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.

This thesis aims to investigate fluoxetine, a widely prescribed SSRI antidepressant, for its role in the pathogenesis of NAFLD and uncover novel mechanisms by which it may contribute to drug-induced steatosis. We demonstrated that increased hepatic lipid accumulation was mediated, in part, via elevated serotonin production. The inhibition of hepatic serotonin synthesis prevented lipid accumulation in fluoxetine-treated hepatocytes demonstrating a causal role for serotonin in fluoxetine-induced hepatic steatosis. Interestingly, in several studies, serotonin signaling has been shown to impact prostaglandin biosynthesis. As prostaglandins have been implicated in the development of NAFLD, and fluoxetine has previously been shown to alter the production of prostaglandins I assessed the role of prostaglandins in fluoxetine-induced hepatic lipid accumulation. Fluoxetine treatment increased mRNA expression of prostaglandin biosynthetic enzymes, increased production of prostaglandin 15-deoxy-Δ12,14PGJ2 (PPARG agonist), and elevated PPARG targets involved in fatty acid uptake. Fluoxetine-induced lipid accumulation, 15-deoxy-Δ12,14PGJ2 production, and the expression of PPARG lipogenic genes were attenuated with a PTGS1 specific inhibitor. Taken together these findings suggested that fluoxetine-induced lipid accumulation was mediated via PTGS1 and its downstream product 15-deoxy-Δ12,14PGJ2. Given that Pparg was elevated following fluoxetine treatment, and PPARG regulates microRNA involved in hepatic lipid accumulation, my final project focused on PPARG’s role in altered miRNA expression. Indeed, fluoxetine treatment increased the miRNA expression of miR-122, an effect that was attenuated when fluoxetine treatment was combined with the PPARG antagonist GW9662, suggesting a fluoxetine-PPARG-miR122 axis contributing to hepatic steatosis. While these studies have only been performed in vitro, an understanding of the molecular changes associated with SSRI treatment may lead to the development of strategies to prevent the increased risk of adverse metabolic outcomes associated with the use of SSRI antidepressants. / Dissertation / Doctor of Philosophy (Medical Science) / In adults, major depressive disorder (depression) is one of the most common psychiatric illnesses. Recent data suggests that there are more than 4.1 million Canadians who currently suffer from depression. Depression is commonly treated using selective serotonin reuptake inhibitor (SSRI) antidepressants. While these antidepressants do help manage depressive symptoms, they can also cause unwanted side effects including a build-up of fat in the liver, leading to fatty liver disease. The goal of my research is to understand the link between SSRI use and the development of fatty liver disease. This thesis investigates the effects of fluoxetine (Prozac®), a commonly used SSRI antidepressant, on molecular pathways that can lead to the development of fatty liver disease. An understanding of the molecular changes with SSRI treatment may lead to the development of strategies to prevent the harmful effects of SSRI antidepressants on the liver.

Thesis (MSCMedSc)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in the world. The disease spectrum of NAFLD extends from steatosis (types 1,2) to non-alcoholic steatohepatitis (NASH) with inflammation (types 3,4). The aims of the study were 1) to analytically validate high-throughput real time polymerase chain reaction (RT-PCR) assays for three selected single nucleotide polymorphisms (SNPs), FTO rs9939609 (intron 1 T>A), TNF-α rs1800629 (-308 G>A) and PPARγ rs1801282 (Pro12Ala, 34 C>G), and 2) to perform genotype-phenotype association studies in relation to biochemical abnormalities, disease severity and age of onset. A total of 119 patients with fatty liver identified on ultrasound, including 88 histologically confirmed NAFLD patients, and 166 control individuals were genotyped for the three selected SNPs. RT-PCR validated against direct sequencing as the gold standard was used for detection of genetic variation. All three SNPs were in Hardy Weinberg equilibrium in the study population, except for a deviation in genotype distribution detected for PPARγ rs1801282 in the NAFLD patient subgroup (p<0.001). After adjustment for age and gender, the risk-associated FTO rs9939609 A-allele was detected at a significantly higher frequency in the Caucasian compared with Coloured patients (p=0.005). The opposite was detected for the risk-associated TNF-α rs1800629 A-allele, which occurred at a significantly higher frequency in the Coloured compared with Caucasian NAFLD patients (p=0.034). The onset of fatty liver disease symptoms was on average 5 years younger in the presence of each risk-associated TNF-α rs1800629 A-allele (p=0.028). When considered in the context of an inferred genotype risk score ranging from 0-6, disease onset occurred on average 3 years earlier (p=0.008) in the presence of each risk-associated FTO A-allele, TNF-α A-allele or PPARγ C-allele. After adjustment for age, gender and race, no differences in genotype distribution or allele frequencies were observed between histologically confirmed NAFLD (types 1,2) and NASH (types 3,4) patients, while the minor allele frequency for the TNF-α rs1800629 was significantly higher in the total NAFLD (types 1-4) (p=0.047) as well as NASH subgroup (NAFLD types 3,4) (p=0.030) compared with obese patients without a histologically confirmed NAFLD diagnosis. A significant correlation was furthermore observed between the number of TNF-α rs1800629 A-alleles and increasing CRP levels (p=0.029), with a favourable reduced effect in the presence of low- to moderate alcohol intake. The average waist circumference of physically active NAFLD patients was 12% lower than in physically inactive patients (p=0.004). In view of the results presented in this study, the inclusion of the selected SNPs, and in particular the pro-inflammatory TNF-α rs1800629 polymorphism, may be considered as part of a comprehensive cardiovascular risk evaluation of NAFLD patients. Ultimately, early detection of patients with fatty liver disease symptoms and effective intervention based on the underlying disease mechanism may prevent progression from NAFLD to NASH, shown to be an independent risk factor for cardiovascular diseases. / AFRIKAANSE OPSOMMING: Nie-alkoholiese lewervervetting (NALV) is die mees algemene kroniese lewersiekte in die wêreld. Die siektespektrum van NALV strek van steatose (vervette lewer tipes 1,2) tot steatohepatitis met inflammasie (NASH tipes 3,4). Die doel van die studie was 1) om analities die hoë omset polimerase kettingreaksie (RT-PKR) metode te valideer vir die geselekteerde enkel nukleotied polimorfismes (ENPs) FTO rs9939609 (intron 1 T>A), TNF-α rs1800629 (-308 G>A) en PPARγ rs1801282 (Pro12Ala, 34 C>G), en 2) om genotipe-fenotipe assosiasie studies uit te voer ten opsigte van relevante biochemiese abnormaliteite, graad van die siekte en aanvangsouderdom. ’n Totaal van 119 pasiënte met vervette lewers is geïdentifiseer met behulp van ultraklank, insluited 88 histologies-bevestigde NALV pasiënte, en 166 kontrole individue. Hierdie pasiënte is gegenotipeer vir die 3 geselekteerde ENP’s. RT-PKR gevalideer met direkte DNA volgorde bepaling as die goue standaard, is gebruik vir opsporing van genetiese variasie. Al die ENP’s was in Hardy Weinberg ekwilibrium in die studie populasie, behalwe vir ’n afwyking in genotipe verspreiding waargeneem vir PPARγ in die NALV subgroep (p<0.001). Nadat aanpassings gemaak is vir ouderdom en geslag, is die risiko-geassosieerde FTO rs9939609 A-alleel waargeneem teen ’n betekenisvol hoër frekwensie in die Kaukasiese pasiënte in vergelyking met Kleurling pasiënte (p=0.005). Die teenoorgestelde is waargeneem vir die risiko-geassosieerde TNF-α rs1800629 A-alleel wat voorgekom het teen ’n betekenisvol hoër frekwensie in die Kleurling NALV pasiënte, in vergelyking met Kaukasiese NALV pasiënte (p=0.034). Die aanvang van NALV was gemiddeld 5 jaar vroeër in die teenwoordigheid van elke risiko-geassosieerde TNF-α rs1800629 A-alleel (p=0.028). Met inagneming van ’n genotipe risiko telling tussen 0–6, het aanvang van siekte gemiddeld 3 jaar vroeër voorgekom (p=0.008) in die teenwoordigheid van elke toenemende risiko-geassosieerde FTO A-alleel, TNF-α A-alleel en PPARγ C-alleel. Nadat aanpassings gemaak is vir ouderdom, geslag en ras, is geen verskille waargeneem in genotipe verspreiding of alleel frekwensies tussen histologies bevestigde NALV (tipes 1,2) en NASH (tipes 3,4) pasiënte nie, terwyl die minor alleel telling vir die TNF-α rs1800629 betekenisvol hoër was in die totale NALV (tipes 1–4) (p=0.047) asook die NASH subgroep (NALV tipes 3,4) (p=0.03) in vergelyking met vetsugtige pasiënte sonder ’n histologies bevestigde diagnose. ‘n Statisties beteknisvolle korrelasie is verder waargeneem tussen die aantal TNF-α rs1800629 A-allele en toenemende CRP vlakke (p=0.029), met n gunstige verlaagde effek in die teenwoordigheid van lae alcohol gebruik. Die gemiddelde middellyf-omtrek van fisies aktiewe NALV pasiënte was 12% minder as fisies onaktiewe pasiente (p=0.004). Na aanleiding van die resultate van hierdie studie behoort insluiting van geselekteerde ENP’s, en in besonder die pro-inflammatoriese TNF-α rs1800629 polimorfisme, as deel van ’n omvattende kardiovaskulere risiko evaluasie oorweeg te word. Aan die einde van die dag mag vroeë identifikasie van NALV pasiente en effektieve intervensie gebasseer op die onderliggende siekte meganisme, vordering tot NASH verhoed wat getoon is om ’n onafhanklike risiko faktor vir kardiovaskulêre siekte te wees. / Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo do presente estudo foi avaliar se o Bezafibrato, um agonista PAN-PPAR, é capaz de aliviar a doença não alcoólica do fígado gorduroso (NAFLD) na prole de machos de mães C57BL/6 obesas. Fêmeas virgens foram alimentadas com uma dieta HL (hiperlipídica, 49% de lipídios) ou uma dieta C (controle, 10% de lipídios) por oito semanas antes do acasalamento e durante os períodos de gestação e lactação. A prole de machos foi subdividida em quatro grupos: C (dieta controle para as mães e filhotes); C/BZ (dieta controle para as mães e filhotes com tratamento com Bezafibrato[100mg/Kg]); HL (dieta HL para as mães e dieta controle para os filhotes); e HL/BZ (dieta HL para as mães e dieta controle para os filhotes com tratamento com Bezafibrato [100mg/Kg]). O tratamento com Bezafibrato começou na 12 semana e se manteve por três semanas. Análise do metabolismo, bioquímica, estereológica e por western-blotting foram realizadas. A dieta HL causou um fenótipo de sobrepeso nas mães e acarretou em uma intolerância oral à glicose com aumento da glicemia de jejum. A prole HL apresentou hiperfagia, ganho de massa corporal, altos níveis de triglicerídeo hepático e plasmático, esteatose hepática e aumento da expressão de proteínas lipogênicas concomitante com diminuição do receptor ativador de proliferação peroxissomal alfa (PPAR&#945;), que é responsável pela &#946;-oxidação e aumento do receptor ativador de proliferação peroxissomal gama (PPAR&#947;) e do elemento regulador de esterol ligante da proteína 1 (SREBP-1c) proteínas envolvidas na lipogênese hepática. Por outro lado, o tratamento com o Bezafibrato reverteu o quadro da programação metabólica no fígado, com uma melhora dos parâmetros morfológicos, bioquímicos e moleculares do fígado dos animais, com um aumento da ativação de PPAR&#945; em associação a uma diminuição do PPAR&#947; e não alterando a expressão de SREBP-1c. Em conclusão, nós demonstramos que o tratamento com Bezafibrato melhora a NAFLD causada pela obesidade materna. / The aim of the present study was to evaluate whether Bezafibrate , a PAN-PPAR agonist, could attenuate non-alcoholic fatty liver disease (NAFLD) of male offspring from obese C57BL/6 dams. Dams were fed on a HF (high-fat, 49% lipids) diet or SC (standard chow; 10% lipids) diet for 8 weeks before mating and during gestation and lactation periods. Male offspring were subdivided into 4 groups: SC (standard-chow for dams and offspring); SC/BZ [standard-chow for dams and offspring with treatment with BZ (100mg/Kg)]; HF (high-fat diet for dams and standard-chow for offspring); HF/BZ [high-fat diet for dams and standard-show for offspring with treatment with Bezafibrate (100mg/Kg)]. Treatment with Bezafibrate started at 12th week and was maintained for 3 weeks. Metabolic measurements, biochemical analysis, stereological tools and western-blotting were performed. The HF diet yielded an overweight phenotype and an increase in oral glucose tolerance and fasting glucose of dams. The HF offspring presented hyperphagia, body mass gain, high levels of plasmatic and hepatic triglycerides, impairment of glucose metabolism, hepatic steatosis and high expression of lipogenic proteins concomitant to decreased expression of PPAR&#945;, which is responsible for &#946;-oxidation. On the other hand, treatment with Bezafibrate reverted hepatic outcomes of metabolic programming, with an improvement of morphological, biochemical and molecular parameters of animals livers, with an increase of PPAR&#945; activation in association with a decrease of PPAR&#947; expression and no changes in SREBP-1c expression. In conclusion, we demonstrated that treatment with Bezafibrate improved NAFLD caused by maternal obesity.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo do presente estudo foi avaliar se o Bezafibrato, um agonista PAN-PPAR, é capaz de aliviar a doença não alcoólica do fígado gorduroso (NAFLD) na prole de machos de mães C57BL/6 obesas. Fêmeas virgens foram alimentadas com uma dieta HL (hiperlipídica, 49% de lipídios) ou uma dieta C (controle, 10% de lipídios) por oito semanas antes do acasalamento e durante os períodos de gestação e lactação. A prole de machos foi subdividida em quatro grupos: C (dieta controle para as mães e filhotes); C/BZ (dieta controle para as mães e filhotes com tratamento com Bezafibrato[100mg/Kg]); HL (dieta HL para as mães e dieta controle para os filhotes); e HL/BZ (dieta HL para as mães e dieta controle para os filhotes com tratamento com Bezafibrato [100mg/Kg]). O tratamento com Bezafibrato começou na 12 semana e se manteve por três semanas. Análise do metabolismo, bioquímica, estereológica e por western-blotting foram realizadas. A dieta HL causou um fenótipo de sobrepeso nas mães e acarretou em uma intolerância oral à glicose com aumento da glicemia de jejum. A prole HL apresentou hiperfagia, ganho de massa corporal, altos níveis de triglicerídeo hepático e plasmático, esteatose hepática e aumento da expressão de proteínas lipogênicas concomitante com diminuição do receptor ativador de proliferação peroxissomal alfa (PPAR&#945;), que é responsável pela &#946;-oxidação e aumento do receptor ativador de proliferação peroxissomal gama (PPAR&#947;) e do elemento regulador de esterol ligante da proteína 1 (SREBP-1c) proteínas envolvidas na lipogênese hepática. Por outro lado, o tratamento com o Bezafibrato reverteu o quadro da programação metabólica no fígado, com uma melhora dos parâmetros morfológicos, bioquímicos e moleculares do fígado dos animais, com um aumento da ativação de PPAR&#945; em associação a uma diminuição do PPAR&#947; e não alterando a expressão de SREBP-1c. Em conclusão, nós demonstramos que o tratamento com Bezafibrato melhora a NAFLD causada pela obesidade materna. / The aim of the present study was to evaluate whether Bezafibrate , a PAN-PPAR agonist, could attenuate non-alcoholic fatty liver disease (NAFLD) of male offspring from obese C57BL/6 dams. Dams were fed on a HF (high-fat, 49% lipids) diet or SC (standard chow; 10% lipids) diet for 8 weeks before mating and during gestation and lactation periods. Male offspring were subdivided into 4 groups: SC (standard-chow for dams and offspring); SC/BZ [standard-chow for dams and offspring with treatment with BZ (100mg/Kg)]; HF (high-fat diet for dams and standard-chow for offspring); HF/BZ [high-fat diet for dams and standard-show for offspring with treatment with Bezafibrate (100mg/Kg)]. Treatment with Bezafibrate started at 12th week and was maintained for 3 weeks. Metabolic measurements, biochemical analysis, stereological tools and western-blotting were performed. The HF diet yielded an overweight phenotype and an increase in oral glucose tolerance and fasting glucose of dams. The HF offspring presented hyperphagia, body mass gain, high levels of plasmatic and hepatic triglycerides, impairment of glucose metabolism, hepatic steatosis and high expression of lipogenic proteins concomitant to decreased expression of PPAR&#945;, which is responsible for &#946;-oxidation. On the other hand, treatment with Bezafibrate reverted hepatic outcomes of metabolic programming, with an improvement of morphological, biochemical and molecular parameters of animals livers, with an increase of PPAR&#945; activation in association with a decrease of PPAR&#947; expression and no changes in SREBP-1c expression. In conclusion, we demonstrated that treatment with Bezafibrate improved NAFLD caused by maternal obesity.

This thesis focuses on the effects of n-3 polyunsaturated fatty acids (n-3 PUFA) on development of non-alcoholic fatty liver disease (NAFLD) in experiment, on prevalence of this condition in patients with type 2 diabetes mellitus and metabolic syndrome and also on non-invasive diagnostics. The aim was to study the effect of n-3 PUFA on NAFLD development in an experimental model and based on analysis of a group of patients with type 2 diabetes and metabolic syndrome to assess the prevalence of this condition. Lastly we aimed to evaluate non-invasive diagnostic methods of liver fibrosis and NASH. We demonstrated beneficial effects of n-3 PUFA administration on NAFLD development in a C57/Bl6 mice high fat methionin-cholin defficient dietary model of NAFLD. n-3 PUFA administration led to biochemical improvement, decrease of lipid accumulation in the liver as well as improvement of histology. These effects are determined by complex modulation of lipid metabolism, mainly due to decrease in availability of fatty acids for triglyceride synthesis in the liver, changes of adipokine levels and amelioration of proinflammatory status in the liver. In a group of type 2 diabetics we found NAFLD prevalence of almost 80%, 14% of these patients had also signs of liver fibrosis or cirrhosis. Non-invasive methods...

This thesis focuses on the effects of n-3 polyunsaturated fatty acids (n-3 PUFA) on development of non-alcoholic fatty liver disease (NAFLD) in experiment, on prevalence of this condition in patients with type 2 diabetes mellitus and metabolic syndrome and also on non-invasive diagnostics. The aim was to study the effect of n-3 PUFA on NAFLD development in an experimental model and based on analysis of a group of patients with type 2 diabetes and metabolic syndrome to assess the prevalence of this condition. Lastly we aimed to evaluate non-invasive diagnostic methods of liver fibrosis and NASH. We demonstrated beneficial effects of n-3 PUFA administration on NAFLD development in a C57/Bl6 mice high fat methionin-cholin defficient dietary model of NAFLD. n-3 PUFA administration led to biochemical improvement, decrease of lipid accumulation in the liver as well as improvement of histology. These effects are determined by complex modulation of lipid metabolism, mainly due to decrease in availability of fatty acids for triglyceride synthesis in the liver, changes of adipokine levels and amelioration of proinflammatory status in the liver. In a group of type 2 diabetics we found NAFLD prevalence of almost 80%, 14% of these patients had also signs of liver fibrosis or cirrhosis. Non-invasive methods...

Les patients atteints de stéatose hépatique non alcoolique (NAFLD) développent fréquemment des manifestations cardiovasculaires. Bien que souvent liées à l'obésité, ces anomalies peuvent également se développer chez des patients non obèses atteints de NAFLD impliquant que cette pathologie hépatique joue, en soi, un rôle dans la pathogenèse des complications cardiaques. Pour répondre à cette question et étudier les mécanismes sous-jacents indépendamment de toutes perturbations métaboliques et comorbidités préexistantes, nous avons utilisé un modèle murin arborant une déficience mitochondriale hépatique associée à un défaut d'assemblage du complexe IV de la chaîne respiratoire. Ce modèle murin avait préalablement été caractérisé au niveau hépatique mettant alors en évidence le développement d'une stéatose microvésiculaire et un profil lipidomique similaire à celui observé chez les patients atteints d'une NAFLD sans obésité. L'identification des mécanismes qui sous-tendent le développement et la progression de la NAFLD sans obésité et de ces répercussions extra-hépatiques ne faisant pas l'objet d'un très grand nombre d'études fondamentales, l'objectif principal était donc d'étudier l'axe foie-coeur. Dans le cadre des travaux de ce mémoire, nous avons cherché à approfondir la caractérisation hépatique, préalablement faite à l'âge de 5 semaines et ayant fait l'objet de publications par des laboratoires collaborateurs. Nous avons par la suite investigué la glycémie, l'insulinémie et le profil des lipoprotéines plasmatiques pour finir par l'analyse du métabolisme et de la fonction cardiaque. L'ensemble de ces expériences ont été faites en prenant en compte l'impact non négligeable du sexe sur la physiopathologie de la NAFLD. Nos résultats ont dévoilé un important remodelage phénotypique sexe-dépendant allant au-delà des lésions hépatiques. Les mâles un peu plus que les femelles présentaient une hypoglycémie à jeun et une sensibilité accrue à l'insuline. Ils présentaient un léger dysfonctionnement diastolique soutenu par un remodelage des lipoprotéines circulantes et dans une certaine mesure, par un remodelage du lipidome cardiaque. À l'inverse, les femelles ne manifestaient aucun dysfonctionnement cardiaque, mais présentaient des déficiences cardiométaboliques soutenues par une altération de l’intégrité et la fonction mitochondriale, un remodelage des lipoprotéines circulantes et une accumulation intracardiaque de triglycérides. À la lumière de ces résultats, cette étude souligne que les défauts métaboliques dans le foie peuvent entraîner des anomalies significatives et dépendantes du sexe affectant à la fois le phénotype mitochondrial/métabolique et la fonction contractile indépendamment de l'obésité. Ce modèle expérimental pourrait s'avérer utile dans la compréhension des mécanismes sous-jacents à la variabilité liée au sexe dans la progression de la NAFLD chez l'homme non obèse. / Cardiac abnormalities often develop in patients with non-alcoholic fatty liver disease (NAFLD). Although frequently linked to obesity, these abnormalities can also develop in patients with lean-NAFLD, implying that the liver pathology per se plays a role in the pathogenesis of cardiac complications. To address this question and investigate the underlying mechanisms independent of any pre-existent metabolic disruptions and comorbidities, we used a murine model of hepatic mitochondrial deficiency associated with a defect in the assembly of respiratory chain complex IV. This mouse model had previously been characterized at the hepatic level, showing the presence of microvesicular steatosis, and a lipidomic profile similar to that observed in patients with lean-NAFLD. Because few fundamental studies have adressed the identification of mechanisms underlying the development and progression of lean-NAFLD and its extrahepatic repercussions, the main aim was to study the liver-heart axis. As a part of this master's project, we sought to deepen the hepatic characterization of this mouse model, previously done at 5-weeks of age, and published by collaborators. We then investigated glycemia, insulinemia and plasma lipoprotein profile, and finally examined cardiac metabolism and function. All these experiments were done in consideration of the non-negligible impact of sexe on the pathophysiology of NAFLD. Our results unveiled a sex-dependent multi-faceted phenotypic remodeling that went beyond liver damage. Males, slightly more than females, showed fasting hypoglycemia and increased insulin sensitivity. They exhibited mild diastolic dysfunction supported by remodeling of the circulating lipoproteins, and to some extent remodeling of cardiac lipidome. Conversely, females did not manifest cardiac dysfunction, but exhibited cardiometabolic impairments supported by impaired mitochondrial integrity and function, remodeling of circulating lipoproteins, and intracardiac accumulation of triglycerides. In light of these findings, this study underscores that metabolic defects in the liver can result in significant sex-dependent abnormalities that affect both the mitochondrial/metabolic phenotype and contractile function independent of obesity. This experimental model may prove useful to better understand the mechanisms underlying the sex-related variability in the progression of lean-NAFLD in humans.

INTRODUÇÃO: A esteato-hepatite não alcóolica (NASH) é considerada problema de saúde pública, dada sua crescente incidência e seu possível papel na carcinogênese hepato-celular. Terapias atuais envolvem alterações de dieta e estilo de vida, mas têm seu resultado prejudicado pela baixa aderência dos pacientes. Abordagens farmacológicas ainda são precárias. Uma grande dificuldade no manejo de NASH reside no limitado entendimento de sua fisiopatologia, que parece envolver complexas alterações metabólicas e inflamatórias. Ácidos graxos poli-insaturados ômega-3 (AGPIs n-3) são reconhecidos por suas propriedades moduladoras do metabolismo lipídico e da inflamação, e estão diminuídos em pacientes com NASH. O uso clínico de AGPIs n-3 tem mostrado benefício no controle da esteatose e na produção de marcadores da resposta metabólica e inflamatória em NASH, embora com algumas observações contraditórias. A compreensão de mecanismos moleculares modulados por AGPIs n-3 em NASH podem ser úteis para identificar alvos moleculares que auxiliem no desenho de intervenção farmacológica efetiva. Nesse sentido, ciências ômicas são particularmente úteis para a compreensão de mecanismos moleculares com alto valor translacional para a prática clínica e podem contribuir para a identificação desses alvos. OBJETIVO: O presente estudo avaliou a resposta proteômica hepática e lipidômica plasmática de pacientes com NASH perante o tratamento com AGPIs n-3. MÉTODO: As avaliações proteômicas e lipidômicas foram desenvolvidas por espectometria de massas e/ou cromatografia gasosa em amostras de biópsias hepáticas e plasma coletadas de pacientes envolvidos em estudo preliminar, realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. O referido estudo envolveu pacientes adultos, de ambos os sexos e com diagnóstico de NASH tratados diariamente, durante 6 meses, com 3 cápsulas contendo mistura de óleo de linhaça e óleo de peixe [0,315 g AGPIs: sendo 0,065 g de ácido eicosapentaenoico (EPA), 0,050 g de docosahexaenoico (DHA) e 0,2 g alfa linolênico (ALA) por cápsula]. Pacientes, após o tratamento com AGPIs n-3, que apresentaram altas concentrações plasmáticas de ALA e/ou DHA e/ou baixas de ácido araquidônico (AA) mostraram melhora parcial das alterações de histologia hepática. No presente estudo, avaliamos as vias proteômicas e marcadores lipidômicos resultantes do tratamento com AGPIs n-3. Isto foi feito por meio da comparação, antes (grupo AT) e depois do tratamento (grupo DT), de pools de tecido hepático (análise por interactoma) e amostras de plasma (OPLS-DA). RESULTADOS: Foram identificadas proteínas hepáticas, exclusivamente e/ou alteradamente expressas, no grupo DT, relacionadas com vias de matriz celular, metabolismo lipídico, de estresse oxidativo, e de retículo endoplasmático e respiração celular. Com excessão da via de matriz celular, a análise do interactoma revelou alteração funcional significativa das vias moduladas por essas proteínas. Em conjunto, essas alterações foram sugestivas de diminuição de lipotoxicidade, estresse oxidativo e respiração anaeróbia, e aumento de respiração aeróbia após tratamento com AGPIs n-3. Estas modificações são marcadores potenciais de melhora de função de retículo endoplasmático e mitocondrial. Em adição, após o tratamento com AGPIs n-3, o perfil lipidômico plasmático mostrou-se alterado com significativo aumento de glicerofosfolípides, ALA e EPA, e diminuição de ácido araquidônico (n-6) e da razão AGPIs n-6/n-3. Estes dados são concordantes com potencial melhora das funções de retículo endoplasmático e mitocondriais. CONCLUSÃO: O tratamento com AGPIs n-3 em pacientes com NASH influenciou favoravelmente o perfil proteômico hepático e lipidômico sistêmico. Em conjunto, essas alterações sugerem melhora da função de retículo endoplasmático e mitocondrial, com potencial impacto na homeostase celular, por meio da modulação de diferentes vias biológicas / INTRODUCTION: Non-alcoholic steatohepatitis (NASH) is considered a public health problem, given its increasing incidence and its possible role in hepatocellular carcinogenesis. Current therapies involve diet and lifestyle changes, but its applicability suffers from low patients adherence. Pharmacological approaches are still missing. A main difficulty in the NASH management lies in the limited understanding of its pathophysiology, which seems to involve complex metabolic and inflammatory disturbances. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are recognized for its modulatory properties on lipid metabolism and inflammation and are decreased in patients with NASH. The clinical use of these PUFAs has shown benefit in controlling steatosis and the production of metabolic and inflammatory response markers in NASH, despite some conflicting reports. Understanding mechanisms modulated by n-3 PUFAs in NASH may be useful for identifying molecular targets that could assist in the design of effective pharmacologic interventions. In this sense, omics sciences are particularly useful for understanding molecular mechanisms with high translational value to clinical practice and may contribute to the identification of these targets. AIM: This study evaluated the liver proteomic and plasma lipidomics responses of patients with NASH towards treatment with n-3 PUFAs. METHODS: The proteomic and lipidomic evaluations were studied by mass spectrometry and / or gas chromatography in samples from liver biopsies and plasma collected from patients enrolled in a preliminary clinical trial of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. This study involved adult patients of both sexes diagnosed with NASH treated daily for 6 months, with 3 capsules containing a mixture of linseed and fish oils [0.315 g PUFAs: 0.065 g eicosapentaenoic acid (EPA) , 0.050 g docosahexaenoic (DHA) and 0.2 g alpha linolenic acid (ALA) per capsule]. Patients, after treatment with n-3 PUFAs, with higher concentrations of ALA and DHA and lower arachidonic acid (AA) showed improvement of liver histology alterations. In the present study we evaluated the proteomics pathways and lipidomics markers resulted from treatment with PUFAs n-3. This was performed by comparing, before (BT group) and after (AT group) treatment, liver tissue pools (analysis interactome) and plasma samples (OPLS-DA). RESULTS: It was identified, in a way exclusive and altered, the expressed liver proteins in AT group, related to pathways of cellular matrix, lipid metabolism, oxidative and endoplasmic reticulum stress and cellular respiration. With the exception of cell matrix, the analysis of the interactome revealed substantial functional alterations of the pathways modulated by these proteins. Together, these changes were suggestive of decreased lipotoxicity, oxidative stress and anaerobic respiration and increased aerobic respiration following treatment with PUFAs n-3. These modifications are potential markers of endoplasmic reticulum and mitochondrial functions improvement. In addition, after treatment with n-3 PUFAs, the lipidomics profile was modified, with significant increase in glycerophospholipids, ALA and EPA and decrease of arachidonic acid (AA) and n-6/n-3 AGPIs ratio. These findings are concordant with potential improvement of reticulum endoplasmic and mitochondrial functions. CONCLUSION: In patients with NASH the treatment with n-3 PUFAs favorably influenced hepatic proteomic and systemic lipidomics profiles. Together, these changes suggest improved endoplasmic reticulum and mitochondrial functions, with potential impact on cellular homeostasis through the modulation of different biological pathways

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