eventually evolve to cirrhosis and end stage complication. Liver biopsy has long been considered the gold standard of reference to diagnose NAFLD but it is costly and invasive. Recently, non-invasive methods have been proposed. Aims and methods: The aim of this study was to investigate the accuracy of non-invasive methods including (Ultrasound, computed tomography scan, Xenon-133 scan, Hepatic steatosis index, Fibroscan, NAFLD fibrosis score, APRI index, and FIB-4 index) and their combination to diagnose steatosis and to diagnose significant liver fibrosis (>F2) and cirrhosis (F4) as compared to liver biopsy. We conducted a retrospective study of 114 NASH patients (79 males, mean age 49.6±10.6). All had adequate liver histology. Results: The distribution of fibrosis stage was as follows: F0-F1= 50%, F2=16.8%, F3=19.2%, F4=14%. The distribution of steatosis grade was as follows: grade 0-1=16%, grade2=53.3%, grade3=30.7%. The following tests correlated with fibrosis: APRI index (r=0.554), FIB-4(r=0.555), NAFLD fibrosis score (r=0.473), Fibroscan(r=0.586) and Hepatic Steatosis Index (HSI) (r=0.245). The FIB-4 and APRI index showed the best diagnostic accuracy for significant fibrosis as indicated by an Area Under the Curve (AUC) of 0.801 and 0.782, respectively. The FIB-4 showed the best AUC= 0.886 for cirrhosis. None of the following tests US, CT, HSI, and xenon-133 scan were considered correlated significantly. The best combination algorithm for the detection of cirrhosis was gender and FIB-4 with an AUC of 0.8937. Conclusion: this study demonstrates that non-invasive methods for liver fibrosis are accurate to diagnose >F2 and F4. Severe steatosis cannot be reliably diagnosed by non-invasive methods. Notably, a combination of FIB-4 and gender significantly improves the performance of the single method for cirrhosis. These methods may help reducing the number of liver biopsies stratifying NASH patients that should start a screening program for HCC and esophageal varices.
Contexte : La stéatose hépatique non alcoolique (SHNA) est l'une des causes les plus répandues des maladies du foie à l'échelle mondiale. Il s'agit d'un spectre de maladies qui se caractérise par une stéatose hépatique macrovésiculaire allant de la stéatose hépatique simple (stéatose) à la stéatohépatite non alcoolique (NASH). La NASH peut éventuellement évoluer vers une cirrhose et des complications en phase terminale. La biopsie du foie a longtemps été considérée comme la norme de référence par excellence pour le diagnostic de la SHNA, mais elle est coûteuse et invasive. Des méthodes non invasives ont récemment été proposées. Objectifs et méthodes : La présente étude avait pour objectif d'évaluer la précision de certaines méthodes non invasives (notamment les ultrasons [US], la tomographie par ordinateur [TO], la scintigraphie au xénon 133, l'indice de stéatose hépatique (ISH), la technique Fibroscan, le score de fibrose de SHNA, l'indice de ratio entre l'aspartate aminotransférase et les plaquettes [APRI] et l'indice FIB-4) et de l'utilisation combinée de ces méthodes pour le diagnostic de la stéatose et pour le diagnostic d'une fibrose hépatique significative (> F2) et de la cirrhose (F4), par comparaison à la biopsie du foie. Nous avons réalisé une étude rétrospective sur 114 patients atteints de NASH (79 patients de sexe masculin, âge moyen de 49,6 ans ± 10,6). Tous ces patients présentaient une histologie hépatique adéquate.Résultats : La répartition des stades de fibrose était la suivante : F0 F1 = 50 %, F2 = 16,8%, F3 = 19,2 %, F4 = 14 %. La répartition des stades de stéatose était la suivante : stade 0-1 = 16 %, stade 2 = 53,3 %, stade 3 = 30,7 %. Les tests suivants ont été mis en corrélation avec la fibrose : l'indice APRI (r = 0,554), l'indice FIB-4 (r = 0,555), le score de fibrose de SHNA (r = 0,473), la technique Fibroscan (r = 0,586) et l'indice de stéatose hépatique (r = 0,245). L'indice FIB-4 et l'indice APRI ont offert la meilleure précision diagnostique en ce qui concerne la fibrose significative, comme l'indiquent la surface sous la courbe (SSC) de 0,801 et la SSC de 0,782 respectivement. L'indice FIB-4 a présenté la meilleure SSC, soit 0,886, pour ce qui est de la cirrhose. Aucun des tests suivants, c'est à dire les tests aux US, la TO, l'ISH, et la scintigraphie au xénon 133, n'était considéré comme étant corrélé significativement. Le meilleur algorithme de combinaison pour le dépistage de la cirrhose était le sexe et l'indice FIB-4 avec une surface sous la courbe de 0,8937. Conclusion: cette étude démontre que les méthodes non invasives de diagnostic de la fibrose hépatique sont précises en ce qui concerne les stades > F2 et F4. La Stéatose sévère ne peut être diagnostiqué de façon fiable par des méthodes non invasives Notamment, une combinaison de l'indice FIB-4 et du sexe améliore considérablement le rendement de la méthode unique en ce qui a trait à la cirrhose. Ces méthodes pourraient aider à réduire le nombre de biopsies du foie visant à stratifier les patients atteints de NASH qui devraient entreprendre un programme de dépistage du carcinome hépatocellulaire (CHC) et des varices œsophagiennes.

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of liver conditions ranging from hepatic steatosis through steatohepatitis to cirrhosis. Its prevalence has been estimated at between one-in-five and one-in-three of the adult population depending on country and diagnostic criteria used. Prevalence increases with degree of obesity, and is very common in those with Type 2 diabetes (T2DM). Rising prevalence of obesity and T2DM, particularly in younger people, will ensure that NAFLD remains a growing clinical concern for the future. Lifestyle modification, which encompasses diet, weight loss, physical activity, and/or exercise related behaviours, is the primary recommended therapy for NAFLD, especially in the absence of approved pharmaceutical agents. Despite lifestyle modifications being central to the management of NAFLD, the evidence base upon which these guidelines are based is lacking, and this is particularly true for physical activity and exercise. The focus of this thesis is on defining, exploring and developing the evidence for physical activity and exercise in NAFLD with a view to improving clinical care. The work contained within this thesis demonstrates that low levels of physical activity are prominent in people with NAFLD and that targeting this with resistance exercise therapy confers benefits to both liver lipid and the factors promoting its accumulation. It also highlights alterations in cardiac structure and function in people with NAFLD in the absence of overt cardiac disease, which may provide a therapeutic avenue in which to decrease cardiac disease risk in people with fatty liver. Over the duration of the work described in this thesis, the number of studies reporting on exercise and liver fat in people with NAFLD has increased markedly. The new information contained within this thesis contributes to this body of knowledge and, over time, will improve the management of a condition that is an increasing burden to the people of the Western world.

The first chapter (Introduction) of the thesis summarises the pathogenesis of NAFLD and its associated risk factors such as type 2 diabetes and cardiovascular disease. Moreover, it describes: a) the potential beneficial effects of long chain omega-3 fatty acid treatment [docosahexaenoic acid (DHA) plus eicosapentaenoic acid (EPA)] in NAFLD; b) the effect of genotypes patatin-like phospholipase domain-containing protein-3 (PNPLA3 I148M) and the transmembrane 6 superfamily member 2 protein (TM6SF2 E167K), on the level of DHA and EPA enrichment and end of study liver fat percentage after DHA+EPA treatment; and c) the effect of fatty acid desaturase (FADS) and Elongase (ELOVL) polymorphisms influencing omega-3 fatty acid metabolism. The second chapter describes the overall aim of this thesis. The aim of my research was to investigate in patients with NAFLD: a) the effect of long-chain omega-3 fatty acid treatment on liver fat percentage and liver fibrosis biomarkers; b) the effect of genotypes influencing NAFLD severity on treatment with DHA+EPA; and c) the effect of genotypes influencing omega-3 fatty acid metabolism in NAFLD. The third chapter describes in details the design and methods used in my research. Chapter four highlights my novel results from the WELCOME study. This chapter describes the baseline and end of study characteristics of the WELCOME study participants and shows the results of the DHA+EPA treatment on liver fat percentage and liver fibrosis biomarkers. This chapter also describes the association between DHA erythrocyte enrichment and decrease in liver fat percentage after DHA+EPA treatment. Chapter five illustrates the association between PNPLA3 I148M and DHA erythrocyte enrichment percentage and end of study liver fat percentage after DHA+EPA treatment. The chapter shows that PNPLA3 I148M was associated with higher end of study liver fat percentage and lower DHA tissue enrichment. Chapter six shows the negative association between FADS polymorphisms and omega-3 fatty acid metabolism in NAFLD. The chapter also shows that there was a gene-DHA+EPA interaction between the minor allele of the FADS1 rs174556 and Δ-5 desaturase activity after treatment with DHA+EPA. Finally, chapter seven, summarises my results in the context of current evidence and knowledge about the subject matter.

Non-alcoholic fatty liver disease (NAFLD) is now the most common liver disease worldwide. Given that NAFLD can progress from steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and potentially hepatocellular carcinoma, early diagnosis and accurate disease staging are primary clinical concerns. Hypothesizing that a subset of liver proteins will exhibit differential expression in NAFLD and that these proteins may represent candidate disease biomarkers; the aims of this project were to use proteomics to identify differentially expressed proteins both in an in vitro and an in vivo model of NASH. Preliminary studies developed and characterised both models used here; experiments utilized a relative quantitative proteomic approach with isobaric tags for relative and absolute quantitation labelling combined with nano-liquid chromatography and tandem mass spectrometry.

Background and aims: The booming prevalence of obesity and diabetes in young age and the increased consumption of alcohol in adolescents lead to the development of Non-Alcoholic Fatty Liver Disease (NAFLD) and Alcoholic Liver Disease (ALD). Task 1 of this work is focused on the development of an in vivo model of pediatric NAFLD and in the study of the therapeutic properties of Silymarin. The task 2 investigated the source of MIF, a cytokine involved in the pathogenesis of ALD. Materials and Methods: NAFLD in vivo study: C57BL/6 male and female mice were exposed to HFHCD or chow diet (CTRL diet) for 16 weeks. Biochemical and biomolecular analysis were performed to follow the progression of the damage. In the second phase Silymarin properties were evaluated (33mg/animal/day). ALD in vitro study: MIF expression was measured in HuH7 and macrophages cells in response to 50mM ethanol. The ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif-/- bone marrow chimeras. MIF was measured in serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH). Results: NAFLD in vivo study: HFHCD induced immediately a significant body weight gain in both genders. Males presented an early epididymal fat-pads hyperplasia and after week 12, a significant hepatomegaly, with alteration of glycemia, insulinemia, lipid profile, and ALT. Comparable body/blood changes were observed in females after week 16. Liver histology showed in both genders a mixed macro-microvesicular steatosis. Inflammatory foci were observed only in males, confirmed also by an increase in MCP-1 and TNF-α mRNA. Conversely, females had no signs of inflammation but rather presented enhanced lipid peroxidation (MDA) and a reduced GSH/GSSG ratio, signs of oxidative stress. By week 8 both genders developed progressive fibrosis. HFHCD+Silymarin decreased liver and visceral fat weight and improved ALT and lipid profile. HFHCD→CTRL diet reverted all altered parameters under study. Histologically, Silymarin slightly reduced inflammatory foci and fibrosis. ALD in vitro study: HuH7, but not THP-1 macrophages, released MIF in response to ethanol challenge in cell culture. In chimeric mice expressing MIF in non-myeloid cells (Mif-/- →WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and cytokine/chemokine mRNA expression. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→ Mif-/-) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH. Conclusions: Data collected suggested that our juvenile NAFLD model had a faster and more aggressive liver injury progression compared with published adult models, with different molecular mechanisms between males and females. Silymarin exerted some beneficial effects without requiring lifestyle improvements, relevant aspect considering the general low compliance of obese subjects in modifying their nutritional behavior. ALD project provided evidence that hepatocyte-derived MIF was critical to the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH. Altogether these new findings can lead to new therapeutic perspective.

Fatty liver has previously often been associated with excessive alcohol consumption. During the last two decades, the interest in fatty liver occurring in non-drinkers i.e. non-alcoholic fatty liver disease (NAFLD) has increased dramatically. Today, NAFLD is considered as the most common liver disease in the developed world. It is strongly associated with obesity, insulin resistance, and hypertension. Thus, NAFLD is considered as the hepatic manifestation of the metabolic syndrome. The spectrum of NAFLD includes: simple fatty liver without necroinflammatory activity; non-alcoholic steatohepatitis (NASH), a condition characterised by hepatocellular injury, inflammation, and fibrosis; cirrhosis; and in some individuals hepatocellular carcinoma. The degree of steatosis in liver biopsies is usually assessed by a morphological semiquantitative approach in which the pathologist uses a four-graded scale: 0–3 or none, slight, moderate and severe. In this thesis we show that there is a considerable inter- and intraindividual variation in such scoring methods and that a more standardised and quantitative approach is preferable. The area/volume of fat in liver biopsies is greatly overestimated when assessed semiquantitatively. Moreover, the point counting technique has a better reproducibility than visual evaluation and should be preferred in estimates of liver steatosis. The long-term clinical and histopathological course of 129 consecutively enrolled NAFLD patients was studied. Mean follow-up (SD) was 13.7 (1.3) years. Survival of NASH patients was reduced compared with a matched reference population. These subjects more often died from cardiovascular and liver-related causes. Seven patients (5.4%) developed end-stage liver disease, including 3 patients with hepatocellular carcinoma. Most NAFLD patients will develop diabetes or impaired glucose tolerance in the long term. Progression of liver fibrosis is associated with more pronounced insulin resistance and significant weight gain. During follow-up, 17 patients had been prescribed a statin. At follow-up, patients on medication with statins had significantly higher BMI. Diabetes was significantly more common among patients on medication with statins and they had significantly more pronounced insulin resistance. However, they exhibited a significant reduction of liver steatosis at follow-up as opposed to patients not taking statins. Although patients under statin treatment exhibited a high risk profile for progression of liver fibrosis, only four patients on statin treatment progressed in fibrosis stage. It is concluded that statins can be prescribed safely in patients with elevated liver enzymes because of NAFLD. Alcohol consumption was evaluated with a validated questionnaire combined with an oral interview. In a multivariate analysis moderate alcohol consumption, particularly when frequency of heavy episodic drinking was analysed, consistent with the diagnosis of NAFLD to be set, was independently associated with fibrosis progression in NAFLD. The NAFLD activity score (NAS) is a newly proposed system to grade the necroinflammatory activity in liver biopsies of NAFLD patients. We evaluated the usefulness of the NAS in predicting clinical deterioration and fibrosis progression in our cohort of NAFLD patients. Although the NAS was independently associated with future risk of progressive fibrosis in NAFLD, the clinical usefulness of the score was limited due to significant overlap in clinical development between NAS-score groups.

Once thought to be a benign accumulation of hepatic fat, non-alcoholic fatty liver disease (NAFLD) is now recognised as a prevalent complication of obesity with serious consequences if left untreated. This includes liver inflammation and impaired glucose metabolism, and with time may result in cirrhosis and organ failure. While basic diagnostic tools exist, such as ultrasound and serum protein markers, these are often unreliable and warrant the use of more invasive techniques such as tissue biopsy. Given most patients only become symptomatic once the disease is advanced, there is an urgent need to develop a better diagnostic strategy for NAFLD. This project aims to address the issue by exploring a novel biomarker candidate: extracellular vesicles (EVs). These submicron vesicles can be isolated from the circulation and tracked back to their parent cells by nature of their surface markers. EVs are constitutively released by all cell types, however, their abundance and molecular cargo are shown to be changed in various conditions and disease states, which also affects the functions they mediate as “intercellular messengers”. Using rodent models of diet-induced NAFLD, we aim to determine whether plasma or liver-derived EVs could be effectively used as a marker for the progression of NAFLD, and whether suitable interventions exist to slow or even reverse the disease. Finally, using in vitro models of hepatocyte EV uptake, the project will also investigate how EVs impart signals within the liver, in a paracrine fashion and from extraheptic tissues

Thesis (Ph.D.)--Indiana University, School of Health, Physical Education and Recreation, 2007.
Source: Dissertation Abstracts International, Volume: 68-07, Section: B, page: 4315. Adviser: Janet P. Wallace. Title from dissertation home page (viewed Apr. 15, 2008).

Abstract : Pro-inflammatory cytokines play a key role in pathogenesis of obesity and non-alcoholic fatty liver disease (NAFLD). IL-15 is a pro-inflammatory cytokine, which signals through a receptor complex composed of the IL-15 receptor (IL-15R) alpha chain, the IL-2/IL-15R beta chain and the common gamma chain. The functions of IL-15 have been extensively described in immune cells but less is known about its functions in others tissues such as the liver. The aim of this thesis is to investigate the role of IL-15 in fatty liver disease. C57BL/6 wildtype (WT) and IL-15 knockout (Il15[superscript -/-]) mice were maintained on high fat diet (HFD) or normal control diet (NCD). After 16 weeks, body weight, liver mass, fat accumulation in the liver, serum lipid levels and gene expression in the liver were evaluated. Intrahepatic lymphocytes (IHL) were also analysed. Primary hepatocytes were stimulated with IL-15 and chemokines gene expression was studied. IHLs were examined in WT, Il15[superscript -/-] and Il15ra[superscript -/-], as well as in macrophage- and hepatocyte-specific Il15ra[superscript -/-] mice. We found that IL-15 deficiency prevents weight gain and accumulation of lipids in the liver. Circulating levels of cholesterol and non-esterified fatty acids were elevated in WT mice but not in Il15[superscript -/-] mice. Hepatic expression of chemokines such as Ccl2, Ccl5 and Cxcl10 was increased in WT mice under HFD, but not in Il15[superscript -/-] mice. The livers of Il15[superscript -/-] and Il15ra[superscript -/-] mice also showed decreased expression of Tnfa and iNOS, and macrophage markers Cd68 and F4/80. Accordingly, stimulation of primary hepatocytes with IL-15 induced chemokine gene expression in WT but not in Il15ra[superscript -/-] hepatocytes. Furthermore, hepatocyte-specific ablation of IL-15Rαreduced infiltration of NK and NKT cells in the liver, suggesting that IL15Rα expression in the hepatocytes is needed for the recruitment and/or maintenance of the NK cell population in the liver. In conclusion, IL-15 promotes fat accumulation in the liver, and this is associated with increased inflammatory response in the liver. Increased availability of IL-15 in obesity may stimulate hepatocytes to secrete chemokines that promote hepatic inflammation resulting in fatty liver disease. IL-15Rα expression in hepatocytes appears to play a role in the maintenance of NK, NKT and iNKT cells. // Résumé : Les cytokines pro-inflammatoires jouent un rôle important dans la pathogenèse de l’obésité et la stéatose hépatique. L'IL-15 est une cytokine pro-inflammatoire qui est trans-présentée par l'IL-15Rα aux chaines IL-2/IL-15Rβ et γc. La fonction de l'IL-15 a été largement décrite dans les cellules immunitaires, mais ses fonctions dans d'autres tissus sont moins connues. Le but de ce mémoire est d'élucider le rôle de l'IL-15 dans la stéatose hépatique. Les souris C57BL/6 de type sauvage (WT) et Il15[indice supérieur -/-] ont été soumises à un régimehyperlipidique (HFD) ou à un régime normal. Après 16 semaines, le poids corporel, lamasse hépatique, l'accumulation de lipides dans le foie, les taux de lipides sériques et l'expression des différents gènes reliés à l’inflammation et au métabolisme dans le foie ont été évalués. Les lymphocytes intra-hépatiques (IHL) ont été également étudiés. Des hépatocytes primaires ont été stimulés avec IL-15, et l'expression génique de chimiokines a été déterminée. Les populations de IHLs ont été également caractérisées chez les souris WT, Il15[indice supérieur -/-] et Il15ra[indice supérieur -/-], ainsi que chez des souris dont la déficience dans l’expression d’IL-15Rα est ciblée aux macrophages ou aux hépatocytes. Nos résultats montrent que la déficience en IL-15 empêche l'accumulation de lipides dans le foie. Les taux de cholestérol et d’acides gras non estérifiés dans le sang étaient élevés chez les souris WT, mais pas chez les souris Il15[indice supérieur -/-]. L'expression hépatique des chimiokines Ccl2, Ccl5, Cxcl10 et des marqueurs de macrophages était augmentée chez les souris WT sous HFD, mais pas chez les souris Il15[indice supérieur -/-]. La stimulation des hépatocytes primaires avec l'IL-15 induit l'expression des gènes des chimiokines chez les hépatocytes WT, mais pas chez les Il15ra[indice supérieur -/-]. En outre, nous avons trouvé une infiltration réduite des cellules NK et NKT dans le foie des souris déficientes en Il15ra[indice supérieur -/-] dans les hépatocytes, ce qui suggère que l'expression d’IL15Rα chez les hépatocytes est nécessaire aurecrutement des cellules NK, NKT et / ou à leur maintien. En conclusion, nous proposons que l’IL-15 favorise l'accumulation de lipides dans le foie, et que ceci est associée à une réponse inflammatoire accrue. La disponibilité accrue de l'IL-15 dans l'obésité pourrait stimuler les hépatocytes à secréter des chimiokines ce qui favorise l'inflammation hépatique et conduirait à la stéatose hépatique. L’expression de l'IL-15Rα dans les hépatocytes semble jouer un rôle principal dans l’infiltration des cellules NK, NKT et iNKT dans le foie.

Recent findings, including our own work, demonstrated that intestinal microbiota species produce bioactive metabolites that engage host cellular pathways. Microbiota-derived metabolites have also been detected in circulation and in the, setting up the intriguing possibility that these bacterial products could directly interact with host cellular pathways at distant sites. The study described in this abstract investigates the hypothesis that gut microbiota dysbiosis perturbs the balance of immunomodulatory microbiota metabolites, which exacerbates liver inflammation in steatosis. We utilize a multi-omic approach to identify microbiota-dependent immunomodulatory metabolites and characterize their effects on liver inflammation and metabolic function. In summary, we show that the levels of AAA-derived microbiota metabolites are significantly depleted in a diet model of liver steatosis, and that these metabolite can act directly on hepatocytes to modulate inflammatory pathways. Our results also show that the microbiota metabolites are ligands for the AhR, which could provide a mechanistic link for the observed anti-inflammatory effects. Taken together, our findings support the hypothesis that dysbiosis of the gut microbiota could predispose the liver to inflammation in diet-induced steatosis through an altered microbiota metabolite profile. Prospectively, additional insights into the mechanisms underlying the link between microbiota dysbiosis and NAFLD could provide novel strategies to treat or prevent the progression of fatty liver diseases through the use of probiotics or postbiotics.

Non-alcoholic fatty liver disease (NAFLD) is characterised by the accumulation of lipid in liver. Liver is the principal organ involved in drug biotransformation. The central hypothesis of this project is that alterations in the liver environment due to lipid accumulation aggravate sensitivity of hepatocytes to toxicity of some commonly prescribed drugs (paracetamol, alcohol, phenobarbital, cisplatin or doxorubicin). The aim of this study was to investigate the effect of lipid overloading (steatosis) on drug cytotoxicity in the liver model Huh7 cell line. Hepatic steatosis was induced in Huh7 cells by exposing cells to 300 μM FFA mixture or 1 mM oleic acid. Impact of steatosis on drug toxicity was examined by co-treating the cells with FFA and paracetamol, alcohol, phenobarbital, cisplatin or doxorubicin. Cell viability MTT assay showed that neither 300 μM FFA mixture nor 1 mM OA caused significant reduction in cell viability after 24 h incubation. However, a significant reduction seen after incubation for 48 h, therefore for the subsequent experiments the time frame chosen was 24 h. The results showed that 300 μM FFA mixture did not induce a significant amount of intracellular lipid, whereas 1 mM OA induced significant amount of intracellular lipid. The subsequent experiments were carried out to test the hypothesis that lipid-loaded Huh7 cells are more sensitive to drug toxicity. Co-treatment with FFA and either paracetamol, ethanol or doxorubicin resulted in further reduction in cell viability. Phenobarbital resulted in enhanced cell viability, and no significant changes in cell viability observed after co-treatment with cisplatin. To investigate mode of cell death, caspase3/7 activity was measured as mediator of apoptosis. Generally, an advance apoptosis was observed in steatotic cells treated with the different drugs. Reactive oxygen species were measured as a possible trigger of apoptosis. A significant amount of ROS were generated by FFAs, and generally, drug treatment induced a higher significant amount of ROS in the steatotic Huh7 cells. To elucidate the specific changes observed upon treatment of steatotic cells with hepatotoxic drugs, a single FFA dose (300 μM FFA mixture) and a single drug (doxorubicin) were selected to identify major alterations in gene expression. The microarray data confirmed alterations in oxidative stress-related genes. Such changes were functionally relevant as confirmed by cellular assay and In-Cell Western blot. In conclusion, the hypothesised effect of steatosis on drug toxicity has been confirmed and steatosis may enhance drug toxicity through changing cellular oxidative state and activating the apoptotic pathway.

Abstract Obesity, insulin resistance, type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD) form a dangerous quartet which threatens human health all over the world. About 25% of adults around the world have NAFLD, which poses risks for cardiovascular and metabolic well-being and may develop into liver cirrhosis and hepatocellular carcinoma. Apart from lifestyle modification, treatment options for NAFLD are scarce. This thesis presents atrial fibrillation (AF) as a new complication of NAFLD among general population of 958 individuals aged 40-60 years participating in the OPERA study. Even after multiple-adjustments for confounding factors, ultrasound-based NAFLD predicted the development of AF during about 16 years of follow-up. Moreover, the association between AF and liver fibrosis in 76 individuals aged 64-82 years in a cross-sectional setting is presented. The thesis also shows that individuals with metabolic syndrome (MetS), with or without NAFLD, are at increased risk of cardiovascular events, T2D and the increase of left ventricular mass index in a study population of 958 individuals aged 40-60 years during a 20-year follow-up. In other words, NAFLD without MetS does not seem to expose to these three cardiometabolic complications. The thesis also shows that rifampicin-activated pregnane X receptor (PXR), a member of the nuclear receptor superfamily of ligand-activated transcription factors with several endobiotic and xenobiotic activators, increases serum levels of cholesterol, phospholipids and certain fatty acids, assessed by nuclear magnetic resonance metabolomics technique, in a randomized, open, placebo-controlled trial among 34 young and healthy individuals. These serum lipids are considered toxic lipids and capable of transforming hepatosteatosis into steatohepatitis and even more severe forms of NAFLD. Moreover, rifampicin-activated PXR has no effect on serum triglycerides, that are non-toxic lipids, or triglyceride accumulation in the liver, assessed by magnetic resonance imaging, in 15 young and healthy individuals. In conclusion, this thesis advances the knowledge in the pathogenesis, lipid metabolism, complications and heterogeneous nature of NAFLD. These may have implications for patient care and follow-up
Tiivistelmä Maailmanlaajuisesti noin 25% täysi-ikäisistä henkilöistä sairastaa alkoholinkäyttöön liittymätöntä rasvamaksaa. Sen tiedetään altistavan sydän- ja verisuonisairauksille, aineenvaihduntahäiriöille, maksakirroosille ja jopa maksasyövälle, mutta elämäntapahoitoa lukuun ottamatta hoitomahdollisuudet ovat toistaiseksi vähäisiä. Tässä väitöskirjassa osoitetaan ensimmäistä kertaa alkoholinkäyttöön liittymättömän rasvamaksan ennustavan itsenäisesti eteisvärinän ilmaantuvuutta noin 16 vuoden seurannan aikana 958 tavallisen keski-ikäisen ihmisen aineistossa osana OPERA-tutkimusta. Lisäksi väitöskirjassa osoitetaan maksan sidekudosmuodostuksen ja eteisvärinän välillä olevan yhteys poikkileikkausasetelmassa 76 iäkkään ihmisen muodostamassa aineistossa. Väitöstutkimuksessa havaittiin myös, että metabolista oireyhtymää sairastavilla henkilöillä on suurentunut tyypin 2 diabeteksen, sydän- ja verisuonisairauksien sekä vasemman kammion koon suurentumisen riski noin 20 vuoden seurannan aikana 958 tutkittavan henkilön aineistossa riippumatta siitä, onko heillä alkoholinkäyttöön liittymätön rasvamaksa. Toisin sanoen alkoholin käyttöön liittymätön rasvamaksa ilman metabolista oireyhtymää ei lisää edellä mainittujen kolmen sairauden riskiä. Väitöstutkimuksessa esitetään lisäksi, että rifampisiinilla aikaansaatu maksan pregnane X -reseptorin aktivaatio johtaa seerumin fosfolipidien, tiettyjen rasvahappojen sekä usean eri kolesterolityypin lisääntymiseen 34 terveen nuoren henkilön aineistossa. Kirjallisuudessa näiden seerumin rasva-aineiden on esitetty aiheuttavan alkoholin käyttöön liittymätöntä maksatulehdusta ja jopa rasvamaksan vakavimpia muotoja. Toisaalta rifampisiini ei lisännyt seerumin triglyseridipitoisuutta eikä aiheuttanut magneettitutkimuksella mitattuna triglyseridien kertymistä maksaan 15 terveen nuoren henkilön aineistossa. Tämä väitöstutkimus antaa lisätietoa rasvamaksan kehittymisestä, rasva-aineenvaihdunnasta ja komplikaatioista sekä korostaa rasvamaksan monimuotoista luonnetta. Nämä löydökset saattavat parantaa rasvamaksaa sairastavien henkilöiden hoitoa ja seurantaa

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease characterised by the accumulation of fat in the liver. It is estimated that 33 % of the UK population have NAFLD with 2-5 % progressing to non-alcoholic steatohepatitis (NASH). Due to a lack of an outright therapy for NAFLD, treatment has been mainly focussed on managing the conditions associated with the disease such as obesity, diabetes mellitus and hyperlipidaemia. This study aimed to investigate the means by which hepatocyte protection is conferred by Gentiana plants (Gentiana lutea, Gentiana macrophylla, Gentiana scabra and Gentiana rigescens) used in herbal medicine for the management of non-alcoholic fatty liver diseases (NAFLD). The role played by some of the inherent Gentiana phytochemicals including: gentiopicroside, sweroside and swertiamarin in promoting hepatocyte protection against the cytotoxic effects of fatty acids were also investigated. Gentiana species: lutea, macrophylla, rigescens, and scabra are known to protect and enhance hepatocyte viability via their antioxidant, anti-inflammatory and bitter components including: amarogentin gentianine, iso-orientin, swertiamarin, gentiopicroside, and sweroside. This study was necessitated due to a lack of adequate research on the hepatoprotective effects of the above-named Gentiana species and phytochemicals with special emphasis on their effect on mitochondrial respiration in the presence of fatty acids. At the time of submission, this was the first study to utilise the seahorse mitochondria stress assay to investigate the Gentiana species as well as phytochemicals: gentiopicroside, sweroside and swertiamarin. It was also found that the most abundant phytochemical in all four Gentiana species was gentiopicroside (up to 4.6% g/g), followed by swertiamarin (0.21–0.45% g/g), and sweroside (0.03- 0.4 % g/g). Furthermore, it was also observed that the methanolic extracts of all four Gentiana protected HepG2 and THLE-2 cells by inhibiting arachidonic acid from diminishing cell replication but showed a mitogenic effect mostly observed in gentiopicroside, Gentiana lutea and Gentiana macrophylla. It was concluded that phytochemicals: gentiopicroside, sweroside and swertiamarin play key roles in the hepatocyte protection exerted by methanolic extracts of Gentiana lutea, Gentiana macrophylla, Gentiana scabra and Gentiana rigescens against the cytotoxic effects of fatty acids. This protection is conferred by enhancing mitochondrial function in terms of increasing maximal respiratory capacity in response to high influx of fatty acids, promoting ATP production as well as scavenging ROS produced as a result of high fatty acid influx and increased mitochondrial respiration. However, the mitogenic effect observed in gentiopicroside and Gentiana macrophylla requires further studies using unmodified primary hepatocytes to gain better understanding.

Non-alcoholic fatty liver disease (NAFLD) is characterised by the accumulation of fat in the liver and is associated with liver-related morbidity and mortality. Nevertheless, the leading cause of death in these patients is cardiovascular disease (CVD). Excess abdominal visceral adipose tissue (VAT) is frequently expressed in NAFLD and is considered a pivotal feature in the pathogenesis ofNAFLD which is predictive of CVD. Endothelial dysfunction is an early manifestation in the development of atherosclerosis and is characterised bya diminished bioavailabilty of the anti-atherogenic molecule NO, which is secreted by the endothelium of all blood vessels throughout the vascular tree. Limited pharmacological treatment is available to reduce hepatic fat, therefore, lifestyle modification interventions comprised of structured exercise and diet are recommended as a non-pharmacological management strategy to reduce hepatic fat in NAFLD. The primary aim of this thesis was to explore nitric oxide (NO)-mediated endothelial function at different levels of the vascular tree in NAFLD patients and to establish whether supervised exercise training has a sustained therapeutic impact on endothelial function. Thirty-two NAFLD patients (21 males, 11 females, 48±2yrs, BMI 31±lkg/m2) and eighteen matched controls (8 males, 10 females, 48±2yrs, BMI 30±lkg/m2) underwent magnetic resonance imaging (MRI) to quantify abdominal VAT and proton magnetic resonance spectroscopy (lH-MRS) to determine intrahepatocellular triglyceride content (IHTC). Brachial artery flow mediated dilatation (FMD) (as an index of endothelial NO function) was also assessed. IHTC (27.2±3.0 vs. 2.9±0.4%) and abdominal VAT (S.4±0.3vs. 3.4±0.2 1) were elevated in NAFLD patients when compared with controls (PO.OS). Impairment in FMD remained in NAFLD patients following independent covariate adjustment for abdominal VAT (S.O±O.S vs. 7.3±0.7%, P=0.01). These fmdings indicate that excess IHTC and abdominal VAT do not explain endothelial dysfunction in NAFLD. Twenty NAFLD patients were randomly assigned to either 16-weeks of supervised moderate intensity (30-60% HRR, 30-4S min, 3-S times per week) exercise training (n=13, SO±3yrs, BMI 30±lkg/m2) or to 16-weeks of conventional care lifestyle advice (n=7, 47±6yrs, BMI 31±2kg/m2). Supervised exercise training induced a greater improvement in FMD when compared with conventional care (3.6±0.6 vs. 0.3±0.8%, P=0.004). There was no significant difference between the effect of exercise and conventional care on IHTC or abdominal VAT (P>O.OS). These data suggest that supervised exercise training is an effective management strategy in NAFLD capable of improving conduit artery endothelial function independent of IHTC and abdominal VAT. In order to explore the longevity of the exercise-induced improvements in conduit artery endothelial function, a 12-month follow up assessment was performed in 9 of the NAFLD patients (S males, 4 females, SO±Syrs, BMI 30±lkg/m2) who completed the 16- week supervised exercise training intervention. The exercise-induced improvement in FMD (S.1±0.8 vs. 7.9±0.8%; P=0.004) was abolished 12 months following the cessation of supervised exercise training (7.9±0.8 vs.S.O±O.S%; P=0.02), returning to a similar level observed at baseline (S.l±0.8 vs. S.O±O.S%; P=0.9S).These findings indicate that in order to chronically sustain exercise-induced improvements in endothelial function in NAFLD patients, long-term exercise supervision and guidance is required. Cutaneous NO-mediated microvessel function reflects generalised microvascular function and provides a translational model to investigate pre-clinical disease, but has not been previously investigated in NAFLD. NO-mediated vasodilatation in the cutaneous microvessels was examined in 13 NAFLD patients (7 males, 6 females, 50±3yrs, BM! 31±lkg/m2) and 7 matched controls (3 males, 4 females, 48±4yrs, BM! 30±2 kg/nr'). Microdialysis fibres were embedded into the skin of the forearm and laser Doppler probes placed over these sites. Both sites were then heated to 42°C, with saline solution infused in one probe and L-NG-monomethyl arginine (L-NMMA) through the second. Following baseline assessment, 11 NAFLD patients were randomly assigned to l o-weeks of supervised moderate intensity exercise training (n=6, 45±5yrs, BM! 31±1kg/m2) or to 16-weeks of conventional care (n=5, 51±3yrs, BM! 30±21kg/m2). The NO contribution to skin blood flow in response to incremental heating was not different between NAFLD patients and controls (P=0.47) at baseline. However, significant differences were evident in NO contribution between the exercise training and conventional care group (P=O.Ol), suggesting that supervised exercise training improves cutaneous NO-mediated microvascular endothelial function in NAFLD patients. This thesis suggests that supervised exercise training has a direct therapeutic impact on endothelial function in NAFLD which may decrease the risk of future heart disease and stroke. As a cardioprotective management strategy in NAFLD, exercise training is superior to that of current conventional care pathways, however, in order to chronically sustain the exercise-induced improvements in endothelial function, long term exercise supervision and guidance is required.

Sphingomyelin (SM) is ubiquitous in human diets. The studies in this thesis investigated the effects of pure egg SM supplementation on the risks of non-alcoholic fatty liver disease (NAFLD) and atherosclerosis development in various mouse models. High fat-fed C57BL/6 mice, a model for NAFLD, was supplemented with different doses of dietary SM for various duration. The results showed that SM supplementation significantly reduced hepatomegaly, total hepatic lipids, particularly hepatic cholesterol and triglyceride, in a dose-dependent manner after 4 weeks. All of these parameters are risk factors of hepatic steatosis, the first stage of NAFLD. Dietary SM was also supplemented to either high fat-fed or chow-fed apoE knockout mice, a model for atherosclerosis. Results indicated that dietary SM did not increase circulating SM, a risk factor of atherosclerosis, nor atherosclerosis development after 16 weeks. At week 19, dietary SM was shown to significantly reduced atherosclerosis development in chow-fed apoE knockout mice. Collectively, studies in this thesis provided evidence that dietary SM can be used to prevent NAFLD in mice. This work lays the groundwork for future investigations into the potential therapeutic benefit of dietary SM on NAFLD and atherosclerotic lesion development in humans.

Non Alcoholic Fatty Liver Disease (NAFLD) is a worldwide increasing disease but still many questions about its evolution, the need of a screening and the availability of effective specific treatments are open. Aims of this PhD project were: 1) the evaluation of NAFLD natural history in a subgroup of NAFLD affected diabetic patients enrolled during the daily clinical activity of a splenohepatology ecoDoppler laboratory in order to identify, if present, predictive factors of “evolutive NAFLD”; 2) the experimental evaluation, in High Fat Diet (HFD) fed rats, of the potential therapeutic effect of 3 molecules targeting respectively: a) lipid metabolism (Apolipoprotein A analogue compound -L4F), b) insulin sensitivity (peroxisome proliferator activated receptor delta agonist –PPARd agonist) c) endothelial function (EET Analog). We developed two studies: a clinical observational study and an experimental study. Clinical study: 100 patients with type 2 diabetes were evaluated as far as steatosis is concerned. Among them, 80 had sonographic signs of steatosis. There was no difference in the prevalence between male and female patients. 21 type 2 diabetic patients with liver steatosis were reevaluated after 6 years without any specific treatment. Liver steatosis increases only in less than 1/3 of non-obese diabetic patients and demonstrates that in the majority of them sonographic degree of steatosis improves or recovers concurrently with biohumoral parameters. The presence of increased levels of serum AST and ferritin and lower pulsatility index of haepatic artery seems to be correlated to a worse prognosis and may be used to identify those patients who deserve a higher surveillance. Experimental study: 30 male Wistar rats (4-5 weeks old, 150 grams body weight) were purchased from Charles River Laboratories. 24 rats have been fed with HFD for 8 weeks. After 8 weeks of diet animals have been divided in 4 groups: 7 untreated (HFD); 7 treated with L4F (L4F), 7 treated with PPARd agonist (PPARd) and 3 treated with EET Analog (EET). Treatments lasted 6 weeks. We demonstrate that HFD induced NAFLD reproduces splanchnic haemodynamic alteration of liver steatosis in humans and shows an activation of innate immune system also at early degree of steatosis without hepatic inflammation and fibrosis. The activation of innate immune system can be evaluated by the analysis of lipopolysaccharide (LPS) stimulated/unstimulated CC motif chemockine ligand 2 (CCL2) production in cultured peripheral blood mononuclear cells (PBMCs). PPARd agonist and L4F improved HFD induced liver steatosis and reduced CCL2 production in PBMCs but preserved the ability of PBMCs to react to LPS stimulation EETA administration didn’t improved liver steatosis and further decreased portal vein velocity and reduced the ability of PBMCs to react to LPS stimulation.

Elevations in the liver enzyme alanine transaminase (ALT), traditionally regarded as a highly specific indicator of liver injury, are now recognised as a novel marker of impaired metabolic health. Elevated ALT has a positive, linear association with the metabolic syndrome and its individual components including truncal adiposity, type 2 diabetes and dyslipidaemia. Prospective data indicates that elevated ALT predicts incident metabolic syndrome and type 2 diabetes in a dose dependent relationship, and may predict incident cardiovascular mortality, although the latter relationship appears non-linear. Similarly, elevations in the liver enzyme gamma-glutamyltransferase (GGT), may also represent impaired metabolic health, but used alone is less specific than ALT. Despite these robust data, the prevalence of elevated ALT and associated mortality outcomes for people with elevated liver enzymes in the Australian population is largely unstudied. ALT is also the enzyme most strongly correlated with hepatic fat accumulation, termed non-alcoholic fatty liver disease (NAFLD), itself frequently referred to as “the hepatic manifestation of the metabolic syndrome”. NAFLD is an important public health problem in developed countries, where it affects up to 30% of people, and 2-4% may progress to liver cirrhosis, hepatocellular carcinoma or liver transplantation. Longitudinal studies indicate that the most important risk factor for disease progression is advanced fibrosis identified at liver biopsy, however it is not feasible to perform biopsy at a widespread level. Therefore, non-invasive scoring systems have been developed and have good accuracy in high risk populations, but their diagnostic accuracy in a population with a low prevalence of advanced fibrosis has not been studied. Furthermore, to effectively treat people with advanced fibrosis and reduce disease progression, interventions that are supported by high Abstract 9 quality evidence on the benefits and harms should be instituted. However, many advocated lifestyle interventions in NAFLD are based on observational data alone, which is subject to selection bias and confounders, and evidence based management is further compromised by the conspicuous lack of information provided on the harms of interventions. In this body of work, a range of epidemiological techniques were used to explore these evidence gaps in the identification, treatment and counselling of people at high risk of metabolic liver disease. In Chapter 2, a cross sectional study established the prevalence of elevated ALT in the Australian population. In Chapter 3, these data were extended by a longitudinal, prospective cohort study to determine if there was an excess risk of cardiovascular and all-cause mortality for individuals with elevations in ALT and GGT. In Chapter 4, a diagnostic test study assessed the accuracy of non-invasive fibrosis scoring systems for detection of people with advanced fibrosis in the general population. To then support evidence informed decision making in NAFLD, Chapter 5 explores the evidence base for standard lifestyle recommendations in NAFLD, and in Chapter 6, a methodological study assessing the balance of reporting of benefits and harms in systematic reviews was undertaken.Prevalence of elevated alanine transaminase (ALT) in Australia and its relationship to metabolic risk factors: a cross sectional study of 9,447 people. Elevated alanine transaminase (ALT) is a strong and reproducible marker of metabolic syndrome, and may help to identify individuals at risk of future metabolic disease, but there is little data from the Australian population. We aimed to determine the prevalence of elevated ALT and its association with metabolic risk factors in the general population. We utilised data from a large, population based cross sectional study (N=9,447) from a nationwide survey. We used standard thresholds [defined as ≥ 40 IU/L (males) and ≥ 30 IU/L (females) and lower thresholds (ALT as ≥ 30 IU/L for males and ≥ 19 IU/L for females), and evaluated for independent predictors of elevated ALT in logistic regression models. Analyses were weighted using population weights. Using standard thresholds, elevated ALT levels were found in 11.2% of the Australian population overall. People with elevated ALT were younger (43 vs 46 years) with more truncal adiposity (100 vs 91 cm), higher levels of pro-atherogenic lipids and glucose and exercised less (120 vs 160 minutes per week, p<0.05 for all analyses). Regression analyses indicated that younger age, male sex, diabetes, triglycerides, apolipoprotein B and waist circumference were independent predictors of elevated ALT. We estimated the population attributable fraction of elevated ALT due to truncal obesity at 47%, which suggests that approximately half of the elevated ALT seen in the Australian population is associated with truncal obesity. Overall, these data provide a benchmark for elevated ALT within the Australian population, and indicate a high prevalence of elevated ALT that is strongly associated with metabolic risk factors. Individuals with elevated ALT should be evaluated for co-existent metabolic disorders. Abstract 11 Outcomes for people with elevated liver enzymes in the general population. Elevated liver enzymes are associated with metabolic risk factors, but whether this confers an elevated risk of cardiovascular or all-cause mortality is unclear. In this longitudinal study, we aimed to determine the excess risk of all-cause and cardiovascular mortality in older people with elevated liver enzymes (alanine transaminase, ALT and gamma glutamyltransferase, GGT). We utilised data from a large, prospective, population based study of 2,061 people aged 50-99 years with linkage to a national death registry. Participants were categorised as having elevated enzymes using standard thresholds (for males, GGT >51 and ALT >40 IU/L, and GGT >33 and ALT >31 IU/L for females), and we included people with elevations in both enzymes to increase detection of those most at risk of metabolic liver disease. Adjusted Cox proportional hazards models assessed the association of elevated enzymes and mortality with long duration follow up. Over a median follow up of 10 years (20,145 person years), 701 people died, including 203 (34%) from cardiovascular disease. Cox regression models adjusted for sex, age, smoking and alcohol intake indicated that people with elevated enzymes had an increased risk of all-cause mortality that was modified by age (test for interaction p=0.01). Age-stratified analyses demonstrated no increased risk at younger ages (age ≤ 59 years, H.R. 0.46 95% confidence interval 0.06-3.49), but increased risk with age; age 60-69, H.R. 1.05 (0.53-2.07), age 70-79 years, H.R. 1.54 (0.81-2.93), and age ≥ 80 years, H.R. 3.53 (1.55-8.04). Similarly, the risk of cardiovascular mortality with elevated enzymes was also modified by, and increased with age (test for interaction p=0.02); age 70-79, H.R. 3.15 (1.37-7.23), age ≥ 80, H.R. 6.86 (2.44-19.30). These data suggest that in community dwelling elderly persons, an elevation in ALT and GGT are associated with an excess risk of all-cause and cardiovascular mortality which increases with age. Identification of people with NAFLD and advanced fibrosis from within the general population. Despite the high prevalence of NAFLD within developed countries, only a small proportion will progress to liver cirrhosis and failure, particularly those with advanced fibrosis. Fibrosis is most accurately detected using liver biopsy or transient elastography, however these tests are not widely available. Therefore, non-invasive fibrosis scoring systems which use a combination of available variables such as ALT, BMI and platelet count are increasingly used. The diagnostic accuracy of non-invasive scores was established in tertiary liver clinics where the prevalence of advanced fibrosis is high, at up to 30%, and whether they are equally accurate in a low prevalence population has not been studied. We used data from a population based, cross-sectional study, where participants underwent clinical assessment, fasting blood tests and transient elastography as the reference standard for advanced fibrosis. We assessed test performance characteristics of three scores, the NAFLD fibrosis score, FIB-4 and Aspartate Transaminase to Platelet Ratio Index (APRI), using standard thresholds. To calculate diagnostic test characteristics, we constructed a 2 by 2 table with the presence or absence of advanced fibrosis according to the transient elastography reading against the presence or absence of advanced fibrosis according to the scoring systems. Area under the receiver operating curve was calculated to assess overall diagnostic accuracy. The response rate was 36%. After exclusions, 922 underwent transient elastography, of which 749 had a valid reading and fifteen had advanced fibrosis (2%). The specificity of non-invasive scores for advanced fibrosis approximated 100% (95% CI 99-100) with a negative predictive value of 98% (95% CI 97-99) for all systems. The sensitivities were low, at 7% (95% CI 0-32) to 13% (95% CI 2-40). Positive predictive values ranged from 50% (95% CI 7-93) to 67% (9-99). Negative likelihood ratios approached one (0.87, 95% CI 0.71-1.06 to 0.93, 95% CI 0.82-1.07), and positive likelihood ratios were uninformative due to the small number of people with positive scores. These data suggest that when non-invasive fibrosis scores are used in the general population, their specificity and negative predictive values are very high and can reliably exclude those without advanced fibrosis without need for further testing, with consequent cost savings and avoidance of unnecessary procedures. However positive results are most likely to be false positives, and should prompt further testing. Lifestyle recommendations for people with NAFLD- which have the highest level of evidence? Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of the metabolic syndrome as it is tightly linked with the global obesity epidemic. Indeed, some propose that NAFLD may even precede development of the metabolic syndrome, although confirmatory studies are lacking. Pharmacological therapies for NAFLD including Vitamin E or pioglitazone may improve surrogate outcomes such as fibrosis, although there are no data on long term clinical outcomes, and concerns remain regarding long term safety. Thus lifestyle modification with diet and exercise counselling remains standard of care, but commonly recommended interventions such as the Mediterranean diet, omega 3 polyunsaturated fatty acids supplements or increased intake of monounsaturated fats are Abstract 14 largely derived from observational data with low validity. We conducted a review to evaluate which diet and exercise recommendations were underpinned by a high level of evidence, in order to prioritise these recommendations for clinical use. We found that no trials have been conducted with clinical endpoints. Trials of the Mediterranean diet and omega 3 polyunsaturated fatty acids show improvement in surrogate outcomes only, such as liver fat. Improvement in surrogate outcomes is also seen for aerobic, anaerobic and combined modality exercise, and a reduction in sedentary time also appears important. Overall, however, there remains a substantial lack of high quality evidence to support lifestyle recommendations. To provide a sound evidence base for NAFLD management, longer duration trials of standardised lifestyle regimes, and inclusion of robust clinical endpoints are essential. Quality of reporting of systematic reviews within gastroenterology- is there balanced reporting of benefits and harms? Systematic reviews are an integral component of evidence-based health care, however reviews tend to focus on purported benefits of therapies without due consideration to potential harms. Information on both outcomes is needed to adequately inform patients. We assessed the reporting of harms in systematic reviews relevant to clinical gastroenterology. We identified all systematic reviews of randomised trials of gastroenterology interventions published from 2008 to 2012 in highly cited gastroenterology and general medical journals. We adapted the Consolidated Standards of Reporting Trials (CONSORT) guidelines for harms and assessed qualitative and quantitative parameters of harms reporting. Regression analyses determined predictors of more Abstract 15 comprehensive harms reporting. In total, 78 systematic reviews were identified, with 72 published in gastroenterology journals and six in general medical journals. Overall, one in three systematic reviews (26/78, 33%) did not refer to harms of the intervention anywhere in the article. Less than half of the studies included adverse events as an outcome measure, and data on absolute rates of adverse events were only provided in 28%. Most (65%) did not include any figures or tables on adverse event; however, all included these on efficacy outcomes with an average of three tables and figures per study. Regression analyses indicated that the use of reporting guidelines was significantly associated with better harms reporting (P = 0.04). These findings suggest that the reporting of harms in systematic reviews pertinent to gastroenterology is largely inadequate and highly asymmetrical compared with the reporting of benefits. We suggest that systematic review authors routinely assess both efficacy and harms outcomes of an intervention, and that reporting guidelines specifically targeting harms reporting be developed.

Les maladies du foies non-alcoholiques (NAFLD) représentent un large spectre de pathologies qui comprennent la stéatose ou la stéatohepatite non alcoolique (NASH). Avec un nombre de patients diagnostiqué en constante augmentation, les NAFLD sont devenues un problème majeur de santé publique dans le monde. Un hypothése de « multiples hits » à été décrite pour regrouper l’ensemble des dérégulations métaboliques associées à la transition de la stéatose vers la NASH. Cette étape est critique au cours de la pathogénèse des NAFLD. En effet dans le cas où la NASH n’est pas prise en charge, elle peut se transformer en une fibrose ou une cirrhose et finalement en un cancer hepatocellulaire (CHC). Ainsi, l’analyse des évènements précoces au cours des NAFLD sont essentiels pour comprendre son évolution vers des pathologies plus dommageables. Dans le foie, plusieurs populations de cellules sont impliquées dans le métabolisme et les fonctions hépatiques ainsi que dans la surveillance immunitaire. Parmi celles ci, le groupe 1 ILC est enrichie dans le foie et peut rapidement induire une réponse immunitaire en produisant des cytokines ou en induisant la mort cellulaire. Le groupe 1 ILC hépatique est composé des cellules NK (Natural Killers) et des ILC1 (Cellules Lymphoïdes Innées de type 1), deux populations de cellules qui partagent un phénotype semblable. Cependant, elles forment bien deux lignages distincts avec des caractéristiques uniques. Nous proposons donc d ‘étudier le(s) role(s) des cellules NK et des ILC1 au cours des étapes précoces des NAFLD.Dans cette étude, nous avons démontré les cellules NK et les ILC1 subissent des modifications hétérogènes au cours des étapes précoces des NAFLD. Alors que les ILC1 présentent une diminution de leurs marqueurs d’activation et d’inhibition et de production de granzyme B, nous détectons une accumulation de cellules NK produisant de l’interféron gamma (IFNg). Ces modifications sont induites rapidement au cours de la stéatose et sont même antérieures au recrutement et à l’activation d’autres cellules immunitaires du foie. Nous soulignons également l’importance du système immunitaire intestinal au cours de l’inflammation hépatique et proposons que la lamina propria de l’intestin puisse servir de réservoir de cellules NK au cours de ce processus. Finalement, nous avons démontré que l’IFNg, secrétée entre autre par les cellules NK induise des dommages au cours de la stéatose mais n’est pas impliquée dans le recrutement ou les modifications observées sur le groupe 1 ILC. Cette étude apporte de nouveaux éléments sur les étapes précoces des NAFLD ainsi que le rôle du groupe 1 ILC au cours de l’inflammation hépatique. Cette étude souligne également l’importance de la distinction entre les cellules NK et les ILC1 au cours des pathologies du foie
Non-alcoholic fatty liver diseases (NAFLD) is a spectrum of liver pathologies that encompass diseases such as steatosis or non-alcoholic steatohepatitis (NASH). With a constant increase of patients diagnosed, NAFLD is becoming a major concern of public health worldwide. A “multiple hits” hypothesis has been described to regroup the metabolic disorders that are associated with the transition from steatosis to NASH. This transition is a critical step during NAFLD pathogenesis as untreated NASH can further develop into fibrosis, cirrhosis and ultimately to hepatocellular carcinoma (HCC). Thus, the analysis of early events occurring during during NAFLD is critical to understand its evolution to more severe pathologies. In the liver, diverse cell populations are involved in hepatic metabolism, function and immune surveillance. Among them, the group 1 ILC is enriched in the liver and can quickly induce an immune response by producing cytokines or inducing cell death. Hepatic group 1 ILC is composed of Natural Killer (NK) cells and Innate Lymphoid Cells 1 (ILC1), two cell populations that share a similar phenotype. Nevertheless they constitute two distinct cell lineages that have unique features. Here we propose to study the roles of NK cells and ILC1 during the early stages of NAFLD.In this work, we demonstrated that NK cells and ILC1 diverge in phenotype and function during the early stages of NAFLD pathogenesis. While ILC1 showed a down-regulation of inhibitory markers and down-regulation of granzyme B, we detected an increase of interferon gamma (IFNg) secreting NK cells. These modifications were found shortly after the induction of steatosis and preceded other hepatic immune cell recruitment or activation. Our work highlighted the role of the immune intestinal populations during liver inflammation and identified the intestinal lamina propria as a potential source of NK cells during this process. Finally, we demonstrated that IFNg is inducing liver damage, but is not involved in hepatic group 1 ILC recruitment or modification in our model of steatosis.This study brings new insights on the early events of NAFLD and the role of hepatic group 1 ILC during liver inflammation. It also underlines the importance of distinguishing the roles of NK cells and ILC1 in liver pathologies

Non-alcoholic fatty liver disease (NAFLD) is a major chronic liver disease and thus a main reason for liver-related morbidities and mortality. NAFLD covers a wide range of diseases starting with steatosis and frequently progressing to non-alcoholic steatohepatitis (NASH), which is an independent predictor for the development of the hepatocellular carcinoma (HCC). Nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme of the mammalian NAD salvage pathway, recycles nicotinamide to nicotinamide mononucleotide (NMN), which is further converted to nicotinamide adenine dinucleotide (NAD). NAD is not only an important redox partner but also a crucial co-substrate for NAD-dependent enzymes such as sirtuin 1 (SIRT1). Thus, NAD metabolism might be involved in the progression of NAFLD by regulating many cellular processes, such as apoptosis, de novo lipogenesis, glycolysis and gluconeogenesis, in the liver. Interestingly, tumor cells have a high NAD turnover due to their rapid proliferation and high activity of NAD-dependent enzymes. For these reasons, I hypothesized that the NAD salvage pathway is dysregulated during the progression of non-alcoholic fatty liver disease. Therefore, the first study of the present work deals with the role of the NAD salvage pathway in a diet-induced mouse model of hepatic steatosis. In mice fed a high-fat diet for 11 weeks hepatic NAMPT mRNA, protein abundance and activity as well as NAD levels were increased. Additionally, SIRT1 protein abundance was upregulated indicating a higher SIRT1 activity. This could be confirmed by detecting decreased acetylation or transcription of SIRT1 targets. For example, p53 and nuclear factor κB (NF-κB) were less acetylated demonstrating lower activity of key regulators of apoptosis and inflammation, respectively. In the second study of this thesis NAMPT activity was inhibited by applying its specific inhibitor FK866 in hepatocarcinoma cells to investigate whether or not NAMPT inhibition could be a potential novel therapeutic approach in HCC treatment. Hepatocarcinoma cells were more sensitive to NAMPT inhibition by FK866 than primary human hepatocytes, presenting a high number of apoptotic cells after FK866 treatment. FK866 induced NAD and ATP depletion which was associated with activation of the key regulator of energy metabolism 5’-AMP-activated protein kinase (AMPK) and decreased activity of its downstream target mammalian target of rapamycin (mTOR). This thesis shows that the NAD salvage pathway is involved in hepatic steatosis and HCC. During hepatic steatosis NAD metabolism is upregulated to potentially protect against adverse effects of the massive hepatic lipid accumulation. To repress the progression to NASH it might be useful to maintain the hepatic NAD levels during early disease stages by administration of NAD precursors, such as NMN. However, hepatocarcinoma cells have a higher activity of NAMPT and NAD-dependent enzymes. NAMPT inhibition by FK866 could be a potential therapeutic approach in HCC, especially due to the fact that NAD depletion is selectively induced in hepatocarcinoma cells, but not in primary human hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) now affects around one in four adults in the human population and parallels the global increase in obesity. Within the spectrum of NAFLD, simple steatosis is associated with insulin resistance and type 2 diabetes while progression to steatohepatitis (NASH) is associated with an increased risk of liver cirrhosis and all-cause mortality. The molecular pathology of NAFLD is incompletely understood, however observational studies in human cohorts suggest the regulation of DNA methylation may play a role. 5-hydroxymethylcytosine (5hmC) is a cytosine modification generated from 5- methylcytosine (5mC) by the Ten eleven translocase isoenzymes (Tets) as part of a demethylation process. The aim of this project was to examine the role of Tet enzyme activity on the pathogenesis and progression of NAFLD. Detailed characterisation of two established murine dietary interventions allowed the selection of a NAFLD mouse model which broadly recapitulated the metabolic, histological and transcriptional features of human disease. Using DNA immunoprecipitation coupled with whole genome next generation sequencing and RNA micro expression arrays I examined the effect of high fat diet feeding (HFD) on hepatic DNA 5hmC levels within annotated gene regions. Whilst the global 5hmC profile was not altered by HFD, there was profound genic enrichment of 5hmC in upregulated mediators of cholesterol synthesis and transport (Lss, Sc4mol, Fdps, Hsd17b7, Cyp17a1, Mvd, Cyp1a2, Dhcr7 and Apoa4) with no enrichment in genes with other pathological functions (drug detoxification, inflammation, cell cycle regulation). Induced peaks of 5hmC enrichment were subsequently abolished following rescue of the NAFLD phenotype by conversion to control diet. Cross species validation was performed in vitro utilising embryonic stem cell derived hepatocytes challenged with a cocktail of high energy substrates. My in vivo findings were broadly replicated with specific 5hmC enrichment in genes synthesising lipotoxic molecules (PLIN2, CIDEC, APOA4, ACADVL, HMGCS2, APOA5, CYP2J2, IGFBP1, PPAP2C, ACSL1, APOC3, ANGPTL4, NRG1) with no enrichment in upregulated genes of alternative function. To determine whether or not the 5hmC enrichment seen is of functional relevance, I studied Tet1-/- C57BL/6J mice. Tet1-/- mice are grossly normal in appearance, however loss of Tet1 conferred a striking resistance to diet induced obesity with reduced body fat mass, improved insulin-sensitivity and near complete absence of NAFLD compared to wild type littermates. Furthermore, the HFD fed Tet1-/- liver transcriptome showed a ‘protective’ profile, with suppression of genes for lipid synthesis, inflammation and fibrosis. Thus, in multiple cross-species models of NAFLD, over nutrition induces genic hydroxymethylation specifically within activated genes driving the synthesis and transport of lipid molecules. Such changes are reversible with resolution of the NAFLD phenotype strengthening functional association. Tet1 deficiency conveys an obesity and NAFLD resistant phenotype. I therefore introduce Tet1 mediated hydroxymethylation as a novel mechanism for NAFLD pathogenesis.

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.

Abstract: La NAFLD (Nonalcoholic fatty liver disease) è la più comune malattia epatica cronica dei paesi occidentali con un incidenza ancora maggiore dell’epatite virale C e del danno epatico causato dall’abuso di alcool. Dal punto di vista istologico la NAFLD include la steatosi semplice (nonalcoholic fatty liver, NAFL) nella sua forma iniziale, che può evolvere in steatoepatite (NASH), caratterizzata dalla presenza di infiltrato infiammatorio e/o di fibrosi, potenzialmente progressiva in cirrosi ,epatocarcinoma (HCC) ed insufficienza epatica. Sono stati identificati diversi eventi intracellulari che legano la NAFLD e la sua progressione a NASH, compreso accumulo di lipidi, disfunzione mitocondriale e stress ossidativo. Evidenze emergenti indicano che l’accumulo epatico sia di acidi grassi liberi che di colesterolo possano svolgere un ruolo determinante nello sviluppo della NAFLD; in particolare, l’eccesso epatico di colesterolo può portare ad un aumento della formazione di ossisteroli, prodotti di ossidazione del colesterolo, i quali potrebbero contribuire al danno epatico. Un’analisi dei campioni epatici di ratti Wistar maschi alimentati con una dieta ricca di acidi grassi (HF), ricca di colesterolo (HC) e una dieta combinata HF+HC mostra che, il modello HF determina steatosi epatica semplice, il modello HC è caratterizzato da degenerazione balloniforme degli epatociti, mentre il modello HF+HC presenta tutte le caratteristiche istologiche della NASH. Effettuando un’analisi lipidomica dei lipidi epatici abbiamo osservato un aumento del colesterolo libero nel modello HC, mentre nel modello HF+HC si riscontravano livelli epatici aumentati sia di colesterolo libero che di acidi grassi liberi. Per quanto riguarda gli ossisteroli epatici, ciò che abbiamo osservato è stato un aumento degli ossisteroli Cholestane-3β,5α,6 β-triol (triolo), 7-Ketocolesterolo, 7α e 7β idrossicolesterolo nelle diete HC e HF+HC, rispetto al controllo e alla dieta HF. Per verificare il meccanismo molecolare coinvolto nella disfunzione mitocondriale e nella tossicità epatocellulare, cellule di epatocarcinoma HuH7 e colture primarie di epatociti isolati da fegato di ratto sono state esposte all’acido palmitico e/o all’acido oleico, con e senza Triolo. Ciò che abbiamo osservato è che, tale ossisterolo induceva apoptosi in cellule co-esposte con i due acidi grassi e ciò era associato ad una alterazione dell’attività respiratoria mitocondriale e ad una down-regolazione dei geni PGC1-α, TFAM e NRF1. In conclusione i nostri dati mostrano che l’accumulo a livello epatico di acidi grassi liberi o ossisteroli induce di per sé una bassa tossicità epatocellulare. Al contrario, l’accumulo epatico di acidi grassi e ossisteroli tossici come il Triolo sono determinanti nell’alterazione della biogenesi e della funzionalità mitocondriale, contribuendo alla patologia epatica della NAFLD.

La malaltia del fetge gras no alcohòlic (MFGNA) inclou un espectre histològic que va des de la esteatosi simple (SS) a l'esteatohepatitis no alcohòlica (EHNA), sent aquesta última freqüentment progressiva. Donat que l'acumulació hepàtica de lípids sembla ser un mecanisme crucial en la patogènesi de la MFGNA, una millor comprensió dels mecanismes subjacents que condueixen a l'acumulació inicial de lipíds hepàtics podria ser de gran interès per controlar la progressió de la malaltia. El sistema endocannabinoide (SE), mitjançant principalment els receptors cannabinoides CB1 i CB2, sembla ser que juga un paper important en la patogènesi de la MFGNA modulant el metabolisme lipídic. Així, la hipòtesi establerta és que, en pacients amb MFGNA, l'expressió de gens i factors de transcripció implicats en la regulació del metabolisme lipídic hepàtic, així com l'expressió dels receptors cannabinoides pot estar alterada; i aquesta alteració podria estar relacionada amb l'aparició de dany hepàtic. En conseqüència, vam evaluar l'expressió hepàtica d'alguns gens clau involucrats en la síntesi de novo d'àcids grassos (AG), captació i transport d’AG, oxidació d’AG, inflamació, així com l’expressió de CB1 i CB2 en una extensa cohort de dones obeses mòrbides amb MFGNA segons la seva histologia hepàtica. La principal troballa ha estat que, en el fetge de dones obeses mòrbides amb SS, els gens clau involucrats en la síntesis de novo d’AG presenten una relació inversa amb el grau histològic d’esteatosi, suggerint que la via lipogènica podria estar disminuïda en etapes avançades d’SS. Respecte al SE, els nostres resultats suggereixen un paper perjudicial de CB1 en la MFGNA, mentre que el rol de CB2 no és del tot aclarit.
La enfermedad del hígado graso no alcohólico (EHGNA) incluye un espectro histológico que va desde la esteatosis simple (SS) a la esteatohepatitis no alcohólica (EHNA), siendo esta última frecuentemente progresiva. Dado que la acumulación hepática de lípidos parece ser un mecanismo crucial en la patogénesis de la EHGNA, una mejor comprensión de los mecanismos subyacentes que conducen a la acumulación inicial de lípidos hepáticos podría ser de gran interés para controlar la progresión de la enfermedad. El sistema endocannabinoide (SE), mediado principalmente por los receptores cannabinoides CB1 y CB2, parece juegar un papel importante en la patogénesis de la EHGNA modulando el metabolismo lipídico. Así, la hipótesis establecida es que, en pacientes con EHGNA, la expresión de genes y factores de transcripción implicados en la regulación del metabolismo lipídico hepático, así como la expresión de los receptores cannabinoides puede estar alterada; y esta alteración podría estar relacionada con la aparición de daño hepático. En consecuencia, evaluamos la expresión hepática de algunos genes clave involucrados en la síntesis de novo de ácidos grasos (AG), captación, transporte y oxidación de AG, inflamación, así como la expresión de CB1 y CB2 en una extensa cohorte de mujeres obesas mórbidas con EHGNA según su histología hepática. El principal hallazgo fue que, en el hígado de mujeres obesas mórbidas con SS, los genes clave involucrados en la síntesis de novo de AG presentan una relación inversa con el grado histológico de esteatosis, sugiriendo que la via lipogénica podría estar disminuida en estadios avanzados de esteatosis. Respecto al SE, nuestros resultados sugieren un papel perjudicial de CB1 en la EHGNA, mientras que el papel de CB2 no es del todo esclarecido.
Non-alcoholic fatty liver disease (NAFLD) encompasses a histological spectrum from simple steatosis (SS) to non-alcoholic steatohepatitis (NASH), with the latter being more frequently progressive. Due to lipid accumulation in the human liver seems to be a crucial mechanism in the NAFLD pathogenesis, an improved understanding of the underlying mechanisms leading to the initial hepatic lipid accumulation could be of great interest for controlling the progression of NAFLD. It has also been reported that the endocannabinoid (EC) system, mediated mainly by CB1 and CB2 cannabinoid receptors, plays an important role in NAFLD pathogenesis by modulating lipid metabolism. We therefore hypothesized that in patients with NAFLD, the expression of genes and transcription factors involved in the regulation of hepatic lipid metabolism, as well as the expression of cannabinoid receptors could be altered; and this alteration may be related to the onset of liver damage. Consequently, we wished to further investigate the fatty acid (FA) metabolism and the EC system by evaluating the hepatic expression of some key genes involved in de novo synthesis FA, FA uptake and transport, FA oxidation, and inflammation; as well as CB1 and CB2 expression in an extensive cohort of morbidly obese (MO) women according to their liver histology. The major finding is that, in liver of MO women with SS, the key genes related to de novo fatty acid synthesis have an inverse relationship with the histological degree of simple steatosis; suggesting that the lipogenic pathway seems to be down-regulated in advanced stages of SS. Regarding the EC system, our results suggest a deleterious role of CB1 in NAFLD, while the role of CB2 is not fully clarified.

Introduction Non-alcoholic fatty liver disease (NAFLD) is common in adults with diabetes mellitus. In addition, it tends to occur as non-alcoholic steatohepatitis (NASH) and/or with liver fibrosis more frequently than in the general population and this has important implications for morbidity and mortality. While simple steatosis due to NAFLD can be determined by non-invasive means with reasonable ease and reliability, establishing the presence and severity of NASH and liver fibrosis degree requires more sophisticated approaches, which are in need of further development. Furthermore, our understanding and interpretation of currently available clinical measures of NAFLD and its severity need improvement. Aims This thesis aimed to explore the frequency of NAFLD overall, and severe fibrosis due to NAFLD specifically, in cohorts with diabetes mellitus using non-invasive techniques and to define the clinical and biochemical associations with these. It also sought to determine broader clinical associations with liver enzymes, the most commonly used clinical markers of NAFLD, with the objective of improving our understanding of other factors that might influence these measures. The work also planned to investigate the use of both established and novel circulating biomarkers for grading liver disease severity in NAFLD. The integration of these biomarkers into liver fibrosis screening algorithms was explored. Methods Data sourced from 4 complimentary and relevant study populations were used in this thesis. One cohort was developed by a process of deliberate clinical recruitment, while data from the other three cohorts were obtained by either sub-analysis of completed trial data, post-hoc analysis of archived samples, or an audit of a pre-existing clinical database. Given the significant differences between cohorts, no direct comparisons were made between them. Results Steatosis was present in 84% of those with type 2 diabetes from a tertiary diabetes centre who had causes of liver disease other than NAFLD excluded. Non-invasive measure of liver fibrosis by transient elastography (TE) was consistent with severe liver fibrosis in ~ 1 in 5 of this group. Across cohorts, markers of fibrosis in NAFLD, but not steatosis per se, were associated with a more severe diabetes phenotype overall and with peripheral neuropathy specifically as a novel finding in this thesis. Collectively across cohorts, ALT was associated with steatosis but not fibrosis. It was higher in the presence of adverse metabolic features, although it was also noted to be related to other factors. These factors included important negative associations with age after 50 years, higher exercise capacity, full-time employment and higher level of education completed. Consistent with these findings, ALT had a prospective, inverse relationship with cardiovascular events and cardiovascular mortality in a cohort of 9795 individuals with type 2 diabetes in a community-based setting. Across cohorts, GGT was associated with non-invasive markers of NAFLD and liver disease severity in NAFLD, as well as with the presence of adverse metabolic features. It was higher in those assessed as having decreased level of physical and emotional wellbeing by the SF-36 and, consistent with this, higher GGT was independently associated with all-cause and cardiovascular mortality in the same 9795 with type 2 diabetes. Additionally, fenofibrate use was associated with decreases in GGT and ALT with time, when compared to placebo, in this group. Changes in weight, HbA1c and triglycerides were also associated with significant changes in these liver enzymes. Liver stiffness measurement by TE appeared to be a reasonable screening tool for liver fibrosis in those with type 2 diabetes from a tertiary diabetes centre who had liver disease due to causes other than NAFLD excluded, although repeat measure may improve assessment. Algorithms with serial assessment by NAFLD fibrosis score, followed by a biomarker for fibrosis, such as circulating Fibroblast Activation Protein (cFAP), and then TE are likely to be clinically useful for triaging those who may require specialist assessment and liver biopsy. Related to FAP, circulating dipeptidyl peptidase 4 (DPP4) activity was significantly associated with cytokeratin M30 Apoptope, a validated marker of hepatocyte apoptosis. Conclusions NAFLD is a real and present complication of diabetes. Moreover, it is likely to increasingly contribute to liver and perhaps systemic morbidity and mortality due to escalating rates of obesity, an aging population and also better treatments for other diabetes-associated illnesses. It is timely to work towards the development of adequate biomarkers for NAFLD severity and become familiar with their use in people with diabetes in order to stratify appropriate investigations and to initiate care in those at risk for unfavourable health outcomes. This stratification will also help to facilitate the development of effective treatments for NAFLD. Such treatments may not only improve liver pathology but could influence the outcomes in diabetes overall.

Previous research has suggested a role for vitamin D in non-alcoholic fatty liver disease (NAFLD) pathogenesis. Several observational studies have observed low vitamin D status (25OHD) with poorer histological findings. The principal aims of this study were to assess diet and lifestyle, 25OHD status, gene variants in vitamin D metabolism in UK children, and separately examine the effect of vitamin D in an in vitro NAFLD model. Dietary results from the case control study (n=32) indicated vitamin D intakes of paediatric patient with biopsy-proven NAFLD and ultrasound-cleared obese patients were 1.7μg/day and 3.5μg/day, respectively, well below the new UK recommendation. Children failed to meet current UK government recommendations for physical activity. In our UK paediatric biopsy-proven NAFLD cohort (n=103), the majority of patients presented with deficient (< 25nmol/L, 25.5%) or insufficient (< 50nmol/L, 80.8%) mean serum 25OHD levels. Furthermore, patients had significantly lower 25OHD levels during winter months in comparison to summer (p=0.0001) and autumn (p=0.0026), while 25OHD levels were non-significantly lower in NASH compared to non-NASH patients (p=0.0576). We observed that single nucleotide polymorphisms (SNPs) involved in vitamin D metabolism were associated with poorer liver histology grading; specifically, three SNPs were associated with increased steatosis and one with increased inflammation score in Caucasian patients. Finally, LX-2 cells, an immortalised human hepatic stellate cell line, demonstrated significantly reduced cell proliferation (p=0.0005) with increasing doses of 1α,25(OH)2D3 after 10 days of incubation in clonogenic assays. In conclusion, we found that NAFLD children have extremely low levels of 25OHD throughout the year, with little dietary contribution. In addition, several vitamin D related SNPs were associated with poorer histological findings. These novel data suggest an important role for vitamin D in the pathogenesis and progression of NAFLD in a paediatric population.

USS dataset and n=3 ,059 in the blood-based outcomes dataset). For example, the odds of USS liver fat per 100 kcal increase in energy intake at age 3 years were 1.79 (95% CIL 1.14 to 2.79). Associations of absolute macronutrient intake with the liver outcomes were inconsistent, and attenuated to the null after adjustment for total energy intake. Physical activity at ages 12 and 14 years was inversely related to risk of NAFLD in adolescence (n=I,292 in the USS dataset and n=2,612 in the blood-based outcomes dataset), for example the odds of liver fat per 15 minute increase in time spent in moderate to vigorous physical activity at age 12 was 0.55 (95%CI: 0.32 to 0.94). The majority of these observed associations attenuated towards the null upon adjustment for fat mass at the time the liver outcomes were assessed, suggesting that these associations may be, in part, mediated by adiposity in adolescence. These results suggest that risk of NAFLD is common in children and adolescents and that risk of NAFLD is affected by modifiable exposures throughout infancy, childhood and early adolescence. Public health initiatives aimed at promoting healthy growth and adiposity gain from infancy, through a healthy diet and physical activity, are warranted for the prevention of NAFLD.

The term non-alcoholic fatty liver disease (NAFLD) has been used as a general name for conditions ranging from simple steatosis to steatohepatitis, advanced fibrosis and cirrhosis. End-stage NAFLD cirrhosis carries a substantial risk for early hepatocellular carcinoma development. Therefore, there is an increasing need for new therapeutic approaches for the treatment of this liver disease. Here, we examined the effect of the thyroid hormone triiodothyronine (T3) and the agonist of the thyroid hormone receptor β isoform (TRβ), GC-1, on fatty liver and steatohepatitis induced in rodents by a choline-deficient, methionine-restricted (CMD) diet. Male Fischer rats fed the CMD diet for 1 week developed a marked fatty liver and mild hepatitis. Concurrent administration of T3 resulted in a complete prevention of the fatty change associated with increased mitochondrial and peroxisomal fatty acid beta-oxidation. To investigate whether T3 could also reverse fully established fatty liver, rats were fed the CMD diet for 10 weeks and then cofed T3 for 1 week. Coadministration of T3 resulted in a complete regression of liver steatosis associated with a decrease of lipid peroxidation, cyclooxygenase-2 expression, and activation of phospho-STAT3 and phospho-SAPK/JNK. Finally, additional experiments showed that GC-1, which has no significant side effects on heart rate, prevented and reverted CMD-induced fat accumulation. These results indicate that TR agonists have the potential to inhibit or reverse hepatic steatosis induced by a nutritional model.

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.

Type 2 diabetes is an established risk factor for non-alcoholic fatty liver disease (NAFLD). Despite this, there are few data investigating NAFLD within large populations of people with Type 2 diabetes. In the initial chapters of this thesis NAFLD is defined and the pathological processes linking it to Type 2 diabetes are described. The epidemiological evidence that underpins current understanding of prevalence, risk factors and progression of the condition is reviewed in detail. Subsequent chapters describe research undertaken within the Edinburgh Type 2 Diabetes Study on the epidemiology of NAFLD in this population. The prevalence of NAFLD was determined within 939 subjects, aged 61 - 76 years, with well-characterised Type 2 diabetes. NAFLD was defined by the presence of ultrasounddiagnosed steatosis with detailed exclusion of secondary causes for liver disease. Ultrasound gradings were validated in a subgroup of 58 participants using 1Fi magnetic resonance spectroscopy, the non-invasive gold-standard method for hepatic lipid quantification, and intra- and inter-observer variability in ultrasound grading was calculated. The final prevalence of NAFLD, 42.6%, was higher than in the general population, but lower than in the few studies that have been performed in populations with Type 2 diabetes. It is likely that this was due to comprehensive screening for secondary causes of liver disease and validation of the ultrasound measure which resulted in re-categorisation as "normal" subjects initially graded as having mild steatosis. Independent predictors of NAFLD were diabetes variables or components of the metabolic syndrome. The utility of conventional "liver function tests" in detecting hepatic steatosis and NAFLD was examined. Although liver enzyme levels (alanine aminotransferase, aspartate aminotransferase and gamma-glutamyltransferase) were significantly higher in participants with hepatic steatosis compared with those with normal liver on ultrasound, values remained within the normal range in the majority of cases. The negative predictive values of normal levels were therefore low, calling into question their utility as screening tests for liver disease in the diabetic population. The prevalence of advanced liver disease had not previously been studied in a population with Type 2 diabetes, and was investigated in our population using surrogate markers of hepatic fibrosis. Hyaluronic acid (HA) levels were significantly elevated in 5.7% of subjects and 0.4% had ultrasound-diagnosed cirrhosis. Hepatocellular carcinoma was detected in 0.2% of participants. Liver enzyme markers were poorly predictive of high HA levels. Clinical risk scores identified a large proportion of subjects as potentially having severe fibrosis, but these scores have not previously been validated in populations with Type 2 diabetes and therefore have to be interpreted with caution. NAFLD has previously been shown to be predictive of cardiovascular disease in the context of Type 2 diabetes, but mechanisms underlying this have not been fully elucidated. In the current study the association of hepatic steatosis and NAFLD with non-classical cardiovascular risk factors - clot formation and lysis dynamics, prothrombotic mediators and markers of inflammation - was investigated. NAFLD was significantly associated with a longer clot lysis time and higher levels of complement C3 and plasminogen activator inhibitor type 1. Following adjustment for these prothrombotic compounds, the association of NAFLD and clot lysis time was lost, and it is therefore hypothesised that NAFLD may influence clot lysis time via increased production of these mediators.

Obesity induced, non-alcoholic fatty liver disease (NAFLD), describes the spectrum from steatosis-to-cirrhosis. NAFLD is now the commonest cause of chronic liver disease in affluent populations. Its prevalence is increasing in tandem with rising obesity rates. However, given the exponential rise in obesity and NAFLD disease prevalence, availability of cheap energy dense foods may not alone be the sole determinant of these rising rates: maternal obesity – through developmental programming - may also be involved. A growing body of epidemiological evidence suggests that perinatal factors may contribute to chronic disease in adulthood via programming. Programming is the process whereby an insult/stimulus during a critical period of development induces permanent structural and/or physiological changes. The role of developmental programming in NAFLD is unknown. The aims, therefore were to determine if maternal obesity during gestation and/or lactation transmits a predisposition to NAFLD in offspring, to evaluate the natural progression of such programmed disease, to examine the relative contributions of maternal obesity and the post-natal environment on offspring NAFLD and to investigate the mechanisms therein. Using a rodent diet induced obesity-programming model, our initial studies confirmed that maternal obesity, through developmental programming, was indeed involved in the pathogenesis of NAFLD with the lactation period most susceptible to these programming effects. Moreover, there was an interaction between in utero exposure to an obesogenic environment and a post-weaning obesogenic environment to cause exacerbated offspring NAFLD. Mechanistically, perturbed hepatic innate immune function and disrupted peripheral circadian rhythms were observed in offspring with NAFLD programmed by maternal obesity. Finally, these results also provide a novel and uniquely pathophysiologically relevant model of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis. To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes. Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis. To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes. Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity.

Thesis (MScMedSc)--Stellenbosch University, 2011.
ENGLISH ABSTRACT: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disease in Western countries and is considered the hepatic manifestation of the Metabolic Syndrome (MetS). Its heterogeneous nature ranges from hepatic steatosis through steatohepatitis to advanced fibrosis and cirrhosis where the ingestion of significant amounts of alcohol has been excluded. The disease profile of NAFLD and its necro-inflammatory subset Nonalcoholic Steatohepatitis (NASH) were described in the parent study, which provided a clinically well-characterised patient cohort for the present investigation. South African patients with NASH had significantly higher mean serum cholesterol and triglyceride levels than those with fatty liver only. The objective of this study was to implement a high-throughput real-time polymerase chain reaction (PCR) method in our laboratory to enable the assessment of cardiovascular genetic risk factors in NAFLD patients. The specific aims were to determine the clinical utility and perform analytical validation of each mutation included in the multi-gene cardiovascular disease (CVD) screening assay. The Pathology Supported Genetic Testing (PSGT) concept developed at our department provides a practical approach to personalized medicine. The CVD multi-gene screen analyses key metabolic pathways relating to atherogenic dyslipidaemia, chronic inflammation, hypercoagulation and iron dysregulation implicated in insulin resistance, which is known to be a universal factor in the pathogenesis of NAFLD. Deleterious low-penetrance mutations in the APOE (APOE2 and E4 alleles), MTHFR (677C>T and 1298A>C), F2 (20210G>A), FV (1691G>A, Leiden) and HFE (C282Y and H63D) genes were included for analysis due to their important role as genetic contributors to these biological processes. A total of 178 patients diagnosed with NAFLD and 75 controls were studied using direct DNA sequencing and a RT-PCR system for mutation detection. In addition, two patients with high ferritin levels were included as case studies. A significant association was found between HFE mutations and elevated Alanine Transaminase (ALT) levels in the NAFLD population (p = 0.04). This discovery is interpreted as the identification of a subset of patients at greater risk of developing progressive liver damage who would benefit most from genetic testing to direct more aggressive therapy at an earlier stage. The necessity of an integrative, systems-based network approach was demonstrated to more accurately distinguish between Hereditary Haemochromatosis (HH) and Insulin Resistance-associated Hepatic Iron Overload (IR-HIO) syndrome in obese patients. The PSGT approach to personalized medicine facilitates diagnosis of CVD subtypes, prevention of cumulative risk and the formulation of gene-based intervention programs tailored to the needs of the patient. These findings support the clinical utility of the CVD multi-gene test to guide chronic disease risk management in patients with NAFLD. The HFE mutation detection component of this test is of particular relevance in directing an effective treatment strategy in patients with a medical history of CVD and/or high iron stores.
AFRIKAANSE OPSOMMING: Nie-Alkoholiese Vettige Lewer Siekte (NAFLD) is die mees algemene kroniese lewer siekte in Westerse lande en word bestempel as die hepatiese manifestasie van die Metaboliese Sindroom (MetS). Die heterogene natuur van NAFLD strek van hepatiese steatose deur steatohepatietis tot gevorderde fibrose en sirrose waar grootskaalse alkohol inname uitgesluit is. Die siekte-profiel van NAFLD en sy nekro-inflammatoriese subtipe Nie-Alkoholiese Steatohepatietis (NASH) is reeds beskryf in die ouer studie, wat ‗n klinies goed-gekarakteriseerde pasiënt groep vir die huidige ondersoek daar gestel het. Suid-Afrikaanse pasiënte met NASH het beduidend hoër gemiddelde serum cholesterol en trigliseried vlakke in vergelyking met slegs vettige lewer. Die doel van hierdie studie was om ‗n hoë deurvoer rieëltyd polimerase kettingreaksie (RT-PCR) metode in ons laboratorium te implimenteer om kardiovaskulêre genetiese risiko faktore in NAFLD pasiënte te ondersoek. Die spesifieke mikpunte was om die kliniese nut en analitiese geldigheid van elke mutasie wat ingesluit is in die multi-geen kardiovaskulêre siekte (KVS) siftings toets vas te stel. Die Patologie Ondersteunde Genetiese Toetsing (PSGT) konsep wat by ons departement ontwikkel is, verskaf ‗n praktiese benadering tot persoonlike medisyne. Die KVS multi-geen toets analiseer belangrike metaboliese weë verwant aan atherogene dyslipidemie, kroniese inflammasie, oormatige bloedstolling en yster disregulering wat betrokke is by insulien weerstand wat bekend is as ‗n universele factor in the patogenese van NAFLD. Nadelige lae-penetrasie mutasies in die APOE (APOE2 en E4 allele), MTHFR (677C>T en 1298A>C) F2 (20210G>A), FV (1691G>A, Leiden) en HFE (C282Y en H63D) gene was ingesluit vir analise as gevolg van hul belangrike rol as genetiese bydraers tot die bogenoemde biologiese prosesse. ‗n Totaal van 178 pasiënte gediagnoseer met NAFLD en 75 kontroles is bestudeer deur gebruik te maak van direkte DNA volgordebepaling en ‗n RT-PCR metode vir mutasie opsporing. Twee pasiënte met verhoogde ferritien vlakke is ook as gevalle studies ingesluit. ‗n Beduidende assosiasie is gevind tussen HFE mutasies en verhoogde Alanien Transaminase (ALT) vlakke in die NAFLD studiepopulasie (p = 0.04) wat aanduidend is van ‗n subgroup van pasiënte wat die meeste baat sal vind uit genetiese toetsing om meer aggressiewe behandeling te rig op' n vroeër stadium. Die noodsaaklikheid van 'n geïntegreerde, stelsels-gebaseerde netwerk benadering is gewys om meer akkuraat te onderskei tussen Oorerflike Hemochromatose (HH) en Insulien Weerstand-geassosieerde Hepatiese Yster Oorlading (IR-HIO) sindroom in vetsugtige pasiënte. Die PSGT benadering tot persoonlike medisyne formuleer geen-gebaseerde intervensie programme aangepas tot die behoeftes van die pasiënt ek maak diagnose van KVS-subtipes en voorkoming van kumulatiewe risiko moontlik. Hierdie bevindinge ondersteun die kliniese nut van die KVS multi-geen toets om riglyne vir die risikobestuur van kroniese siektes soos NAFLD daar te stel. Die HFE mutasie opsporings komponent van hierdie toets is van besondere belang om 'n effektiewe strategie vir die behandeling van pasiënte met 'n mediese geskiedenis van KVS en/of hoë yster vlakke daar te stel.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide health problem which includes steatosis (triglyceride accumulation alone), non-alcoholic steatohepatitis (NASH, with liver inflammation), fibrosis, cirrhosis and hepatocellular carcinoma. Liver fibrosis, which is a reversible response, is the final phase of most chronic liver disease and is characterized by accumulation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). Glucocorticoids (GCs) regulate many aspects of metabolism involved in NAFLD. Also, GCs limit HSC activation in vitro. Tissue GC levels are regulated by 11β- hydroxysteroid dehydrogenase-1 (11β-HSD1) which converts inactive 11- dehydrocorticosterone (DHC) into active corticosterone. Previous studies demonstrate that 11β-HSD1 deficiency improves fatty liver in obesity models, but the role of 11β-HSD1 in mechanisms involved in the progression and/or resolution of hepatic injury is largely unknown. I hypothesized that 11β-HSD1 modulates fibrotic and inflammatory responses during hepatic injury and/or the resolution phase. First I sought to address if the levels of 11β-HSD1 during different models of liver injury are dysregulated. In mice, 11β-HSD1 was down-regulated in choline deficient diet (CDD) induced steatosis, methionine and choline deficient diet (MCDD) induced NASH, carbon tetrachloride (CCL4) induced liver fibrosis and thioacetamide (TAA) induced liver fibrosis. In CCL4 injured livers, the down regulation of 11β- HSD1 was observed around the scar area. To test if 11β-HSD1 plays a key role in modulating liver inflammation and fibrosis responses in NAFLD and liver fibrosis I used initially11β-HSD1 knockout (KO) mice. 11β-HSD1 KO showed higher HSC activation only in the High fat feeding model but not in CDD and MCDD models. In the CCL4 injury model, despite reduced hepatocellular injury, 11β-HSD1 KO mice showed enhanced collagen deposition during peak injury and increased fibrotic gene expression during the early resolution phase although unaltered inflammatory markers during both peak injury and resolution. To further dissect cell-specificity on the effect of 11β-HSD1, I repeated the CCL4-injury model using the hepatocyte-specific 11β-HSD1 KO (Alb-HSD1). Alb-HSD1 mice did not show increased susceptibility to fibrosis compared to control littermates suggesting that the 11β- HSD1 possibly modulates fibrotic response by affecting HSC function. To mechanistically address how GCs inhibit HSC activation in vitro I studied the effects of 11β-HSD1 on HSC in vitro. 11β-HSD1 expression was down-regulated during ‘spontaneous’ HSC activation, and 11β-HSD1 deficiency enhanced susceptibility to activation. The GC (11-DHC)’s inhibitory effect on HSC activation was reversed by 11β-HSD1 inhibition. Finally, to address the clinical relevance of 11β-HSD1 in hepatic injury and/or resolution a selective 11β-HSD1 inhibitor, UE2316, was used. UE2316 induced a pro-fibrotic phenotype in ob/ob mice and CCL4-treated C57BL/6 mice, but had no effect when administered only during injury resolution. In conclusion, 11β-HSD1 deficiency causes increased activation of HSCs following diet and chemical injury and promotes liver fibrosis. Effects of 11β-HSD1 inhibitors, which are a potential treatment for metabolic syndrome, are perhaps offset by adverse outcomes in liver.

Thesis (Ph. D.)--George Mason University, 2007.
Title from PDF t.p. (viewed Jan. 18, 2008). Thesis director: Ancha Baranova Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biosciences. Vita: p. 213 Includes bibliographical references (p. 188-212). Also available in print.

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.

碩士
中山醫學大學
生化暨生物科技研究所
102
Non-alcoholic fatty liver disease (NAFLD), a leading cause of liver damage, comprises of a spectrum of liver abnormalities including the early fat deposition in the liver (hepatic steatosis) and advanced nonalcoholic steatohepatitis (NASH). In addition, alcoholic liver disease (ALD) also lead to similar liver syndrome with NASH.In previous studies, mulberry leaves had been demonstrated to possess anti-oxidantant promoting effects. In this study, we have examined the effect of water extracts of mulberry leaves (MLE) on HFD-induced hepatic steatosis in rats. We used two animal models to do our research: NASH and ALD models. The rats were fed either a rodent normal chow, chow containing high-fat diet (HFD), or HFD containing 0.5% or 1% and 2% MLE in the diet for 10 weeks. Moreover, we first fed the HFD diet for 4 weeks to induce hepatic steatosis, then treated with MLE (0.5% or 1% and 2% in the diet) in the HFD diet for 6 weeks. In ALD model, we fed Lieber-DeCarli alcohol liquid feed for 8 weeks. We assayed antioxidant enzyme activity, such as SOD, catalase, GSH peroxidase. Furthermore, we also used the ELISA kits to measure the inflammatory factors, such as IL-6, TNF-α and the fat-relative factors, as adiponectin, leptin. Our data indicated that both NASH and ALD model are inclusion of 0.5%, 1% and 2% MLE in the HFD diet decreased liver fat content, liver weight, hepatic oxidative products, and prevented hepatic steatosis significantly. In serum, the AST, ALT and total triglyceride, total cholesterol decreased significantly in treating MLE group. MLE increased anti-oxidant enzyme activities in liver of HFD-induced rats. We also found that inflammatory factors TNF-d and the fat-relative factors, such as adiponectin, leptin, reduced in the treating MLE groups. These findings suggested that MLE effectively prevented and caused the regression of experimental hepatic steatosis. In the future, we could find the way to do more research about finding more effective compounds from mulberry leaves so that we would have more solution about treating liver disease.

Non-alcoholic fatty liver disease (NAFLD) is highly prevalent. 20-30% of patients with NAFLD progress to non-alcoholic steatohepatitis (NASH), characterised by steatosis, ballooning degeneration of hepatocytes, inflammation and fibrosis, but the cause(s) of this transition remains unclear. Foz/foz (Alms1 mutant) mice develop hyperphagia and obesity-related NAFLD. High fat (HF)-feeding induces NASH transition by 12 weeks, and fibrosis by 24 weeks. The aims of this research were to investigate the cause and effect of hepatic cholesterol dysregulation in foz/foz mice, its pathogenic significance in NASH, and the potential of dietary and pharmacological approaches to treat NASH. After 24 weeks HF-feeding, hepatic free cholesterol (FC) and cholesteryl ester (CE) content were markedly increased in foz/foz mice. Hepatic cholesterol accumulation was associated with increased expression of low-density lipoprotein receptor (LDLR), but not cholesterol synthesis. Bile acid (BA) biosynthesis genes were suppressed, as was expression of cholesterol and BA export proteins. Transcriptional regulators of cholesterol metabolism were differentially expressed in foz/foz mice with NASH: SREBP-2, a nuclear regulator of LDLR increased significantly, while LXR-a and HNF-4a decreased. Importantly, expression of liver receptor homolog-1 (LRH-1), while significantly elevated in HF-fed WT mice, was unaltered in HF-fed foz/foz mice. To test whether hyperinsulinaemia was responsible for some or all of these changes, experiments were conducted in primary murine hepatocyte cultures. Insulin activated both SREBP-2 and LDLR, while simultaneously decreasing BA export proteins and LRH-1 expression. Removing dietary cholesterol in HF-fed foz/foz mice ameliorated liver injury, whereas increasing dietary cholesterol accentuated injury. Steatohepatitis severity correlated with hepatic FC and CE, but not hepatic triglyceride or free fatty acids. Hepatic cholesterol loading activated nuclear factor (NF)-kB and c-Jun N-terminal kinase (JNK), and increased serum monocyte chemotactic protein (MCP)-1. Finally, quantitative analysis of sirius red staining demonstrated a positive relationship between hepatic cholesterol content and fibrosis severity. To establish whether pharmacological modulation of cholesterol turnover could modulate NASH outcome, HF-fed foz/foz and WT mice were administered atorvastatin and/or ezetimibe after NASH onset. Both agents significantly lowered hepatic CE and FC, serum ALT, total cholesterol and HDL and normalised liver size in foz/foz mice. Hepatocellular apoptosis and expression of inflammatory mediators was suppressed as was hepatic macrophage infiltration. While histological steatosis and ballooning scores were unaltered, lobular inflammation and liver fibrosis were significantly reduced in drug-treated foz/foz mice. In foz/foz mice, NASH is associated with profoundly disordered hepatic cholesterol turnover. Insulin-induced activation of SREBP-2 and suppression of LRH-1 likely result in increased LDLR expression and suppression of genes involved in cholesterol biotransformation and export. Consequently, cholesterol accumulates within the livers of foz/foz mice. The evidence from both dietary and pharmacological intervention experiments is that hepatic cholesterol mediates hepatocellular apoptosis, inflammatory cell recruitment and liver fibrosis in this model. Conversely, inhibition of cholesterol uptake/redistribution and biosynthesis, and dietary cholesterol restriction, reduces liver injury, inflammation, and fibrosis in foz/foz mice with NASH. These findings strongly support the hypothesis that hepatic cholesterol, secondary to insulin resistance, acts as a lipotoxic mediator of pro-fibrotic liver injury and inflammation in experimental NASH. -- provided by Candidate.

Doutoramento em Motricidade Humana, na especialidade de Actividade Física e Saúde
The present thesis includes five original studies focusing on body composition (BC) in non-alcoholic fatty liver disease (NAFLD) patients, mainly in a clinical perspective, directed to clinical practice. The first study of this thesis aimed at analyzing the relation between body fat (BF) content and distribution, as assessed by dual energy x-ray absorptiometry (DXA), and cardiac autonomic control, more specifically with heart rate recovery after a maximal exercise test, which is an indirect clinical marker of parasympathetic reactivation, also known to be a strong risk factor for overall and cardiac mortality. The second study focused on the utility of waist circumference (WC) measurement, as a predictor of both BF content and distribution, and also on the comparison of different WC measurement protocols based on biological criteria, protocols’ precision and practical criteria, aiming to identify a preferential measurement protocol to be used in NAFLD patients. The third and fourth studies focused on the influence of using different WC measurement protocols in the relation of both waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) with BF content and distribution, as assessed by DXA, and aimed to identity a preferable measurement protocol. The fifth study focused on body indexes and circumferences usefulness as surrogates of BF content and distribution and aimed at identifying if there is a preferential clinical predictor of both BF content and distribution, as compared to the commonly used body mass index, in NAFLD patients. The results obtained confirmed the strong relation between BC and cardiac autonomic control and showed that BF distribution is more important than BF content in explaining cardiac autonomic control variation. It was also possible to conclude that WC measured just above the iliac crest seem preferable to be used in NAFLD patients, either singly or included in body indexes such as WHR or WHtR, mostly due to practical criteria but also because of it strong correlation with both BF content and distribution. WHtR appears to be the best BF content and distribution surrogate to be used in clinical practice with NAFLD patients. WC alone is a good practical alternative, when simplicity and time saving are important instrument/method selection criteria.
RESUMO: A presente tese integra cinco investigações originais que se centram no estudo da composição corporal (CC) em pacientes com doença do fígado gordo não-alcoólico (DFGNA), numa perspetiva eminentemente clínica e direcionada para a prática. Um primeiro estudo visou analisar a relação da quantidade e distribuição da massa gorda corporal (MG), avaliada por densitometria por raio-X de dupla energia (DXA), com o controlo autonómico cardíaco, mais especificamente com um indicador indireto da reativação do sistema nervoso parassimpático, que é a frequência cardíaca de recuperação após um esforço máximo, que também é um forte fator de risco para mortalidade. O segundo estudo visou avaliar a utilidade da medição do perímetro da cintura, isoladamente, como preditor da quantidade e distribuição de MG, em pacientes com DFGNA, e comparar os resultados e os procedimentos da medição do perímetro da cintura realizada segundo diferentes protocolos de medição de modo a identificar um protocolo preferencial. O terceiro e quarto estudo pretenderam avaliar o impacto da utilização do perímetro da cintura obtido segundo diferentes protocolos de medição na performance da razão cintura/anca e da razão cintura/altura, enquanto indicadores clínicos, duplamente indiretos, de quantidade e distribuição de MG. O quinto e último estudo deste trabalho teve como objetivo avaliar a relação de perímetros e índices corporais com a quantidade e distribuição de MG, em pacientes com DFGNA, e procurou identificar a existência de alternativas preferenciais à utilização do índice de massa corporal. Os resultados encontrados no presente trabalho permitem confirmar que a CC está fortemente relacionada com o controlo autonómico cardíaco, em pacientes com DFGNA, e que, nessa relação, a distribuição de MG parece ser mais determinante do que a sua quantidade absoluta e relativa. Também foi possível concluir que o perímetro da cintura medido imediatamente acima da crista ilíaca parece ser a melhor metodologia para ser utilizada com esta população, sobretudo por razões de ordem prática, mas também pelo seu desempenho na relação com quantidade e distribuição de MG, quer quando utilizado isoladamente como quando integrado em índices corporais, como a razão cintura/ anca ou a razão cintura/ altura. A razão cintura/altura parece ser a melhor alternativa para ser usada como preditor da quantidade e distribuição de MG em pacientes DFGNA, sendo que o perímetro da cintura também é uma boa alternativa sobretudo por razões de ordem prática.

Shen, Jiayun.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 166-199).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Non-alcoholic fatty liver disease (NAFLD) comprises a series of liver lesions ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and is a major cause of chronic liver disease that may progress to cirrhosis and hepatocellular carcinoma. Despite the risk factors related with the metabolic syndrome, the mechanisms of disease are not entirely known. Further, several promising molecules with different mechanisms of action are currently in development to treat NASH, although reported efficacy to date has been limited. Therefore, a better understanding of NAFLD pathogenesis and strategies targeting diverse therapeutically responsive nodes should pave the way to find novel and sustainable treatments eagerly waited for disease management. In that regard, we have shown that receptor interacting protein kinase 3 (RIPK3), an indispensable component of necroptosis, is increased in liver samples of human and experimental NASH, resulting in deleterious disease phenotypes including development of hepatic tumorigenesis. Intriguingly, RIPK3 was also found to be a crucial regulator of lipid and mitochondrial energy homeostasis, a role that is yet to be further explored. In turn, as key regulators of hepatic metabolism, farnesoid X receptor (FXR) and G protein-coupled receptor 5 (TGR5) influence NAFLD pathogenesis. In this regard, INT-767, a dual FXR and TGR5 agonist, constitutes a promising therapeutic strategy. Similarly, miR-21 genetic ablation and concomitant use of obeticholic acid (OCA) strongly improved diet-induced NAFLD and NASH through targeting PPARα and FXR, respectively. This further justifies the therapeutic potential of simultaneously activating nuclear receptors and inhibiting miR-21, which points towards multiple targeting approaches that deserve further attention. The main goal of the work presented in this thesis was to elucidate the role of cell death linked to mitochondrial bioenergetic and metabolic function in human and experimental NAFLD and NASH, and to further evaluate the therapeutic relevance of multiple targeting approaches in the resolution of the disease. First, we explored the role of RIPK3 in mitochondrial bioenergetics and hepatic lipid metabolism. Our results showed decreased expression and activities of mitochondrial respiratory chain complexes, downregulation of key mitochondrial biogenesis and function meditators, namely PGC1α, NRF1, NRF2, TFAM and MFN2, as well as increased mitochondrial DNA damage in diet-induced NASH mice. In vitro, murine hepatocytes exposed to palmitic acid (PA) displayed significantly altered mitochondrial morphology, membrane potential and respiration capacity, resulting in higher mitochondrial reactive oxygen species overload. Interestinly, Ripk3 depletion reverted deleterious mitochondrial dysfunction and halted NASH phenotype both in vitro and in vivo. Functional studies in PA exposed cells established a link between RIPK3 and lipid droplet- associated perilipin 1 (PLIN1), which modulated lipid droplet metabolism. This RIPK3-driven altered lipid droplet metabolism was also observed in diet-induced NASH in mice, as well as in a large cohort of human NAFLD patients. We also sought to evaluate the therapeutic potential of FXR and TGR5 dual agonist INT-767 and inhibition of miR-21, alone or in combination, in a pre-clinical model of NASH. INT-767 and anti-miR-21 significantly diminished hepatic lipid accumulation, fibrosis and necroinflammation, while ameliorating lipid and cholesterol homeostasis. Both treatments were effective at improving dysregulated mitochondrial function and halting NASH-associated carcinogenesis, decreasing the formation of pre-neoplastic liver nodules. Noteworthy, the combination of both treatments afforded better protective effects than each strategy alone. Overall, the results of this thesis revealed a key function of RIPK3 in regulating mitochondrial bioenergetics and mitochondria-associated lipid homeostasis in lipid-loaded cells, diet-induced NASH in mice and NAFLD in patients, suggesting RIPK3 as an attractive therapeutic target that deserves further investigation. In addition, simultaneous targeting of more than one therapeutically responsive metabolic nodes of NASH pathogenesis could represent an effective therapeutic approach for better disease resolution. These results highlight the key role of liver cell mitochondrial metabolism in NAFLD and NASH, provide potential therapeutic targets and suggest possible translation into future applications.

BACKGROUND: As the prevalence of metabolic risk factors in the American population has increased over time, so too has the diagnoses of non-alcoholic fatty liver disease (NAFLD). Within this spectrum of disease lies the potential for silent progression towards cirrhosis, leaving the patient with few options for treatment. Currently, the standard of care remains counseling on diet and exercise with the goal of reversing disease progression by addressing the underlying risk factors. LITERATURE REVIEW: Recent studies have shown that a correlation exists between low levels of serum 25-hydroxyvitamin D and hepatic injury from NAFLD. This has become an active area of research, due in part to the anti-inflammatory and immunoregulatory properties of vitamin D. The prospect of a simple and cost effective intervention that can exert its effects on the mechanisms behind the development of NAFLD is interesting and warrants further research. PROPOSED PROJECT: This proposal is for a double-blind, randomized, experimental study of vitamin D3 (cholecalciferol) versus placebo in a patient population of those with both clinically proven NAFLD and concomitant vitamin D deficiency. Liver fibrosis will be measured and staged with the use of FibroScan elastography. The statistical analysis thereafter will determine if a clinically significant reduction in hepatic fibrosis exists, compared with the results of the placebo group. CONCLUSIONS/SIGNIFICANCE: Should vitamin D prove to be an effective treatment option in reversing the progression of NAFLD, clinicians would be equipped with a simple and safe tool to augment their management of the patient. For those that experience barriers (i.e. lower socioeconomic status, other comorbidities, etc.) preventing them from improving diet and exercise, vitamin D would serve as an alternative therapy to aid in reducing their disease burden. Easier methods to treat their disease now projects improved quality of life years later.