Au cours des deux dernières décennies, la stéatose hépatique non alcoolique (NAFLD) a été une maladie épidémique croissante, non seulement dans les pays occidentaux mais également dans le monde entier en raison de l’augmentation continue des modes de vie sédentaires, de l’obésité, et de la résistance à l’insuline. La prévalence mondiale de la NALFD est actuellement estimée à 25% dans la population générale adulte. NALFD est composé d’un éventail d’affections hépatiques s’étendant du foie gras non-alcoolique (NAFL), stéatohépatite non-alcoolique (NASH), fibrose avancée et cirrhose qui peut progresser au carcinome hépatocellulaire (HCC). L’inflammation induite par NASH peut moduler l’activation des cellules stellaires hépatiques (CSH) et donc influencer la progression de la fibrose hépatique. Le rôle de l’inflammation de type 3, qui est caractérisée par la production des cytokines IL-17A et IL-22, dans la fibrose de type NAFLD demeure incompris. Dans cette thèse, nous avons évalué le rôle d’IL-22 et d’IL-17A dans la fibrose liée à la NAFLD. Des biopsies cliniques de foie NAFLD humain et un modèle murin in vivo de NAFLD ont été utilisés et des expériences in vitro ont été effectuées. Nous avons démontré que l’expression hépatique d’IL-22 est plus élevée chez les femmes et chez les femelles avec NAFLD versus les hommes et les mâles. Nous avons identifié les neutrophiles et les cellules T, y compris les cellules T Th17, Th22 et γδ, en tant que principaux producteurs d’IL-22 chez les sujets féminins et les souris atteintes de NAFLD. De plus, nous avons démontré que l’absence de la signalisation endogène du récepteur IL-22 (modèle IL-22RA1 knockout) chez les souris femelles avec NAFLD, aggravait les lésions hépatiques, l’inflammation et la fibrose, comparé aux mâles. Cet effet hépatoprotecteur dépend des mécanismes anti-apoptotiques médiés par la signalisation du récepteur IL-22 qui favorisent la survie des hépatocytes et réduisent au minimum les dommages au foie. Nous avons également montré que l’expression hépatique d’IL-22BP est régulé à la hausse chez les souris femelles avec NAFLD comparé aux mâles. Dans ces femelles, le ratio d’ARN messager hépatique de l’IL-22 envers celui de l’IL-22BP est corrélé positivement avec les gènes en aval de cible d’IL-22 (gènes anti-apoptotiques et antioxydants). Par ailleurs, nous avons prouvé que les neutrophiles intrahépatiques produisent l’IL-17A in situ dans notre modèle NAFLD et ceci correspondait fortement avec la progression de la fibrose de foie et les dommages hépatiques. Nous avons fourni des preuves préliminaires que l’IL-17A peut induire des pièges extracellulaires de neutrophiles (NET) in vitro, et la signature de NETs est impliquée dans la progression de la fibrose hépatique dans notre NAFLD. Pris ensemble, Ces résultats démontrent qu’identifié un nouveau rôle de l’inflammation de type 3 dans la fibrose liée au NAFLD, où l’action de l’IL-22 est dépendante du sexe et possède des 4 fonctions hépatoprotectrices contre la fibrose du foie chez les femelles, alors que l’IL-17A agit en tant que cytokine profibrogénique et favorise la fibrose de foie. / Non-alcoholic fatty liver disease (NAFLD) is a growing epidemic, not only in western countries but also worldwide due to the continuous rise in sedentary lifestyles, obesity, and insulin resistance over the last two decades. The global prevalence of NALFD is currently estimated to be 25% in the general adult population. NAFLD is comprised of a spectrum of liver disease ranging from non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), advanced fibrosis, and finally cirrhosis that can progress to hepatocellular carcinoma (HCC). NASH-induced inflammation can modulate hepatic stellate cells (HSCs) activation and hence influence hepatic fibrosis progression. The role of type 3 inflammation, which is characterized by the production of the cytokines IL-17A and IL-22, in NAFLD-related fibrosis remain not clear. In this thesis, we evaluated the role of IL-22 and IL-17A in NAFLD-related fibrosis using clinical liver biopsies from a NAFLD human cohort, an in vivo NAFLD mouse model and in vitro experiments. We report that hepatic IL-22 expression had sexually dimorphic differences in both humans and mice with NAFLD where it was elevated in females versus males. We identified intrahepatic neutrophils in female subjects with NAFLD as well as T cells, including Th17, Th22, γδ T cells, in female mice with NAFLD as major producers of IL-22. In addition, we demonstrated that lack of endogenous IL-22 receptor signaling (IL-22RA1 knockout model), exacerbated liver injury, inflammation, and fibrosis in female but not male mice with NAFLD. This hepatoprotective effect was dependent on IL-22 receptor signaling-induced anti-apoptotic signals that promote hepatocyte survival and minimize liver damage. We also demonstrated that hepatic IL-22BP expression was upregulated in female mice with NAFLD compared to males, and the hepatic IL22/IL-22BP mRNA ratio positively correlated with IL-22 downstream target genes (anti-apoptotic and antioxidant genes) in those females. Moreover, we showed that intrahepatic neutrophils produce IL-17A in situ in our NAFLD model and this was strongly correlated with progression of liver fibrosis and liver injury. We provided preliminary evidence that IL-17A can induce neutrophil extracellular traps (NETs) in vitro, and that NETs are implicated in liver fibrosis progression in our NAFLD model. Taken together, we identified a novel role for type 3 inflammation in NAFLD-related fibrosis, where IL-22 act in sex-dependent manner and provided hepatoprotective functions against liver fibrosis in females, while IL-17A act as profibrogenic cytokine and promotes liver fibrosis through enhancing NETs.

<p>  </p> <p><strong>ABSTRACT</strong></p> <p>The worldwide obesity prevalence has almost tripled since 1973 according to the World Health Organization. In the United States, the disease is especially prevalent, with a recorded prevalence of 41.9 percent in 2017, as reported by the National Health and Nutrition Examination Survey. Obesity is associated with an increased risk of heart disease, type 2 diabetes, stroke, non-alcoholic fatty liver disease (NAFLD), and certain types of cancers, including prostate cancer (PCa). While obesity is preventable and reversible, it is a relapsing disease that requires long-term intervention. Furthermore, accumulating evidence shows obesity is not simply a matter of lack of willpower but the re-wired and altered biology that may need medical treatment. Therefore, researchers have been searching for effective approaches to treat obesity and obesity-related diseases. To this end, my research focuses on exploring the role of the sterol O-acyltransferase (SOAT) enzyme and how the inhibition of the enzyme benefits the treatment of obesity and PCa. In addition, I also studied the molecular signatures of NAFLD, with a special focus on altered lipid metabolism using proteomics and determined the protein oligomerization profiles. The major lines of research are summarized in the following and discussed in greater detail in chapters 2 to 5. </p> <p>SOAT enzyme catalyzes the conversion of free cholesterol into its storage form, cholesteryl ester. Our group previously showed that increased SOAT1 expression is associated with increased adipogenesis <em>in vitro</em> and increased adiposity in adipose tissue. When SOAT1 activity was blocked using the pharmacological inhibitor avasimibe, lipid droplet formation and expansion during adipogenesis were suppressed. We further showed that non-orally administered avasimibe led to significant fat mass loss in diet-induced obese (DIO) mice with concomitant food intake suppression and decreased expression of lipogenic genes in adipose tissue. Based on the promising use of avasimibe as an anti-obesity medication, I sought to answer whether avasimibe can enhance the weight loss effect of glucagon-like peptide-1 receptor agonist (GLP-1RA) by accelerating fat mass loss (chapter 2). We found subcutaneous administration of avasimibe can significantly potentiate the weight-reducing effect of the GLP-1RA in DIO mice.</p> <p>Inspired by the lipid droplet modulatory role of the SOAT1 enzyme, I also expanded my dissertation project to cancer (chapter 3). I found that low SOAT1 expression is associated with favorable patient outcomes among PCa patients who had previously undergone anti-hormone therapy. Since the current treatment option, anti-androgen drug enzalutamide, induces mechanisms of resistance in a short period, I hypothesized that blockage of SOAT1 activity using avasimibe would enhance the enzalutamide action and help overcome the resistance. To test this hypothesis, I characterized lipidomic signatures of PCa cells in response to enzalutamide and avasimibe treatments. Then, I tested the anti-cancer effect of the combined treatment in cell cultures and in xenograft tumors in nude mice. I found the combined treatment was significantly more effective in inhibiting cancer cell proliferation and tumor growth than each drug treatment alone. These findings provide insights into molecular signatures associated with enzalutamide treatment outcomes and can serve as a prelude to developing a therapeutic regimen targeting cholesterol metabolism. </p> <p>Among the comorbidities of obesity, NAFLD is very common in obese adults and the prevalence is close to 50–90%. We launched the third project (chapter 4) where we compared the liver proteome from lean mice and DIO mice. To date, most of the omics studies on DIO have been monolithic and very few have explored the multi-omic aspects of fatty liver tissue. To address this gap, we integrated global proteomics, phosphoproteomics and lipidomics to determine molecular signatures of the fatty liver. We identified a range of biological pathways that were altered, and we showed how the alterations in lipid content and amount were correlated with the alterations in the liver proteome and phosphoproteome. The results shed light on the interrelated nature of these biological processes. This was hypothesis-generating study that provided extensive data that could guide future investigations. </p> <p>We followed up on the third project and employed extensive measures to determine the protein oligomerization profiles of the fatty liver (chapter 5). Understanding the modes of protein oligomerization is important since proteins typically exert their biological functions by interacting with other proteins to form protein complexes. We used size exclusion chromatography (SEC) to fractionate liver proteins into 32 fractions based on their size and conducted label-free quantitation of each fraction using mass spectrometry. We successfully obtained elution profiles of individual proteins for subsequent comparison. Our approach enabled the identification of 1172 proteins found common in four liver samples (two lean livers and two fatty livers) for correlation profiling. We discovered that protein elution profiles were highly conserved in the fatty livers despite the metabolic disease state. At the same time, we identified several proteins with different elution profiles between the lean and the fatty livers, which could potentially mediate the hepatic dysfunctions displayed in NAFLD. This study delivers novel pieces of information about protein complex formation in fatty livers.</p> <p>The four research projects included in this dissertation explored obesity and obesity related diseases. Cholesterol accumulation is manifested as lipid metabolism is altered during the progression of obesity in adipose tissue and in PCa. Pharmacological inhibition of SOAT is responsible for cholesterol accumulation was effective in weight management in DIO and demonstrated anti-cancer effect in PCa models together with enzalutamide. These findings suggest that SOAT could be a therapeutic target for diseases featuring cholesteryl ester accumulation. Subsequent projects explored the liver proteome and revealed. In the subsequent projects, liver proteome showed a clear distinction between lean mice and obese mice. The identified proteins in these studies could facilitate the development of targeted therapies for treating NAFLD.</p>

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Introduktion: Icke-alkoholorsakad fettleversjukdom (NAFLD) kännetecknas av hepatisk fettinlagring i frånvaro av alkoholmissbruk. För en andel av patienterna leder sjukdomen till leverinflammation och fibros och benämns då icke-alkoholorsakad steatohepatit (NASH). Denna variant av sjukdomen kan utvecklas till levercirros eller levercancer. Farmakoterapi vid NAFLD är ett forskningsområde som utvecklas snabbt och som domineras av agonister på farnesoid X receptorer (FXR) och peroxisomproliferatoraktiverade receptorer (PPAR). Dessa receptorer är inblandade i lipid- och glukosmetabolism och modulerar även inflammatorisk signalering. Syfte: Detta arbete syftar till att undersöka om FXR-agonister och PPAR-agonister har terapeutisk potential vid NAFLD/NASH. Metod: Litteratursökningen utfördes på databasen PubMed. Totalt granskades tre studier på FXR-agonisten obetikolsyra och tre studier på PPAR-agonisterna saroglitazar, elafibranor och lanifibranor. Resultat: Behandling med 25 mg obetikolsyra ledde till minskad sjukdomsaktivitet hos en större andel patienter jämfört med placebo (45 % vs 21%; p &lt; 0,0002 i FLINT och 36% vs 24%; p = 0,0012 i REGENERATE) och även till minskad leverfibros hos en större andel patienter jämfört med placebo (35% vs 19%; p = 0,004 i FLINT och 23% vs 12% p = 0,0012 i REGENERATE). Behandling med obetikolsyra ledde till ökat LDL-C och minskat HDL-C. Behandling med PPAR-α/γ agonisten saroglitazar gav en signifikant sänkning av leverenzymvärden (p &lt; 0,001) jämfört med placebo. PPAR-α/δ agonisten elafibranor orsakade tillbakagång av NASH för en större andel patienter jämfört med placebo enligt en post-hoc analys (19% vs 12%; p = 0,045). Pan-PPAR agonisten lanifibranor minskade sjukdomsaktiviteten i högre utsträckning än placebo (55% vs 33%; p = 0,007) och uppfyllde även sekundära effektmått på tillbakagång av NASH och minskning av leverfibros. Samtliga PPAR-agonister hade positiva effekter på patienternas lipidprofil och insulinkänslighet. Slutsats: Båda klasser av läkemedel ger en kliniskt meningsfull förbättring av leverhistologin vid NASH. PPAR-agonister har dessutom gynnsamma metabola effekter. Följaktligen har både FXR-agonister och PPAR-agonister terapeutisk potential vid NAFLD/NASH.

Using a novel, adipose-liver, two-organ, human-on-a-chip system, the metabolic disease non-alcoholic fatty liver disease was modeled. This model was then used to test the effects of the gut microbiome on NAFLD progression. Two products of the gut microbiome, Trimethylamine-n-oxide and butyrate, were selected as representatives of potentially harmful and potentially beneficial compounds. A dose response, adipocyte and hepatocyte monocultures controls, and HoaC systems were run for 14 days. Through this experimentation, it was found that a dysbiosis of the gut microbiome could be influencing NAFLD progression. Additionally, further development and discovery regarding adipose-liver systems was added to the ongoing conversation of HoaC systems and their usages.

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<p>In obesity, nonalcoholic fatty liver disease (NAFLD) has been associated with the development of hepatic insulin resistance. Acetyl coA carboxylase (ACC), which exists as two separate isoforms (ACC1 and ACC2), is an important metabolic enzyme which controls the production of the metabolic intermediate malonyl coA, and hence, fat metabolism. AMP-activated protein kinase (AMPK) has been shown to inhibit ACC activity by phosphoryating ACC1 at Ser79 and ACC2 at Ser221. The objectives of this were to determine the physiological importance of AMPK phosphorylation of ACC as it relates to the development of NAFLD and insulin resistance.</p> <p>We examined the metabolic phenotype of C57Bl6 mice with a targeted ACC1 Ser79 to Ala and ACC2 Ser221 to Ala double knock-in mutation (ACC DKI), which would inhibit AMPK phosphorylation of ACC and compared them to wild-type (WT) mice. Basic body characteristics, assessment of insulin sensitivity, and assessment of liver steatosis were used.</p> <p>ACC DKI body mass and energy expenditure were not different compared to WT. Liver ACC activity and malonyl coA were higher in ACC DKI mice. The livers of ACC DKI mice displayed greater triacylglycerol accumulation and aggregation of neutrophils. ACC DKI mice were insulin resistant as shown by: higher fasting blood glucose and insulin, glucose and insulin intolerance, liver insulin resistance, and impaired insulin-stimulated glucose disposal rate.</p> <p>In summary, we have shown that the phosphorylation of ACC1 Ser79 and ACC2 Ser221 is critical for maintaining ACC activity and malonyl coA levels in the liver. The dysregulation of this pathway results in liver fat accumulation and the development of insulin resistance. These studies demonstrate that AMPK phosphorylation of ACC is essential for maintaining metabolic homeostasis.</p> / Master of Science (MSc)