UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE MEDICINA TESIS DOCTORAL Análisis de los factores de riesgo para el progreso y la regresión de la enfermedad de hígado graso no alcohólica: aspectos cualitativos de la dieta, alcohol y actividad física Analysis of the risk factors for progression and regression of nonalcoholic-fatty liver disease: qualitative aspects of diet, alcohol and physical activity MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Olga Estévez Vázquez DIRECTORA Yulia Nevzorova © Olga Estévez Vázquez, 2023 Directora: Yulia Nevzorova Facultad de Medicina Universidad Complutense de Madrid Análisis de los factores de riesgo para el progreso y la regresión de la enfermedad de hígado graso no alcohólica: aspectos cualitativos de la dieta, alcohol y actividad física Analysis of the risk factors for progression and regression of nonalcoholic-fatty liver disease: qualitative aspects of diet, alcohol and physical activity TESIS DOCTORAL Olga Estévez Vázquez UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE MEDICINA TESIS DOCTORAL Análisis de los factores de riesgo para el progreso y la regresión de la enfermedad de hígado graso no alcohólica: aspectos cualitativos de la dieta, alcohol y actividad física Analysis of the risk factors for progression and regression of nonalcoholic-fatty liver disease: qualitative aspects of diet, alcohol and physical activity MEMORIA PARA OPTAR AL GRADO DE DOCTORA PRESENTADA POR Olga Estévez Vázquez DIRECTORA Yulia Nevzorova La comprensión es una calle de doble sentido Eleanor Roosevelt Acknowledgements 11 Agradeciminetos Acknowledgements Acknowledgements 12 Acknowledgements 13 En estas páginas, intentaré brevemente resumir los nombres de todas las personas que me han acompañado en esta peripecia. Cabe recordar que, el orden de los factores no altera el valor del producto, y sin entrar en matemáticas, este listado es organizable de 1000 y una formas, al ser difícil ponderar ciertos conceptos intangibles. First, I would like to thank to Dra. Yulia Nevzorova, my PI and thesis director for bringing me the opportunity of joining her lab, supporting me during these years and always trust on me. Letting me learn from your knowledge and allowing me to be part of several publications and congress has helped me to grown as a scientist. Your grant has appeared in my life at a critical point, and when is hard to choose a way in the path, small details make the difference. También me gustaría agradecerse al Dr. Javier Cubero su apoyo durante esta etapa. Tu esfuerzo y trabajo son clave para el crecimiento de nuestro grupo. Gracias por tu cercanía, tus consejos y por ser el jefe con el espíritu más joven. I would also need to thank Prof. Özlen Konü, for her hospitality and kindness. Letting me the opportunity of being part of her nice lab at Bilkent University of Ankara and giving me the opportunity of having my first contact with the amazing zebrafish world. Thanks to Prof. Gülçin Çakan, for taking me as part of her team at IBG (Izmir Biomedicine and Genome Center) and putting that much effort on this project. I really appreciate the invest on time and facilities you have done for helping me. Al departamento de Inmunología de Complutense, al que pertenezco. Por estar siempre disponibles y decididos a ayudar. Porque ante cualquier duda, tienes la tranquilidad de que en la cuarta planta encontrarás la respuesta. Sobre todo, tengo que agradecerles su apoyo a mis compis del lab: A Carlos, por tu cercanía, por habernos ayudado a crecer como grupo y por tener tan buen ojo para formar equipo. Esperemos que los jueves siempre sigan siendo de memes, que la gente no deshonre la paella y que nadie se vuelva a disfrazar de fallera. A Arantza, por ser mi guía de ruta, por nuestras vidas paralelas. Por haber sido compañera de carrera, piso y trabajo. Pocas cosas más importantes se me ocurre que se puedan compartir, sin ti no hubiese llegado hasta aquí, en el más literal de los sentidos. Por sentar los cimientos y trabajar los inicios de este lugar. Gracias por estar Acknowledgements 14 ahí en los buenos y malos ratos y a ver si dejas de hacer documentos. #check #Boston #oPorriño #lab #losabia A Raquel, por su paciencia y capacidad de análisis. Por ser mi mentora, la que acumula más trienios de sabiduría y por saber poner luz en el camino cuando más hace falta. Por poder siempre contar con ella… menos para tomar algo o tareas extracurriculares, gracias por ser la compañera más fiel. A Marina, por poner orden en lo terrenal y lo mental. Por ser la segoviana más maja de Castilla y de Tetuán. Por ser mi guía en la cinematografía, y lograr que vea de un tirón Titanic, sin casi ni ir al baño. Por tu apoyo incondicional y porque sigas siendo siempre tan lindi, llevando el color por donde vayas. Gracias también a la ya postdoc Laura, cuya risa inconfundible ya puede ser reconocida por los montes suizos. Siempre disponible para ofrecer su ayuda física e intelectual, ha sido un verdadero placer compartir poyata contigo. Que el futuro te depare grandes logros, mucha suerte en tu camino. A Álex, por ser mi mejor compañero de mesa, el que nunca pone límites a mis papeles y puede trabajar en un campo lleno de minas. Por ser mi técnico de informática y por no juzgar. Sigue siendo siempre un bebé así de bueno, pero déjame ganar alguna vez al 3 en raya. #tucu #hipsdon’tlie Thanks to Hang Hang for your contagious joy and positivity. For teaching us that there is always a gap for taking a rest, just the intention is needed. For being so independent and brave, we have a lot to learn from you. A Héctor, el último en incorporarse a filas. Espero que el camino esté ahora un poco más llano que con las primeras piedras de los cimientos, un placer pasarte el relevo y que la suerte te acompañe. Thanks to the “Chinese crew”, Marker, Hui, Feifei and Kang. You were a guide for me at my first months in the lab, always available for lending me a hand and key part of the foundation of this lab. It has been a pleasure to learn from all of you a bit more about your culture and other ways of working, for making this place more international. I hope we can meet each other again at some point. Gracias a Nuria, compañera en los inicios de esta etapa y a todos los que se han paseado por estas estancias. A Sebas por ser el más glotón o a Jose por cuidar los ratones en verano. Acknowledgements 15 I really need to thank to Bilkent lab members, specially to Güneş and Ruya, for being my teachers in a totally new field for me and helping me in every single detail. You are an amazing team!. Specially thanks to Güneş for being more than a labmate, for taking care of me also after the working hours and giving me the opportunity of joining her gang (Mohammad, Umut, Pedram, Eylül, Melis, Selin…). I really fill lucky of have met such a great group. Thanks also to IBG people, especially Merve and Ebru for making me feel like at home from the very first day, being the best guide tours of Izmir. Thanks to Emine for being my best working hands, taking time for me and for continue working on the project together with Kalid. To Esra for letting me take part of such a special day and to Metan for his kindness and eternal smile. Really waiting for meeting all of you again somewhere around the world! Por supuesto también tengo que dar gracias a mi familia. A mi madre y a mi padre por haberme apoyado en mis decisiones, por ayudarme a crecer, avanzar y progresar. Por estar siempre al otro lado del teléfono, o del andén de Vigo Urzáiz, según se tercie, y aconsejar con la experiencia de los años a la espalda de la forma más certera. A mi abuela Maruja y a mi abuelo José, por haberme dado tanto amor, cuidarme con tanta ternura y por haber tenido siempre confianza en mí. Porque sé que estaríais orgullosos de verme hoy. A mi tía Olga, a Luis y la prima Mari Carmen, por vuestra sabiduría, generosidad y apoyo incondicional Gracias a mis gallegos y sobre todo a mis gallegas. A las gallegas de Vigo: las Julitas. Lore, Isa, Prin, Vero y Fari. Porque sois mi alegría cada vez que vuelvo por casa, por hacerme sentir que los años no pasan y ser mi punto de referencia. Gracias a Lore, por ser mi amiga más longeva, por haber crecido conmigo. A Ta, por sus locuras y sus historias surrealistas. A l@s galleg@s de Madrid (por genética y por contagio). A Samu, Araña (again), Nenita, Ana, María, Dios, Rebesa, Irene, Laura y Luci. A las madrileñas que van para gallegas Sasha y Paloma. Por sacarme los findes de paseo, llevarme a tomar café con cosas para mojar, comida vegana e inviegna o unos yodas y lo que surja. Por alegrarme la vida en general. Espero que sigamos aumentando la check list #6ºC #lanovia #goloshina #jaroliso Acknowledgements 16 A Samu, por ser o meu comodín mais socorrido, a persona mais nomeada nas miñas conversacións. Por esa capacidade que tes de sacarlle importancia as cousas, e ver sempre o lado positivo, facendo todo sinxelo. Por ser tan manitas, alimentarme con receitas exóticas, e sobre todo por ser tan uaba. A Carlos V (Nenita), por ser mi compi de piso más versátil. Aunque ahora ya eres mayor e independiente, echo de menos esas noches de Wyoming y First dates. A mis biólogas Inés, Carlota y Andrea, porque la distancia no consiga separarnos. Me inspirasteis emprender este camino y por vuestra valentía al empezar el vuestro. A Sol, por su pasión por la ciencia y por la vida. Espero veros pronto. Y para cerrar filas, gracias a la música, al deporte, al cine y al teatro y a las calles de Madrid, por hacerme sentir. Gracias, el espectáculo debe continuar. “The show must go on- Queen” Table of contents Table of Contents 1. Resumen ...............................................................................................................23 2. Summary ...............................................................................................................29 3. Abbreviations .......................................................................................................35 4. Introduction ..........................................................................................................43 4.1. Anatomy of the liver ........................................................................................43 4.1.1. The lobule: functional unit of the liver ......................................................44 4.1.2. Blood and bile circulation in the liver .......................................................44 4.1.3. Liver zonation ..........................................................................................45 4.1.4. Biliary tree................................................................................................46 4.2. Functions ........................................................................................................47 4.3. Histology of the liver........................................................................................48 4.3.1. Parenchymal cells....................................................................................48 4.3.1.1. Hepatocytes......................................................................................48 4.3.1.2. Cholangiocytes or biliary cells ..........................................................49 4.3.2. Non-parenchymal cells ............................................................................49 4.3.2.1. The sinusoidal endothelial cells (LSECs) .........................................49 4.3.2.2. Kupffer cells (KCs)............................................................................50 4.3.2.3. Hepatic stellate cells (HSCs) ..........................................................51 4.3.2.4. Hepatic immune cells .......................................................................51 4.4. Space of Disse* ............................................................................................52 4.5. Liver related diseases .....................................................................................53 4.5.1. Chronic liver disease (CLD).....................................................................53 4.5.2. Non-alcoholic fatty liver disease- metabolic associated fatty liver disease / non-alcoholic steatohepatitis (NAFLD- MAFLD/NASH) .........................................53 4.5.2.1. Epidemiology of NAFLD ...................................................................54 4.5.2.2. Pathophysiology of NAFLD ..............................................................55 4.5.2.3. Risk factors.......................................................................................57 A. Diet ..........................................................................................................57 B. Sedentary lifestyle ...................................................................................59 C. Alcohol- consumption...........................................................................61 D. Gut dysbiosis........................................................................................63 4.5.3. Strategies for NAFLD prevention/ reversion ...........................................64 4.5.3.1. Lifestyle modification ........................................................................64 4.5.3.2. Diet ...................................................................................................64 4.5.3.3. Physical activity ................................................................................65 4.5.3.4. Pharmacotherapy and clinical interventions .....................................67 4.5.3.5. Prebiotics, probiotics and synbiotics.................................................67 4.5.4. Animal models for NAFLD study..............................................................68 4.5.4.1. Mice and rats ....................................................................................68 4.5.4.2. Zebrafish...........................................................................................70 4.5.4.3. Others...............................................................................................72 5. Open questions ....................................................................................................75 6. Objectives .............................................................................................................79 7. Materials and methods ........................................................................................83 7.1. Materials .........................................................................................................83 7.1.1. Chemicals ................................................................................................83 7.1.2 Standard kits and enzymes .....................................................................85 7.1.3. Standard buffers and media ....................................................................86 Table of contents 18 7.1.4. Immunoblotting gels ................................................................................. 89 7.1.5. Immunostaining and immunoblotting antibodies ...................................... 90 7.1.5.1. Primary antibodies ............................................................................... 90 7.1.5.2. Secondary antibodies ....................................................................... 92 7.1.6. Primer sequences used for qRT-PCR ..................................................... 92 7.1.7. Diets ....................................................................................................... 93 7.1.7.1. Mice .................................................................................................. 93 7.1.7.2. Zebrafish ........................................................................................... 94 7.1.8. Composition diets .................................................................................... 94 7.1.8.1. Mice ...................................................................................................... 94 7.1.8.2. Zebrafish .............................................................................................. 95 7.1.9. Instrument and equipment ....................................................................... 95 7.1.10. Software ............................................................................................... 97 7.1.11. General materials ................................................................................. 98 7.2. Methods .......................................................................................................... 99 7.2.1. Animal maintenance ................................................................................ 99 7.2.1.1. Mice .................................................................................................. 99 7.2.1.2. Zebrafish ......................................................................................... 100 7.2.2. Animal strains ........................................................................................ 100 7.2.2.1. Mice ................................................................................................ 100 7.2.2.2. Zebrafish ......................................................................................... 100 7.2.3. Development of preclinical models ........................................................ 100 7.2.3.1. Mice ................................................................................................ 100 7.2.3.1.1. Western diet (WD) ...................................................................... 100 7.2.3.1.2. Western diet withdrawal ............................................................. 101 7.2.3.1.3. DUAL diet: DUAL withdrawal and combination with physical exercise 102 7.2.3.1.4. Physical exercise ........................................................................ 103 7.2.3.2. Zebrafish ......................................................................................... 104 7.2.3.2.1. DUAL model in larval zebrafish .................................................. 104 7.2.3.2.2. DUAL model in adult zebrafish ................................................... 106 7.2.4. Basal glucose and glucose tolerance test ............................................. 107 7.2.4.1. Mice ................................................................................................ 107 7.2.4.2. Zebrafish ......................................................................................... 108 7.2.5. Food/water intake and body weight measurement ................................ 108 7.2.6. Animal dissection ................................................................................... 109 7.2.6.1. Mice ................................................................................................ 109 7.2.6.2. Zebrafish ......................................................................................... 110 7.2.7. Hepatic triglycerides (TG) content- mice ............................................... 110 7.2.8. Histological analysis .............................................................................. 111 7.2.8.1. Tissue processing and embedding- mice, zebrafish ...................... 111 7.2.8.2. Hematoxylin and eosin (H&E) staining- mice, zebrafish ................ 112 7.2.8.3. Sirius red (SR) staining- mice ......................................................... 112 7.2.8.4. Oil Red O (ORO) staining- mice ..................................................... 112 7.2.8.5. Immunohistochemistry (IHC) staining- mice ................................... 113 7.2.8.6. Immunofluorescence (IF) staining- mice ........................................ 114 7.2.8.7. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining- mice .................................................................................... 114 7.2.8.8. Nile red in vivo staining- larvae zebrafish ....................................... 115 7.2.8.9. BODIPY in vivo staining- larvae zebrafish ...................................... 115 7.2.8.10. Imaging and staining analysis ........................................................ 115 7.2.9. RNA isolation and analysis .................................................................... 116 7.2.9.1. RNA isolation .................................................................................. 116 7.2.9.2. Reverse transcription from RNA to complementary DNA (cDNA) .. 116 19 Table of contents 7.2.9.3. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) ..117 7.2.10. Protein isolation and analysis ............................................................119 7.2.10.1. Protein extraction and quantification ..............................................119 7.2.10.2. Immunoblotting assay (WB) ...........................................................119 7.2.11. Lipidomic analysis: lipid extraction and quantification........................120 7.2.12. Magnetic resonance imaging (MRI) ...................................................121 7.2.13. Stool analysis .....................................................................................122 7.2.14. Statistical analysis..............................................................................122 8. Results ................................................................................................................127 8.1. Western diet (WD) is associated with the development of obesity and metabolic syndrome (MS)........................................................................................127 8.2. WD consumption triggered hepatomegaly....................................................130 8.3. WD feeding induced lipidome alterations in murine livers ............................133 8.4. WD altered the balance between fat storage and oxidation in the liver ........135 8.5. WD with high levels of PA and trans fat increased the risk of NAFLD- associated hepatitis and fibrosis..............................................................................136 8.6. The WD withdrawal, reversed NAFLD in all treated groups independently of the diet composition.................................................................................................141 8.7. WTD, specially in combination with exercise (WTD+EXER) reversed MS caused by short-term DUAL diet feeding.................................................................145 8.8. WTD and WTD+EXER reduced hepatomegaly and hepatic steatosis caused by short-term DUAL feeding ....................................................................................148 8.9. Cell death induced by DUAL diet, was ameliorated after WTD and WTD+EXER.............................................................................................................150 8.10. Short-term DUAL diet feeding lead to hepatic inflammation, reversed by WTD and WTD+EXER ............................................................................................152 8.11. Pathological changes in the intestine caused by DUAL diet, were reverted by WTD and WTD+EXER........................................................................................154 8.12. Short-term DUAL diet feeding leads to bacterial dysbiosis, that can be reverted by WTD and WTD+EXER according to 16S rRNA sequencing analysis ..157 8.13. Only the combination of WTD +EXER, was able to ameliorate MS caused by DUAL long- term feeding ....................................................................................159 8.14. WTD and WTD+EXER strategies, were able to reverse hepatomegaly and hepatic steatosis after long-term DUAL feeding ......................................................162 8.15. WTD and WTD+EXER could partially reduce lipid metabolism abnormalities caused by DUAL diet ...............................................................................................164 8.16. Cell death and compensatory proliferation caused by DUAL diet, was ameliorated by WTD and especially by the combination of WTD+EXER................165 8.17. WTD+EXER was able to reduce hepatic inflammation triggered by long- term DUAL feeding ..................................................................................................167 8.18. WTD+EXER was able to decrease hepatic stellate cell activation and fibrogenesis .............................................................................................................170 8.19. WTD and WTD+EXER reverted the intestinal changes caused by DUAL diet feeding 171 8.20. Influence of DUAL diet in morphology of zebrafish larvae ........................172 8.21. Nile red staining revealed no significant changes in TG accumulation after DUAL feeding in 10 dpf AB larvae ...........................................................................173 8.22. DUAL diet feeding has triggered hepatic lipid accumulation in 10 dpf FABP zebrafish larvae .......................................................................................................174 8.23. DUAL diet feeding triggers hepatic steatosis in adult AB zebrafish ..........175 9. Discussion ..........................................................................................................179 10. Conclusions........................................................................................................199 20 Table of contents 11. References ..........................................................................................................203 12. Appendix .............................................................................................................218 12.1. Publications ...............................................................................................218 12.2. Participation in conferences ......................................................................219 12.3. Membership of Scientific Societies............................................................225 12.4. Most Outstanding Awards .........................................................................226 12.5. Others........................................................................................................226 Resumen RESUMEN Resumen 22 Resumen 23 1. Resumen Introducción y objetivos La enfermedad del hígado graso no alcohólico (NAFLD, por sus siglas en inglés), que va desde el hígado graso simple, pasando por la esteatohepatitis no alcohólica (NASH, por sus siglas en inglés) y la fibrosis hasta la cirrosis, tiene una elevada prevalencia y está presente en más del 20 % de la población. Esta elevada prevalencia de NAFLD probablemente se deba a las epidemias contemporáneas de obesidad, patrones dietéticos poco saludables y estilo de vida sedentario. La pieza clave de cualquier régimen de tratamiento para pacientes con NAFLD es la modificación del estilo de vida centrada en los cambios en la dieta y actividad física. El objetivo principal de nuestro estudio fue describir el efecto de la dieta y el estilo de vida en el desarrollo de NAFLD. En primer lugar, nuestro objetivo es probar si los aspectos cualitativos de la dieta, como los niveles de ácido palmítico (AP) y la fuente de grasa, son factores de riesgo para NAFLD. En segundo lugar, utilizamos un modelo murino DUAL, sinérgico de NASH y consumo de alcohol, y estudiamos el impacto biológico del ejercicio físico y los cambios en la dieta sobre la regresión de la esteatohepatitis y la fibrosis. Finalmente, confirmamos nuestro hallazgo en un modelo animal alternativo: el pez cebra (Danio rerio). Métodos En la primera parte de nuestro estudio, alimentamos a ratones macho C57BL/6 con tres tipos de dieta occidental (WD) con diferente contenido de PA y origen de grasa durante 14 semanas: 1. LP-WD: baja concentración de PA (fuente principal de grasa) aceite de maíz y soja); 2. HP-WD: alta concentración de PA (principal fuente de grasa: aceite de palma); 3. HP-Trans-WD: alta concentración de PA (principalmente grasas trans). Como control se usó una dieta de comida normal (CD). Como segundo enfoque, aplicamos el modelo DUAL (10 % de alcohol en agua potable azucarada junto con una WD) a C57BL/6J durante un período corto (10 semanas) y largo (23 semanas), seguido por el reemplazo con dieta para comer y agua normal (WTD), o por WTD en combinación con sesiones de cinta durante 20 días (WTD+EXER). Síndrome metabólico (SM), parámetros séricos, hígado, tejido adiposo blanco epididimal (eWAT) e histología del intestino, se analizaron en detalle. Resumen 24 Finalmente, para obtener resultados confirmados en el modelo DUAL murino, se realizó el estudio de prueba en el pez cebra. La alimentación DUAL se aplicó desde los 6 hasta los 9 dpf a larvas y peces adultos de 1,5 días de edad durante 20 días. Se realizaron imágenes in vivo y análisis de muestras. Resultados 1. La alimentación con los tres tipos de WD provocó un aumento de peso significativo, hipertrofia de los adipocitos, hepatomegalia, alteraciones del metabolismo de los lípidos y esteatohepatitis. Los grupos alimentados con HP demostraron obesidad más prominente, hipercolesterolemia, daño hepático más fuerte y fibrosis. Solo la alimentación con HP-Trans-WD resultó en intolerancia a la glucosa y elevación de las transaminasas séricas. El retiro breve de la dieta mejoró totalmente las alteraciones metabólicas y los síntomas correspondientes de NAFLD en todos los animales tratados. Sin embargo, la inflamación hepática leve aún era detectable en los grupos de abstinencia de HP en relación con los controles. 2. En comparación con los ratones alimentados DUAL durante 10 semanas, los grupos WTD y WTD+EXER mostraron una disminución significativa en la masa corporal, el peso del hígado y la grasa, una mejora notable en la morfología del hígado y la grasa, esteatosis hepática atenuada con un contenido mejorado de triglicéridos hepáticos (TG), disminución del daño hepático, inflamación, permeabilidad intestinal y esteatosis. Sin embargo, después de la alimentación DUAL a largo plazo, el reemplazo de la dieta sin ejercicio físico resultó solo en una mejora significativa de los parámetros séricos de daño hepático y colesterol sérico, una ligera disminución del peso del hígado y una ligera disminución del contenido de TG en el hígado. Es importante destacar que la obesidad, la hipertrofia celular e-WAT, la hepatomegalia, los marcadores centrales de la inflamación hepática (niveles de CD45 y F4/80), así como la activación de las células estrelladas y la permeabilidad intestinal fueron reversibles solo en el grupo de retiro de la dieta en combinación. con ejercicios físicos después de la alimentación a largo plazo. 3. La exposición a corto plazo de la dieta DUAL a larvas de pez cebra indujo cambios morfológicos significativos y acumulación de grasa en el hígado. El pez cebra adulto expuesto a la dieta DUAL desarrolló un aumento de peso y una notable acumulación de grasa en el hígado. Conclusiones Resumen 25 La modificación de la dieta, sola o en combinación con ejercicio físico, en pacientes con esteatohepatitis en estadios iniciales podría considerarse como una terapia no farmacológica eficaz, reduciendo el peso corporal, la hepatomegalia, la esteatosis y la inflamación hepática. Sin embargo, solo una combinación de cambios en la dieta y actividad física puede conducir a la mejoría clínica en las etapas avanzadas de la esteatohepatitis. Además, las recomendaciones dietéticas para prevenir y controlar la NAFLD deben centrarse no solo en la cantidad sino también en la calidad de la dieta. Resumen 26 Summary SUMMARY Summary 28 Summary 29 2. Summary Introduction and objectives Non-alcoholic fatty liver disease (NAFLD), which ranges from simple fatty liver, through non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis, is present in more than 20% of the general population. The high prevalence of NAFLD is probably due to the contemporary epidemics of obesity, unhealthy dietary pattern, and sedentary lifestyle. The cornerstone of any treatment regimen for patients with NAFLD is lifestyle modification focused on dietary changes and physical activity. The main objective of our study was to outline the effect of diet and lifestyle factors on the development of NAFLD. First, we aim to test whether qualitative aspects of diet such as levels of Palmitic acid (PA) and the fat source, are risk factors for NAFLD. Second, we used a synergistic DUAL murine model of NAFLD and alcohol consumption to study the biological impact of physical exercise and dietary changes on the regression of steatohepatitis and fibrosis. Finally, we confirmed our finding in an alternative animal model, zebrafish (Danio rerio). Methods In the first part of our study, we feed C57BL/6 male mice with three types of Western diet (WD) with different PA content and fat origin for 14 weeks: 1. LP-WD—low concentration of PA (main fat source—corn and soybean oils); 2. HP-WD—high concentration of PA (main fat source—palm oil); 3. HP-Trans-WD—high concentration of PA (mainly transfat). Normal chow diet (CD) was used as control. As a second approach we applied DUAL model (10% alcohol in sweetened drinking water together with a WD) to C57BL/6J for short (10 weeks) and long (23 weeks) period followed by either the replacement with chow diet and normal water (WTD), or by WTD in combination with treadmill sessions for 20 days (WTD+EXER). Metabolic syndrome (MS), serum parameters, liver, epididymal white adipose tissue (eWAT) and intestine histology, were analysed in detail. Finally, in order to confirm the results, obtained in murine DUAL model the trial study in zebrafish was performed. The DUAL feeding was applied from 6dpf up to 9 dpf to larvae and 1.5-day old adult fish for 20 days. Live imaging and analysis of samples was performed. Summary 30 Results 1. Feeding with all three types of WD caused significant weight gain, adipocytes enlargement, hepatomegaly, lipid metabolism alterations, steatohepatitis. Feeding with HP groups demonstrated more prominent obesity, hypercholesterolemia, stronger hepatic injury, fibrosis. Only the feeding with HP- Trans-WD resulted in glucose intolerance and elevation of serum transaminases. Brief diet withdrawal totally ameliorated metabolic alterations and corresponding symptoms of NAFLD in all treated animals. However, mild hepatic inflammation was still detectable in HP withdrawal groups relative to the controls. 2. Compared to the 10 weeks DUAL-fed mice, WTD and WTD+EXER groups showed significant decrease in body mass, liver and fat weight, remarkable improvement in liver and fat morphology, attenuated hepatic steatosis with improved hepatic triglyceride (TG) content, diminished hepatic injury, inflammation, intestinal permeability and steatosis. Nevertheless, after the long- term DUAL feeding the diet replacement without physical exercise resulted only in the significant improvement of serum parameters of liver damage and serum cholesterol, while caused slight decrease of liver TG content and liver weight. Importantly, obesity, eWAT cellular hypertrophy, hepatomegaly, the central markers of the hepatic inflammation (CD45 and F4/80 levels), as well as activation of stellate cells and gut permeability were reversible only in the group of the diet withdrawal in combination with physical exercises after long-term feeding. 3. Short-term exposure of DUAL diet to zebrafish larvae induced significant morphological changes and accumulation of fat in liver. Adult zebrafish exposed to DUAL diet, developed an increased body weight and remarkable fat accumulation in the liver. Conclusions Dietary modification, alone or combination with physical exercise in patients with the initial stages of steatohepatitis might be considered as an efficient non-pharmacological therapy, reducing body weight, hepatomegaly, steatosis, and liver inflammation. However, only a combination of dietary changes and physical activity can lead to the clinical improvement at the advance stages of steatohepatitis. Moreover, the dietary Summary 31 recommendations to prevent and manage NAFLD should focus not only on quantity but also quality of the diet. Summary 32 Abbreviations ABBREVIATIONS Abbreviations 34 Abbreviations 35 3. Abbreviations ACC Acetyl-CoA Carboxylase ACOX Acyl- Coenzyme A Oxidase ACSM American College of Sports Medicine ADH Alcohol dehydrogenase ALD Alcoholic liver disease ALDH Aldehyde dehydrogenase ALIOS American Lifestyle-Induced Obesity Syndrome ALT Alanine aminotransferase ANOVA Analysis of variance AP Alkaline phosphatase APS Ammonium persulfate α-SMA Alpha-smooth muscle actin ASH Alcoholic steatohepatitis AST Aspartate aminotransferase AUC Area under the curve BD Bile duct BMI Body mass index BSA Bovine serum albumin BSEP Bile salt export pump BW Body weight CAT Catalase CD36 Cluster of differentiation 36 CD45 Cluster of differentiation 45 cDNA Complementary DNA CE Cholesteryl ester ChREBP Carbohydrate response element binding protein CLD Chronic liver disease CLS Crown-like structures Abbreviations 36 CPT-1c Carnitine Palmitoyltransferase–1c CT Threshold cycle CTRL Control CV Cava vein Cyc Cyclin CYP2E1 Cytochrome P450 2E1 dH2O Distilled H2O DAMP Damage-associated molecular pattern DG Diglycerides DGAT Diacylglycerol Acyl Transferase DHH Department of Health and Human Services DMSO Dimethyl sulfoxide DNL De novo lipogenesis dpf Days post fertilization DTT Dithiothreitol E3 Embryo media EASL European Association for the Study of the Liver ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid EHGNA Enfermedad del hígado graso no alcohólico ER Endoplasmic reticulum EtOH Ethanol eWAT Epididymal white adipose tissue FA Fatty acid FASN Fatty Acid Synthase FAO Fatty acid oxidation FC Free cholesterol FELASA Federation for Laboratory Animal Science Associations FFA Free fatty acid FXR Farnesoid X receptor Abbreviations 37 GAPDH Glyceraldehyde-3-phosphate dehydrogenase GLP Glucagon-like peptide-1 GS Glutamine synthetase GTT Glucose test tolerance H2AX Histone 2AX HA Hepatic artery HCC Hepatocellular carcinoma HDL High-Density Lipoprotein HIT High intensity training HMGB1 High-mobility group box-1 protein HSCs Hepatic stellate cells H&E Hematoxylin and eosin HPC Hepatic progenitor cell HP-Trans-WD Western diet with high concentration of PA, mainly Trans-fat HP-WD Western Diet with High concentration of PA IF Immunofluorescence IFN- γ Interferon-gamma IHC Immunohistochemistry IHL intrahepatic lipid content IL-6 Interleukin 6 IOD Integrated optic density IP Intraperitoneal IR Insulin resistance KCs Kupffer cells LDH Lactate dehydrogenase LDL Low-Density Lipoprotein LPS Lipopolysaccharide LP-WD Western Diet with Low concentration of PA LSECs Liver sinusoidal endothelial cells LW Liver weight Abbreviations 38 MAFLD Metabolic associated fatty liver disease MCP-1 Monocyte chemoattractant protein 1 MD Mediterranean diet MET Metabolic equivalent MetS Metabolic syndrome MetOH Methanol mRNA Messenger RNA MRI Magnetic resonance imaging MS Metabolic Syndrome MSC Mesenchymal stem cells NAFL Non-alcoholic fatty liver NAFLD Non-alcoholic fatty liver disease NAS NAFLD activity score NASH Non-alcoholic steatohepatitis NK Natural killer NKT Natural killer-T-cells NP40 Nonidet P-40 NPC Non-parenchymal cells NTCP Sodium taurocholate cotransporting polypeptide OD Optical density O/N Overnight ORO Oil red O OS Oxidative stress OUT Operational taxonomic unit PA Palmitic acid P-ACC Phospho-Acetyl-CoA Carboxylase PBS Phosphate buffered saline PCNA Proliferating cell nuclear antigen PC Phosphatidylcholine PCR Polymerase chain reaction Abbreviations 39 PE Phosphatidylethanolamine PEMT Phosphatidylethanolamine N-Methyltransferase PI Phosphatidylinositol PPARa Peroxisome proliferation-active receptor alpha PPARg Peroxisome Proliferator activated receptor gamma PS Phosphatidylserine PV Portal vein PVDF Polyvinylidene Difluoride qRT-PCR Quantitative real-time polymerase chain reaction rcf Relative centrifugal force rRNA Ribosomal ribonucleic acid ROS Reactive oxygen species RT Room temperature SCD1 Stearoyl-CoA desaturase SCFA Short-chain fatty acid SD Standard deviation SDS Sodium dodecyl sulfate SR Sirius red SREBP1c Sterol regulatory element-binding protein-1c T2DM Type 2 diabetes mellitus TBS Tris-buffered saline TG Triglycerides TLC Thin Layer Chromatography TLR Toll-like receptor TEMED Tetramethylethylenediamide TNF-α Tumor necrosis factor alpha TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling VLDL Very low-density lipoprotein WAT White Adipose Tissue WB Western blot Abbreviations 40 WD Western diet WHO World Health Organization Introduction INTRODUCTION Introduction 42 Introduction 4. Introduction 4.1. Anatomy of the liver The liver is the largest organ and gland in vertebrates, representing around 2% of the body weight (1.3 kg- 1.7 kg) in human [1] and approximately 4- 5% of the body weight in mice [2]. It is covered by a visceral peritoneum and a connective tissue layer [3] and located inferior to the diaphragm, occupying most of the right part of the abdominal cavity close to the stomach and gut [3]. The liver is a multilobed organ anatomically divided into four lobes: right, left, caudate, and quadrate, reflecting the distribution of the major branches of the blood vessels [1-3] (Figure 1). Figure 1. Liver anterior- posterior view and hepatic lobule architecture. According to morphological anatomy, the liver is divided into four lobes: left and right when is observed from the anterior view, and caudate and quadrate can be distinguished from the posterior view. The blood circulates from the portal vein into hepatic sinusoids and flows out through the central vein. Created with BioRender.com. Introduction 44 4.1.1. The lobule: functional unit of the liver The hepatic lobule constitutes the structural and functional unit of the liver. It has hexagonal shape and contains a portal triad formed by a portal vein (PV), hepatic artery (HA) and bile duct (BD), in each corner of the lobular hexagon, and the central vein (CV) located in the middle of the lobe [5] (Figure 2). 4.1.2. Blood and bile circulation in the liver Blood enters the hepatic sinusoids via the portal vein and hepatic artery and flows along the cords of hepatocytes to the central vein. Bile circulates in the opposite direction through bile canaliculi before entering the bile duct [1, 4] (Figure 2). Oxygenated blood arriving directly from the aorta via the hepatic artery represents only 25% of the incoming blood supply, while the remaining 75% of blood arrives trough the portal vein. Unlike most veins, the portal vein does not drain into the heart, delivering venous blood from the gastrointestinal tract directly to the liver through the hepatic sinusoids. The portal vein accomplishes two tasks: it supplies the liver with metabolic substrates, and it ensures that ingested substances are first processed and detoxified by the hepatocytes before reaching the systemic circulation [5]. Blood from branches of the HA and PV are mixed in the sinusoids and circulates through them until been collected in a CV, which drains into the hepatic vein. The hepatic vein subsequently drains into the inferior vena cava [4, 6] (Figure 2). While blood moves through the lobe into the central vein, the cells use the oxygen and process nutrients generating waste products. This gradient formed across the lobe sinusoids, leads to different hepatocyte functions based on their location, creating a liver zonation [6]. Introduction 45 Figure 2. Blood stream circulation in the liver sinusoids. Blood arrives to the liver though the PV (deoxygenated and nutrient rich blood from the intestinal tract) and HA (oxygenated blood with low nutrient content). Both oxygenated and deoxygenated blood, are mixed in the sinusoids, while flowing through the CV. Bile is flowing in the opposite direction through bile canaliculi until BD. Blood from the CV will circulate out of the liver through the hepatic vein draining into the inferior vena cava. Created with BioRender.com 4.1.3. Liver zonation As blood flows directionally toward the central vein, hepatocytes take up oxygen, nutrients, and metabolize hormones, shaping their microenvironment by creating a gradient along the periportal-pericentral axis. According to this gradient, three different zones can be distinguished in the liver lobe (Figure 3): A. Periportal: it is the region surrounding the hepatic arteries and portal veins. It is the first area were hepatocytes regenerate after liver damage due to their proximity to oxygenated blood and nutrients. This zone plays a significant role in oxidative metabolisms such as beta-oxidation, gluconeogenesis, bile formation, cholesterol formation and amino acid catabolism. B. Mid- lobular: is a transition zone in between the periportal and pericentral areas, and its hepatocytes are key contributors for liver regeneration. Introduction 46 C. Pericentral: adjacent to the central vein, is the lowest perfused zone due to its distance from the portal triad. It plays a role in multiple functions as detoxification, ketogenesis, glycolysis or lipogenesis [7]. Figure 3. Liver lobule and hepatic zonation. Liver parenchyma is constituted by a mass of cells, which are perfused with blood vessels and bile ducts. The oxygenated blood flows through the hepatic artery, while deoxygenated, nutrient enriched but undetoxified blood arrives by the portal vein, into the hepatic sinusoids and circulates into the central vein. According to this flow procedure, three different zones are described: periportal, mid-lobular and pericentral. Modified from [7]. Created with BioRender.com. 4.1.4. Biliary tree The biliary tree is composed of extrahepatic and intrahepatic bile ducts, lined by mature epithelial cells called cholangiocytes, that contains peribiliary glands deep within the duct walls [8]. The cholangiocytes are responsible of modifying the bile, that is transported along the biliary tree. Bile is the excretion product of the hepatocytes, that is collected and transported by the bile canaliculi, which are formed by the apical sides of two adjacent hepatocytes in the hepatic plate [2]. It is an aqueous secretion, constitute of 95% of water, in which several Introduction 47 endogenous solids are dissolved, including bile salts, bilirubin cholesterol or vitamins, as well as exogenous drugs, xenobiotics and toxins originated from hepatocytes. Bile fluid is necessary for the appropriate absorption of lipids from the digestion [3], and protects the organism from infections by excreting immunoglobulin A (IgA), inflammatory cytokines, and stimulating the innate immune system in the intestine [9]. It forms a secretory route for harmful exogenous lipophilic substances, such as bilirubin, bile salts and is the major route for cholesterol elimination [2]. The gallbladder is the responsible organ of bile storage, and is connected to the liver by the bile ducts [2]. The canaliculi drain into an intralobular bile duct that collects the bile from each lobule. This intralobular ducts merge into the hepatic duct that contacts with the bile duct, that collects into the gallbladder [10]. 4.2. Functions The liver is an essential organ for a great variety of functions, including metabolism, digestion, vitamin storage or protein synthesis. It forms the first line of defense for drug and alcohol detoxification and is an important immunological organ. The liver plays a major role in maintaining glucose homeostasis by storing (glycogenesis), or releasing (glycogenolysis/gluconeogenesis) glucose by interacting with insulin and glucagon respectively [11]. When nutrients are available, insulin is secreted from pancreatic β-cells and promotes hepatic glycogen synthesis and lipogenesis. However, during fasting periods, insulin levels are decreased, and glucagon is secreted from pancreatic α-cells to promote hepatic glucose production to meet body energetic demands [12]. The liver is also fundamental for lipid and cholesterol metabolism. It is a critical organ for digestive absorption and performs uptake, synthesis, packaging, and secretion of lipids and lipoproteins. The liver’s biliary synthesis and secretion system enables efficient absorption of lipids from digestion. It can use fatty acids as an internal energy source through oxidative pathways, though it can also provide energy to other organs from the ketogenic products. This ability to provide ketones as an energetic substrate is necessary for organisms undergoing extreme fasting or consuming extremely low levels of dietary carbohydrates. It can also package excess lipid for secretion and storage in other tissues, such as adipose [5]. Introduction 48 Moreover, major player of protein and amino acid metabolism, being responsible for the production of 85-90% of circulating proteins, the processing of amino acids for energy, and disposal of nitrogenous waste from protein degradation in the form of urea metabolism. Albumin is the most abundant of these secreted proteins and is essential for maintenance of blood volume, and transport of critical molecules as lipids and hormones [5]. The liver also plays a key role in the blood coagulation process since it is the site of synthesis of all clotting factors and their inhibitors. Liver damage is commonly associated with variable impairment of hemostasis, which is the process of blood clot formation to prevent excessive blood loss due to vessel injury. After endothelial injury, the platelets adhere to subendothelial matrix covering the injured area and forming a platelet plug by the link to fibrinogen molecules [13]. Injury or damage in the blood vessels, cause vascular spasms, that can trigger vasoconstriction eventually stopping the blood flow. At this stage, collagen fibers will release ATP and inflammatory mediators for the recruitment of macrophages. Furthermore, the thrombogenicity of the extracellular matrix (ECM) increases, promoting platelet adhesion and aggregation. Vasoconstriction will expose collagen from the damage area encouraging platelets to the adhesion, forming a platelet plug, and sealing the injured area [14]. 4.3. Histology of the liver The liver is composed of parenchymal cells, which include hepatocytes and cholangiocytes, and non-parenchymal cells, including sinusoidal endothelial cells (LSECs), resident macrophages as Hepatic stellate cells (HSCs) and Kupffer cells (KCs), and other immune cells (Figure 4). 4.3.1. Parenchymal cells 4.3.1.1. Hepatocytes Hepatocytes are the most abundant cell population in the liver, constituting about 80% of the liver parenchyma [4]. They are radially organized in the liver lobule and separated from the blood by an endothelial wall, leaving open the space of Disse* [5], a thin perisinusoidal area between the endothelial cells and hepatocytes filled with blood plasma [15]. Introduction 49 Hepatocytes perform a wide array of metabolic, secretory, and endocrine functions. They form an extensive endoplasmic network adjacent to numerous mitochondrias and Golgi apparatuses, reflecting their high metabolic activity including β-oxidation, lipogenesis, glucose release and use, amino acid uptake and ammonia detoxification, bile and cholesterol formation, and synthesis of certain plasma proteins such as albumin and fibrinogen [3]. Hepatocytes are stable cells and rarely divide in the normal state, as they are quiescent in the G0 phase of the cell cycle. Under physiological or pathological conditions, the liver can regrow to normal size even after resection of 90% of the liver volume, due to its strong regeneration ability. Most liver diseases are characterized by hepatocyte damage and decompensation of liver regeneration, which may eventually develop into liver cancer [16, 17]. 4.3.1.2. Cholangiocytes or biliary cells Cholangiocytes, also known as biliary cells, represent a minor 3-5% proportion of all liver cells. They are the epithelial cells lining the intrahepatic and extrahepatic bile ducts, which participate in bile production and homeostasis, and responsible for approximately 30% of total bile flow [18]. Biliary cells can be activated by endogenous and exogenous stimuli and are involved in the modification of bile volume and composition [19]. They also contribute to liver regeneration when hepatocyte regeneration is impaired [18]. Bile modification occurs through coordinated transport of ions, solutes, and water across the cholangiocyte apical and basolateral plasma membranes. This process is modulated by hormones, peptides and nucleotide, among others, intracellular signaling pathways and regulatory cascades [19]. 4.3.2. Non-parenchymal cells 4.3.2.1. The sinusoidal endothelial cells (LSECs) The sinusoidal endothelial cells (LSECs) are the most abundant non-parenchymal cell type in the liver, representing approximately 15-20% of liver cells [20-22]. They are highly specialized cells, that constitute the interface between blood cells on one side, and both hepatocytes and hepatic stellate cells on the other side. The fenestrated endothelium provides a semipermeable barrier between hepatic parenchyma and the blood, that can act as a dynamic and selective sieve for soluble molecules, lipoproteins and virus particles, with small diameter, excluding passage of larger molecules [20]. Introduction 50 They have the highest endocytosis capacity of human cells [23], having their cytoplasm packed with vesicles and organelles associated with uptake, transport and degradation of endocytosed material [20]. Figure 4. Liver cell types. Schematical representation of the different cell types that are present in the liver parenchyma. Created with BioRender.com 4.3.2.2. Kupffer cells (KCs) Kupffer cells (KCs) are non-parenchymal cells that account for approximately 15% of the total liver cell population and constitute the largest population of resident macrophages in the body (80- 90%) [24], playing a critical role in the innate immune response. They present a surface with many cytoplasmic extensions (filipodia, lamellipodia and pseudopodia), and a cytoplasm with numerous lysosomes and cellular inclusions [25]. Their strategical localization in the hepatic sinusoid allows them to efficiently phagocytize pathogens or undesirable material entering from the portal or arterial circulation, like coagulation products, thrombocyte aggregates or cancer cells [26]. Under physiological conditions, they are the first innate immune cells, and they protect the liver from infections. Under pathological conditions, they are activated by different components and can differentiate into M1-like (classical) or M2-like (alternative) macrophages [25]. KCs actively migrate to their target and injury site upon activation, secreting a cocktail of cytokines that results in the recruitment of white bloods cells and Natural killer (NK) cells, which contributes to the local healing process [27]. Introduction 51 4.3.2.3. Hepatic stellate cells (HSCs) Hepatic stellate cells (HSCs) are liver resident macrophages located in the space of Disse*, between the LSECs and the hepatic epithelial cells, and constitute about 5-7% of the cells in the liver [28]. Quiescent HSCs (qHSCs) store vitamin A, and upon activation they lose their retinol reservoir. The activation of these cells leads to the transition from a quiescent vitamin A- storing cell, to an activated HSCs (aHSCs), a proliferative fibrogenic myofibroblast-like phenotype responsible for secretion of ECM into the space of Disse [29]. If the imbalance persists, the expansion of the fibrotic scar followed by the vascularized septae, leads to cirrhosis and/or end-stage hepatocellular carcinoma (HCC) [15]. While qHSC become activated they suffer morphological changes, passing from star-shaped cells into fibroblast or myofibroblasts [30]. Hepatic fibrosis is a wound-healing response to long-term liver injury, including chronic viral infection, excessive alcohol consumption, non-alcoholic steatohepatitis (NASH) or autoimmune liver disease. Is derived from the excessive accumulation of ECM proteins and collagen fibers in the liver parenchyma that disrupts liver architecture by forming a fibrous scar. Advance stages of liver fibrosis stages lead to cirrhosis, that result in hepatocellular dysfunction, increase the intrahepatic resistance to the blood flow and can require liver transplantation [31]. 4.3.2.4. Hepatic immune cells Every minute, the human liver receives 1.5 L of blood as well as a massive load of harmless dietary and commensal antigens, to which it must remain tolerant. It is also exposed to a variety of viruses, bacteria, parasites, and metastatic tumor cells, therefore needs mechanisms to override immune tolerance [2]. Hepatic immune cells are located along sinusoids and in portal tracts [32], and they constitute a major fraction of the body’s innate (native) immune capacity, and a small component of its acquired (adaptive) immune response [32]. The major components of the hepatic immune system are innate lymphocytes, which include a variety of T cells and non-T cells, as the natural killer (NK) cells, that can respond rapidly to conserved ligands. In addition to the lymphocytes, populations of hepatic resident cells like KCs or HSCs, play an important role on liver immune system [2]. Introduction 52 NK cells constitute 40% of total lymphocytes in the liver of humans [33], therefore understanding their role in liver homeostasis and inflammation is crucial. NK cells are important in early innate immune response but can also regulate innate and adaptive responses. Due to their cytotoxic function, they are important in the immune response against hepatotropic viral infections but are also involved in the inflammatory processes of autoimmune and fatty liver diseases [34]. They can lyse infected or cancerous cells and produce pro-inflammatory cytokines such as interferon gamma (IFNγ). Hence, they provide a potent pro-inflammatory and cytotoxic defence against viral challenge, but its dysregulation can lead to chronic inflammation and disease. The function of NK cells is tightly regulated via signalling through both activating and inhibitory receptors expressed on their surface [34]. 4.4. Space of Disse* The space of Disse, is a thin perisinusoidal area between the endothelial cells and hepatocytes filled with blood plasma, that has acquired great importance in liver disease. It has been originally described by the German anatomist and histologist Joseph Disse in 1880, as an area where several cell types are located: NK cell, mesenchymal stem cells (MSC), KCs and HSCs. It provides the perfect microenvironment for the stem cells niche in the liver and the interchange of nutrients between cells (Figure 4, 5) [15]. This perivascular niche is constituted by extracellular matrix proteins, sinusoidal endothelial cells, liver parenchymal cells and sympathetic nerve endings, stablishing a microenvironment that is suitable to maintain HSC and to control their fate. The stem cell niche integrity is important for the behavior of HSC in the normal, regenerative, aged and diseased liver [35]. Introduction 53 Figure 5. Schematic representation of liver cell components in normal and pathological conditions. Under pathological conditions, liver architecture is modified associated with the activation of the KCs and HSCs, followed by the infiltration of immune cells as neutrophils and monocytes, and the recruitment of NK cells. Modified from [15]. Created with BioRender.com 4.5. Liver related diseases 4.5.1. Chronic liver disease (CLD) Chronic liver disease (CLD) accounts for approximately 2 million deaths per year, being the 10th cause of death worldwide [36, 37] and representing a major health problem [38]. This disease is characterized by a progressive deterioration of liver functions, like metabolism, detoxification or protein synthesis, for a period longer than six months [39]. CLD is characterized by a continuous process of inflammation, oxidative stress destruction, and regeneration of liver parenchyma, which leads to fibrosis, cirrhosis and finally to the development of HCC [40]. There is a broad spectrum of etiologies that leads to CLD, which includes high fat food, sugary soft drink intake, prolonged alcohol abuse, infections, toxins, autoimmune diseases, genetic and metabolic disorders [41, 42]. 4.5.2. Non-alcoholic fatty liver disease- metabolic associated fatty liver disease / non-alcoholic steatohepatitis (NAFLD- MAFLD/NASH) Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the western world, and its prevalence increases in parallel to the global rise in obesity, type 2 diabetes mellitus (T2DM) and MS, affecting to people of any age [39, 43]. NAFLD is divided into two major subtypes: Non-alcoholic fatty liver (NAFL or simple steatosis), and Nonalcoholic steatohepatitis (NASH). While NAFL is the non-progressive form that rarely develops into cirrhosis, NASH is the progressive form of NAFLD, that can lead to cirrhosis and HCC. NASH is characterized by the presence of steatosis, ballooning degeneration and lobular inflammation, with or without peri-sinusoidal fibrosis, on liver histology [44] (Figure 6). Introduction 54 Figure 6. Non-alcoholic fatty liver disease (NAFLD) spectrum. A healthy liver is considered when lipid accumulation in hepatocytes is lower than 5%. If the disease percentage of fat increases, the disease can progress into Non-alcoholic fatty liver (NAFL), or more advance stages as, Non-alcoholic steatohepatitis (NASH) characterized by steatosis, ballooning, and inflammation, with or without fibrosis. Disease can be reversible until this stage, while last stages of the disease, cirrhosis, and HCC, are irreversible. Created with BioRender.com. In 2020, a group of experts have achieved an agreement regarding that NAFLD term, does not accurately reflects the current knowledge about this disease. Because this disease not only caused liver damage but also affects most of the organs and generates a metabolic dysfunction the term Metabolic Associated Fatty Liver Disease (MAFLD), more accurately reflects the disease and its global affection [45] . 4.5.2.1. Epidemiology of NAFLD One quarter of the global population is estimated to have NAFLD, percentage that can range from 13% in Africa to 42% in southeast Asia, while in Spain the prevalence of NAFLD reached 25.8% [46]. Although, the incidence of NASH is projected to increase up to 56% in the next 10 years in countries as China, France, Germany, Italy, Japan, UK, USA or Spain [47]. NAFLD is already the fastest global growing cause of HCC, while its incidence is rapidly rising worldwide. Introduction 55 In Europe NAFLD prevalence exceeds 25% in adults, being higher in those with Metabolic syndrome (MS) related comorbidities. More than half of the obese European adults have NAFLD (57%), while the relatively low rates among non-obese adults (14%) remain clinically relevant [48]. Besides, prevalence in adults is higher in men than women [49]. Increased incidence of NAFLD is expected to have a proportionate impact on healthcare resources and costs associated with advanced liver disease. NAFLD has a wide range of economic implications for affected populations and societies, including both direct medical expenses or indirect costs associated with consequences such as job loss. Evidence shows that NAFLD progression is associated with substantial health-care costs, socioeconomic losses, and reduced quality of life, meaning that there are both social and economic arguments for acting on NAFLD [50, 51]. Efforts to mitigate disease burden are critical and should be linked to strategies that ameliorate the growth of obesity and diabetes mellitus (DM) at both national and global levels [52]. Early intervention could help reduce the burden of disease, associated health-care costs and economic losses [50, 51]. 4.5.2.2. Pathophysiology of NAFLD NAFLD is characterized by the presence of hepatic steatosis in more than 5% hepatocytes, in addition to any of the following three criteria: overweight or obesity, presence of type 2 Diabetes Mellitus (T2DM) or evidence of metabolic dysregulation [53- 55]. The pathogenesis of NAFLD involves a “two-hit” process. The first hit is insulin resistance (IR) which causes liver steatosis as result of increased hepatic lipogenesis and impaired free fatty acid (FFA) degradation [56]. The second caused by lipotoxicity is responsible of oxidative stress, mitochondrial dysfunction, proinflammatory cytokines release contributing to hepatic injury, inflammation, and fibrosis [57]. Although most patients with NAFLD have simple steatosis, this term represents a spectrum of liver disease progressing through NASH and fibrosis, to cirrhosis and end stage liver failure. NASH encompasses a heterogeneous range of histology with a variable spectrum of inflammatory and fibrotic changes on liver biopsies. Although the histological definition of NASH does not require fibrosis, fibrosis is more predictive of disease-related morbidity and mortality. Steatosis rarely progresses to advanced liver Introduction 56 disease, whereas NASH, particularly with significant fibrosis, has the potential to progress to cirrhosis, stage liver failure and HCC [55] (Figure 7). Figure 7. Pathophysiology of NAFLD/NASH. The most common risk factors of NAFLD/NASH are diet-induced obesity and insulin resistance that trigger macrovesicular steatosis, lobular inflammation, hepatocellular ballooning, activation of HSCs and fibrosis. Moreover, key cellular pathways are activated, such as ER stress, lipotoxicity and de novo lipogenesis, oxidative stress, apoptosis and fibrogenic pathways. Additionally, intestinal dysbiosis plays a pivotal role in disease progression. CCL2, C-C motif chemokine ligand 2; ER, endoplasmic reticulum; FFAs, free fatty acids; IL-6, interleukin 6; LPS, lipopolysaccharide; ROS, reactive oxygen species; TNF, tumor necrosis factor [58]. The excessive hepatic accumulation of lipids results from an increased fatty acid input/output balance due to changes in the lifestyle associated with high energy uptake, sedentarism, and MS. The increased FFA uptake in the liver results from high energy diet, increased lipolysis of adipose tissue, and de novo hepatic lipogenesis, while output mechanisms related with fatty acid oxidation and very low-density lipoproteins secretion (VLDL) remain insufficient to compensate for the accumulation of triglycerides [59, 60]. Saturated fatty acids in the liver induce lipotoxicity associated with increased Introduction 57 endoplasmic reticulum stress [61, 62] and lipid oxidation, increasing the oxidative stress in the liver [63]. In addition, poor diet in patients with NAFLD may contribute to changes in the microbiota, related with low production of short chain fatty acids (SCFAs), increased intestinal permeability, and translocation of luminal bacteria or its products to the liver where they will encourage an inflammatory environment. Consequently, hepatic stellate cells will activate and release collagen, contributing to fibrosis and progression of the disease [63]. 4.5.2.3. Risk factors All recent studies confirm that NAFLD is a complex disease that is affected by metabolic and environmental factors, along with genetic and epigenetic predisposition, involving multiple organs and diverse mechanisms. Genetic, epigenetic factors, ethnicity or gender, diet, life style, alcohol consumption, hepatotoxic drugs, as well as smoking can strongly influence the development and pathogenesis of NAFLD [43] (Figure 8). The exact contribution of each factor in the development of this disease is unknown and may even vary by geographic location [64]. Besides, different studies have shown a strong association between NAFLD and comorbidities such as obesity, MS, T2DM and dyslipidemia [65]. A. Diet NAFLD is often referred to as a self-inflicted disease, implying the influence of personal behavioural and dietary choices. However, the impact of the surrounding “fast and processed food” obesogenic environment on the choices of children and adults should not be neglected [56]. Processed or ready-to-consume food products are characteristically energy-dense, fatty, sugary or salty. At the same time, they are hyper- palatable, cheap and attractive, therefore highly dominant, and they comprise the majority of the total energy intake among young adults currently [66, 67]. The food industry and especially the fast-food industry, extensively uses the types of lipids with long shelf life that are easy and inexpensive to produce [68]. Processing natural liquid vegetable oils, by adding hydrogen, modifies their texture from liquid to solid forms and results in the development of trans-fats (or partially hydrogenated fats). Trans Fatty Acids (FA) give food a desirable taste and texture. The main examples of Introduction 58 industrial Trans FA are margarines, commercially baked products, deep-fried fast foods, packaged snack foods and other prepared food [69]. According to the U.S. Food and Drug Administration (FDA), in 1994- 1996 we consumed about 5.6 g of trans-fat per day [70]. In the early 1990s, researchers began to identify the adverse health effects and reported that intake of Trans FA increased the risk of coronary heart disease by altering the ratio between Low-Density Lipoprotein (LDL) cholesterol and the “good” High-Density Lipoprotein (HDL) cholesterol [71]. Increased consumer awareness of the health implications of Trans FA resulted in local and state efforts to limit or ban their use [9]. “Anti trans-fat” measures introduced by governments accelerate the use of an accessible alternative, palm oil, obtained from the fruit of the palm tree (Elaeis guineensis). Palm oil is abundant, low-cost and chemically stable. Currently, it is the most widely produced edible vegetable oil in the world [72]. It is rich in saturated FAs, especially Palmitic Acid (PA), unlike most of the vegetable oils, which are rich in unsaturated fat. For this reason, palm oil is semisolid at room temperature, making it suitable for the formulation of processed food [73]. Recently, the impact of palm-oil consumption on the heart, especially in the development of coronary artery disease, was a matter of considerable debate. The main argument against the use of palm oil is the fact that it contains saturated PA, which may case elevation of total cholesterol and LDL levels [74]. During the last decades, energy intake has increased along with the consumption of animal fat and energy-dense foods, while fiber intake has decreased. This dietary shift contributes to the rise of non-communicable diseases, including obesity, T2DM, cardiovascular disease and cancer. A poor diet was found to be the leading risk factor of death and third leading risk factor for disability-adjusted life-years loss in the United States [75]. Due to the increasing trends in overweight and obesity, in both adults and children, there is a strong focus on dietary overconsumption and energy restriction. However, obesity and its associated metabolic disorders, are preventable and many strategies exist to achieve successful weight loss by improving dietary habits and energy balance [75]. It has been demonstrated that high-caloric diets, especially those rich in saturated, trans fatty acids, cholesterol and fructose-rich, increase visceral fat and the incidence of NAFLD [76]. Moreover, certain dietary patterns, such as a western diet, has been associated with NAFLD, independent of gender, family income and physical activity level Introduction 59 [77, 78]. The “western dietary pattern” consists of a high intake of fast food, soft drinks, processed meat, high-fat dairy products, hydrogenated fats, mayonnaise, salty snacks, sugar-sweet desserts, organ meats, and refined grains [79]. High consumption of soft drinks increases the risk of NAFLD due to the high caloric content and/or the excessive amount of sugar, such as fructose, in these drinks, which may contribute to increase the risk of MS [78]. Moreover, refined grains, white bread, and sugar-sweets desserts, which are constituents of the "western dietary pattern", rapidly increase the insulin and glucose levels in blood, which cause IR, diabetes, and obesity [80]. This rapid increase in blood sugar enhances the rate of de-novo synthesis and increases fat in liver cells [81]. Figure 8. Main factors that contribute to NAFLD development. Created with BioRender.com. B. Sedentary lifestyle There is a strong support for the role of sedentarism as a primary contributor to the development and progression of NAFLD. Most cross-sectional studies suggest that reduced habitual physical activity is associated with hepatic fat independent of age, sex, and body mass index (BMI). Introduction 60 Physical inactivity increases the risk for a positive energy balance, contributing to IR, promoting adipose increase and hyperinsulinemia. This may result in ectopic lipid storage in the liver, in part through increased FFA uptake, activation of de novo lipogenesis, or triglycerides (TG) export suppression. Moreover, low aerobic fitness has a direct impact on the phenotype of the liver, increasing the susceptibility of NAFLD. Under conditions of physical inactivity, the liver exhibits reductions in hepatic mitochondrial content and function, reducing the oxidative capacity and making the liver more susceptible to excess lipid accumulation [82]. Increasing physical activity promotes health benefits in patients with NAFLD, independent of body weight reduction. It can reduce the risk of developing IR, dyslipidaemia, T2DM or reduce blood pressure, as well as MS [64]. Exercise could also mediate its beneficial effects directly on the liver and indirectly via extrahepatic pathways, forming a dose-response relationship with NAFLD in terms of prevalence and disease severity [83]. Physical activity, either aerobic and resistance training, effectively reduce hepatic steatosis and reduce the NAFLD-associated cardiovascular risk [83] and have an essential role in weight reduction and maintenance, reduces hepatic steatosis and NAFLD-associated cardiovascular diseases [84]. A recent study has analyzed a cohort of NAFLD patients, with dietary interventions but without any added physical activity. They have tried to establish the effect of different dietary modifications on intrahepatic lipid content (IHL), liver fibrosis, and liver function. The study showed Mediterranean diet (MD), mainly consisted of vegetables, fruits, legumes, nuts, and unsaturated dietary fats from vegetable oils, without energy restriction, leads to significant reduction of IHL. However, the diet without exercise did not lead to significant changes in liver enzymes, lipid profile, fasting glucose or insulin levels, or IR [85]. Growing evidence highlights the need for physical activity in reducing the body weight, improve liver histology and reduce fibrosis in NAFLD patients [86]. Weight loss achieved through physical activity improves hepatic and peripheral insulin sensitivity, but physical activity, regardless of the effects on body mass, also decreases proinflammatory and oxidative stress markers and improves liver enzymes. In addition, it might also affect the gut microbiota and modulate the liver inflammatory response and NASH progression [87]. Introduction 61 Physical exercise may also have effect in gut microbiota. It has been demonstrated that athletes had a higher diversity of gut micro-organisms, however mechanism in still unclear [87] (Figure 9). Intestinal dysbiosis, changes in bacterial composition or distribution, is associated with a reduction in the production of short-chain fatty acids (SCFAs) and increased intestinal permeability. Translocation of bacteria or its products to the liver result in activation of immune cells, hepatocytes, LSECs, and release of proinflammatory cytokines. Production of reactive oxygen species (ROS) and proinflammatory cytokines drive the activation of HSCs and deposition of collagen inducing fibrosis and progression of liver disease from simple steatosis to steatohepatitis, cirrhosis and HCC [88]. Figure 9. Beneficial effects of physical exercise. Exercise (aerobic, resistance and combined activities) influences liver enzymes ad intrahepatic TG. Physical activity is associated with reduction of TG and transaminases (ALT and AST) levels. Moreover, physical activity can reduce oxidative stress (OS) and reactive oxygen species (ROS) overproduction, via de up regulation of antioxidant enzymes and hepatic inflammation. NAFLD is also associated with dysbiosis, loss of microbiota diversity, moreover there are few studies suggesting the practice of physical exercise can improve the bacterial diversity. Modified from [89]. C. Alcohol- consumption According to the World Health Organization (WHO), about 2 billion people consume alcohol worldwide and more than 75 million are diagnosed with alcohol use disorders. Between 4% and 25% of the global disease burden of specific cancers is attributable to alcohol [36]. Introduction 62 Global alcohol consumption has decreased in the past few decades, whereas the consumption remains high, with values of 10 L of pure alcohol consumed per adult each year in Europe [90]. Alcoholic liver disease (ALD) is a major cause of liver disease worldwide that can led from simple fatty liver to alcoholic steatohepatitis (ASH), fibrosis and leading to cirrhosis and HCC [39, 91]. Early stages of the disease which include simple steatosis, or hepatitis, characterized by an inflammatory environment, can be reversible. However more advances stages turn into irreversible, as cirrhosis that also leads to portal hypertension [29]. There is a large discrepancy in the definition of a ‘‘drink”, regarding the grams of alcohol included, varying from 8 to 16 g. According to the Dietary guidelines for Americans, one standard drink of ‘‘pure” alcohol is defined as 14 g. European Association for the Study of the Liver (EASL) in 2018 has suggested to standardise the measure to 10 g, to facilitate comparisons among studies, as has been used by the WHO [92]. EtOH could be metabolized into acetaldehyde by three different systems: alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1) enzymes or the catalase (CAT), a peroxisomal enzyme that also catalyzes the removal of ROS. Acetaldehyde is further metabolized to acetate by the action of the aldehyde dehydrogenase (ALDH) [93, 94]. EtOH metabolism generates gut dysbiosis, inflammation and disturbs permeability causing an increase in circulating FFA and bacterial-derived products such as lipopolysaccharides (LPS), that are recognized by pathogen-recognition receptors, like Toll-like receptors (TLRs) and activates KCs. Thus, KCs resulting in the activation of proinflammatory cytokines (TNF-α, IL-1 and IL-6) that activates HSCs and leads to accumulation of ECM proteins in the liver tissue with synergistic effects on hepatic damage, fibrosis development and cirrhosis [58, 95, 96] (Figure 10). Introduction 63 Figure 10. Pathophysiology of ALD. EtOH is metabolized into acetaldehyde by ADH, CYP2E1 and catalase (a peroxisomal enzyme that catalyzes the removal of ROS). Acetaldehyde is metabolized to acetate by the ALDH. As a result, EtOH metabolism leads to gut dysbiosis, inflammation and increased permeability that affects lipid accumulation (caused by increased circulating free fatty acids), hepatic immune cell infiltration, hepatocyte damage, cholestasis, and fibrosis. ADH, alcohol dehydrogenase; ALDH, acetaldehyde dehydrogenase; CCL2, C-C motif chemokine ligand 2; CYP2E1, EtOH: ethanol; cytochrome P450 isoenzyme 2E1; ER, endoplasmic reticulum; FFAs, free fatty acids; IL-6, interleukin 6; LPS, lipopolysaccharide; ROS, reactive oxygen species; TNF, tumor necrosis factor; VLDL, very low-density lipoprotein [58]. D. Gut dysbiosis Dysbiosis is defined as a gut microbial imbalance, that, can include an increase in the proportion of small bowel bacteria, alteration in the relative proportion of benevolent microbes to pathogenic ones, and the translocation of colonic bacteria [97]. Lately, there is a growing interest in gut microbiota and the restoration of dysbiosis. Microbiota composition is adversely affected in patients with NAFLD, as gut microbiota in healthy individuals mostly has a balance among the different populations, this balance is disrupted regarding major phyla. Many of these bacterial groups of gut microbiota have been shown to either increase or decrease in NAFLD and produced toxic metabolites, inflammation, oxidative stress and leads to liver cirrhosis. In recent years, advances in Introduction 64 technology have shown that human gut microbiota is extremely variable in abundance and composition and play significant role in supporting the human health and its alteration can contribute to the development of many diseases like obesity, NAFLD and HCC. Probiotics are commonly used as evidence suggests that could regulate intestinal microbiota and can be preferred as a novel preventing or treatment approach for NAFLD and other chronic liver diseases [98]. 4.5.3. Strategies for NAFLD prevention/ reversion Currently, treatment options for NASH are limited. Lifestyle changes with weight loss are the foundation of treatment, but it is hard to achieve and maintain and is often not enough in morbidly obese patients. However, it is believed that a combination of treatment as lifestyle adjustments, increasing physical activity and alcohol cessation, can be beneficial for NAFLD patients [99]. 4.5.3.1. Lifestyle modification A 5% weight loss has been shown to reduce liver steatosis, a loss of 7% (or more) improves NASH histology, and 10% or greater weight loss can have an effect in hepatic fibrosis regression. Weight loss can be more successfully achieved with a combination of moderate dietary restriction paired with physical activity [100]. Diet, weight loss and physical activity are the cornerstone of every treatment for NAFLD and are recommended by both the American and European associations for the study of the liver [101-103]. 4.5.3.2. Diet Low-carbohydrate/higher-protein diets have been associated with metabolic benefits, independent of weight loss. Caloric restriction promotes fat mobilization from the liver [104]. A healthy diet with a reduction of caloric intake and high-glycaemic index (GI) foods, increased consumption of monounsaturated fatty acids, omega-3 fatty acids, fibers, and specific protein sources such as fish and poultry are suggested to have beneficial effects [105]. The Mediterranean diet (MD) is the most recommended diet pattern for NAFLD management [106], as it has shown the ability to improve liver steatosis in several studies, independent of caloric restriction. The MD is traditionally plant based (whole grains, legumes, fruit, vegetables), lower in carbohydrates (limited simple sugars and Introduction 65 refined carbohydrates), and rich in monounsaturated (mostly olive oil) and omega-3 fats, and incorporates limited fish, red meat, poultry and eggs [104, 107]. Unsaturated fatty acids (Omega 3), which can reduce total cholesterol and has a protective role against NAFLD, are abundant in fish [108, 109]. The high consumption of fruits and vegetables increases the intake of antioxidant vitamins, such as vitamins A, C or E, which have a protective role against NAFLD development [110] and oxidative stress [111]. Although, fruits and vegetables represent good sources of dietary fibers, which have an inverse association with IR and reduce the risk of NAFLD [79]. Patients should be encouraged to analyze the influence of cultural factors, financial concerns, lifestyle choices and personal preferences on their diets. Meals should be nutritionally balanced and should be composed of lean meat, fruit, vegetables, and whole grain products [99]. However, there are social barriers to patient’s adherence to a healthy diet, including social priorities and rivalries, family’s food habits, poor social support, social impasses, and dominant food patterns [112]. A study that has done in Spain, has detected that the main barriers to healthy eating were irregular working hours, busy lifestyle, willpower, and unappealing food. Conversely, the prevention and health promotion aspects are the main perceived benefits [113]. The best diet is the one a patient can follow, based on individual preferences and eating behaviors. 4.5.3.3. Physical activity Current guidelines recommend 150-200 min/week of moderate-intensity aerobic physical activities in three to five sessions. Aerobic training is defined as any activity that uses large muscle groups, can be maintained continuously and is rhythmic in nature. The majority of NAFLD exercise studies apply aerobic training as cross-training, cycling, rowing, running, walking and other rhythmic exercise for aerobic training [114], however anaerobic activities as yoga, tai-chi, and pilates offer alternative modalities of exercise beyond traditional aerobic or strength training [115]. Multiple large comparative effectiveness studies, demonstrate equivalency between exercise modalities, it is most important to consider patient compliance with exercise training and how likely they are to achieve a specific outcome when choosing a modality of exercise to prescribe [114]. Introduction 66 According to the intensity of the exercise, low, moderate, and high- (vigorous) intensity exercise is defined by different metabolic equivalents (METs). Low intensity exercise is performed from 2.0 to 2.9 METs, moderate-intensity 3.0 to 5.9, and ≥ 6 for high-intensity [46]. High- intensity Interval Training (HIIT) is a method of exercise training that incorporates both strength and aerobic exercise with extreme intensity variation across low, moderate and high-intensity. This time-efficient system makes it attractive for patients with NAFLD who have identified time limitations as a barrier to exercise practice. Moderate-intensity is to be recommended over low-intensity exercise training, however, a recommendation suggesting moderate-intensity exercise over vigorous-intensity exercise or HIIT cannot be made from the available evidence [114]. The optimal duration and frequency of exercise training has not been established specific to NAFLD patients. Evidence-based guidelines from the Department of Health and Human Services (DHHS) from EEUU and the American College of Sports Medicine (ACSM), at least 150 minutes each week can be recommended [116]. In terms of duration, not surprisingly, a longer duration of exercise leads to better outcomes and regular physical activity for more than 4 months significantly improves metabolic parameters in NAFLD, more than if the activity is discontinuous [48]. It has been also suggested that the age of NAFLD patients may be an important variable in improving the levels of serum transaminases, and clinically young patients may have greater benefits from exercise than older patients. Specifically, exercise can decrease both ALT and AST levels in young NAFLD patients, while with aging, this efficacy was progressively reduced [116]. The barriers to physical activity in developed countries are usually related to motivational and internal barriers, while in developing ones there are lack of facilities, places and educational programs. Nevertheless, lack of interest and motivation to do exercises is the most important items of “internal barriers”. In obese patients, tiredness because of overweight is often reported, while osteoarthritis in older women is also a major reason. Evidence show that obesity has strong association with knee osteoarthritis and weight reduction would be a preventive strategy. Therefore, osteoarthritis and joint pain should be paid attention in designing exercises for middle aged and old patients Therefore, NAFLD patients should undergo adequate exercise sessions as soon as possible upon diagnosis of the disease [117]. Introduction 67 4.5.3.4. Pharmacotherapy and clinical interventions Apart from lifestyle modifications such as weight loss, a Mediterranean diet and physical activity, only a few NAFLD-specific pharmacological treatment options such as Vitamin E and Pioglitazone are considered by current international guidelines. However, recently randomized controlled trials with Glucagon-like peptide-1 (GLP-1) agonists, Farnesoid X receptor (FXR) and Proliferator-activated receptor (PPAR) ligands as well as other agents have been published and may expand the therapeutic options for NAFLD in the near future [118]. In morbidly obese patients, bariatric surgery is considered the most effective treatment for obesity as well as the accompanying diseases. Bariatric surgery promotes much greater weight loss than conservative treatment, regardless of the applied surgical technique [119]. 4.5.3.5. Prebiotics, probiotics and synbiotics Diet is considered one of the main drivers that modulates the composition of gut microbiota, affecting human metabolism. A disruption in the homeostasis of gut microbiota may lead to dysbiosis, which is commonly reflected by a reduction of the beneficial species and an increment in pathogenic microbiota. Gut microbiota is a highly dynamic entity and presents a constant flow in its composition. Due to the anatomical and functional interactions by the PV, gut and liver are in close relation, thus altered intestinal microbiota might affect liver function. Dysbiosis is directly related with an increased intestinal permeability as a consequence of some aspects, including the epithelial barrier deterioration, small intestinal bacterial overgrowth, tight junctions’ alteration, and even the whole bacterial translocation, causing endotoxemia, which might reach and damage the liver through the PV. Alterations of the permeability of the gut barrier, affects the immune system of the host or the alteration of bacterial balance regarding sensibility of certain species to drug presence [117]. Prebiotics and probiotics are microbial therapies that can manipulate intestinal microbiota. Prebiotics are nondigestible food ingredients that promote growth of beneficial microorganisms in the intestine. Probiotics are live microorganisms that, administrated in adequate amounts, confer a health benefit to the host. Moreover, synbiotics are a combination of prebiotics and probiotics, that their use can maximize the effect of both prebiotics and probiotics [120]. Introduction 68 The intestinal effects of prebiotics, probiotics, and synbiotics have been attributed to several mechanisms, such as delayed macronutrient absorption, bacterial fermentation by product absorption (short-chain fatty acid), bile acid interactions, improved barrier function to decrease toxic product filtration (trimethylamine or lipopolysaccharide) and enhanced immune system to reduce intestinal inflammation. All this effects, can promote weight loss, improve liver function, and elicit an anti-inflammatory and hypolipidemic effect [120]. Several studies and clinical trials have encouraged the use of probiotic supplementation as promising and safe therapeutic approach however, mechanisms that link prebiotics or probiotics with decreased serum hepatic enzymes are not fully elucidated [121]. 4.5.4. Animal models for NAFLD study Although intense research during the last years has led to considerable improvements in the understanding of NAFLD pathogenesis, effective therapies are still lacking. A better understanding of the mechanisms implicated in the initiation, progression, and resolution of this disease is crucially needed. For this purpose, animal models are essential to study the NAFLD pathophysiology, identify potential therapeutic targets, and to evaluate the impact of therapies. The ideal animal model for the study of NAFLD, should recapitulate all aspects of the pathogenesis in humans, the typical histological features and progression, and the metabolic background as obesity, IR, hyperglycemia, hyperinsulinemia, dyslipidemia or altered adipokine profiles [122]. Compared to clinical research, the use of animal models offers several advantages, as the possibility of collecting several samples at different time-points realizing sequential studies, fast disease development, ability to control and reduce variables, possibility of genetic modification that allows the study of specific genes and signaling pathways. Moreover, compared to in vitro systems, animal models allows the study of the liver as a complete organ, interactions in between cells and cells with the matrix, and the crosstalk of the liver with the rest of the body, including immune, vascular, metabolic, and endocrine interactions [122, 123]. 4.5.4.1. Mice and rats Mice (Mus musculus) and rats (Rattus) have long served as the preferred species for biomedical research animal models due to their anatomical, physiological, and genetic Introduction 69 similarity to humans. They are also nowadays the best standardized models for NAFLD study. Advantages of rodents include their small size, easy maintenance, short life cycle, and abundant genetic resources. It also has economical advantages as mice and rats are relatively small and require little space or resources to maintain, have short gestation times but relatively large numbers of offspring, and rapid development to adulthood and relatively short life spans. For example, mice have a gestation period of approximately 19-21 days, they can be weaned at 3-4 weeks of age and reach sexual maturity by 5-6 weeks of age, allowing large numbers of mice to be generated for studies fairly quickly [124]. Mouse models have been extensively used in many studies of NAFLD pathogenesis however, they have some limitations as not all mouse models can replicate the full spectrum of human NAFLD regarding both the metabolic characteristics and histological pattern. For example, mice exhibit significant differences in lipid metabolism versus humans, as they carry most of the plasma cholesterol in HDL, while humans carry much of it in LDL. However, it is still reasonable to assume that many of the Western diet (WD) harmful effects for mice may also affect humans, both in terms of MS and NAFLD [125]. There are several modified diets that can be used for mice feeding, in terms of developing a NAFLD model. One of the widely used models is High fat diet (HFD) enriched with both fructose and cholesterol and referred as Western diet (WD). C57BL/6J mice treated with WD diet develop obesity, MS, steatosis, oxidative stress, moderate lobular inflammation, and initial fibrosis [58, 122]. Recently we established a physiological, fast and innovative experimental model of CLD- DUAL model which synergistically combines the effects of alcohol and WD. In our innovative DUAL model, we overcome the natural mouse aversion to alcohol by incorporating D-glucose in the water. Sweetened water successfully masked the taste of alcohol increasing alcohol intake. Additionally, the consumption of WD significantly elevated ethanol (EtOH) intake as it has been shown in previous reports, suggesting the existence of a positive correlation between EtOH and fat whereby each nutrient stimulates consumption of the other [126]. This finally resulted in higher daily caloric consumption, robustly increases body weight, and intensifies liver damage in DUAL-fed animals. Altogether, preclinical DUAL model induces the progression to advanced liver fibrosis and cirrhosis in the context of key risk factors for the human condition (i.e., alcohol Introduction 70 consumption, obesity, MS), naturally mimicking human pathology. Importantly, it is an easy, affordable, highly reproducible, time-efficient diet which does not require any special skill or expensive equipment and therefore can be further used for the development of much needed therapeutic options [127]. 4.5.4.2. Zebrafish Zebrafish (Danio rerio) is another animal model frequently used in NAFLD research field. The zebrafish liver is organized in 3 contiguous lobes (2 lateral and 1 ventral) and instead of a portal architecture, hepatocytes in fish livers are arranged in tubules, with bile ductules coursing between 2 rows of hepatocytes. The cellular basis for liver diseases appears to be similar between mammals and fish, however differences in the microenvironment may contribute to species-specific responses to injury. Furthermore, cells in zebrafish livers appear similar to mammals and perform many of the same functions, including bile secretion, glycogen and lipid storage, insulin response, or secretion of serum proteins [128, 129]. It also has a higher proportion of non- parenchymal cells (immune cells) compared to hepatocytes (Figure 11). Organogenesis is underway in nearly all systems by 24 hours postfertilization (hpf) and the embryo is swimming by 2 days postfertilization (dpf). By 5 dpf, all major organ systems are established, and larvae are ready to feed, hence zebrafish liver is fully formed in 5 days [130]zdsr5j. In adult stage, the trilobular zebrafish liver, similar to mammals, plays an important role in the metabolic homeostasis of the body including the processing of carbohydrates, lipids, proteins and vitamins. In addition, it also plays a key role in detoxification and the synthesis of serum proteins such as albumin or fibrinogen. A clear distinction can be made between the male and female liver in the adult zebrafish, as female hepatocytes are very basophilic as a result of the production of vitellogenin [131]. Using zebrafish for studying NAFLD has multiple benefits, including low cost, fast maturation, easy genetic modification, and feasibility of experimental manipulation [132]. Zebrafish possess orthologues of critical lipid metabolic genes including microsomal triglycerides (TG) or microsomal triglyceride transfer protein (mttp), gene families, or LDL receptor (ldlr), and their expression pattern appear to be comparable to humans [133]. Moreover, the transparency and whole-body real-time monitoring make the larval zebrafish a promising model to study NAFLD [134]. Introduction 71 In addition to wildtype zebrafish models, transgenic reporter line fabp10a:mCherry (fatty acid binding protein 10a: monomeric Cherry), is used to label hepatocytes. Identification of the liver specific fabp10a, and its promoter sequence allowed successful development of liver reporter lines that drive the reporter gene expression in hepatocytes [135]. Zebrafish NAFLD models also include hepatocyte enlargement and ballooning, TG accumulation or ROS increase. However, the interaction between hepatocytes and other cell types, and the progression to more severe steatohepatitis that involves inflammation and fibrosis, remain to be explored in this model. The number of hepatobiliary diseases studied in zebrafish is growing [136]. New tools and approaches are being developed, including gene editing technologies to mutate genes of interest, large-scale chemical screens, and advanced imaging approaches to observe, mark, and track hepatic cells. Zebrafish researchers are set to not only increase our understanding of the mechanisms of liver disease but also identify new therapeutic targets and test candidate compounds for efficacy and safety before they are tested in patients [129]. Figure 11. Differences in between unit structures of the mouse and zebrafish livers. Both mouse and zebrafish livers are composed of histological units with polygonal shape, still there are several differences in between both. In mouse, bile ducts are located in periportal areas, whereas in zebrafish are randomly distributed through the hepatic lobules. The bile transport in mouse goes along bile canaliculi along the Introduction 72 hepatocytes, whereas zebrafish form a reticulate network of preductules. Hepatic zonation is also a characteristic of mammals, not confirmed in zebrafish liver. Regarding PV, in mouse it is a single blood vessel that branches before entering the liver, though in zebrafish, multiple PVs enter the liver independent from the intestine. Figure modified from [137]. Created with BioRender.com 4.5.4.3. Others In summary, Each animal model has its own advantages and disadvantages that must be considered carefully before initializing a study, however all the scientific researchers should follow the ethical guidelines while selecting the animal models for their research or training purpose in educational institutions [138]. The selection of appropriate animal models continues to be one of the key questions faced in this field. Although significant progress has been made, the research in hepatology should continue to establish animal models anatomically and physiologically as close to human as possible to allow for translation of the experimental results to human medicine [58]. Open questions OPEN QUESTIONS Open questions 74 Open questions 75 5. Open questions Along with the increase of obesity and type 2 diabetes (T2DM), NAFLD has become the most common chronic liver disease in industrialized countries. Overweight, lack of exercise, alcohol consumption are the most important risk factors. So far, no specific drug therapy has been approved for this illness, neither for steatohepatitis nor for fatty liver in general. Despite that the pathogenesis of NAFLD is becoming increasingly better understood many questions have not yet been answered: 1. Currently, transfat and PA consumption remain a major source of concern within health policies regarding the reduction of cardiovascular and metabolic-disease risk. However, evidence of the consequences of transfat and PA consumption in the development of NAFLD is scarce and even controversial. 2. Whether steatohepatitis is reversible remains a relevant clinical question and is a constant matter of debate. Can the lifestyle modifications, including increased physical activity and dietary changes, be the chosen treatment, even for advanced stages of NAFLD? 3. What are the underlying molecular mechanisms of exercise and diet interventions on steatohepatitis? Do they exert their health effects via similar or different pathways? 4. There is increasing evidence that gut permeability and the dysbiosis of gut microbiota contribute to the development of NAFLD. Does the underlying mechanism of the beneficial effects of exercise and diet on NAFLD also involve regulation of microbiome? 5. Mice represent the largest group of NAFLD models despite their incomplete resemblance to human. Can the experimental results made in mice be further confirmed in completely different animal model such for example Zebrafish? Open questions 76 Objectives 76 OBJECTIVES Objectives 78 Objectives 76 6. Objectives In the present study, we aimed to evaluate three general aims: 1. To analyse if the type of the fat and the level of PA in the diet can be a risk factor for NAFLD development and regression. 1.1. To test the effect of WDs with different PA concentrations for the development of obesity, MS, steatohepatitis, liver damage and hepatic fibrosis in murine model of NAFLD. 1.2. To test if WD withdrawal is able to reverse murine NAFLD independently of the diet composition. 2. To evaluate the biological impact of dietary modifications alone or in combination with aerobic physical exercise on the progression DUAL- diet induced steatohepatitis and fibrosis. 2.1 To test the effects of diet withdrawal after short-term application of DUAL diet. 2.2 To analyze if metabolic and hepatic changes induced by the long-term DUAL diet are correspondingly reversible. 3. Develop an experimental preclinical model of DUAL diet in adult and larval zebrafish (Danio rerio). Objectives 80 Material and methods 81 MATERIAL AND METHODS Material and methods 82 Material and methods 83 7. Materials and methods 7.1. Materials 7.1.1. Chemicals Reagent Manufacturer 20% w/v Glucose solution Braun GmbH, Krönberg, Germany 30% Acrylamide/bis solution Bio-Rad, CA, USA 4X Laemmli buffer Bio-Rad, CA, USA Acetic acid (glacial) 100%- CH3COOH AppliChem, Darmstadt, Germany c (NH4)2S2O8 Bio-Rad, CA, USA BODIPY™ 493/503 Thermo Fisher Scientific, MA, USA Bovine serum albumin, lyophilized powder, ≥96% (BSA) Sigma-Aldrich, MO, USA Calcium chloride dihydrate- CaCl2. 2H2O Carbo Erba, Emmendingen, Germany Chloroform- CHCl3 Fisher Scientific, Boston, USA Complete mini Roche, Basel, Switzerland D (+)-glucose- C6H12O6 Sigma-Aldrich, MO, USA D (+)-sucrose- C12H22O11 AppliChem, Darmstadt, Germany Dako faramount aqueous mounting medium Agilent, California, USA Direct red 80 (Sirius red)- C45H26N10Na6O21S6 Sigma-Aldrich, MO, USA Di-sodium hydrogen phosphate 7- hydrate for analysis- Na2HPO4 · 7H2O AppliChem, Darmstadt, Germany Dithiothreitol (DTT)- C4H10O2S2 Thermo Fisher Scientific, MA, USA Dodecyl sulphate sodium salt (SDS) for analysis- C12H25NaO4S AppliChem, Darmstadt, Germany Eosin Y- C20H6Br4Na2O5 Sigma-Aldrich, MO, USA Ethanol absolute for analysis- C2H5OH AppliChem, Darmstadt, Germany Ethylenediamine-tetra acetic acid (EDTA) salt solution, 0.5 M- C10H16N2O8 Sigma-Aldrich, MO, USA Formaldehyde solution 4% (PFA) – CH2O AppliChem, Darmstadt, Germany Goat serum Thermo Fisher Scientific, MA, USA Material and methods 84 Hematoxylin Solution for clinical diagnosis, Mayer’s- C16H14O6·12H2O AppliChem, Darmstadt, Germany Hydrochloric acid technical grade 37%- HCl AppliChem, Darmstadt, Germany Hydrogen peroxide 30%- H2O2 AppliChem, Darmstadt, Germany Isoflurane- C3H2ClF5O Solvet, Segovia, Spain Isopropanol- C3H8O AppliChem, Darmstadt, Germany Isopropanol- C3H8O Scharlab Chemicals, Barcelona, Spain Magnesium chloride hexahydrate- MgCl2. 6H2O EMSURE®, Sigma-Aldrich, St. Louis, USA Methanol (MetOH) BioChemica- CH3OH AppliChem, Darmstadt, Germany Nile red Thermo Fisher Scientific, MA, USA Normal horse serum blocking solution, 2.5% Vector Laboratories, Petersburg, UK Non-fat dried milk powder AppliChem, Darmstadt, Germany Nonidet® P-40 (NP40) AppliChem, Darmstadt, Germany Oil red O (ORO) Sigma-Aldrich, St. Louis, USA Paraplast Plus Embedding Medium Leica, Wetzlar, Germany Phosphate-buffered saline (PBS) buffer 1X, Dulbecco’s- Powder AppliChem, Darmstadt, Germany PhosSTOPTM Roche, Basel, Switzerland Picric acid- C6H3N3O7 Sigma-Aldrich, St. Louis, USA Precision plus protein standards Bio-Rad, CA, USA Protease Inhibitor Cocktail Sigma-Aldrich, St. Louis, USA Ponceau S solution Sigma-Aldrich, St. Louis, USA Potassium chloride > 99.0%- KCl Merck, NY, EEUU Restore western blot stripping buffer 500 mL Thermo Fisher Scientific, MA, USA Roti®-Histokitt Carl Roth, Karlsruhe, Germany Sodium bicarbonate- NaHCO3 Sigma-Aldrich, St. Louis, USA Sodium citrate- Na3C6H5O7 AppliChem, Darmstadt, Germany Sodium chloride for molecular biology - NaCl AppliChem, Darmstadt, Germany Sodium deoxycholate (SDS)- C24H39NaO4 Sigma-Aldrich, St. Louis, USA Material and methods 85 Sodium hydroxide- NaOH AppliChem, Darmstadt, Germany Tetramethylethylenediamine (TEMED)- (CH₃)₂NCH₂CH₂N(CH₃)₂ Bio-Rad, CA, USA Tissue-Tek® O.C.T.™ compound Sakura, Barcelona, Spain Tricaine methanesulfonate- C10H15NO5S Sigma-Aldrich, St. Louis, USA Tris/glycine buffer 10x Bio-Rad, CA, USA Tri-sodium citrate 2-hydrate - HOC(COONa)(CH2COONa)2 · 2H2O AppliChem, Darmstadt, Germany Tris- C4H11NO3 AppliChem, Darmstadt, Germany Triton X-100- C14H22O(C2H4O)n(n=9-10) AppliChem, Darmstadt, Germany TRIzol™ reagent Thermo Fisher Scientific, MA, USA Tween-20- C26H50O10 Sigma-Aldrich, St. Louis, USA Vectashield® mounting medium with DAPI Vector Laboratories, Petersburg, UK Water for molecular biology- H2O AppliChem, Darmstadt, Germany Xylene- C6H10 Carl Roth, Karlsruhe, Germany 7.1.2 Standard kits and enzymes Kit/Assay Manufacturer Reference ECL™ prime western blotting detection reagents Amersham Thermo Fisher Scientific, MA, USA RPN2232 High-capacity cDNA reverse transcription kit Thermo Fisher Scientific, MA, USA 4374966 ImmPACT ® DAB Substrate Kit, Peroxidase (HRP) Vector Laboratories, Petersburg, UK SK-4105 In situ cell death detection kit, fluorescein Roche, Basel, Switzerland 11684795910 Lipopolysaccharides (LPS) ELISA Kit Antibodies-online.com, Aachen, Germany ABIN6574100 Pierce BCA protein assay kit (Reagent A: contains sodium carbonate, sodium Thermo Fisher Scientific, MA, USA 23227 Material and methods 86 bicarbonate, pierce BCA detection reagent in 0.1 N sodium hydroxide. Reagent B: bright blue clear solution free of particulate matter) OneScript Plus cDNA Synthesis kit Abm, Canada G236 Triglycerides liquicolor mono kit RAL, Barcelona, Spain GN90125 Triglycerides-LQ, Quantitative determination of triglycerides Spinreact, Girona, Spain MI41031 Enzyme Manufacturer Reference BLOXALL® endogenous blocking solution, peroxidase and alkaline phosphatase Vector Laboratories, Petersburg, UK SP-6000-100 RealQ Plus 2x Master Mix Ampliqon, Odense, Denmark A323402 SYBR GreenERTM qPCR super mix Thermo Fisher Scientific, MA, USA 11762500 7.1.3. Standard buffers and media - PBS: 10X PBS Volume/ Quantity PBS buffer (1X, Dulbecco’s)- Powder 9.95 g dH2O 1 L 1X PBS Volume 10x PBS 100 mL dH2O 900 mL - TBS and TBS-T buffers: Material and methods 87 10X Tris-buffered saline (TBS) Volume/Quantity Tris 24.2 g NaCl 80.0 g dH2O 1 L 1X TBS with tween (TBS-T)* Volume 10x TBS 100 mL Tween-20 0.5 mL dH2O 900 mL *TBS-T buffer solution pH should be adjusted at 7.6 value before being used. - Hepatic triglycerides homogenization buffer: Hepatic triglycerides homogenization buffer* Volume/Quantity PH value Tri-sodium citrate 2- hydrate 390.00 g pH = 7.5 EDTA 110.00 mg D (+)-sucrose 21.35 mg dH2O 250 mL *Hepatic triglycerides homogenization buffer should be prepared in advance and store at 4ºC to use. - Picro-Sirius red (SR) solution: SR solution Volume/Quantity Direct Red 80 0.25 g Picric acid * 250 mL Acidified water Volume Acetic acid 1.5 mL dH2O 300 mL *Picric acid is an explosive product in contact with the oxygen - Oil red O (ORO) solution: ORO stock solution* Volume/Quantity Material and methods 88 ORO powder 0.5 g Isopropanol 100 mL ORO working solution Volume ORO stock solution 48 mL dH2O 32 mL *ORO stock solution should be prepared in advanced and perfectly mixed during 2 h warming it. Working solution should be filtered three times with Whatman paper to avoid solid particles. Both solutions must be kept in dark. - TUNEL buffer solution 50 mM Na-citrate: 50 mM Na-citrate solution (TUNEL) Volume/Quantity PH value 150 mM Na-citrate 8.8 g pH = 6.0 Triton-X-100 0.2 mL 1x PBS 200 mL - RIPA and RIPA complete buffers: RIPA buffer Volume Final concentration SDS 10% 1.0 mL 0.1 % Sodium deoxycholate 0.5 g 0.5 % NP40 0.5 mL 0.5 % NaCl 5M 3.0 mL 150 mM Tris HCl, pH 7.8, 1 M 5 ml 50 mM EDTA 0.25 M 2.0 mL 250 mM dH2O 100.0 mL RIPA buffer complete Volume/Quantity RIPA buffer 9 mL DTT 20 μL PhosSTOP 1 tablet Protease Inhibitor Cocktail 1 mL - Western blot buffers: running and transfer Material and methods 89 Running buffer Volume Tris/glycine buffer 100 mL 10% SDS 10 mL dH2O 1 L Transfer buffer* Volume Tris/glycine buffer 100 mL MetOH 200 mL dH2O 1 L *Transfer buffer should be stored at 4ºC to use. - Tricaine solution for zebrafish anaesthesia: Stock Tricaine solution (50 mg/ml) Volume/Quantity Tricaine 2.5 g dH2O 50 ml - Embryo/ egg/ E3 medium for larvae zebrafish maintenance: E3 medium (60X)* Volume/Quantity NaCl 17.2 g KCl 0.76 g CaCl2. 2H2O 2.9 g MgSO4. 6H2O 4.9 g dH2O 1L *E3 should be autoclaved before using. 7.1.4. Immunoblotting gels - Stacking gel: Stacking gel (5%)* Volume dH2O 4.5 mL 30% acrylamide/bis solution 1.3 mL 10% APS 80 μL 10% SDS 80 μL Tris/HCl pH = 6.8 2.0 mL Material and methods 90 TEMED 8.0 μL Total volume 8.0 mL Separating gel* 7% 10% dH2O 10.46 mL 7.9 mL 30% acrylamide/bis solution 4.14 mL 6.7 mL 10% APS 200 μL 200 μL 10 SDS 200 μL 200 μL Tris/HCl pH = 8.8 5.0 mL 5.0 mL TEMED 12.0 μL 8.0 μL Total volume 20 mL 20 mL *Volume represented in both stacking and separating gel tables, is valid for the preparation of 2 gels. 7.1.5. Immunostaining and immunoblotting antibodies 7.1.5.1. Primary antibodies Product Host Manufacturer Reference Dilution 4-HNE (4- hydroxynonenal) Rabbit Abcam ab46545 1:100 (IHC) α-SMA (alpha-smooth muscle actin) Rabbit Abcam Ab32575 1:1000 (WB) 1:100 (IHC) BIP (binding immunoglobulin protein) Rabbit Cell Signaling 3177S 1:1000 (WB) CD45 (cluster of differentiation 45 ) Rat BD 550539 1:100 (IF) COLLAGEN I Rabbit Abcam Ab34710 1:200 (IF) CPT1-C (carnitine O- palmitoyltransferase1-C) Mouse Santa Cruz sc-514555 1:500 (WB) Material and methods 91 CYP2E1 (cytochrome P450 ) Rabbit Abcam ab28146 1:1000 (WB) F4/80 Rat BIO-RAD MCA497 1:150 (IF) FASN (fatty Acid Synthase) Rabbit Cell Signaling 273/3189S 1:1000 (WB) GAPDH (gliceraldehyde- 3-phosphate dehydrogenase) Mouse Bio-Rad MCA4739 1:5000 (WB) Ki67 Rabbit Abcam ab16667 1:100 (IF) Ki67 Rabbit Abcam ab15580 1:500 (IHC) PPAR α (peroxisome proliferator-activated receptors) Mouse Santa Cruz sc-398394 1:500 (WB) P-ACC (phospho-Acetyl- CoA Carboxylase) Rabbit Cell Signalling 3661S 1:1000 (WB) PCNA (proliferating cell nuclear antigen) Mouse Life Technologies 13-3900 1:1000 (IHC) 1:1000 (WB) SRBP-1 (sterol regulatory element- binding transcription factor 1) Mouse Santa Cruz sc-17755 1:1000 (WB) OCCLUDIN Rabbit ThermoFisher 71-1500 1:200 (WB) PHALLOIDIN Amanita phalloides Sigma-Aldrich P5282 1:500 (IF) VIMENTIN Rabbit Cell Signaling Technology 5741S 1:100 (IHC) Material and methods 92 7.1.5.2. Secondary antibodies Product Host Manufacturer Reference Dilution Anti-mouse HRP Goat Bio-Rad STAR207P 1:5000 (WB) Anti-mouse IgG, biotinylated Goat Vector Laboratories BA-9200 IHC Anti-rabbit IgG, HRP Goat Cell Signaling Technology 70745 1:3000 (WB) Anti-rabbit IgG, biotinylated Goat Vector Laboratories BA-1000 IHC Anti-rabbit IgG (H+L) Alexa Fluor 488 Donkey Invitrogen (Paisley, UK) A-21206 1:400 (IF) Anti-rat IgG (H+L) Alexa Fluor 488 Goat Invitrogen A-11006 1:500 (IF) 7.1.6. Primer sequences used for qRT-PCR Gene FORWARD (5’ – 3’) REVERSE (5’ – 3’) Acc CTGAGATTGAGGTAATGAAGATG G AGCCTGTTGAACTTTACTGGG Acox TAACTTCCTCACTCGAAGCCA AGTTCCATGACCCATCTCTGTC Apob TCCAGGTACGAACTCAAGC CACGGTATCCAGGAACAACTC Bsep TCTGACTCAGTGATTCTTCGCA CCCATAAACATCAGCCAGTTGT Cylin E GAAAAGCGAGGATAGCAGTCAG CCCAATTCAAGACGGGAAGTG Cyp27a1 AGGGCAAGTACCCAATAAGAGA TCGTTTAAGGCATCCGTGTAGA Gapdh TGTTGAAGTCACAGGAGACAACC T AACCTGCCAAGTATGATGACATC A Material and methods 93 Il-6 GCTACCAAACTGGATATAATCAG GA CCAGGTAGCTATGGTCTCC AGAA Mcp1 TTAAAAACCTGGATCGGAACCAA GCATTAGCTTCAGATTTACGGGT Ntcp CAAACCTCAGAAGGACCAAACA GTAGGAGGATTATTCCCGTTGTG Scd1 GTTCCAGAATGACGTGTACGA GGCTTGTAGTACCTCCTCTG Srebp1 ACAGTGACTTCCCTGGCCTAT GCATGGACGGGTACATCTTCAA Pparα ATTCGGCTGAAGCTGGTGTAC CTGGCATTTGTTCCGGTTCT Pparγ CACAATGCCATCAGGTTTGG GCTGGTCGATATCACTGGAGATC Tlr-2 CACCACTGCCCGTAGATGAAG AGGGTACAGTCGTCGAACTCT Tlr-4 TGGCTGGTTTACACATCCATCGG T TGGCACCATTGAAGCTGAGGTCT A Tnf- α CCTCTTCTCATTCCTGCTTGTGG GAGAAGATGATCTGAGTGTGAGG Zo-1 GCTTTAGCGAACAGAAGGAGC TTCATTTTTCCGAGACTTCACCA 7.1.7. Diets 7.1.7.1. Mice Diet type Abbreviation Reference Manufacturer Chow diet CTRL LASQC diet® Rod18-H Altromin, Lage, Germany Low palmitic western diet LP-WD D16010101 Research Diets, New Brunswick, NJ High palmitic western diet HP-WD D18121807 Research Diets, New Brunswick, NJ Material and methods 94 7.1.7.2. Zebrafish Diet type Abbreviation Reference Manufacturer Control diet, FID ZEBRA CTRL No ref- formula Sparos, Olhão, Portugal Western diet, FID ZEBRA WD No ref- formula Sparos, Olhão, Portugal 7.1.8. Composition diets 7.1.8.1. Mice CTRL LP-WD HP-WD HP-Trans- WD DUAL Main nutrients (kcal%) Protein 16 20 20 20 20 Fat 10 40 40 40 40 Carbohydrate 73 40 40 40 40 - Main Ingredients of WDs % Diet LP-WD HP-WD HP-Trans- WD DUAL Corn Oil 15 10 0 0 Soybean Oil 2.8 2.8 2.8 2.8 Palm Oil 0 5 0 0 Primex Shortening (Trans fat) 0 0 15.9 0 Primex Shortening- Z (non trans-fat) 15.9 Lard 2.2 2.2 2.2 2.2 Fructose 22.1 22.1 22.1 22.1 Cholesterol 2 2 2 22.1 High palmitic trans western diet HP-Trans-WD D09100301 Research Diets, New Brunswick, NJ DUAL model western diet DUAL D16022301 Research Diets, New Brunswick, NJ Material and methods 95 - Concentration of PA in WDs % Diet LP-WD HP-WD HP-Trans- WD DUAL C16, Palmitic (g) 13.6 30.3 29.8 Total Fat (g) 180 180 180 180 7.1.8.2. Zebrafish % Diet* CTRL- Sparos WD Fishmeal 20 20 Squid meal 34.6 34.6 Palm oil 6 Cholesterol 2 Fructose 8 % Diet CTRL- Sparos WD Fat 12.6 20.5 Fiber 7.7 0.4 Energy MJ/ kg 20.6 22 *Diameter for zebrafish adults feeding was in between 200 and 400 micrometres (µm) however larval feeding was performed by less than 200 µm smashed by the use of a mortar. 7.1.9. Instrument and equipment Instrument/Equipment Manufacturer Amersham Imager 600 GE Healthcare, Amersham, UK Antigen 2100-Retriever Aptum Biologics, Hants, UK Centrifuge 5415 D Eppendorf Hamburg, Germany Centrifuge Z233M-2 and refrigerated Hermle Labor Technik GmbH, Wehingen, Germany Cryostat CM1950 Leica Biosystems, Wetzlar, Germany Densitometer GS-800 Bio-Rad, CA, USA Material and methods 96 Drying and sterilizing ovens natural convection J.P. selecta, Barcelona, Spain Embedding center, dispenser + hot plate Leica EG1160 Leica Biosystems, Wetzlar, Germany FastPrep-24TM5G MP Biomedicals, Illkirch-Graffenstaden, France Gel chamber MINI-Protein Bio-Rad, CA, USA Glucometer Accu-Chek Roche, Basel, Switzerland Glucometer Contour Plus Elite Ascensia, Basel, Switzerland Glucose reactive strips Accu-Chek Roche, Basel, Switzerland PowerPac™ Basic Power Supply Bio-Rad, CA, USA Glass homogenizers Omni International, Inc. Kennesaw, GA, USA LI-COR Odyssey Leica Biosystems, Wetzlar, Germany LightCycler 96 Instrument Roche, Switzerland Macroscope DISKUS Z16 APO Leica Biosystems, Wetzlar, Germany Microscope SZX10 with SC50 Camera, SZX2-ILLB (stereomicroscope) Olympus, Tokyo, Japon Manual microtome RM2125 RTS Leica Biosystems, Wetzlar, Germany Microcentrifuge IR 220 VAC Carl Roth, Karlsruhe, Germany Microscope DMI6000B, Leica AF6000 LX, Live Cell Imaging Leica Biosystems, Wetzlar, Germany Microscope Eclipse Ci optical Nikon, Tokyo, Japon Microscope Fluorescence SZX2-ILLB Olympus, Tokyo, Japon Microscope Leica MZ10F, zebrafish larvae imaging Leica Biosystems, Wetzlar, Germany Microplate reader SpectraFluor Plus TECAN, Mannedorf, Switzerland Microscope, ZEISS Axio Lab.A1 Carl Zeiss Microscopy GmbH, Jena, Germany Material and methods 97 MRI Biospin 7T equipment Bruker, Germany NanoDropTM One Microvolume UV-Vis spectrophotometer Thermo Fisher Scientific, MA, USA NanoDrop ND-1000 Thermo Fisher Scientific, MA, USA Retriever (2100) Aptum Biologics, Hants, UK SPECTROstarNano spectrophotometer BMG LABTECH, Ortenberg, Germany Stereomicroscope SZX10 equipped with SC50 camera, imaging color of zebrafish Olympus, Tokyo, Japon Stereomicroscope SZX16 equipped with XC50 camera, imaging fluorescence of zebrafish Olympus, Tokyo, Japon T100 Thermal Cycler Bio-Rad, CA, USA Thermal Cycler PCR 2720 Applied Biosystems, USA Thermomixer Eppendorf Hamburg, Germany Treadmill for exercise training Harvard Apparatus, Holliston, Massachusetts Vortex Reax 200 Dismalab, Madrid, Spain Water bath HI1210 Leica Biosystems, Wetzlar, Germany Water bath Precisdig JP SelectaTM, Barcelona, Spain 7.1.10. Software Software Manufacturer Applied Biosystems 7300 Real-Time PCR System software Thermo Fisher Scientific, MA, USA AxioVision SE64. Rel.4.9 Carl Zeiss Microscopy GmbH, Jena, Germany Material and methods 98 BioRender BioRender.com (2023) CLC Genomics Workbench QIAGEN, Venlo, The Netherlands EZBiocloud Seoul, Republic of Korea GraphPad Prism version 8.1 San Diego, CA, USA Image Lab software, Version 6.1.0. Bio-Rad, CA, USA ImageJ Version 1.53t LOCI, University of Wisconsin, USA LAS X software, image analysis Leica Biosystems, Wetzlar, Germany Microsoft Office Microsoft, NM, USA MNova software Mestrelab, Santiago de Compostela, Spain Quantity One software Bio-Rad, CA, USA SILVA (v132) Bremen, Germany. 7.1.11. General materials Product Manufacturer 1.1 mL serum-gel polypropylene microtubes Sarstedt Inc, Nümbrecht, Alemania Cell culture plate sterile, 96 Well, flat- bottom with lid Greiner Bio-One, Kremsmünster (Austria) Coverslips Hirschmann, Eberstadt Germany Cryogenic tubes Thermo Fisher Scientific, MA, USA Dako pen Agilent, California, USA Eppendorf reference 2G single-channel, fixed, volume (10 μL, 100 μL, 1000 μL) Eppendorf Hamburg, Germany Eppendorf tubes (1.5 mL, 2.0 mL) Thermo Fisher Scientific, MA, USA Falcon tube (15 mL, 50 mL) Labbox, Barcelona, Spain Gavage needle Kent Scientific, Torrington, USA Graduated filter tip (sterile) 10, 100, 1000 μL Starlab, Barcelona, Spain Histological cassettes with lid Labolan, Navarra, Spain Material and methods 99 7.2. Methods 7.2.1. Animal maintenance 7.2.1.1. Mice All mice strains were maintained in the animal facility of the Faculty of Biology at Complutense University of Madrid (UCM) in a temperature-controlled room with 12 h light/dark cycle and as free access to food and water, according to the guidelines of the Federation for Laboratory Animal Science Associations (FELASA). All animal procedures were carried out according to Spanish legal requirements and animal protection law and approved by the authority of environment conservation and consumer protection of the Regional Government of Madrid (PROEX- 125.1/20, 397.2/21). Histological cassettes, plastic, 24*24*5mm Labolan, Navarra, Spain MicroAmp fast optical 96 well reaction plate, 0.1mL Thermo Fisher Scientific, MA, USA MicroAmp optical adhesive film Thermo Fisher Scientific, MA, USA Micropipete tips (100 μL, 200 μL and 1 mL) Grenier Bio-One, Kremsmünster (Austria) Mini Trans-Blot® electrophoretic transfer cell Bio-Rad, CA, USA Pipettes (5 mL, 10 mL, 25 mL) Grenier Bio-One, Kremsmünster (Austria) Polyvinylidene difluoride (PVDF) membrane (0.45 μm or 0.22 μm) Bio-Rad, CA, USA Reactive strips Roche, Basel, Switzerland Slides. Microscope KP Silan printer slides. Adhesive. Ready to use. Klinipath, Duiven, Netherlands Syringes 1 mL BD Plastipak, New Jersey, USA Syringe 1 mL with 27 x 13 needle BD Plastipak, New Jersey, USA Whatman® paper Bio-Rad, CA, USA Material and methods 100 7.2.1.2. Zebrafish Zebrafish (Danio rerio) wild- type were raised under standard laboratory conditions with 14-h light/ 10- dark cycle at a temperature of 28.5 ± 1ºC. Fish were fed with a standard diet including freeze-dried rotifer, fresh artemia culture, and flakes. Juvenile fish were fed 5 times a day (Rotifer, baby powder, and artemia) and adult fish were fed 2 times a day (artemia and fish flakes). For embryo and larvae experiments, fish were kept at 28.5 °C incubators in an E3 fish or embryo medium. Aquariums were filled by filtered water coming from the system. All the experiments and protocols have been approved by the Animal Ethics Committee of Bilkent University, Ankara, Turkey. 7.2.2. Animal strains 7.2.2.1. Mice For our studies, we have used 10 weeks old male C57BL/6J mice, obtained from Janvier Labs, France. 7.2.2.2. Zebrafish AB zebrafish were used at embryo and adult stages, both males and females, and FABP (fatty acid binding protein) males and females, were just used at embryo stage. Embryos were treated from 6 days post fertilization (dpf) up to 10 dpf, while adult zebrafish has just started in animals older than 1.5 years old. 7.2.3. Development of preclinical models 7.2.3.1. Mice 7.2.3.1.1. Western diet (WD) Mice were randomly assigned into 4 groups with a sample size of 7 to 9 animals in each. Control (CTRL) group was fed with chow diet and normal water. Three treated groups received different types of Western Diet (WD) (7.1.7 Diets - Material and methods section) and sweetened drinking water containing 6.75% D-glucose, for 14 weeks* (Figure 12). Adding glucose (in the drinking water) together with fructose (as a Material and methods 101 component of the WD) lead to more harm as glucose potentiates fructose absorption from the gut, while fructose catalyzes glucose uptake and storage in the liver [139]. Figure 12. A schematic description of the WD feeding: CTRL group was fed with chow diet and normal water while the experimental groups were fed with three different types of WD, differentiated by the total amount of palmitic acid (PA), in their components and the origin of the fat from their ingredients. Low palmitic western diet (LP-WD), high palmitic western diet (HP-WD) and HP-trans- WD (HP-trans- WD). Created with BioRender.com. 7.2.3.1.2. Western diet withdrawal For the withdrawal study, mice were exposed to WD and sweetened drinking water for 14 weeks, followed by 20 days of chow diet and normal water. The corresponding controls received chow diet and normal water for the equivalent amount of time (17 weeks) (Figure 13). Material and methods 102 Figure 13. Scheme that shows WD withdrawal procedure. CTRL and WD experimental groups were fed as previous described*, during 14 weeks. After this feeding period, WD and sweet water was removed from the three treated groups and replace by chow diet and normal filtrated water for 20 days. Created with BioRender.com. 7.2.3.1.3. DUAL diet: DUAL withdrawal and combination with physical exercise Mice were randomly assigned into 4 groups with a sample size of 5 to 9 animals in each. A DUAL diet feeding was performed for 10 and 23 weeks. DUAL diet is composed by a Western diet (WD- D1602230) and 10% v/ v absolute EtOH in drinking sweetened water (6.75% D (+)-glucose). WD is enriched in fat and cholesterol. Regular rodent chow diet (Rod18-H) and filtered tap water was used as corresponding control diet. In is important to take into account, that for the introduction of the EtOH in the drinking water, adaptation period should be performed by increasing EtOH concentration gradually (1%, 2%, 4%, 5% until 10% v/v at week 4). This adaptation period is included in the total total length of the treatment. Low EtOH intake due to natural aversion in mice is the main limiting factor for DUAL feeding. In this model the taste of alcohol is masked by adding sweet substrate to the drinking water. Moreover, fructose (as a component of the WD) and glucose (in the drinking water) together lead to more harm as glucose potentiates fructose absorption Material and methods 103 from the gut, while fructose catalyzes glucose uptake and storage in the liver [139]. Mice usually consume alcohol up to 25% vol/vol [140]. In WITHDRAWAL (WTD), after either 10 or 23 weeks of DUAL diet, western diet food was replaced by regular chow diet and water with ethanol and glucose, was changed to the normal tap water. Moreover in DUAL removal plus physical exercise mice, replacement of the diet was follow in paralel by a non volunteer physical exercise training, 5 days per week (Figure 14). Figure 14. A schematic description of DUAL diet in a murine model. C57BL/6J wild type (WT) male mice, were fed with either chow diet and filtered tap water, or western diet (WD) plus 10% ethanol in sweetened water (6.75% glucose). Created with BioRender.com. Upper right: BIDI code with Treadmill machine video for physical exercise. 7.2.3.1.4. Physical exercise After DUAL diet removal, food and water replacement by regular chow diet and filtered tap water was combined with non-volunteer physical exercise 5 days per week (Monday- Friday). Exercise training was performed every day, using a two-lane Treadmill (Harvard Apparatus) for mice. Material and methods 104 Treadmill equipment consists of a rolling belt with adjustable speed (up to 150 cm/s) and slope (from -25 to 25 degrees) and a control unit. The rolling belt is built with especially selected materials to guarantee the best performance under conditions of intensive use and the minimum operations of maintenance, as well as simplicity in keeping it clean. The lanes (corridors of activity for the animal) have sufficient width for the subject to correct its errors in coordination, thereby allowing an exact measurement of the fatigue without deficiencies in motor coordination. The treadmill unit controls the speed of the belt, shows measured data in its touchscreen display and provides electrical shock to the grid. The electrical shock supplied by the grid has a constant intensity (from 0 to 2 mA), which is the electrical power that circulates through the animal and only depends on the value of the mA chosen and not of the subject (quantity of body mass in contact with the bars or perspiration). Procedure was performed according to the following protocol: - Week 1: 10 minutes (min) training at 10 metres (m)/ min - Week 2: 20 min at 10 m/ min - Week 3: 30 min at 15 m/min 7.2.3.2. Zebrafish 7.2.3.2.1. DUAL model in larval zebrafish - AB zebrafish DUAL model AB zebrafish breeding couples were set at midday (2:30-3:30 p.m.) into breeding tank, keeping a separator in between males and females. The following day in the early morning, 8:30 a.m., separator was removed. About 3-4 hours later, 11:30-12:30, while embryos were already released into the water, embryos were collected into a petri dish filled with embryo media (E3). Empty or dead embryos were discarded before keeping the plates into the incubator at 28.5ºC. AB and FABP zebrafish larvae were placed into 6 wells petri dish plates filled with embryo medium, at 5 dpf. Experimental trial has started at 6 dpf, after 1 day adaptation period, up to 10 dpf. Fish were fed two times per day with commercial Sparos food, with pellet size smaller than 200 µm, that was also smashed by the use of a mortar in order to obtain Material and methods 105 smaller particles and help animal feeding. 25 larvae were place in each of the wells and E3 media was daily replace in all feeding trials (Figure 15). Both constant and intermittent feeding procedures, were tried. For intermittent feeding, powder food was placed into the well, by the use of a forceps, and kept for 15 minutes, followed by E3 media removal and replaced with clean one, with or without EtOH. In order to set up, the ideal conditions for DUAL model in zebrafish, pilot studies with different EtOH concentrations were tried: 0.15%, 0.25%, 1% and 1.5% from 6dpf. DUAL group was fed by WD and either 0.15% or 0.25% EtOH diluted in the embryo media. The corresponding control groups were fed by either control powder food (Sparos), control food and 0.15% or 0.25% EtOH E3 media or just WD. Exposure solution was changed every 24 hours and the dead embryos were removed in order to prevent the contamination of the surviving embryos. Figure 15. Scheme of AB larval feeding procedure. 6dpf larvae were place into 6 wells plates filled with E3 with or without treatment. DUAL fish were kept in E3 media with either 0.15 or 0.25% vol/vol EtOH and fed with WD. The correspondent control groups were run in parallel. CTRL group was fed with control powder food (Sparos), as well as EtOH group, that has been kept in 0.15% or 0.25% EtOH E3 media. WD larvae were fed with the correspondent WD powder food. Created with BioRender.com. Material and methods 106 - FABP zebrafish DUAL model FABP larvae zebrafish were place into 6 well plates (25 larvae per well) filled by E3 media. Treatment has been started at 6dpf until 10 dpf, by daily feeding and replacement of E3 fresh media alone or in combination with EtOH. Daily control of the morphology, movement and behaviour of the larva was done, and life imaging by the use of a fluorescence microscope was performed at 10 dpf (Figure 16). WD groups was fed by Sparos WD while in DUAL, WD feeding was combined with 0.25% EOH in the media. CTRL group was fed with control diet (CTRL), as well as EtOH in with 0.25% EtOH in was included into the embryo media solution. Figure 16. Scheme of FABP feeding procedure. 6dpf larvae were place into 6 wells plates filled with embryo media with or without treatment. CTRL group was fed with control powder food, as well as EtOH group, that was also kept in 0.25% EtOH E3 media. WD larvae were fed with the correspondent WD powder food along with DUAL fish, that were also immerse in 0.25% EtOH media. Created with BioRender.com. 7.2.3.2.2. DUAL model in adult zebrafish 1.5 years old AB fish were removed from the system and placed into independent aquarium, including 6 males and 6 females AB adult zebrafish were placed in each of them, filled with filtered water. Each of the tanks was containing an independent water filter and a heater, to keep a constant temperature of 28.5 ± 1ºC. Adaptation was performed during 3 days in the new aquarium, by keeping their regular feeding trial. Upon this period, Sparos food, either control or WD, was introduce into the Material and methods 107 regime. Fish were treated with a DUAL diet consisting of WD (Sparos) and 0.25% vol/vol EtOH absolute, diluted in water. Controls were fed with either normal diet only (Sparos), WD only, or EtOH. Food pellet size for adult feeding was in between 200 and 400 µm (Figure 17). 2 litres of the water with diluted ethanol solution were daily replaced, to avoid any decrease in the ethanol concentration due to evaporation and to maintain a clean housing environment. Whole water tank replacement was performed 2 times per week, to avoid abrupt changes in the fish environment and minimize stress levels. Figure 17. A schematic description of DUAL diet in a zebrafish adult model. 12 AB adult zebrafish (6 females and 6 males), were placed at independent aquariums. DUAL fish were fed with WD dry flake and 0.25% EtOH in the water, while the corresponding control groups were fed with either control dry flake (Sparos) (CTRL), CTRL diet + 0.25% EtOH in the water or WD, in parallel. Created with BioRender.com. 7.2.4. Basal glucose and glucose tolerance test 7.2.4.1. Mice At the end of the treatment, glucose levels in blood were measured after 12 h overnight fasting by glucose tolerance test (GTT). An amount of 7.5 g/kg body mass of 20% glucose solution was gently administrated by intraperitoneal (IP) injection, in order to perform the glucose tolerance test (GTT) to determine possible individuals with impaired glucose tolerance and type diabetes mellitus (T2DM) [141]. Glucose levels were measured using an Accu-Chek glucometer (Roche) and reactive strips at 0, 5, 30, 60, 90 and 120 minutes using established protocols [141]. For the IP injection, the area was disinfected with 70% EtOH, and 1 mL syringe with 27G needle was introduced in the lower part of the abdomen to avoid the damage to the Material and methods 108 urinary bladder, caecum and other abdominal organs. Syringe was inserted by a 40º angle to horizontal plane and pulled back to ensure negative pressure prior to injecting. The total volume injected per mouse was calculated according to the BW, being the maximum allowed volume of administration per mice < 10 mL/kg [141]. 7.2.4.2. Zebrafish At the end of the treatment, basal glucose levels of adult fish were tested after 6 h fasting. Measured was performed by cutting the dorsal fin, and blood drop was collected by the use of a glucometer (Contour Plus Elite glucometer- Ascensia- Diabetes Care). 7.2.5. Food/water intake and body weight measurement - Mice Food and water intake were measured once per week. Food was weekly weighted (g) and water volume was measured (mL) and recorded. Approximately 60 g of food were kept weekly in every cage, and periodically refilled with storage food (4ºC). Total kcal ingested were calculated according to the energetic value of food and drinking water. The equivalences were: Chow diet = 3 kcal/g, WD = 4.5 kcal/g, D- glucose = 4 kcal/g and EtOH = 7 kcal/g. Moreover, to monitor and control the body weight (BW) of the mice, we have weighed them every week, on the same day and at approximately the same time in the afternoon. Body surface of the mice was calculated using the data from the sacrifice day and by the formula: Body mass index (BMI) was calculated by dividing the BW in grams by the body length: Body surface= 0. 007184 ∗ 𝑏𝑜𝑑𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 (𝐵𝑊)𝑖𝑛 𝑘𝑔 ∗ 0.425 ∗ 𝐵𝑜𝑑𝑦 𝑙𝑒𝑛𝑔𝑡ℎ (𝑐𝑚)!.#$% BMI= !"($) &'() +,-./01 Material and methods 109 - Zebrafish Larvae fish were fed ad libitum with powder food included in the E3 media. Both intermittent (15 minutes feeding 2 times a day, by replacement with E3 media after feeding period) and constant, by keeping the food in the E3 media until the following day were tried, feeding trials were performed. Adult feeding was performed 2 times per day, according to the amount of fish included in each of the aquarium and the BW at the initial point of the trial, and following the recommendations given by the manufacturer, that corresponds to a 5% of the total biomass. Fish should stop being interested in food following around 1 min, however as CTRL and WD are not isoenergetic and fish from the WD treatment may tend to be satiated easily, an adjustment of feeding based on daily observation should be performed. 7.2.6. Animal dissection 7.2.6.1. Mice Twelve-hours fasted mice were sacrificed by an overdose of isoflurane (Solvet). BW measure of the mice was performed before and after the fasting. Consecutively, mice were opened at the abdomen along the linea alba and blood, and internal organs were collected. - Blood Blood was taken from the vena cava inferior with a 1 mL syringe (27G needle) for serum analysis. Serum was separated in 1.1 mL Serum-Gel Polypropylene microtubes (Sarstedt) by centrifugation at 12000 rpm, 10 min, 4ºC, collected and stored at -80ºC for subsequent determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), cholesterol, lactate dehydrogenase (LDH) and TG levels. Serum was diluted 1:5 for measuring serologic markers and the analysis of blood was performed at the University Hospital RWTH Aachen, Germany, using automated analyzers. - Organs First, liver and epididymal white adipose tissue (eWAT) were removed and weighed as well as intestine samples that were collected, and length was measured. After that, organs were washed with cold PBS and liver images were taken in a MZ16 Stereo Material and methods 110 microscope with a Leica DFC480 digital camera connected to the DISKUS Z16 APO (Leica) macroscope. Liver and eWAT samples were cut into pieces and part of the tissue was stored in plastic cassettes, fixed in 4% formaldehyde (PFA- AppliChem) and embedded in paraffin to perform different stainings as hematoxylin and eosin (H&E), Sirius red (SR), immunohistochemistry (IHC) or immunofluorescence (IF). Moreover 2 pieces of liver tissue were preserved in Tissue-Tek O.C.T.TM (Sakura) compound and kept in -80°C for Oil red O (ORO) and immunofluorescence (IF) staining. Although, some of the tissue pieces were stored in cryotubes (Thermo Fisher Scientific), frozen in liquid nitrogen and kept at -80ºC for RNA and protein isolation, or performing other protocols on demand, like TG content measurement, or lipidomic analysis. Regarding intestine tissue processing, the full intestine, including caecum, was divided into its structural parts: colon, ileum, jejunum, and duodenum. The four parts were at the same time divided into 3 smaller pieces and each of them was preserved for performing a different technique: paraffin inclusion, O.C.T. embedding, or cryopreservation in -80ºC, until RNA or protein extraction. 7.2.6.2. Zebrafish - Larvae At end point of the treatment, they were sacrificed by freezing method, introducing the petri dish into a -20ºC freezer. - Adults Fish were fasted for 6 h and sacrifice by ice shock (tricaine anaesthesia plus ice shock). Body length and weight of the fish was recorded at the end point of the treatment and body mass index was calculated accordingly. Liver and intestine were collected and kept into cryotubes in -80ºC for RNA and protein isolation. Some of the fish were embedded in paraffin and hematoxylin and eosin (H&E) staining was performed. 7.2.7. Hepatic triglycerides (TG) content- mice Hepatic TG concentrations from liver were measured following the manual guide of Material and methods 111 triglycerides liquicolor mono kit. An amount of 21-23 mg of liver tissue was homogenized on ice with a manual glass homogenizer in 1 mL homogenization buffer (standard buffer and media) previously prepared and cooled, or by mechanical homogenization by the use of Lysing matrix tubes and FastPrep TM. The homogenate solution was centrifuged for 5 min at 1200 rpm, 4ºC and the upper two layers were gently mixed and transferred to a new tube. Samples were dilute in homogenization buffer (1:3) and 2 μL were mixed with 200 μL kit reagent in a 96 well- flat bottom plate. After an incubation of 45 min at RT, absorbance was measured at 450 nm in a spectrophotometer (BMG LABTECH). A standard curve was prepared diluting standard solution in dH2O at 50 mg/dL, 75 mg/dL, 100 mg/dL, 150 mg/dL,175 mg/dL; dH2O was used as a blank control. Liver triglycerides content were calculated according to the absorbance of the standard curve and the grams of liver used. 7.2.8. Histological analysis 7.2.8.1. Tissue processing and embedding- mice, zebrafish One or two small pieces of the tissue were included into each cassette and fixed in 4% formaldehyde (PFA) for 48h. In the case of adult zebrafish, head and tail were removed and several PFA injections were performed among the skin, in order to facilitate the fixation of the internal organs. After fixation step, samples were washed with tap water 30 min, by changing the water frequently, before starting the dehydration procedure. If the embedding was not performed right after the fixation, samples were washed in 1x PBS (3 x 10 min) and stored at 4ºC. Next steps of the protocol were followed: - Dehydration: samples were dehydrated in 50% EtOH for 30 min and subsequently in 70%, 85% and 95% EtOH tanks for 1 h in each. Tissue was kept in fresh 95% EtOH tank overnight and the following day cassettes were transferred into 100% EtOH for 30 min, twice. - Cleaning: tissue pieces were kept in absolute EtOH: xylene (1:1) for 30 min and after that, two times in xylene for 30 min. - Wax infiltration: three tanks of wax, previously warmed at 70ºC in the water bath, were used for the wax infiltration, keeping the samples in each tank for 1 h. - Embedding: small pieces of tissue were embedded into paraffin blocks with an Material and methods 112 embedding center, constituted by a dispenser and a hot plate machine (Leica). Paraffin sections were at 5 μm thickness using a manual microtome (Leica) and a water bath (Leica), followed by slices drying at room temperature (RT). 7.2.8.2. Hematoxylin and eosin (H&E) staining- mice, zebrafish Hematoxylin and eosin (H&E) staining was done in paraffin sections of liver, and eWAT mice tissue, and whole staining of adult zebrafish. Deparaffinization and rehydration of the samples were performed as follow: 3 x 5 min xylene, 2 x 5 min 100% EtOH, 2 x 5 min 95% EtOH, 2 x 5 min 70% EtOH, 2 x 5 min dH2O. For nuclear staining, slides were immersed in hematoxylin for 30 sec, rinsed in tap water for 10 min and 2 x 5 min in dH2O. Next the slides were immersed in eosin and followed by dehydration: 2 x 10 sec 95% EtOH, 2 x 10 sec 100% EtOH, 3 x 5 min xylene. Finally, the stained paraffin sections were mounted with a coverslip using Roti®-Histokitt and pictures were acquired in a 20x magnification with an Eclipse Ci optical microscope (Nikon). 7.2.8.3. Sirius red (SR) staining- mice In order to determinate collagen deposition, Sirius red (SR) staining was performed. First, paraffin sections were deparaffinized by incubation in 60ºC for 30 min, followed by 2 x 5 min xylene and rehydrated using a descending percentage of EtOH series (2 x 2 min 100% EtOH, 2 min 95% EtOH, 2 min 80% EtOH and 2 min dH2O). Subsequently, slides were incubated for 1 h in Picro-Sirius red solution. Afterwards, staining was fixed by 4 min incubation in acidified water. Picro-Sirius red solution and acidified water are described in section standard buffer and media. Then, sections were dehydrated in 100% EtOH (2 x 30 seconds) and cleared in xylene (2 x 5 min). Roti®-Histokitt was used for mounting stained paraffin sections and pictures were taken in a 20x magnification using an Eclipse Ci optical microscope (Nikon). Collagen deposition was showed as red fibers in the liver and positive area was quantified via Image J© software (National Institutes of Health). 7.2.8.4. Oil Red O (ORO) staining- mice To detect neutral lipids in liver tissue ORO staining was performed in 7 μm frozen liver sections previously cut in a cryostat (Leica). Samples were fixed in 4% PFA for 25 min (frozen sections were previously air-dried for 40 min) and washed 3 x 5 min in dH2O. Material and methods 113 Subsequently, samples were stained for 40 min with ORO working solution and counterstained with hematoxylin (nuclear staining). After ORO working solution and hematoxylin steps, slides were shortly washed with dH2O and rinsed with tap water by frequent water replacement until water was clear. Slides were mounted with a ready to use Dako faramount aqueous mounting medium (Dako). After staining, pictures were taken with an optical microscope (Nikon), and lipid content was quantified by Image J© software (National Institutes of Health) following the protocol previously described [142]. 7.2.8.5. Immunohistochemistry (IHC) staining- mice Paraffin sections cut to a thickness of 5 μm were preheated at 60ºC for 30 min in the oven and subsequently deparaffinized and rehydrated with xylene and serial EtOH solutions (from 100% to 70%), respectively. After that, antigen unmasking was performed by boiling sections in 10 mM sodium citrate buffer (citrate/PBS-T, pH = 6.0) using a pressure- cooker Antigen 2100-Retriever (Aptum Biologics). When a cycle of the pressure-cooker is completed, slides were removed from the citrate buffer and washed in dH2O 3 x 5 min. Afterwards, samples were surrounded with a Dako Pen that contains a hydrophobic solution, to individually stain each section and all the procedures were performed in a humid chamber to avoid dehydration of the sections. BLOXALL® endogenous blocking solution (Vector Laboratories) was added to the samples during 10 min to inactivate endogenous peroxidase. After removing the blocking solution, samples were washed 2 x 5 min dH2O plus 1 x 5 min PBS. To avoid non-specific binding of immunoglobulins in the tissue, samples were blocked with 50 μL 2.5% normal horse serum blocking solution (Vector Laboratories) for 30 min at RT. After that, sections were incubated with primary antibody diluted in PBS with 1% BSA and 0.3% Triton (antibodies dilution are provided in Immunostaining and Immunoblotting section) for overnight at 4ºC. The following day, slides were washed 3 x 5 min with PBS and incubated with biotinylated secondary antibodies (Vector Laboratories) during 1 h at RT. To eliminate the excess of secondary antibody, slices were washed 3 x 5 min in PBS and signals were developed with DAB (Vector Laboratories). The incubation with DAB ranges from 30 sec to 5 min depending on the target antigen. Positive areas/cells stain in brown when the DAB reacts with the biotinylated secondary antibodies. After that, sections were submerged in dH2O and counterstained with hematoxylin for 40 sec. Dehydrating was performed in increasing percentages of EtOH and then in xylene. Slices were mounted with Roti®- Histokitt, and pictures were taken using an optical microscope (Nikon). Image J© software Material and methods 114 (National Institutes of Health) was used to quantify positive cells. 7.2.8.6. Immunofluorescence (IF) staining- mice Immunofluorescence (IF) stainings were performed in 5 μm frozen liver sections that were air-dried for 20 min, fixed in 4% PFA (AppliChem) and washed with PBS 3 x 5 min. PBS containing 5% goat serum and 0.3% Triton was used for blocking step (1 h, RT) followed by incubation with primary antibody in blocking solution at optimized dilutions overnight at 4°C. List of primary antibodies are provided in Immunostaining and immunoblotting antibodies section. On the second day, slides were washed with PBS 3 x 10 min and incubated with anti-rat secondary antibody labelled with Alexa Fluor 488 (Thermo Fisher Scientific) for 1 h at RT. Then, slides were washed 3 x 10 min in PBS and nuclei were counterstained with Vectashield mounting medium containing DAPI (Vector Laboratories). A humidity box was used to keep the slides wet and prevent them from drying out. Microscopy and image acquisition were performed using an Microscope DMI6000B (Leica) and both LAS X (Leica) and Image J© software (National Institutes of Health) were used to quantify positive cells. 7.2.8.7. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining- mice Cell death was determined with terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. During late stages of programmed cell death, or apoptosis, DNA becomes highly fragmented. This fragmentation provides an opportunity to attach a modified dUTP to the 3’-OH ends of the damaged DNA using the enzyme terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) reaction. The modified dUTP, such as BrdUTP or EdUTP, can then be detected. Frozen 5 μm cryosections from mouse liver were dried in air for 30 min, fixed in 4% formaldehyde at RT for 20 min, followed by 3 x 10 min wash in PBS. After that, slides were incubated in 30% hydrogen peroxide diluted in Methanol (MetOH), (20 mL 30% H2O2 + 180 mL MetOH), followed by 10 min washed in PBS. To have access to the DNA, the nuclei were permeabilized by the use of a 150 mM Na-citrate buffer (8.78 g Na-citrate in 200 ml of PBS and 200 µL of Triton-X-100), with 6 pH value (Standard buffer and media section). Material and methods 115 7.2.8.8. Nile red in vivo staining- larvae zebrafish Nile red staining was performed at 10 dpf. The previous day, AB larvae were O/N fasted and kept in fresh media upon avoiding intervention of any external factors in the staining. Nile red day visualizes deep tissue fat deposition in zebrafish. Larvae were incubated with 0.5 ug/ ml Nile red at room temperature for 1 h in the dark and live images were taken. 7.2.8.9. BODIPY in vivo staining- larvae zebrafish At 10 dpf, BODIPY essay was performed. BODIPY™ 493/503 is a fluorescent lipid analogue to trace lipid in vivo and can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids. Life larvae were incubated in BODIPY solution for 1h, followed by several washing steps, to avoid imaging background, and right after picture record was performed in order to trace the lipid location. 7.2.8.10. Imaging and staining analysis Mounted samples were observed in the microscope (optical - Nikon - and fluorescent - Carl Zeiss Microscopy GmbH - microscopes) and pictures were taken for subsequent image analysis. Images were randomly taken at 20x and 40x magnification and were analyzed in Image J© software (National Institutes of Health). Area was quantified for SR, ORO, αSMA, Collagen I and positive cells were counted for TUNEL, Ki67, CD45 and F4/80 stainings. H&E and SR sections were analyzed by an expert liver pathologist blinded to the dietary condition. Samples were classified according to the NAFLD fibrosis score, which is used to distinguish between patients with non-alcoholic fatty liver disease and different ranges of liver fibrosis (F0-F4) based on METAVIR score system [143]. Fibrosis score F0 No fibrosis F1 Periportal fibrosis: F1a: mild zone fibrosis F1b: moderate zone perisinusoidal fibrosis F1c: periportal fibrosis Material and methods 116 F2 Periportal fibrosis with septa F3 Periportal fibrosis with numerous septa F4 Cirrhosis 7.2.9. RNA isolation and analysis 7.2.9.1. RNA isolation RNA from liver and intestine cryopreserved tissue (mice and zebrafish) was isolated with Trizol (Thermo Fisher Scientific) reagent following the manufacturer’s recommendations. The place to work with RNA should be perfectly cleaned with 70% EtOH, contamination and RNAses are frequently problems in this technique. 50-60 mg of liver tissue was homogenized in a FastPrepTM with Trizol and right after supernatant was collected into a new Eppendorf tube. Samples were centrifuged 10 min, 12000 rcf (relative centrifugal force) at 4ºC. After the centrifugation step samples were separated into a lower red phenol- chloroform, an interphase and a colorless upper aqueous phase. Then, in between 200 to 500 μL from the aqueous phase containing the RNA were transferred to a new tube. To precipitate the RNA, samples were incubated for 15 min with the same amount of the obtained aqueous phase 200-500μL of isopropanol (AppliChem) and centrifuged for 10 min at 12000 rcf at 4ºC. Total RNA precipitate forms a white gel-like pellet at the bottom of the tube, and supernatant was discarded by decantation and micropipette. RNA was washed by resuspending the pellet in 1 mL 70% EtOH and centrifuged 10 min, 12000 rcf at 4ºC, this process was repeated three times. Supernatant was discarded with a micropipette and pellet was dry at RT. Finally, to solubilize the RNA, the pellet was diluted with water for molecular biology and vortexed 20-30 sec at 65ºC, maximum speed in the thermomixer (Eppendorf). Samples were stored at -80ºC. 7.2.9.2. Reverse transcription from RNA to complementary DNA (cDNA) For the complementary DNA (cDNA) synthesis, which consists in the transcription of a single-stranded RNA to cDNA, RNA concentration of each sample was measured in a Nanodrop (Thermo Fisher Scientific) using an optical density (OD) of 260 nm and the Material and methods 117 purity of the sample was determined according to the E260/E280 ratio; a protein free nuclei acid solution typically has a 1.8-2.0 ratio. According to the RNA concentration measurements, each RNA sample was standardized into a 1 μg/μL dilution. Reverse transcription was performed using an Applied Biosystems™ high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) following the manufacturer’s protocol: Reagent Volume RNA (1 μg/μL) 10 μL Water for molecular biology 4.2 μL 10x buffer RT 2 μL 25x dNTPs 0.8 μL 10x random primer 2 μL Enzyme Reverse transcriptase 1 μL Total volume 20 μL Reactions were incubated in a PCR thermocycler (T100 thermal cycler, Bio-Rad) following the next protocol: Finally, samples were removed from the thermocycler and 85 μL of water for molecular biology was added to each of the samples that were preserved in -20ºC upon to be used. 7.2.9.3. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Quantitative real-time PCR (qRT-PCR) is a method used for the amplification and quantification of DNA products. The DNA amplification is monitored at each cycle of the PCR and is based on fluorescent detection. The fluorescent signal increases with increasing PCR cycles. The point at which the fluorescence becomes measurable is called the threshold cycle (CT), so that lower CT values denote higher amounts of the gene of interest. Temperature Time (h:min:sec) 25 ºC 00:10:00 37 ºC 02:00:00 85 ºC 00:05:00 4 ºC Hold Material and methods 118 qRT-PCR was performed in 20 μL reaction volume using SYBR Green Master Mix (Thermo Fisher Scientific, Invitrogen) and a 7300 real time PCR system by the Genomics and Proteomics Facility (Faculty of Biology, UCM). At the end of the PCR, baselines and threshold values were established using AB7300 Real-Time PCR System software (Thermo Fisher Scientific), and the CT values were exported to Microsoft Excel to calculate relative messenger RNA (mRNA) expression according to the ΔΔCT method [87], which determinate the relative quantification of a target gene in comparison to a housekeeping gene (Gapdh). The list of primers used is provided in the Primer sequences used for qRT-PCR section. To perform the qRT-PCR analysis, the reaction mix and program was used as follows: qRT-PCR reaction mix Volume Primer forward 1 μL Primer reverse 1 μL SYBR Green ERTM qPCR super mix 10 μL cDNA 5 μL Water for molecular biology 3 μL Total volume 20 μL PCR program Temperature Time(h:min:sec) Cycles Activation 95 ºC 00:00:10 Denaturing 95 ºC 00:00:15 x 40 Annealing/Elongation 60 ºC 00:01:00 Conservation 4 ºC hold To perform the dissociation curve, the following steps were added to the PCR program: Temperature Time (h:min:sec) 95 ºC 00:00:15 60 ºC 00:01:00 95 ºC 00:00:15 Material and methods 119 7.2.10. Protein isolation and analysis 7.2.10.1. Protein extraction and quantification Frozen liver and colon tissue from mice, were used for protein extraction. Approximately, 50 mg frozen tissue was homogenized on ice with 500 μL RIPA complete buffer (Standard buffer and media number of section), by the use of a FastPrepTM. Homogenized tissue was transferred into a new tube, and kept on ice for 15 minutes, followed by a 10 min centrifugation step 12000 rpm at 4ºC. The upper layer was taken to a new tube and protein quantification was calculated using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), based on a colorimetric detection. For the protein quantification, protein was diluted with PBS buffer up to a 1:20 proportion. Diluted samples were mixed with working buffer, constituted by A:B buffer at a 50:1 proportion in a 96-well plate and incubated during 30 min at 37ºC. The plate was read in a spectrophotometer set to 562 nm and protein concentration was calculated according to the OD562 of the standard curve of BSA. RIPA complete buffer was used as a blank. The final concentration of the samples was adjusted to 4 μg/μL, by mixing them with RIPA and 4X Laemmli buffers. Samples were denatured 10 min at 95ºC shaking in the thermomixer and stored at -80ºC. 7.2.10.2. Immunoblotting assay (WB) Protein expression was detected performing immunoblotting technique or Western blot (WB). Liver and colon samples were defrosted, homogenized by vortex and a short spin. Handmade gels containing 5% polyacrylamide stacking gel and separating gel that range from 7 to 10% according to the molecular weight of the proteins were used (Immunoblotting gels section). An amount of 60-80 μg of tissue were loaded and separated at 100-120 V in running buffer. After the electrophoresis, the separated proteins were transferred from the gel onto a Polyvinylidene Difluoride (PVDF) membrane (0.45 μm) making a sandwich with Whatman paper in a wet chamber for 2h at 300 mA on ice following the standard protocols [144]. Running and transfer buffers were prepared as is indicated in materials section (Standard buffer and media). To confirm the correct transferring of the proteins, Ponceau S solution (Sigma-Aldrich) was used. Then, the membranes were incubated in 5% non-fat dry milk or BSA diluted in 0.1% TBS-Tween (TBS-T) to block non-specific binding sites. Material and methods 120 After 1 h, membranes were incubated with the optimized dilution of primary antibody in 2.5% non-fat dry milk or BSA, shaking overnight at 4ºC. The next morning, to get rid of non- bound antibody, the membranes were washed 3 x 10 min in TBS-T and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody diluted in 2.5% non- fat dry milk or BSA. Subsequently, membranes were washed 3 x 10 min in TBS-T. Target bands were visualized incubating the membrane in Amersham ECL Prime (GE Healthcare) for 2 min and exposing it to an odyssey Fc Imaging system (LI-COR) or Amersham Imager 600 (GE Healthcare) until specific signals were detectable. Primary and secondary antibodies’ dilution are provided in Immunostaining and immunoblotting antibodies section. 7.2.11. Lipidomic analysis: lipid extraction and quantification To prepare lipid extracts from liver, the tissue was homogenized using cold PBS 1X and 1-2 mg of tissue, and extraction was performed by using the Folch method [145]. For this, 1.5 ml of dH2O and 8 ml of chloroform:methanol:HCl (2:1:0.0075, v/v) solution (Scharlau Chemicals, Spain) were added to the liver homogenate, and tubes were vigorously shaken with a vortex for 2 min. Tubes were centrifuged at 1000 g for 15 min at 4 °C to separate the aqueous phase from the organic one. The lower organic phase was transferred to another extraction tube using a glass Pasteur pipette and lipids from the upper phase were reextracted by adding 4 ml of chloroform:methanol:HCl (2:1:0.0075, v/v) to the tubes that were vigorously shaken for 2 min and centrifuged at 1000 g for 10 min at 4°C. The organic phase was mixed with the previously obtained one. For the removal of aqueous contaminant, the chloroform extract was washed with the third part of the total volume of KCl 0.88% solution. Each tube was shaken with a vortex for 30 sec and centrifuged for 10 min at 1000 g and the lower phase was transferred to another extraction tube. Finally, the chloroform in extract was evaporated in a concentrator- evaporator and lipids were dissolved in an appropriate volume of toluene and were kept in glass vials at -20°C in N2 atmosphere until they were processed. The lipid separation was performed by thin layer chromatography (TLC) by six chromatographic developments [146]. First, the silica-gel plates of 20 x 20 cm (“Pre- coated TLC-plates SIL-G25”, Macherey-Nagel) were pre-treated with 1 mM EDTA-Na2 (not less than 4 h) and were left drying overnight and washed with Material and methods 121 chroloform:methanol:dH2O (60:40:10, v/v/v) to remove contaminants. The following day, the plates were activated at 100° C for 30 minutes; after that, 3 μl of pure lipid mixtures (Avanti Polar Lipids, USA and Sigma-Aldrich) of known concentrations and 3 or 8 μl of the lipid extracts were applied (per duplicate). Six different chromatographic developments were consecutively carried out in solutions of decreasing polarity. After each chromatographic development, the plates were dried using hot air. When the chromatographic separations were performed, the plate was stained immersing in a CuSO4 solution at 10% (p/v) in H3PO4 at 8% (v/v) for 10 sec, followed by drying with hot air until the first dots started to be visible. Then, the plates were developed for 3 min at 200°C. The image of the TLC plate was digitalized with the densitometer GS-800 and quantification was performed with the Quantity One software. The integrated optic density (IOD) of each lipid spot, after subtraction of the background, was interpolated in the IOD values of the calibration curves. The results obtained were expressed in μmol per gram of liver or nmol per mg of cellular protein. Lipidomic analysis was performed by Patricia Aspichueta Celaá, Physiology Department, from the University of the Basque Country (UPV). 7.2.12. Magnetic resonance imaging (MRI) The whole study has been carried out on an MRI Biospin 7T equipment (Bruker) using a volume antenna. Two sequences (T1_RARE- Rapid Acquisition with Refocused Echoes), are acquired with and without fat suppression. The parameters of these sequences are: TE / TR = 6.5 / 1500 ms. 8 2 averages. Rare factor 4. Coronal. Select the number of slices and adjust to the size of the animal. Fov = 80x40 mm. Matrix 256x256 pixels. Slice thickness = 0.5 mm. Slice gap = 0.25 mm. The estimated value of fat in the body, was obtained by using ImageJ© software. After a noise filtering process, the volume of segmented fat is measured and, using a density of 0.9 g / ml, and an estimation of the fat mass is obtained. After homogenizing the magnetic field, a proton spectrum of the entire body of the animal is acquired using a PRESS_1H sequence with the following parameters: TE / TR = 16.5 / 2500 ms and 64 averages. The analysis was carried out with the MNova software (Mestrelab). The area under the curve (AUC) of the water and fat peaks was measure, as well as the intensity of these peaks, and the relationship between peaks of these Material and methods 122 values is calculated. MRI analysis was performed at Medical Imaging Laboratory, Experimental Medicine and Surgery Unit from Gregorio Marañón hospital, Madrid. 7.2.13. Stool analysis 10 weeks faecal samples from DUAL, WTD and WTD+EXER feeding mice, along with the CTRL group, were analyzed in order to check their microbiota composition. Samples were kept in -80ºC, upon analysis. Before DNA extraction, purification of the samples is needed, by several centrifugation and filtration steps. Comparison of the bacterial 16S rRNA gene sequence has been used to perform this microbial analysis. From the FasQ files, a taxonomical analysis was performed, by the use of OTUs. Sequence-based analysis assumes that the taxonomic differences between bacteria, are reflected by differences in their 16S gene sequence. To identify and quantify different bacterial species, assumption that certain amount of 16S gene sequence variation exist within species is taken. Generate operational taxonomic units (OTUs), were created based on the assumption that two sequences which are more than 97% similar, belong to the same species. OTU analysis was performed by the software CLC Genomics Workbench and the plugin Microbial Module (Qiagen). A filtration of the quality of the reads by Q20 was performed. For the comparison of the obtained data, two databases were used SILVA (v132) (https://www.arb-silva.de/) y EZBiocloud (https://help.ezbiocloud.net/ezbiocloud-16s-database/). After DNA extraction, samples were analyzed, and FasQ files with bacterial composition in the samples were obtained. Microbiota analysis was performed by Genomic Unit, Biology building from the Complutense University of Madrid, Spain. 7.2.14. Statistical analysis Data are expressed as mean ± standard deviation (SD) with individual data points. GraphPad Prism version 8.1 (San Diego) was used to perform statistical analysis and graphs design. One-way ANOVA followed by Turkey post-hoc test was used. P-values for significance are indicated as follows: * p=0.05; ** p<0.01; *** p<0.001, **** p<0.0001. * Symbol represents the differences intragroup between control groups vs treated. T-test Student was used for the statistical analysis of performed experiments with two groups Material and methods 123 of comparison. P-values for significance are indicated as follows: * p=0.05; ** p<0.01; *** p<0.001, **** p<0.0001. Material and methods 124 Results 125 RESULTS Results 126 Results 127 8. Results I. DIETARY FAT QUALITY AND QUANTITY REPERCUSSION IN NAFLD DEVELEPMENT AND REGRESSION 8.1. Western diet (WD) is associated with the development of obesity and metabolic syndrome (MS) Ten-week-old C57BL/6J male mice were fed with three different WDs and sweetened water for 14 weeks. The three types of WD had a similar content of fat (40%), fructose (22%) and cholesterol (2%). However, the origin of fat and the level of PA was greatly different between the groups. In group 1, the level of PA was low (LP-WD), and the corn and soybean oils were the main source of fat. In group 2, the level of PA was high (HP- WD) due to the palm oil added to the formula. In group 3, the level of PA was also high (HP-Trans-WD), but the trans fats were the main source of the fat (Diets and composition diets from Material and methods section). Glucose was added to the drinking water in order to potentiate fructose absorption from the diet [139]. Mice fed with the control chow diet and filtered tap water were used as controls. Body Weight (BW) steadily increased in all treated groups during the experimental period. Nevertheless, as shown in (Figure 18A, B), throughout the 14-week period the mice fed with LP-WD, on average, gained less weight compared to HP-WD and HP- Trans-WD. Figure 18. Metabolic profile of mice treated with different types of WD and CTRL group. (A) Left: Body weight (BW) curve during the feeding period. Right: BW area Results 128 under the curve (arbitrary units) after 14 weeks of feeding. (B) End point BW after 14 weeks of feeding (n = 7-9). ** for p < 0.01; **** p < 0.0001 Obesity is associated with significant changes in epididymal White Adipose Tissue (eWAT), which has profound systemic and hepatic consequences [147]. Mice fed with all types of WD demonstrated significant expansion of eWAT in comparison to control animals (Figure 19A, B). Magnetic resonance imaging (MRI) confirmed the significant increase of WAT/ body ratio (%) (Figure 19C). With the development of obesity, WAT undergoes a process of tissue remodeling in which adipocytes increase in size (hypertrophy). Consistently, he administration of all types of WDs have led to remarkable hypertrophy and increased the size adipocyte up to 1,5- 2 times after 14 weeks of feeding (Figure 19D). Adipose tissue expansion in obesity is accompanied by inflammatory changes within adipose tissue. Multiple, typical crown-like structures (CLS) composed of macrophages surrounding apoptotic adipocytes [148], were detected in WD-fed groups, while they are rarely present in the controls (Figure 19E). Results 129 Figure 19. WD feeding increases eWAT deposits and triggers hyperplasia. (A) eWAT weight (g) (n = 7-9). (B) eWAT weight–to-BW ratio (%) (n = 7-9). (C) MRI scan image of all the experimental groups. The numbers represent the average WAT deposit in the mice. (D) Size measurement of adipocytes in eWAT tissue (arbitrary units) stained in H&E. (E) Representative eWAT H&E. Asterisks indicate CLS. Scale bar = 100 µm (n = 4-5). Scale bar = 100 µm. ** for p < 0.01, *** p < 0.001; **** p < 0.0001. Results 130 Obesity is the triggering agent for the development of metabolic syndrome (MS). MS is a cluster of conditions including obesity, high blood sugar and dyslipidemia. Hence, the basal glucose levels after 12 h of fasting were not elevated in all WD treated groups compared with CTRL (Figure 20A). However, a Glucose Tolerance Test (GTT) revealed significantly impaired glucose tolerance in mice fed a HP-Trans-WD (Figure 20B, C). In line, serum level of total cholesterol was significantly higher in HP-WD and HP-Trans- WD fed animals compared with LP-WD and CTRL groups (Figure 20D). Figure 20. HP-Trans-WD leads to glucose tolerance impairment, while cholesterol in serum was increased by either HP-WD or HP-Trans-WD. (A) Basal glucose levels in blood after 12 h of fasting (n = 3-5). (B) GTT curve after 14 weeks feeding. (C) Area under the curve GTT (arbitrary units) after 14 weeks. (n= 3-5). (D) Levels of cholesterol in serum (n = 6-8). * for p < 0.05; *** p < 0.001; **** p < 0.0001. 8.2. WD consumption triggered hepatomegaly Obesity, hypercholesterolemia and glucose intolerance are the main features of MS and are closely associated with the progression and severity of NAFLD [149]. Macroscopic examination by necropsy revealed that liver of mice fed with all types of WD is Results 131 significantly enlarged and the hepatic parenchyma is pale yellow in color (Figure 21A). Accordingly, the liver mass and hepatosomatic ratio were increased in all treated groups compared with CTRL mice (Figure 21B, C). Figure 21. WD feeding leads to hepatomegaly and steatosis. (A) Liver macroscopic images after 14 weeks of feeding. Scale bar is represented in cm. (B) Liver weight (g) (n = 7-8). (C) Liver weight–to-BW ratio (%) (n = 7-8). ** for p < 0.01; *** p < 0.001; **** p < 0.0001. The increase in liver weight may be induced by hypertrophy, hyperplasia, or very frequently a combination of the two. In order to study this point, we stained other membrane of liver cells with IF phalloidin staining and revealed that hepatocytes of all WD-fed groups were almost twice enlarged compared to CTRLs. (Figure 22A, C). At the same time, Ki-67 staining (proliferation marker) revealed mild but significantly higher cellular proliferation in all treated groups (Figure 22B, D) indicating that hepatomegaly was caused by a simultaneous combination of hypertrophy and hyperplasia of the hepatic cells. Results 132 Figure 22. WD leads to hepatocyte enlargement an increase cell death. (A) Representative phalloidin-stained liver images (n = 3-4). (B) Ki-67 after 14 weeks of feeding. Positive proliferating cells are stained in green and indicated by arrows. Nuclei are stained in blue using DAPI as a counterstain. (n = 3–4). Scale bar = 100 µm. (C) Quantification of hepatocytes size in phalloidin-stained liver pictures quantified by ImageJ software (n = 3-4). (D) Quantification of ki67 positive cells as % (n = 3-4). ** for p < 0.01; *** p < 0.001; **** p < 0.0001. Careful examination of liver histology revealed that all mice treated with WD develop abnormal accumulation of lipids in the liver compared to control animals. H&E staining showed distended hepatocytes with foamy appearing cytoplasm and multiple lipid vacuoles (Figure 23A). Blinded quantitative analysis performed by an experienced pathologist confirmed that WD-fed animals exhibited micro- and macro-vesicular steatosis in 30–80% hepatocytes mainly located in the periportal regions of the lobules (Figure 23A, C). These findings were confirmed by Oil-Red-O staining in liver cryosections (Figure 23B, D). Results 133 Figure 23. WD feedings leads to a hepatic steatosis, in all treated groups, especially in HP-Trans-WD. (A) H and E representative images. Scale bar = 100 µm. (n= 7-9). (B) Illustrative ORO-stained liver sections. Scale bar = 100 µm. (n = 4-6). (C) Pathological evaluation of hepatic steatosis in mice fed with WDs (n= 4). (D) Quantification of ORO-stained area (n = 4-6). * for p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. 8.3. WD feeding induced lipidome alterations in murine livers In order to identify and quantify the major lipid classes discriminating the pathological statuses of the liver, a quantitative lipidomic analysis was performed. Lipids were extracted from the liver tissue and further separated by Thin Layer Chromatography (TLC). Total hepatic Triglyceride (TG) content was markedly increased in all mice fed with WD compared with CTRL group (Figure 24A). However, the level of Diglycerides (DG) was significantly increased only in mice fed with HP-WD and HP-Trans-WD (Figure 24B). Importantly, hepatic Free Cholesterol (FC) levels significantly increased in both groups fed with a HP diet (Figure 24C). The total Phosphatidylcholine (PC) content was Results 134 decreased (Figure 24D), whilst Phosphatidylethanolamine (PE) levels significantly increased (Figure 24E), and the PC/PE ratio decreased only in mice fed with a trans fat diet (Figure 24F). A trend towards decreased Phosphatidylserine (PS) levels was found in the HP-Trans-WD group (Figure 24G). Figure 24. Hepatic lipidomic analysis in mice treated with WD and corresponding controls. Distribution of lipid classes within hepatic lipids (µmol/g of tissue). (A) Triglycerides (TG). (B) Diglycerides (DG). (C) Free cholesterol (FC). (D) Hepatic phospholipids Phosphatidylcholine (PC). (E) Phosphatidylethanolamine (PE). (F) PC/PE ratio. (G) Phosphatidylserine (PS) (n = 4-5). * for p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Results 135 8.4. WD altered the balance between fat storage and oxidation in the liver Subsequently, we assessed the effects of all three types of WD on major molecular mechanisms that regulate lipid metabolism in the liver. Consistently with previous studies [20], the excessive lipid load in all treated groups induced the expression of Pparγ, early induced lipogenic transcription factor mediating an adipogenic transformation of hepatocytes (Figure 25A). We also found strong increase of Scd1 mRNA expression in all treated animals (Figure 25B). Scd1 desaturase converts saturated FA to monounsaturated FA [150], the major substrate for TG in the liver [151]. Hence, the substantial increase in Scd1 expression was mainly induced by the excessive dietary fat, as mRNA of main regulators of de novo lipogenesis Acc (Figure 25C) and Srebp-1 were not increased after feeding with WDs (Figure 25D). Figure 25. Changes in lipid metabolism in mice fed with WD. (A) Pparγ, (B) Scd1, (C) Acc and (D) Srebp-1 mRNA relative expression to GAPDH after 14 weeks on WD (n = 4-6) * for p < 0.05; ** p < 0.01; *** p < 0.001. Results 136 Furthermore, FASN protein expression was remarkably suppressed in HP-Trans-WD group (Figure 26A). Increased lipid load in the liver resulted in enhanced lipid oxidation only in LP-WD, as observed by raised Acox (Figure 26B) and CPT-1c expression levels. In sharp contrast, the application of both diets with high levels of PA seems to not induce the transcription of genes and proteins for an adequate lipid oxidation (Figure 26C). Finally, decreased Apob levels may indicate the impairment in Very Low-Density Lipoprotein (VLDL) secretion in HP-Trans-WD (Figure 26D). Figure 26. Changes in fatty acid synthesis and beta oxidation. (A) FASN western blot using HSC- 70 as a loading control. Ratio: normalization of FASN expression by densitometry. (B) Respective Acox mRNA expression relative to Gapdh (n = 4-6). (C) CPT-1c WB using GAPDH as loading control. Quantification of Western blot was performed by densitometry using ImageJ software. (D) Apob mRNA expression relative to Gapdh (n = 4-6). ** for p < 0.01. 8.5. WD with high levels of PA and trans fat increased the risk of NAFLD-associated hepatitis and fibrosis Excessive lipid accumulation leads to cell death and inflammation in the liver [152]. Plasma levels of Alanine Aminotransferase (ALT) and Aspartate Aminotransferase Results 137 (AST), important clinical markers of hepatocellular injury were significantly elevated in animals fed with HP-Trans-WD (Figure 27A). Thus, the detection of hepatic in situ cell death revealed significantly higher numbers of TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick- end labeling) positive cells in HP-WD and HP-Trans-WD fed livers in comparison with LP-WD-fed animals and the controls (Figure 27B, D). All WD treated groups showed an increased accumulation of F4/80 positive liver macrophages, as assessed by Immunofluorescence (IF) staining (Figure 27C, E). Additionally, the mRNA expression of Tnf-α was significantly increased only in mice fed either with a HP-WD or a HP-Trans- WD (Figure 27F). Results 138 Figure 27. Hepatitis and hepatic fibrosis in mice on the WD for 14 weeks. (A) ALT and AST measurements in serum after 12 h of fasting (n = 7-9). (B) Representative TUNEL-stained photomicrographs at 14 weeks. (n = 6-7). Scale bar = 100 µm. (C) F4/80 IF staining in liver sections of mice fed for 14 weeks. Positive immune cells are stained in green. Nuclei are stained in blue using DAPI as a counterstain. Arrows indicate F4/80 positive cells, respectively. Scale bar = 100 µm. (n = 4). (D) Quantification of % TUNEL positive cells (n = 6-7). (E) Quantification of % F4/80 positive cells, using ImageJ software (n = 4). (F) Tnf-α mRNA relative expression to Gapdh. * for p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Results 139 Cell death, inflammation and TNF-α overproduction induces the activation of Hepatic Stellate Cells (HSCs) in the liver [153]. Consistently, significant expression of α -Smooth Muscle Actin (α-SMA), a marker of HSCs activation, was detected by Immunohistochemistry (IHC) staining in both groups treated with HP-WD and HP-Trans- WD (Figure 28A, D). Activated HSCs are the major source of Extracellular Matrix (ECM) during progression of fibrosis [154]. Hence, Sirius Red (SR) staining clearly demonstrated that feeding with HP-WD and HP-Trans-WD induced collagen expression in the liver (Figure 28B, E). These findings were additionally confirmed by IF staining for Collagen I (Figure 28C, F). Evaluation of SR samples by a pathologist revealed, an F1a fibrosis score for LP-WD mice, and F1b for both HP-WD and HP-Trans-WD mice (Figure 28G). Results 140 Figure 28. WD triggers hepatic fibrosis in all treated groups besides, in a more accentuated way after HP and HP-Trans feeding. (A) Fibrosis-related stainings in liver and corresponding quantification of positive stained areas after 14 weeks of treatment. Results 141 Representative liver images stained with α-SMA (IHC) (B), SR (C), Collagen I (IF) (n = 4-7). Scale bar = 100 µm. (D) % of α-SMA positive area. (E) Quantification of the % of liver fibrotic area from SR staining. (F) % Collagen I positive area. (n = 4-7). (G) Pathological evaluation of SR staining according to the fibrosis score (n = 4-5). * for p <0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. 8.6. The WD withdrawal, reversed NAFLD in all treated groups independently of the diet composition Lastly, we investigated whether WD withdrawal could ameliorate MS and NAFLD related liver phenotype. The restoration of a chow diet feeding regimen for 20 days was performed after 14 weeks of WD in all treated groups, while CTRL group was maintained on chow diet for 17 weeks in total (Figure 29A). The switch from all different WDs to chow feeding diet, induced a decrease in body weight (Figure 29B, D) and significantly reduced hepatomegaly (Figure 29C, E), compared with the corresponding 14 weeks feeding period of the three different types of WDs that has been used, achieving similar levels of the CTRL group. The plasma levels of transaminases and cholesterol progressively decreased over the duration of withdrawal in all treated groups, and eventually reached almost the same level as in the CTRL animals (Figure 29F). Importantly, WD withdrawal normalized impaired glucose intolerance in mice fed with HP-Trans-WD (Figure 29G, H). Results 142 Figure 29. Effects of WD withdrawal on MS. (A) Mice were fed for 14 weeks with WD followed by 3 weeks of withdrawal. (B) Body and (C) Liver weight (n = 5-6) of the mice after food withdrawal. (D) Reduction of body (E) and liver weight (g) after WD withdrawal (n= 5). (F) AST, ALT and Cholesterol in serum after 12 h of fasting (n = 3-5). (G) Basal glucose levels in blood after 12 h of fasting. (H) Left: GTT curve after the period of withdrawal. Right: Area under the curve GTT (arbitrary units) (n = 3-6). * for p <0.05; ** p < 0.01 Results 143 In light of these metabolic improvements, pathological changes in the liver were explored next. All WD withdrawal groups, showed significant improvement in hepatocellular morphology (Figure 30A) and attenuated hepatic steatosis with improved hepatic TG content (Figure 30B, E, F). However, even after 3 weeks of diet withdrawal, we observed low-grade level of infiltration in both groups treated with high PA (Figure 30C, G). Hence, the withdrawal was capable to revert fibrotic changes in all treated groups (Figure 30D, H). Results 144 Results 145 Figure 30. Effects of WD withdrawal on steatohepatitis and fibrosis. (A) H and E staining of liver sections from each group. Scale bar = 100 µm (n = 3–6). (B) Representative Oil Red O staining of liver cryosections after WD diet withdrawal. Scale bar = 100 µm (n = 3-6). (C) IF staining for CD45. Scale bar = 100 µm (n = 3). (D) SR staining and quantification of positive areas after withdrawal of WD. (n = 4). (E) Quantification of hepatic TG after diet withdrawal. (n = 3-6). (F) Quantification of ORO % of fat area (n = 3-6). (G) Quantification of the % of CD45 positive cells. The number of CD45 positive cells (green, arrows) was quantified and calculated as percentage of total cells (DAPI, blue). (n = 3). (H) Quantification of the % of liver fibrotic are from SR attaining (n = 4). Scale bar = 100 µm (n = 3–6). ** p < 0.01. II. EFFECTS OF WTD AND WTD+EXER IN NAFLD REGRESSION DUE TO DUAL DIET SHORT AND LONG-TERM FEEDING A. SHORT-TERM FEEDING 8.7. WTD, specially in combination with exercise (WTD+EXER) reversed MS caused by short-term DUAL diet feeding In the second part of our study, we used a preclinical DUAL (NAFLD plus alcohol) model in mice [127]. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for different periods of time. First, 10-week-old C57BL/6J male mice, were fed with DUAL diet (WD+ 10% EtOH+ 6.75% glucose), control group (CTRL) was treated in parallel, during 10 weeks (short- term feeding). After DUAL feeding, treatment was replaced by normal filtered water and regular chow diet for 20 more days in WTD and WTD+EXER groups. Moreover, in WTD+EXER group, diet replacement was match by five days weekly non-volunteer physical exercise using a Treadmill platform (Harvard Apparatus) (described in Material and methods section). WTD and remarkably WTD+EXER strategies were able to significantly decrease the BW grow generated by DUAL diet feeding at the end point of the treatment (Figure 31A, B). Obesity is strongly associated with features of MS, including glycemia and dyslipidemia [57]. In fact, the circulating levels of total cholesterol were constantly higher in DUAL-fed Results 146 animals compared with the other groups, nevertheless WTD and WTD+EXER, was able to reverse cholesterol increase, up to similar levels of the CTRL group (Figure 31C). Hence, no significant difference between CTRL animals and treated ones, was detected in basal glucose levels after 6h of fasting (Figure 31D). Figure 31. WTD alone or in combination with EXER were able to ameliorate obesity and dyslipidemia, caused by short-term DUAL feeding. (A) BW curve during the feeding period. (B) End point body weight (g) (n = 5-6). (C) Total cholesterol in serum levels in mg/dl (n = 5-13). (D) Basal glucose levels after 6 h fasting measured in mg/dl (n = 5-7). * for p < 0.05; ** p < 0.01; **** p < 0.0001 Moreover, obesity is associated with profound changes in the function of the eWAT tissue, which has important systemic and hepatic consequences [147]. Mice fed with DUAL diet, accumulated greater eWAT tissue compared to control animals, though WTD and WTD+EXER strategies were able to significantly decrease epididymal fat deposits in mice (Figure 32A, B). The morphometric evaluation of adipocytes, by H&E staining, in WAT from epidydimal fat pad showed that DUAL diet led to hypertrophy of eWAT cells by increased adipocyte size approximately 1.8 times. WTD and WTD+EXER were able to significantly decrease Results 147 hypertrophy of adipocytes, hence the size of cells never returns to CTL-like levels (Figure 32C, D). In order to lo determine the infiltration of immune cells in the eWAT tissue, CD45 (total leucocytes marker) and F4/80 (macrophages) stainings were performed. Both CD45 and F4/80 markers, revealed a mild increase in immune cell infiltration in all DUAL and WTD and WTD+EXER animals (Figure 32E, F). Results 148 Figure 32. WTD and WTD+EXER have reduced adiposity and adipocyte hyperplasia, triggered by short-term DUAL diet feeding. (A) eWAT measurement at end point of the treatment (g) (B) Ratio in between eWAT and BW (%). (C) Quantification of the adipocyte mean area in AU (n = 5-7). (D) Representative H&E staining of the eWAT tissue. Scale bar = 100 µm (E) CD45 and (F) F4/80 IF staining of eWAT paraffin sections. Positive cells are stained in green and DAPI has been used as nuclear counterstain. Scale bar = 100 µm. (n = 4) * for p < 0.05; ** p < 0.01; *** p < 0.001. (n = 3-4). 8.8. WTD and WTD+EXER reduced hepatomegaly and hepatic steatosis caused by short-term DUAL feeding Obesity and MS predispose to the development of fatty liver disease [155]. After 10 weeks of DUAL diet, animals exhibited enlarged livers, which were pale and yellowish in color, indicating lipid accumulation (Figure 33A). Accordingly, the hepatic mass and the hepatosomatic ratio of DUAL mice were increased compared to control. In contrast, WTD and WTD+EXER strategies, were able to decrease liver size up to the levels of the control group (Figure 33B, C). Figure 33. Hepatomegaly caused by short-term DUAL feeding was successfully reverted after WD and WD+EXER. (A) Macroscopic liver images of all treated groups and corresponding control mice. Pictures were taken with a stereomicroscope. Scale bar Results 149 = 1cm. (B) Liver weight of the different groups measured in grams and (C) Ratio between liver weight and BW in %. * for p < 0.05; *** p < 0.001; **** p < 0.0001. H&E staining of liver tissue was performed to evaluate the liver morphology. Importantly macrolipid and microlipid droplets, were detected in liver parenchyma after short-term DUAL feeding, nonetheless after WTD and WTD+EXER livers histology was similar to control group, revealing almost no fat accumulation (Figure 34A). After 10 weeks of feeding DUAL mice displayed profuse positive ORO staining. Remarkably, WTD and WTD+EXER groups did not show significant accumulation of neutral lipids in liver parenchyma at the end point of the treatment (Figure 34B, C). Consistently, hepatic TG were strongly increased in DUAL group, and significantly reduced after WTD and WTD+EXER (Figure 34D). Figure 34. WD and WD+EXER significantly decreased hepatic steatosis caused by DUAL diet. (A) Representative H&E staining of liver sections (n = 5-6). Scale bar = 100 µm (B) ORO staining of liver cryosections, neutral lipids are stained in bright red. Scale bar = 100 µm. (C) ORO quantification as % fat area (n = 4-6). (D) Liver triglycerides measurement as mg TG/ g liver (n = 4). * for p < 0.05; ** p < 0.01; **** p < 0.0001 Results 150 Next, we analysed the lipid metabolism and found a significant decrease in CPT-1c expression in DUAL group after 10 weeks of treatment. This finding indicates a reduction in lipid oxidation in DUAL fed animals, as inhibition of CPT-1c limits the transport of FA from the cytosol to the mitochondria and undergo beta-oxidation, resulting in lipid partitioning and storage [156, 157]. Importantly, WTD and WTD+EXER were able to mildly restore the protein expression levels decreased by DUAL diet, up to similar levels of the CTRL group (Figure 35A). Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that is expressed in tissues that have a high oxidative activity. PPARα plays a key role in metabolism [158] and regulates genes involved in peroxisomal and mitochondrial β- oxidation [159]. PPARα expression levels, were specially decreased in DUAL mice, but slightly restored in after WTD an especially in WTD+EXER group (Figure 35B). Figure 35. Application of WTD in combination with EXER improved beta- oxidation. (A) CPT-1c WB, using GAPDH as loading control. (B) PPARα WB, using GAPDH as loading control. Quantification of Western blot was performed by densitometry using ImageJ software. 8.9. Cell death induced by DUAL diet, was ameliorated after WTD and WTD+EXER Next, we measured clinically relevant liver function tests. Both ALT and AST were significantly increased in DUAL mice compared to control, however WTD and WTD+EXER, were able to reduce the levels of both transaminases to the levels of the control group (Figure 36A, B). To confirm the existence of hepatic hypoxic conditions, we evaluated the amounts of LDH, since its production increases under low oxygen concentrations. Level of LDH was also considerably higher in DUAL mice, and significantly decreased in WTD+EXER mice, compared to DUAL (Figure 36C). In contrast, serum levels of AP were not different in between treated groups compared to Results 151 the CTRL (Figure 36D). All together the alterations of liver function tests denoted hepatocellular pattern of liver injury [160]. Death of hepatocytes may trigger compensatory proliferation in surrounding cells to maintain tissue homeostasis. Accordingly, ki67 staining revealed, that cellular proliferation was higher in DUAL mice compared to control, thought was significantly reduced in WTD mice (Figure 36E, F). Figure 36. WTD and WTD+EXER significantly decreased liver damage and compensatory cell proliferation after short-term DUAL feeding. (A) AST and (B) ALT levels in serum measured in U/L (n = 5-13). (C) LDH and (D) AP levels in serum measured in U/L (n = 5-13). (E) ki67 IHC liver staining. (F) Positive ki67 cells quantification (n = 4). Scale bar = 100 µm. * for p < 0.05; ** p < 0.01. Results 152 8.10. Short-term DUAL diet feeding lead to hepatic inflammation, reversed by WTD and WTD+EXER Fat accumulation and cell death in the liver further caused immune cell infiltration and hepatic inflammation [161]. Short-term DUAL fed mice, showed an increase accumulation of CD45 and F4/80 positive Kupffer cells/ macrophages, as assessed by immunofluorescence (IF) staining. Importantly, the number of infiltrating immune cells were significantly reduced after WTD and WTD+EXER achieving similar levels to the control group (Figure 37A-D). Infiltrating cells actively produce different cytokines and create a proinflammatory microenvironment. Consistently mRNA expression of Tnf-α, was significantly increased in DUAL mice. Hence, we detected that the level of Tnf-α was similar to the control group in WTD and WTD+EXER mice (Figure 37E). Consistently, Mcp-1, an important chemokine for macrophage recruitment to the site of tissue injury [162], was significantly increased after DUAL diet feeding, nevertheless WTD and WTD+EXER were able to decrease it (Figure 37F). Hepatic injury can be amplified by the release of the ligands from Toll like receptors 2 and 4 (Tlr-2 and Tlr-4) [163]. In line, analysis of Tlr-2 and Tlr-4, revealed a significantly increased in DUAL compared to control, hence the expression levels in WTD and WTD+EXER were similar to the control group (Figure 37G, H). Results 153 Figure 37. WD and WD+EXER reduced hepatic inflammation caused by DUAL diet. (A) CD45 staining of liver cryosections. (B) F4/80 staining of liver cryosections. Scale bar = 100 µm. (C) Quantification of CD45 positive cells in % (n = 4). (D) Quantification of F4/80 positive cells in % (n = 4). (E) Tnfα. (F) Mcp-1. (G) Tlr-2 (H) Tlr-4 mRNA relative expression to Gapdh (n = 5-7). * for p < 0.05; ** p < 0.01; **** p < 0.0001. State of liver inflammation leads to the transformation of hepatic stellate cells (HSCs) to myofibroblast, which produce extracellular matrix, that results in liver fibrosis. In a normal situation, fibrosis is a wound healing process that preserves tissue integrity, nevertheless Results 154 progressive fibrosis can become pathogenic [164]. Thought SR staining revealed no significant differences in between the treated groups and the control (Figure 38A, B). Figure 38. Mild fibrosis after short-term DUAL feeding. (A) Representative SR staining microscope images. (B) Quantification of the fibrotic areas (%). (n = 4-6) Scale bar = 100 µm. 8.11. Pathological changes in the intestine caused by DUAL diet, were reverted by WTD and WTD+EXER The digestive system and the liver have a close communication loop, which is known as “gut-liver axis”. There are several mechanisms that are characterized by the alteration of this gut-liver axis, as the impairment of the gut barrier or the increase of intestinal permeability, resulting in endotoxemia and inflammation, changing bile acid profiles and metabolites produced by the microbiome. Gut microbiome is composed by a large number of commensal bacteria that live in human digestive tract, and which are mainly anaerobic [165]. This permeability disruptions known as leaky gut syndrome, represent an aggravating condition for these pathologies [166]. Next, we have analysed the effects of DUAL diet and WTD alone or in combination with exercise on gut morphology, permeability, and microbiome. DUAL diet has led to a significant reduction in intestine length, nonetheless in WTD and specially WTD+EXER intestine has a similar length to the control group (Figure 39A, B). These alterations were mainly caused by changes in the small intestine, as colon length was similar between all groups (Figure 39C). Results 155 In order to elucidate possible alterations in gut permeability, due to disruptions of the tight junctions in between the epithelial cells. OCCLUDIN expression in colon was decreased in all treated groups, showing and slight improvement in WTD+EXER group (Figure 39D). By measuring proliferating cell nuclear antigen (PCNA), we revealed that after 10 weeks of feeding the DUAL diet led to the increased proliferation in colon, which was still upregulated even after WTD or WTD+EXER. (Figure 39E). Furthermore, analysis of the LPS content in serum shows a significant increase in DUAL group compared to control, thought levels were significantly reduced in both WTD and WTD+EXER mice (Figure 39F). Results 156 Figure 39. WTD+EXER reduced changes in gut caused by DUAL diet. (A) Intestine macroscopic pictures of all the treated and control mice (n = 5-7). Scale = 1 cm. (B) Intestine and (C) and colon length measurement in cm (n= 5-7). (D) Occludin WB, using GAPDH as loading control. Ratio normalized to GAPDH by densitometry. (E) PCNA WB, using HSC-70 as loading control. Ratio normalized to HSC-70 by densitometry. (F) LPS in serum, measure as concentration of LPS in ng/ml (n = 4-3). * for p < 0.05; ** p < 0.01. Results 157 8.12. Short-term DUAL diet feeding leads to bacterial dysbiosis, that can be reverted by WTD and WTD+EXER according to 16S rRNA sequencing analysis Leaky gut is closely correlated with dysbiosis of the gut microbiome. 16S rRNA sequencing of stool samples from the different experimental groups was performed. The fastQ files, were analyzed by taxonomic assignation by the use of OTUs (Operational Taxonomic Unit), including a Reads quality control, from CLC Genomics Workbench software. The reference database used were SILVA (v132) and EZBiocloud. At Phylum level, the analysis revealed an alteration in the ratio Firmicutes/ Bacteroidota, finding in DUAL mice an increase in Bacteroides and a decrease in Firmicutes, compared to the control group. This balance looks to be mildly recovered in the WTD and WTD+EXER mice (Figure 40A, B). Alpha diversity index, which is a major indicator to describe the diversity in gut microbiota [167], was calculated in order to determine the variety of bacteria al Phylum levels. Result has shown that the diversity was higher in the three treated groups compared to the CTRL compared to control (Figure 40C). At Family level 16S bacterial analysis also revealed that the most abundant bacteria in all groups were Muribaculaceae and Lachnospiraceae. Importantly, the percentage of Lachnospiraceae was significantly reduced in DUAL stool samples, compared to control (30% vs 19%). However, WTD and WTD+EXER increased Lachnospiraceae to values similar to CTRL group. The decrease of the Lachnospiraceae in the DUAL mice was accompanied by the increase in the percentage of the rest of bacterial families, Bacteroidaceae (12% DUAL vs 5% CTRL), or Tannerellaceae (5% DUAL vs 2% CTRL). The dysbiotic changes in DUAL were significantly reverted after WTD and WTD+EXER (Figure 40D-H). Results 158 Figure 40. Bacterial 16S RNA gene sequence analysis revealed a dysbiosis in DUAL mice, ameliorated by WTD and WTD+EXER. (A) Table summarizing the main Results 159 bacterial phylum that are present in the samples and (B) Representation by stack column diagram the relative abundance at Phylum level regarding the intestinal microbiota composition in the different experimental groups (n = 4-5). (C) Graphical representation of the alpha diversity index at Phylum level, of the bacterias observed in each group of samples. (D) Representation by stack column diagram the relative abundance at Family level regarding the intestinal microbiota composition in the different experimental groups. (E) Family relative abundance in CTRL. (F) DUAL (G) WTD (H) WTD+EXER represented by sectors diagrams (n = 4-5). B. LONG-TERM FEEDING 8.13. Only the combination of WTD +EXER, was able to ameliorate MS caused by DUAL long- term feeding Next, we applied the DUAL diet for the long period of time- 23 weeks. Long-term DUAL diet feeding leads to a significant and constant body mass increase. Importantly, only the combination of WTD+EXER led to a remarkable decrease of body weight, indicating the benefit of physical activity on obesity control (Figure 41A, B). Figure 41. WTD and WTD+EXER, reduced obesity caused by long-term DUAL feeding. (A) BW curve during the feeding period (n=5-10). (B) End point body weight after 23 weeks of feeding (n=5-10). * for p < 0.05; *** p < 0.001; **** p < 0.0001. Overweight is associated with profound changes in eWAT tissue. Though exercise stabilized the body weight in WTD+EXER group, there was no difference in eWAT mass or eWAT/body weight ratio between the DUAL and WTD and WTD+EXER group (Figure 42A, B). Results 160 Obesity has significant systemic consequences such as dyslipidemia and glycemia [57]. Remarkably WTD and WTD+EXER, were able to significantly decrease the levels of serum cholesterol caused by DUAL diet feeding, although not up to CTRL group levels (Figure 42C). Additionally, basal glucose levels, measured after 12h overnight (O/N) fasting, demonstrated that DUAL fed mice developed hyperglycemia. Importantly WTD+EXER has normalised levels of glucose (Figure 42D). Figure 42. Only the combination of WTD with EXER could reduce hypercholesterolemia and hyperglycemia caused by long-term DUAL diet feeding. (A) eWAT weight (g) (n = 5-10). (B) eWAT/ body weight ratio (%) (n = 5-10). (C) Levels of cholesterol in serum (mg/dl) (n = 5-10). (D) Basal glucose levels, calculated after 12 h of fasting (n = 4-10). * for p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. The morphometric evaluation of WAT from epididymal fat pad, showed that only the combination of WTD+EXER, was able to ameliorate the adipocyte hypertrophy caused by long-term DUAL feeding (Figure 43A, B). Consistently, eWAT inflammation (CD45 and F4/80 positive cells) was higher in DUAL animals after long-term feeding. After WTD eWAT inflammation remains high in WTD mice, but remarkably decreased in WTD+EXER group (Figure 43C, D). Results 161 Figure 43. Hypertrophy and immune cell infiltration after long-term DUAL feeding was significantly reduced only WTD+EXER mice. (A) Representative eWAT H&E. Scale bar= 100µm (n= 4-6). (B) Adipocyte area quantification in arbitrary units (n = 4-6). (C) Representative CD45 IF staining of eWAT and (D) Representative F4/80 IF staining in eWAT. Positive immune cells are stained in green and nuclei are stained in blue using DAPI as counterstain. Scale bar 100 µm (n = 4). Scale bar = 100 μm (n = 4). * for p < 0.05; ** p < 0.01; *** p < 0.001 Results 162 8.14. WTD and WTD+EXER strategies, were able to reverse hepatomegaly and hepatic steatosis after long-term DUAL feeding Obesity and MS predispose to the development of fatty liver disease [155]. After long- term, DUAL diet feeding animals exhibited extraordinarily enlarged and yellowish livers. In sharp contrast, WTD and WTD+EXER overcame the gain in liver mass (Figure 44A, B) and accordingly, liver weight/ body weight ratio significantly decreased in both groups, compared to DUAL mice (Figure 44C). WTD+EXER, and in a less robust way WTD, were able to revert the loss of the typical hexagonal shape of the hepatocytes, that the DUAL mice have experienced, as demonstrated by phalloidin staining (Figure 44D, E). Figure 44. WTD and WTD+EXER have reversed hepatomegaly generated by long- term DUAL feeding. (A) Liver macroscopic images after 23 weeks of feeding. Scale = 1 cm (B) Liver weight (g) (n = 5-6). (C) Liver weight-to-BW ratio (%) (n = 5-6). (D) Quantification of hepatocytes size from phalloidin staining performed by ImageJ Results 163 software. (E) Representative phalloidin-stained IF liver images. Scale bar = 100 μm (n = 4). * for p < 0.05; ** p < 0.01, **** p < 0.0001. Next, H&E staining of liver samples, revealed WTD and WTD+EXER, were able to diminish microvesicular and macrovesicular steatosis caused by DUAL diet feeding. Figure 45A). This finding was further confirmed by ORO staining were all treated groups displayed positive ORO staining in comparison with the control mice. However, macro- and microlipid droplets were more profuse in DUAL fed mice, than in WTD or WTD+EXER experimental groups, where lipid accumulation was significantly reduced (Figure 45B, C). Consistently, hepatic TG content was significantly decreased in WTD and WTD+EXER groups, compared to DUAL, achieving similar levels as the control animals (Figure 45D). Figure 45. Mild reduction of hepatic steatosis after WTD and WTD+EXER in long- term DUAL feeding. (A) H&E representative images after 23 weeks of feeding (n = 5- 10). (B) Illustrative ORO-stained liver sections from each group. Scale bar = 100 μm. (C) Quantification of ORO-stained area (n = 5-6). (D) Liver triglycerides quantification (mg TG/ mg liver) (n = 5-8). * for p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Results 164 8.15. WTD and WTD+EXER could partially reduce lipid metabolism abnormalities caused by DUAL diet Dietary FFAs are the main source of TG in the liver. Consistently we found significant upregulation of Cd36 expression (FA translocase) in the livers of long-term DUAL-fed animals. Reasonably, the withdrawal of the fat-rich DUAL diet leads to the reduction of Cd36 level in WTD and WTD+EXER livers to values similar to CTRL group (Figure 46A). In line, Pparγ, another steatogenic role mediating the expression of Cd36 [168], was elevated in DUAL animals and down-regulated WTD and WTD+EXER groups (Figure 46B). Intensive lipid load induces an increase in lipid oxidation. In consistence with previous publication [127], long-term DUAL did not induce rise in carnitine palmitoyltransferase 1c (CPT-1c) protein expression. Moreover, CPT-1c expression in WTD and WTD+EXER was also similar to CTRL mice (Figure 46C). Consistently, PPARα, the transcriptional regulator of genes involved in peroxisomal and mitochondrial β-oxidation was down- regulated in DUAL, WTD and WTD+EXER groups (Figure 46D). Fatty acid synthase (FASN) is a major determinant of de novo lipogenesis [169]. Western blot analysis revealed, that FASN protein expression was remarkably enhanced after DUAL diet and only the combination of WTD+EXER mice was able to revert it to the CTRL-like levels (Figure 46E). Results 165 Figure 46. Impaired lipid metabolism caused by DUAL diet, was ameliorated by WTD+EXER. (A) Cd36 and (B) Pparγ mRNA relative expression to Gapdh (C) CPT-1c and (D) PPARα western blot using GAPDH as a loading control. (E) FASN western blot using HSC-70 as a loading control. Ratio between target protein and the correspondent loading control GAPDH or HSC-70, was calculated (n = 4). 8.16. Cell death and compensatory proliferation caused by DUAL diet, was ameliorated by WTD and especially by the combination of WTD+EXER Excessive fat accumulation in DUAL livers contributes to liver damage and caused modest but significant increases of the plasma levels of ALT, AST (Figure 47A), LDH (Figure 47B) and AP (Figure 47C), which are major clinical indicators of hepatobiliary cellular liver injury. Nonetheless, WTD and the combination of WTD+EXER were able to significantly reduced AST, LDH and AP levels (Figures 47A-C). The precise assay for the measurement of cell death and tissue injury, TUNEL staining revealed a significant increase in cell death in DUAL mice, which was slightly ameliorated in WTD and WTD+EXER groups (Figure 47D, E). Results 166 Figure 47. WTD+EXER significantly reduced cell death caused by DUAL diet feeding. (A) Left: AST and Right: ALT measurements in serum after 12 hours of fasting (U/L) (n = 5-10). (B) AP and (C) LDH levels in serum (U/L), after 12 hours fasting. (D) Representative TUNEL-stained IF images. Scale bar = 100 μm. (E) Quantification of TUNEL positive cells in % (n = 4). * for p < 0.05; ** p < 0.01; **** p < 0.0001. Hepatocyte death could lead to compensatory proliferation in surrounding cells to keep tissue homeostasis. As a result, ki67 immunofluorescence (Figure 48A, C) and Results 167 immunohistochemistry (Figure 48B, D) staining, showed that cell proliferation was higher in DUAL mice and significantly decreased after both WTD and WTD+EXER. Figure 48. Hepatic cell proliferation prompt by DUAL feeding, was reduced by WTD and WTD+EXER. (A) ki67 IF staining in liver after 23 weeks of feeding (n = 5). Scale bar = 100 μm. (B) ki67 IHC staining in liver after 23 weeks of feeding. (n = 4). Scale bar = 100 μm. (C) Quantification of ki67 positive cells in % from IF staining (n = 5). (D) Number of ki67 positive cells in IHC staining (n = 4). Scale bar = 100 μm. * for p < 0.05; ** p < 0.01; **** p < 0.0001. 8.17. WTD+EXER was able to reduce hepatic inflammation triggered by long-term DUAL feeding DUAL mice showed an increased accumulation of CD45 and F4/80 and positive Kupffer cells/macrophages, as assessed by IF staining (Figure 49A-D). However, WTD strategy alone was not effective in reducing the CD45, F4/80 infiltration. Remarkably, only the combination of WTD+EXER could significantly reduce the numbers of CD45 and F4/80 positive cells (Figure 49C, D). Results 168 Infiltrating cells actively produce different cytokines and further contribute to create a pro- inflammatory microenvironment. Consistently, mRNA expression levels of Tnf-α and Mcp-1 were significantly increased in DUAL mice. Both inflammatory markers expression levels were not reverted after WTD alone or WTD +EXER (Figure 49E). Toll like receptors (Tlrs) are a key factor involved in the activation of innate immune signalling pathways [170]. Both Tlr-2 and Tlr-4 genes expression was increased after DUAL diet feeding nevertheless, WTD and WTD+EXER were able to reduce Tlr-2 levels, however none detectable changes were found in the levels of Tlr-4 (Figure 49F). Results 169 Figure 49. Only combination of WTD+EXER reduced hepatic inflammation after long-term DUAL feeding. (A) lllustrative CD45 IF staining in liver cryosections of mice 23 weeks fed mice and (B) F4/80 staining. (C) Quantification of CD45 positive cells in %. (D) Quantification of F4/80 positive cells in %. (E) Left: Tnf-α. Right: Mcp-1 mRNA relative expression to Gapdh. (F) Left: Tlr-2. Right: Tlr-4. * for p < 0.05; ** p < 0.01; *** p < 0.001 Results 170 8.18. WTD+EXER was able to decrease hepatic stellate cell activation and fibrogenesis TNF-α overproduction induces activation of HSCs in the liver [153]. We found strong expression of α-smooth muscle actin (α- SMA), a marker of HSCs activation, using IHC staining and western blot analysis in DUAL-fed animals. WTD+EXER, but not WTD alone, reduced HSCs activation to levels exhibited by the control (Figure 50A, B). Activated HSCs are the major source of ECM during progression of fibrosis. Hence, SR staining clearly demonstrated that feeding a DUAL diet induced severe collagen expression in the liver (Figure 50C, D). Results 171 Figure 50. Neither WTD or WTD + EXER resulted in ECM degradation and remodeling (A) αSMA IHC staining. Activated HSC are represented in brown color. (n = 3-4). Scale bar = 100 μm (B) αSMA western blot using HSC-70 as a loading control. Ratio between α-SMA and HSC-70 was calculated. (C) SR stained areas with collagen fibers staining in bright red (n = 4-7). Scale bar = 100 μm. (D) Quantification of liver fibrotic area in % (n = 4-7). ** for p < 0.01 8.19. WTD and WTD+EXER reverted the intestinal changes caused by DUAL diet feeding Long-term DUAL diet feeding has decreased the total intestine/ body length ratio. Remarkably, WTD and WTD+EXER strategies were able to return intestine length up to Results 172 CTRL level (Figure 51A, B). Regarding colon length, WTD+EXER was able to ameliorate the length changes caused by DUAL diet feeding (Figure 51C). Changes in the gut morphology were accompanied by the defects in gut permeability, as demonstrated by the increase in the LPS levels in serum of DUAL mice, however combination of WTD+EXER, was able to significantly decrease LPS (Figure 51D). Figure 51. Intestinal alterations induced by long-term DUAL diet were reverted by WTD and mainly by WTD+EXER. (A) Intestine pictures of treated and CTRL groups. Scale bar = 1cm. (B) Intestine length measure in cm (n = 5-6). (C) Colon length measurement in cm (n = 5-6). (D) LPS in serum, ng/ml (n = 4-5). Scale bar = 100 μm (n = 5-10). * for p < 0.05; ** p < 0.01. III. LARVAE AND ADULT ZEBRAFISH AS AN ANIMAL MODEL FOR DUAL DIET FEEDING 8.20. Influence of DUAL diet in morphology of zebrafish larvae Lastly, we applied the DUAL diet to Zebrafish (Danio rerio). 6 dpf AB larvae were exposed to DUAL diet with 1 or 1.5% of EtOH content, and its correspondent control groups with control Sparos diet (CTRL), control Sparos diet + 1% EtOH (EtOH 1%) or Western diet (WD), during 3 days from 6 dpf up to 9dpf. At this Results 173 timepoint, E3 media was replaced by fresh one without any treatment included, leaving the larvae for 12 hours O/N fasting. After fasting, when larvae were 10 dpf, Nile red staining was performed in order to detect lipid accumulation in the fish. After ethanol exposure, either 1 or 1.5%, most zebrafish died rapidly during the first 24h. Meanwhile the influence of ethanol in larval morphology, was investigated. Zebrafish embryos, exposed to the different treatments have shown evident hepatotoxic effects, such as darkening and lost transparency in liver. Additionally, Zebrafish embryos exposed to DUAL diet demonstrated spinal curvature most evident after 72h exposure time (Figure 52A). Figure 52. AB larvae images 7 to 9dpf from DUAL fish (1 and 1.5% EtOH) and corresponding control group. (A) Morphology of larvae zebrafish after exposure of 24, 48 and 72 h to either CTRL diet, CTRL+1% EtOH, WD and DUAL with 1 and 1.5% of EtOH. Each experiment was repeated 3 times, (n =15 fish per group). Scale bar = 200 μm. 8.21. Nile red staining revealed no significant changes in TG accumulation after DUAL feeding in 10 dpf AB larvae The influence of the ethanol on the survival rate, has become more critical with the enlargement of the treatment, implying that few larvae per well have survived in ethanol groups (either in combination with control or WD). Therefore, we reduced the of EtOH concentration up to 0.15 and 0.25%. Feeding was started ag 6 dpf lasted up to 9 dpf, and before the sacrifice the larvae were O/N fasted for 12 h. Upon fasting, AB larvae were incubated with Nile red dye during 1 h and pictures were taken under a fluorescence microscope. Evaluation was performed by the quantification of the fluorescence area that corresponds with the localization of the liver region (Figure Results 174 53A). Incubation with Nile red do not show significant changes regarding lipid accumulation in liver parenchyma in between the different experimental groups (Figure 53B-C). Figure 53. Nile red staining of 10dpf AB larvae, revealed no significant differences in liver size and hepatic fat accumulation between experimental groups. (A) Live image of a 10 dpf AB larvae zebrafish with liver and intestinal duct stained in red by Nile red staining. Scale bar = 200 um. (B) Nile red staining images under fluorescence microscope from all experimental groups. (C) Quantification of the liver area measured by ImageJ (n = 10). 8.22. DUAL diet feeding has triggered hepatic lipid accumulation in 10 dpf FABP zebrafish larvae A new experimental trial was performed by using the liver fatty acid-binding protein (FABP) zebrafish. This small cytosolic protein is expressed in various tissues, including the liver and small intestine [171]. This transgenic fish with red fluorescence in the liver, greatly facilitates the observation of this organ in vivo (Figure 54A). Larvae were treated from 6 dpf up to 9 dpf, and at this point they were O/N fasted for 12 h. Results 175 At 10 dpf, BODIPY essay was performed to trace the neutral lipids. Life imaging of the treated larvae show that DUAL diet treatment resulted in fat accumulation in the liver, as revealed by BODIPY labeling of lipid droplets in hepatocytes. TG hepatic content was higher in WD compared to either CTRL or EtOH groups, however EtOH treated fish did not show any relevant lipid accumulation in comparison with the CTRL (Figure 54B-C). Figure 54. BODIPY signal increases after DUAL diet feeding in 10 dpf FABP zebrafish. (A) Scheme of FABP zebrafish, with fluorescence red liver and BODIPY green signal through the intestinal duct and liver area. Time-line for the DUAL feeding of the zebrafish larvae. (B) Top panel: separated green (BODIPY) and red (liver) life images of FABP larvae (Scale bar = 50 μm). Lower panel: magnification of the liver region. (Scale bar = 200 μm). (C) Overlapped images from green and red channels of the different treated groups. 8.23. DUAL diet feeding triggers hepatic steatosis in adult AB zebrafish Finally, adult AB zebrafish, were treated with DUAL diet for 21 days, and its corresponding control groups were run in parallel with either WD, 0.25% EtOH or CTRL diet. Results 176 Body weight measurement at the end point of the treatment has shown a significant body weight increase in DUAL mice compared to CTRL (Figure 55A). Regarding body length, BMI, basal glucose, liver weight and intestine length no significant changes were detected in between groups (Figure 55B-F). At the end point of the treatment, paraffin embedding of the whole fish was performed, followed by H&E staining in order to analyse the histology samples. H&E slides were analysed by an expert fish pathologist and the analysis revealed very intensive micro and macro steatosis in the hepatic tissue of DUAL fed fish (Figure 55G). Figure 55. Representative H&E staining sections from liver parenchyma of adult AB zebrafish, from the different experimental groups. (A) Body weight g. (B) Body length (cm). (C) Calculation of the BMI in kg/m². (D) Basal glucose levels after 12 h of fasting measured from tail blood in mg/ml. (E) Liver weight (g) (F) Intestine length (cm) (n = 6-7). (G) H&E representative images (n = 2-3). Scale bar = 50μm. * for p < 0.05 Discussion 177 DISCUSSION Discussion 178 Discussion 179 9. Discussion Over the past two decades, some light has been shed on a number of aspects of NAFLD pathogenesis and the complex relationships between liver steatosis, obesity and modifiable risk factors, such as food habits and sedentary lifestyle. Despite the increase in knowledge, there is still no universally approved medical treatment for NAFLD patients [4]. The current body of research suggests that the best way to prevent NAFLD is the combination of a balanced diet with regular exercise to achieve a healthy weight. In contrast, excessive fat consumption is strongly related with accumulation of fat in the liver. The quality of fat might be as important as the quantity for NAFLD progression. Although the effects of specific types of dietary fat on cardiovascular diseases have been more or less widely studied, there have been only a limited number of investigations examining the effects specifically on NAFLD in this regard. Consequently, it is critical to investigate the effects of transfat and HP oils as possible triggers to the development of NAFLD, since the consumption of ultra-processed foods, rich in both oils, is increasing worldwide. Therefore, the first aim of the present study was to investigate the effects of three different WDs on the liver and the pathophysiological modulation that these diets perpetrate as a mechanism to develop NAFLD. We showed that the consumption of all types of WDs rich in fat, fructose and cholesterol for 14 weeks predisposed mice to the development of obesity, hepatomegaly, hepatic cell death and steatosis. Even LP-WD promoted obesity, hepatomegaly and significant liver fat accumulation, with modulation of FA metabolism. Thus, only HP-WD and HP- Trans-WD induced hypercholesterolemia and immune cell infiltration resembling the pattern of Nonalcoholic Steatohepatitis (NASH). Moreover, only mice treated with HP- WD and HP-Trans-WD developed significant liver fibrosis, an important hepatic feature of NAFLD that is usually observed in patients with persistent necroinflammatory changes [25]. Our observations are in line with previously published murine models fed with a high fat diet (HFD) containing trans fats—mice receiving American Lifestyle-Induced Obesity Syndrome (ALIOS) diet [26]. ALIOS mice became obese and developed severe hepatic steatosis with associated necroinflammatory changes. Plasma ALT levels increased, as liver TNF- α and procollagen mRNA, indicating an inflammatory and profibrogenic response to injury [25]. Feeding with a high-fructose medium-chain-transfat diet in another study was also associated with obesity, increased hepatic oxidative stress and a steatohepatitis-like phenotype with significant fibrosis [27]. Discussion 180 The mechanisms of hepatic steatosis caused by transfats are the subject of considerable debate. We examined mRNA expression of key lipid metabolic genes involved in FA uptake, export and oxidation. Our data showed that the diet rich in transfat promotes hepatic lipid accumulation by more than one mechanism. We found that HP-Trans-WD in the liver reduced expression of Apob (hepatic TG secretion) and Acox (FA -oxidation). Both decreased FA oxidation and secretion are not able to offset the diet-induced increase in intrahepatic lipid and together contribute to the diet-induced fatty liver and hepatic accumulation of fat. Consistently, it has been demonstrated in vitro [28,29], as well as in human studies [30], that Trans FAs alter secretion and size of Apo-B100 containing particles produced by hepatic parenchymal cells and reduce the expression of FA Oxidation (FAO) enzymes [31]. In line with these observations, an increase of SCD1 activity in HP-Trans-WD was previously reported in obese subjects and associated with lower FA oxidation and higher fat storage [21]. Interestingly, we found that feeding with HP-Trans-WD did not increase the expression of key genes of de novo lipogenesis, but in fact FASN expression was profoundly suppressed. This appears paradoxical because FASN suppression markedly improves steatosis in other experimental models of fatty liver, and FASN antagonists are under development as plausible treatments for hepatic steatosis [32]. Therefore, it seems that in HP-Trans-WD fed animals, FASN suppression is likely a compensatory mechanism designed to reduce lipid synthesis under conditions in which lipid oxidation is reduced and TG export is inactive. Moreover, FASN expression is impaired by hepatic inflammation in experimental models characterized by severe hepatocellular damage and inflammation, as well as in patients with steatohepatitis [33]. Consistently, mice fed with HP-Trans-WD showed high levels of plasma markers of liver injury and significant inflammation in liver tissue, accompanied by increased Tnf-α expression. Concomitant with our observation, recent human studies showed that Trans FAs modulate human macrophage response, increasing the production of TNF-α [34]. Lipidomic analysis revealed a marked step from DG to TG (precursor/product) in HP-Trans-WD. These findings are highly consistent with human lipidomic NAFLD data published by A.J. Sanyal’s group [35]. Moreover, knockdown of Diacylglycerol Acyl Transferase (DGAT) ameliorates the fatty liver in the ob/ob mouse [36], suggesting that DGAT plays an important role in the development of hepatic steatosis in mice fed HP Trans-WD. Several publications also supported the link between altered DG levels and insulin resistance [37]. DG are derived from lipogenesis and membrane phospholipids (PL). The lower level of de novo lipogenesis and the parallel Discussion 181 decrease in PC suggest that the membrane PC may definitely contribute to the observed increase in DG in HP-Trans-WD mice in the current study [34]. Indeed, PC is one of the most abundant PL in mammals and a major component of cellular membrane lipids. Furthermore, PC levels were reported to be decreased in the liver samples of patients with NAFLD [35,38]. In hepatocytes, up to 30% of PC comes from the conversion of Phosphatidylethanolamine (PE) to PC by Phosphatidylethanolamine N- Methyltransferase (PEMT). Hence, the significant decrease of the hepatic PC/PE ratio was previously observed in NAFLD patients [39]. Additionally, a loss-of-function polymorphism in the PEMT gene seems to be associated with susceptibility in NAFLD [39]. Furthermore, PC is the only phospholipid molecule that is known to regulate the assembly and secretion of lipoproteins. It has been found that low hepatic PC levels due to its synthesis disruption impair the VLDL secretion, and significantly decrease the levels of circulating VLDL lipoproteins and result in hepatic accumulation of TG [40], which is absolutely in line with low Apo-B100 gene expression in HP-Trans-WD. Additionally, a low PC/PE ratio possibly leads to a rearrangement of outer monolayer, loss of membrane integrity and increased permeability to pro-inflammatory molecules such as cytokines, initiating the inflammation in HP-Trans-WD mice [41]. Glycerophospholipids are another component of cellular membrane associated with cellular signaling and cellular apoptosis. Decreased levels of PS and PI were previously reported in liver biopsy samples of patients with NASH [38]. Another striking observation was the increase in FC in mice fed HP-Trans-WD. FC is well-known to be highly cytotoxic [42]. FC accumulation leads to liver injury through the activation of signaling pathways in KCs, HSCs and hepatocytes, promoting inflammation and fibrogenesis [43]. Accordingly, we found significant hepatic infiltration of F4/80 positive cells as well as HSC activation in the HP-Trans-WD group. Concomitantly, Trans FA also increases the cellular accumulation and the secretion of free cholesterol by hepatocytes in vitro [30,40]. Notably, we found that only HP-Trans-WD induced a significantly impaired glucose tolerance after 14 weeks of feeding. Actually, transfats already have attracted critical attention as a potential modifiable risk factor for T2DM in several previous studies. Diet enriched in transfat intake has been associated with diabetes, insulin sensitivity and systemic inflammation [44,45]. Animal studies demonstrated that a high transfat diet causes weight gain and impaired insulin sensitivity in mice and monkeys [46,47]. Overall, our findings raise a red flag on the food high in transfats because of the harmful effects on liver and metabolism in general. However, the proposed industrial substitute—palm Discussion 182 oil does not seem to be a good alternative either, mainly because of high saturated FA, especially PA (C16:0 around 44%). PA treatment has been known to induce inflammation and cellular injury in various tissues. For example, Chen et al. showed that for each kilogram of palm oil consumed annually per capita, the mortality due to ischemic cardiomyopathy increases to the equivalent of 68 for 100,000 deaths in developed countries [48]. The effects of saturated PA on the liver are another pressing issue [8,16]. Boland et al. showed that the substitution of transfat with palm oil (GAN diet) resulted in maintained NASH phenotype in both ob/ob and C57BL/6J mice [49]. In our study, we found that animals fed with HP- WD develop obesity, hepatomegaly, steatosis, inflammatory cells infiltration in the liver and fibrosis as HP-Trans-WD. The changes in the lipid metabolism and lipidomic were also very similar between both groups: high Scd and Ppar mRNA expression levels in combination with low FA oxidation (CPT1, Acox), leading altogether to significant fat accumulation in the liver. Moreover, the levels of TG, DG and FC were similarly increased in HP-WD. The main difference between groups was that HP-WD feeding was not associated with glucose intolerance and did not prompt the increase of transaminases as HP-Trans-WD. In addition, the hepatic, metabolic and lipidomic changes were much more pronounced in HP-WD compared with LP-WD fed animals. In accordance, a previous in vitro study showed that saturated PA fatty acids induce hepatocyte lipoapoptosis, and it is more toxic than with unsaturated fatty acids [50]. Lifestyle modifications including diet, exercise and weight loss have been found to be effective in controlling NAFLD. Indeed, we found that WD withdrawal for only 3 weeks resulted in significant improvement in several features of MS and NAFLD, and in general was very efficient in returning most of the analyzed parameters to control-like levels. The withdrawal from all types of WDs promoted lower body mass; decreased visceral fat accumulation; remarkably improved steatosis, serum transaminases, cholesterol and hepatic TG content; and attenuated hepatic fibrosis. Importantly, it also normalized glucose tolerance in HP-Trans-WD. However, the specific lipid composition of the diet seems to also be important in this setting. Liver inflammation in HP and HP-Trans-WD as seen post-withdrawal, is not fully reversible. This, of course, raises concerns, pondering the frequent cycling of diet and weight in the population with obesity. In summary, dietary recommendations to prevent and manage NAFLD should focus not only on quantity but also quality of the diet. Our study clearly demonstrated that modifying Discussion 183 types of fat in the diet can lower risk of NAFLD progression and improve features of MS. Yet, it is very important to understand that “transfat free” and/or “palm oil free” labeling should not indulge people in buying high fat or processed food. Altogether, this study provides important information on the role of different types of fat for development and progression of NAFLD and could be used to inform policy makers discussing the new European Union (EU) regulations to promote better food environments. In another hand, recent epidemiological and experimental evidence has highlighted the dangerous synergism of alcohol and NAFLD in the progression of CLD [172-175]. Epidemiological studies using a large cohorts of patients in Northern Italy [172], France [173], Scotland [174], China [176] and South Korea [175] clearly showed that obese alcoholics have 2-3 times higher risk of developing steatohepatitis and progression to fibrosis or cirrhosis. Hence, obese individuals consuming 15 or more drinks per week have an adjusted relative rate of liver-related death of 18.9 compared to 3.16 in their lean counterparts [174]. One of the two conditions is often predominant, with the other acting as a cofactor of morbi-mortality [91]. Nevertheless, synergy of alcohol and NAFLD is not well described and presents a large grey area in Hepatology field with huge unmet need in pre-clinical and clinical studies. Traditionally, in pre-clinical studies, most ALD and NAFLD researchers use animal models to identify molecular mechanisms of the disease, to exploit molecular targets or test putative pharmacological agents. Knowledge gained from these studies can then be used in further clinical trials for development of novel diagnostic, prognostic, preventative, and therapeutic strategies [177]. Recently we established a new experimental mouse model which physiologically mimics all extrahepatic and intrahepatic features of DUAL ethology CLD (NAFLD + alcohol). In our innovative DUAL model, we overcome the natural mouse aversion to alcohol by incorporating D-glucose in the water. Sweetened water successfully masked the taste of alcohol increasing alcohol intake. Additionally, the consumption of fatty WD significantly elevated ethanol intake as well. Consistently, previous reports suggest the existence of a positive correlation between ethanol and fat whereby each nutrient stimulates consumption of the other [126]. This finally resulted in higher daily caloric consumption robustly increases body weight and intensifies liver damage in DUAL-fed animals. Discussion 184 Another key feature of our DUAL model is obesity with remarkable damage of WAT: hypertrophic adipocytes develop an inflammatory phenotype and CLS, composed of necrotic cells. Those changes are often evident in patients with ALD/NAFLD, and significantly contribute to disease progression [91]. Abdominal obesity is a predominant underlying risk factor for MS [57]. DUAL animals simultaneously to obesity, develop basal hyperglycemia and hypercholesterolemia, three important medical conditions of MS [178]. MS promotes the flux of lipids to the liver and represents an important risk factor for fatty liver disease. Hence, hepatic steatosis induced by DUAL diet takes a short period of time. After 10 weeks of feeding, DUAL mice already developed micro- and macrovesicular steatosis with hepatocytes ballooning. Consequently, the accumulation of FFA in liver induces oxidative stress and leads to inflammation and immune cell infiltration into the hepatic parenchyma. Inflammatory cytokines, produced by immune cells (eg: TNF-a), further activate HSCs and stimulate the production of collagen fibers and extracellular matrix deposition in the liver, leading to fibrogenesis [153]. Mice fed a DUAL diet exhibit advanced accumulation of collagen in portal and bridging areas, already after 23 weeks of feeding. Notably, transcriptomic changes relevant to chronic liver diseases in human were also indicated in DUAL mice. Importantly, the upregulation of TNF, NF-κB [179] and TLR2,4/9 [180, 181] in DUAL animals entirely recapitulated the pathogenesis of humanlike steatohepatitis and correlate with disease progression to advanced fibrosis. Altogether, our novel preclinical model induces the progression to advanced steatohepatitis and fibrosis in the context of key risk factors for the human condition (i.e., alcohol consumption, obesity, MS), naturally mimicking human pathology. Whether obesity, MS, steatohepatitis and hepatic fibrosis are reversible remains a relevant clinical question and is a constant matter of debate. Therefore, we used a synergistic DUAL murine model of NASH and alcohol consumption to study the biological impact of dietary changes and physical exercise on the progression of steatohepatitis and fibrosis. The aim of this study was to test whether switching the diet is sufficient to reverse DUAL-diet induced phenotype, or only the combination of diet withdrawal and physical exercise can be considered as a right therapeutic option. We initiated DUAL diet when the C57BL/6 mice reached 10 weeks of age. After the short- term (10 weeks) or long-term (23 weeks) feeding we either (i) replaced the DUAL diet Discussion 185 with chow diet and normal water for 20 days (withdrawal group (WTD)); or (ii) combined diet withdrawal with daily treadmill sessions for 20 days (WTD+EXER). WTD alone was effective in decreasing the body mass of animals that were fed a DUAL- diet for a short period of time. In contrast, WTD or WTD in combination with treadmill running exhibited a reduction in body weight compared with animals from long-term DUAL-fed group, hence levels remained higher than those of the CTRL mice. The decrease of eWAT weight or the reduction of adiposity was also depending on the duration of feeding. Such as, after the short-term DUAL feeding, both WTD and WTD+EXER could significantly decrease the amount of adipose tissue, after a long-term feeding neither WTD or WTD+EXER have led to the reduction of eWAT mass. Adipose tissue expands primarily through hypertrophic growth of existing adipocytes [182]. In our study we were able to show that switching the diet is sufficient to restore adipocyte size in 10-weeks DUAL fed animals. Whereas only mice subjected to WTD+EXER exhibited a decline in eWAT cells size after the long-term feeding. The adipose tissue displays remarkable plasticity in response to external stimuli such as dietary intervention and exercise. Exercise is known to stimulate lipolysis [183], decrease lipogenic gene expression in adipose tissue [184], and promote angiogenesis [185]. These three mechanisms may explain how exercise promotes smaller, metabolically healthy adipocytes even after long-term feeding. Moreover, smaller adipocytes have been shown to be protective against metabolic decline [186, 187] and adipocyte size may be used as a predictor for the development of insulin resistance in obese individuals [182, 188]. Infiltration of inflammatory immune cells in adipose tissue has been extensively studied and is known to contribute to obesity-related inflammation and macrophage infiltration [189]. A possible explanation is that increasing hypertrophic adipocytes gradually entails the death of adipocytes, which needs to be removed by macrophages [182]. Remarkably, even after long-term DUAL feeding bouts of exercise in combination with WTD could reduce immune infiltration and CLS in adipose tissue of DUAL mice. Macrophages are frequently implicated as a major contributor to adipose tissue inflammation [182]. It also has been reported that expansion of adipose tissue not only increases macrophage infiltration in adipose tissue but also causes a change in Discussion 186 polarization of macrophages, which would convert from type M2 macrophages, with an anti-inflammatory secretory profile, into M1 macrophages, having a proinflammatory secretory profile. M1 are responsible for expression of most proinflammatory cytokines produced in adipose tissue and molecules involved in recruitment of additional macrophages, creating a vicious cycle that amplify activation of inflammatory pathways [190]. Taking this into account, our findings need to be further confirmed by other technical approaches such flow cytometry [191]. DUAL associated obesity after long-term feeding causes increased fibrosis in eWAT which has been linked to metabolic dysfunction by limiting the healthy expansion of adipose tissue [192]. Only the combination of WTD and treadmill training decreased fibrosis in eWAT measured by decreased Sirius red staining. Taken together, we showed that combination of WTD and tread-mill exercises improved adipose tissue cellularity, limit fibrosis and pro-inflammatory immune cell infiltration in WAT. Exercises exerts positive changes to adipose tissue that promote healthier adipocytes and improve metabolic homeostasis. However, there are important questions that remain to be addressed: (1) angiogenesis and capillary density in WAT, (2). characterisation of immune cells phenotype and citokines secretion in WAT, (3) analysis of gene expression related to fatty acid oxidation, lipolysis, and fatty acid synthesis in eWAT, (4) systemic WTD-related or exercise-related signals that WAT cells are responding. More detail future studies are needed to address these topics which will help understand the mechanisms underlying the exercise effect on adipose tissue and metabolic health. Exercise and diet control are fundamental approaches in the treatment of obesity-related metabolic conditions. Therefore, next we analyzed the DUAL-related metabolic disorders such as hypercholesterolemia and hyperglycemia in DUAL animals. After the short-term feeding the increase of serum cholesterol was successfully reduced by WTD and WTD+EXER. After the long-term feeding the cholesterol levels significantly and equally dropped down in both WTD and WTD+EXER groups however, did not reach the level exhibited by the CTRL animals. Hence, previous studies showed [193], that the chief determinants of blood total cholesterol concentrations are dietary intake of saturated fat, polyunsaturated fat, and cholesterol. Cholesterol concentrations are also affected by reduced energy intakes resulting in weight loss and possibly by specific dietary supplements such as fibres and fish oil. Discussion 187 Next important finding was that only dietary modification in combination with treadmill (WTD+EXER) are able to maintain the control of glycaemia is long-term DUAL fed mice. Diet and exercise are considered by all diabetes clinical guidelines to be the foundation for DM2 management. While caloric restriction-induced weight loss may have a large influence on insulin sensitivity, the subsequent loss of fat free mass may compromise glucose uptake, given that skeletal muscles is responsible for a larger portion of glucose uptake. Exercise can augment glucose disposal and improve insulin action, and thus can be a tool to aid in glucose regulation. Muscle contraction and contraction-mediated skeletal muscle blood flow leads to glucose uptake via insulin- dependent and independent mechanisms. Exercise-mediated glucose disposal can decrease circulating blood glucose but may be affected by other determinants of systemic glucose metabolism [194]. An alternative mechanism by which exercise could improve glycaemic control, is via the enhancement of pancreatic β cell activity, which can become compromised as a consequence of overstimulation and excessive insulin secretion in response to a loss of insulin sensitivity. In support of this, it has been reported that exercise training plus weight loss can increase pancreatic β cell function in a linear dose-response manner in adults with prediabetes and T2DM. The components of glucose disposal need to be further considered to better understand the impact of exercise on glucose clearance in DUAL model [195]. It has been reported that diet and lifestyle modification leading to weight loss of 10% or more has been proven to be an effective strategy to achieve resolution of NASH in >90% of patients [196]. In the DUAL liver, both WTD and WTD+EXER remarkably reverted hepatomegaly, steatosis and TG accumulation induced by short term feeding. After the long-term feeding WTD and WTD+ EXER significantly improved the hepatomegaly hence the liver mass did not return to CTRL-like levels. Consistently, the levels of hepatic steatosis and TG accumulation in the liver decreased after WTD and WTD+ EXER but was still higher than corresponding controls. The withdrawal of the fat rich DUAL diet reduced this flux of FFA to the liver. In addition to storage into TG leading to steatosis, FFAs are considered the metabolically and immunologically active form of fat contributing to cell damage and inflammation that are characteristic of NASH [197]. Discussion 188 In agreement with previous animal models [198-200], we found increase in lipogenesis in DUAL mice after long term feeding. In lipogenesis, acetyl-coA derived from the Krebs cycle gets converted into long-chain fatty acids facilitated by a lipogenic enzymes, fatty acid synthase (FASN), which was significantly upregulated in DUAL mice. Interestingly, WTD+EXER combination could reduce the expression of FASN in the liver. Thus, it seems reasonable to suggest that treadmill running could contribute to attenuating hepatic TG accumulation, via suppressed de novo lipogenesis. Epigenetic mechanisms (e.g., reduction of DNA hypermethylation) have been proposed to be responsible for the beneficial effect of physical exercise on the metabolic pathways including de novo lipogenesis [201]. The liver can neutralize metabolically and immunologically active FFA in three major pathways: esterification of FFA into triglycerides and sequestration into lipid droplets (steatosis), excretion in very-low-density lipoprotein, and fatty acid oxidation in hepatocyte mitochondria (β-oxidation) [196]. Consistently with previous animal studies [196], we found that diet modification in combination with treadmill running increase β oxidation in DUAL livers after short-term feeding. Peroxisome proliferator-activated receptor-α (PPARα) is a lipid-sensing member of the nuclear receptor superfamily that regulates the expression of enzymes responsible for mitochondrial fatty acid oxidation and thereby stimulates β-oxidation in the liver Hashimoto et al. (2000) reported that mice with insufficiently expressed PPARα had excessive accumulation of hepatic TG [202]. In our study, DUAL-induced downregulation of hepatic PPARα expression was alleviated with the exercise training. Besides, the hepatic protein expression of carnitine palmitoyl-CoA transferase 1 (CPT- 1), an enzyme necessary for transport of FFA from the cytosol across the mitochondrial membrane was improved in WTD and WTD+EXER groups resulting in a more complete degradation of FFA compared with DUAL animals. All together this indicate that diet modification in combination with exercise training could contribute to the oxidation of FFA via enhanced expression of PPARα and its target genes, such as CPT1a in DUAL mice subjected to a short-term feeding. A different situation was revealed after 23 weeks of DUAL feeding: the protein level of CPT-1c was upregulated in DUAL mice indicating that β-oxidation becomes overwhelmed by the abundance of FFA and mitochondria sustain damage, reactive Discussion 189 oxygen species (ROS) are formed resulting in toxic metabolites capable of causing further hepatic damage. Also, in the pathology of human NASH, multiple markers of this oxidative stress have been shown to be elevated [203]. Importantly, DUAL diet withdrawal alone has not decreased the level of CPT-1c. In contrast CPT-1c level was significantly dropped in WTD+EXER animals to values similar to CTRL group. Previous human studies revealed that aerobic exercise significantly reduced serum levels of ROS production and lipid peroxidation [196]. The expression levels of PPARα, which regulates the oxidation of fatty acids, has being significantly decreased in all treated groups. It is the main transcriptional regulator of the genes involved in lipid metabolism and modulating the rate of biosynthesis-catabolism of FFAs. FFA accumulation is derived also by mobilization of TG from adipose tissue together with a decrease in PPARα activation [204, 205]. The activation of inflammation and the innate immune system, and the role of macrophages as predominant effector immune cells in steatohepatitis are well established [206]. Innate immune activation is thought to be a consequence of hepatocyte damage induced by the above-described toxic consequences of FFA and ROS. Indeed, elevations in ALT, AST, LDH as markers of hepatocyte damage was found in serum of DUAL mice after short and long periods of feeding. After the short-term feeding the increase of serum transaminases was successfully reduced by WTD and WTD+EXER to the levels exhibited by the control mice. After the long- term DUAL feeding WTD alone or in combination with Ex also decrease the levels of transaminases. Hence, on the microscopic level (TUNEL staining) WTD and WTD+EXER could only slightly improve the levels of hepatic damage. Additionally, the increase of AP in blood indicate the hepatobiliary pattern of liver damage [207] after 23 weeks of feeding. Consistently, several human trials demonstrated that various exercise regimens reduced the serum levels of serum transaminases [208-211]. With cellular damage, the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box-1 protein (HMGB1) and mitochondrial DNA can activate pattern recognition receptors on macrophages, which are a major source of inflammatory cytokines leading to amplification of the inflammation [212, 213]. Hepatic inflammation strongly increases after 23 weeks of DUAL feeding. Only the combination of WTD+EXER was not able to decrease the levels of inflammatory CD45 and F4-80 cells infiltrating the hepatic parenchyma. Discussion 190 However, liver macrophages are a heterogeneous population composed of monocyte- derived infiltrating and resident that partially lose their ability to self-renew and are replaced by monocyte-derived KCs during steatohepatitis. We showed that WTD+EXER decrease the total macrophages in the liver. However, the cytokines levels remained high in WTD and WTD+EXER groups. Only pro-inflammatory monocyte-derived activated macrophages secrete harmful cytokines. Therefore, we suggest that WTD+EXER after long term DUAL feeding partially reduced the accumulation of pro- inflammatory macrophages in the liver parenchyma but did not shift macrophages toward a less inflammatory phenotype [214]. The precise mechanisms by which exercise reduces hepatic inflammation in DUAL steatohepatitis requires further analysis. Following the effects of long-term DUAL feeding on the development of steatohepatitis, we have investigated the effects of diet modification and exercises on downstream outcomes, such as hepatic fibrosis. In our experimental settings, WTD and WTD+EXER strategies only partially improve fibrosis. WTD+EXER lowered αSMA levels. However, the accumulation of collagen fibers in liver after 23 week of feeding was still high in WTD and WTD+EXER groups. Growing scientific evidence supports the importance of gut microbiota in metabolic disorders linked to obesity and CLD [215, 216]. Gut microbiota is known to improve the host energy yield from digested food, to alter choline metabolism and to regulate the enterohepatic circulation of bile acids (BAs) [217] . Likewise, changes in the microbiota composition are known to disrupt the intestinal barrier integrity, allowing the subsequent translocation of bacterial products into the portal vein to alter the gut-liver crosstalk, which induces downstream inflammatory pathways involved in progression of steatohepatitis [218]. The complex relationship between gut microbiota and liver opens up an attractive window for seeking successful and safe therapies for fatty liver and steatohepatitis [219]. Our experiments using DUAL diet revealed interesting results. We detected a remarkable reduction of the total gut length in mice fed with DUAL diet for 10 and 23 weeks. In fact, such shortening is one of the biological markers in the assessment of colonic inflammation [220]. Colonic inflammation is likely to have important consequences, potentially resulting in villus shortening (villus atrophy), gap formation in the epithelium and, importantly, permeability defects [221]. The defects in gut permeability, pose the risk of the translocation of luminal bacteria and their products (e.g., LPS, lipoteichoic acid, bacterial DNA) to the blood and might affect other distant organs that drain and Discussion 191 filter translocated bacteria. Consistently, mice fed with DUAL diet revealed reduced levels of tight occluding junctions and significant increased levels of LPS in serum. Nonetheless, diet modification alone or in combination with exercise performance effectively improved diet-induced morphological and functional alterations in mouse intestine, ameliorating altered intestinal barrier permeability and associated gut-liver crosstalk derangement. Increased intestinal permeability leads to translocation of microbes and microbial products (endotoxins from gram-negative bacteria, β-glucan from fungi, DNA) the portal circulation. On reaching the liver, they activate of Toll-like Receptors (TLR2/4) - primary drivers of the immune response during liver disease. Therefore, the analysis of toll-like receptors activation was next to be identified in our DUAL model. Indeed, we found strong upregulation of TLR2 and 4 mRNA in our animals after 10 and 23 weeks of DASH- feeding in comparison to control mice, TLR signaling in liver activates downstream proinflammatory cascade, resulting in activation of NF-kB which initiate and maintain the expression of inflammatory cytokines, oxidative and endoplasmic reticulum (ER) stress, and subsequent liver damage and fibrosis [222]. In our study we showed that diet modification and eexercise prevented LPS influx and demonstrate tendency towards the downregulation of TLR-4 and TLR-2 and 4 in WTD and WTD+EXER groups. Altogether, our data highlight the capacity of exercise to maintain gut integrity and counteract both intestinal and systemic inflammatory response, even in the presence of diet modification. We believe, DUAL-induced alteration of the gut-liver axis, intestinal barrier disruption and inflammatory/metabolic gene deregulation promoted by changes in microbiota. Metagenomic analyses showed an altered gut microbiota profile at phylum, class and genus levels in DUAL-fed mice, leading to microbial imbalance. DUAL intake changed bacteria concentration and diversity, increasing the Firmicutes/Bacteroidetes ratio in comparison to that of the control group, as previously described. The ratio between these two phyla (the Firmicutes/Bacteroidetes (F/B) ratio) has been associated with maintaining homeostasis, and changes in this ratio can lead to various pathologies. For example, increases in the abundance of specific Firmicutes or Bacteroidetes species lead to obesity and bowel inflammation, respectively [223, 224]. Firmicutes bacteria are Gram-positive and play a key role in the nutrition and metabolism of the host. Through their metabolic products, Firmicutes bacteria are indirectly Discussion 192 connected with other tissues and organs and regulate hunger and satiety. In contrast, Bacteroidetes bacteria are Gram-negative and associated with immunomodulation. Their components, lipopolysaccharides and flagellin, interact with cell receptors and enhance immune reactions through cytokine synthesis [225]. In our study, diet and exercise interventions balanced DUAL-induced alterations in microbiota composition, promoting opposite changes in fecal microbiota to those characteristics of obesity and related metabolic alterations. F/B ratio returned to the control levels [226-228]. This finding totally correlates with previous studies which highlighted the capacity of exercise to impact the diversity, composition and functionality of gut microbial populations in in vivo models of obesity and human studies [219, 229, 230]. Based on this data, synergistic administration of probiotics, prebiotics, synbiotics or fecal transplantation in combination with diet modulation/aerobic can be an appropriate therapeutic strategy in patients with CLD and should be definitely tested in DUAL-mice in a future. From a general point of view, lifestyle interventions have certain unique advantages, but also limitations that need to be considered. First of all cost-effectiveness of lifestyle interventions is favourable. Noteworthy, the annual healthcare expenditure for unhealthy diets are estimated to range from 3 to 148€ per capita and from 3-181€ per capita for sedentary life style, moreover unhealthy lifestyle can be attributed to ~6 years of life- expectancy lost [231]. Targeting both aspects by lifestyle interventions does therefore indeed make sense. As outlined above in our study diet and lifestyle interventions significantly improved obesity, metabolism and steatohepatitis in DUAL mice. Presumably, WTD and Ex trigger beneficial health effects more efficient than drugs targeting only a certain mechanism of CLD- development. Nevertheless, based on previous clinical studies, several caveats need to be kept in mind that limit these promising aspects. The adherence to lifestyle interventions declines in parallel with the duration of the intervention, resulting in a rebound-phenomenon that has largely been shown for obesity [232]. In terms of adherence, underestimated factors such as gender, intrinsic and extrinsic motivation (including monitoring of the intervention), socioeconomic status, among others, are also known and thus complicate interpretation of the outcome [231]. Traditionally, in pre-clinical studies, most ALD and NAFLD researchers use mouse Discussion 193 models to identify molecular mechanisms of the disease, to exploit molecular targets or test putative pharmacological agents. Knowledge gained from these studies can then be used in further clinical trials for development of novel diagnostic, prognostic, preventative, and therapeutic strategies [177]. However, mice exhibit significant differences in lipid metabolism versus humans [128]. Therefore, does the murine DUAL model properly reflect the clinical reality of human NAFLD-associated with alcohol? To answer this question, we applied DUAL diet to tropical freshwater vertebrate zebrafish (Danio rerio). The experiments revealed that short-term exposure of DUAL diet to zebrafish larvae could induce morphological changes in gut and in the liver, hepatic and intestine steatosis. Adult zebrafish exposed to DUAL diet regime, developed an increase in body weight accompanied by hepatic steatosis rise, loss of hepatocyte morphology and extrahepatic fat accumulation. Moreover, histopathological analysis of the samples has showed that the incidence and degree of steatosis was more severe in DUAL diet-fed adult and larvae compared to the rest of the groups. The molecular mechanism underlying steatosis increase in zebrafish DUAL model should be further investigated. In conclusion, zebrafish has been emerged as a powerful animal model for many human diseases. In this study we established an animal model for NAFLD and alcohol using larvae and adult zebrafish. Compared to the rodent animal models, zebrafish model offers many advantages, such low cost and easy maintenance, short time period for NAFLD development or no need of extra toxins to induce liver injury [233, 234]. Although anatomical architecture of the zebrafish livers differs from mammals, it has been demonstrated the similarity in pathophysiological manifestations in humans including steatosis, steatohepatitis, fibrosis and neoplasia [235]. Such as, the lipid metabolism in zebrafish, starts in the intestine tract with the help of the bile produce of the liver, cholesterol transport is mediated by lipoproteins and TG are stored in intramuscular adipocytes, subcutaneous and visceral, similar to humans [236, 237]. Moreover, the lipid accumulation can be directly visualized in the liver of the transparent larvae and the chemical agents can be just simply added to the embryo media or water [238-240]. These characteristics made Danio rerio a powerful animal model and a useful tool to elucidate the pathogenesis NAFLD and related disorders as well as therapeutic strategies. Discussion 194 Figure 56. Graphical abstract that summarizes the results obtained from the experimental procedures. (A) All types of WDs, predisposed mice to the development of obesity, hepatomegaly, steatosis, and inflammation, however only HP-WD and HP- Discussion 195 Trans-WD induced hypercholesterolemia, hepatic cell death immune cell infiltration and fibrosis and only HP-Trans-WD induced a significantly impaired glucose tolerance. (B) WTD and WTD+EXER have decreased BW, adiposity, hepatomegaly, steatosis, inflammation, cell death in liver, hypercholesterolemia, gut permeability, and dysbiosis. Moreover, only the combination of WTD+EXER were able to reduce these parameters after long-term feeding. (C) DUAL diet feeding in zebrafish has caused morphological changes and hepatic steatosis in larval zebrafish, while in adults has either increase body weight and hepatic steatosis. Created with BioRender.com. Discussion 196 Conclusions 197 CONCLUSIONS Conclusions 198 Conclusions 199 10. Conclusions 1. The consumption of all types of WDs, independently of the fat source, during 14 weeks predisposed mice to the development of obesity, hepatomegaly, hepatic cell death and steatosis. 2. Only HP-WD and HP-Trans-WD induced hypercholesterolemia and immune cell infiltration resembling the pattern of steatohepatitis. Moreover, only mice treated with HP-WD and HP-Trans-WD developed significant liver fibrosis, an important hepatic feature of NAFLD. 3. Only HP-Trans-WD induced a significantly impaired glucose tolerance after 14 weeks of feeding. 4. The withdrawal from all types of WDs promoted lower body mass; decreased visceral fat accumulation; remarkably improved steatosis, serum transaminases, cholesterol and hepatic TG content; and attenuated hepatic fibrosis. Importantly, it also normalized glucose tolerance in HP-Trans-WD. 5. Liver inflammation in HP and HP-Trans-WD as seen post-withdrawal, was not fully reversible. 6. After the short-term DUAL feeding: diet withdrawal alone or in combination with treadmill running was able to decrease obesity, adiposity, hepatomegaly and steatohepatitis, enhance β-oxidation in the liver, improve intestinal. 7. After the long-term feeding the withdrawal of diet decreased cholesterol and transaminase levels in blood, reduced hepatomegaly. 8. After the long-term feeding the combination of diet withdrawal and treadmill running reduced obesity and adiposity, normalized levels of glucose, cholesterol and transaminases in blood, decreased hepatomegaly and, liver damage, hepatic inflammation, attenuated activation of HSCs and improved gut permeability. 9. Short-term exposure of DUAL diet to zebrafish larvae induced significant morphological, and hepatic steatosis. Application of DUAL diet to the adult zebrafish increased body weight and induced significant hepatic steatosis. Conclusions 200 Final conclusion: Dietary modification, alone or combination with physical exercisers in patients with the initial stages of steatohepatitis might be considered as an efficient non-pharmacological therapy. However, only a combination of dietary changes and physical activity can lead to the clinical improvement at the advance stages of steatohepatitis. 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Dis Model Mech, 2013. 6(5): p. 1213-26 Appendix 217 APPENDDIX Appendix 218 12. Appendix 12.1. Publications Pierluigi Ramadori*, Marius Maximilian Woitok*, Olga Estévez-Vázquez*, Raquel Benedé-Ubieto*, Hector Leal-Lassalle, Arantza Lamas-Paz, Feifei Guo, Jeanne Fabre, Julia Otto, Anna Verwaayen, Johanna Reissing, Tony Bruns, Stephanie Erschfeld, Ute Haas, Daniela Paffen, Leonard J Nelson, Javier Vaquero, Rafael Bañares, Christian Trautwein, Francisco Javier Cubero, Christian Liedtke, Yulia A Nevzorova. Lack of Cyclin E1 in hepatocytes aggravates ethanol-induced liver injury and hepatic steatosis in experimental murine model of acute and chronic alcohol-associated liver disease. Molecular Basis of Disease 2023. Co-first author. (IF 6.63, Q1). DOI: 10.1016/j.bbadis.2023.166646 Feifei Guo, Olga Estévez-Vázquez, Raquel Benedé-Ubieto, Douglas Maya-Miles, Kang Zheng, Rocío Gallego-Durán, Ángela Rojas, Javier Ampuero, Manuel Romero-Gómez, Kaye Philip, Isioma U. Egbuniwe, Chaobo Chen, Jorge Simon, Teresa C. Delgado, María Luz Martínez-Chantar, Jie Sun, Johanna Reissing, Tony Bruns, Arantza Lamas-Paz, Manuel Gómez del Moral, Marius Maximilian Woitok, Javier Vaquero, José R. Regueiro, Christian Liedtke, Christian Trautwein, Rafael Bañares, Francisco Javier Cubero, Yulia A. Nevzorova,*. A Shortcut from Metabolic-Associated Fatty Liver Disease (MAFLD) to Hepatocellular Carcinoma (HCC): c-MYC a Promising Target for Preventative Strategies and Individualized Therapy. Co-first autor. Cancers. 2022. (IF 6.575, Q1). DOI: 10.3390/cancers14010192 Olga Estévez-Vázquez, Raquel Benedé-Ubieto, Feifei Guo, Beatriz Gómez-Santos, Patricia Aspichueta, Johanna Reissing, Tony Bruns, Carlos Sanz-García, Svenja Sydor, Lars P. Bechmann, Eva Maranillo, José Ramón Sañudo, Maria Teresa Vazquez, Arantza Lamas-Paz, Laura Morán, Marina S. Mazariegos, Andreea Ciudin, Juan M. Pericàs, María Isabel Peligros, Javier Vaquero, Eduardo Martinez-Naves, Christian Liedtke, Jose R Regueiro, Christian Trautwein, Rafael Bañares, Francisco Javier Cubero, Yulia A. Nevzorova *. Fat: quality, or quantity? What matters most for the progression of Metabolic Associated Fatty Liver Disease (MAFLD). Biomedicines. 2021. (IF 4.7, Q1). DOI: 10.3390/biomedicines9101289 Appendix 219 Benedé-Ubieto R, Estévez-Vázquez O*, Flores-Perojo V, Macías-Rodríguez RU, Ruiz- Margáin A, Martínez-Naves E, Regueiro JR, Ávila M, Trautwein C, Bañares R, Bosch J, Cubero FJ, Nevzorova YA. Abnormal liver function test in patients infected with coronavirus (SARS-CoV-2): A retrospective single center study from Spain. Journal of Clinical Medicine. 2021. *Co-first author (IF 3.3, Q1). DOI: 10.3390/JCM10051039 Benedé-Ubieto R., Estévez-Vázquez O., Guo F., Chen, C, Singh, Y., Nakaya, H., Gómez del Moral, M., Lamas-Paz, A., Morán, L., López-Alcántara, N., Reissing, J., Bruns, T., Avila, M., Santamaría, E., Mazariegos, M.S., Woitok, M.M, Haas, U., Zheng, K., Juárez, I., Martín-Villa J.M., Asensio, I., Vaquero, J., Peligros, M.I., Argemi, J., Bataller, R., Ampuero, J., Gómez M.R., Trautwein C., Liedtke C., Bañares, R., Cubero, F. J., Nevzorova Y.A. A novel experimental DUAL model of advanced liver damage. Hepatology Communications. 2021, (IF 5.1, Q1). DOI: 10.1002/HEP4.1698 Benede-Ubieto R, Estevez-Vazquez O*, Ramadori P, Cubero FJ, Nevzorova YA. Guidelines and Considerations for Metabolic Tolerance Tests in Mice. Diabetes, metabolic syndrome, and obesity: targets and therapy. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2020. (IF 2.8, Q3). Co-first author. DOI: 10.2147/DMSO.S234665 12.2. Participation in conferences 1. European Association or the Study of the Liver (EALS) congress 2023. “The beneficial effects of the diet withdrawal and physical exercise on steatohepatitis of DUAL etiology”. Olga Estévez-Vázquez, Raquel Benedé-Ubieto, Héctor Leal-Lassalle, Jeanne Fabre, Johanna Reissing, Marina Mazariegos León, Carlos Sanz Garcia, Tony Bruns, Javier Vaquero, Rafael Bañares, Francisco Javier Cubero, Yulia A. Nevzorova. Wien, Austria. Date: 21/06/2023- 24/06/202321. Accepted for poster ner 2896 2. EASL congress 2023 “Microbiota-targeted interventions in the gut-liver axis for chronic liver disease of DUAL etiology”. Raquel Benedé-Ubieto, Olga Estévez- Vázquez, Héctor Leal-Lassalle, Ana Redondo-Urzainqui, José María Herranz, Alexander Tyakht, Viktoria Odintsova, Beatriz Gómez- Santos, Patricia Aspichueta, Johanna Reissing, Tony Bruns, Andreaa Ciudin, Juan M Pericàs, Javier Vaquero, Christian Trautwein, Christian Liedtke, Rafael Bañares, Matías A. Avila, Francisco Appendix 220 Javier Cubero, Yulia A. Nevzorova. Wien, Austria. Date: 21/06/2023- 24/06/2023. Accepted for poster ner 1423 3. Kick-Off Meeting Horizon. San Lorenzo de El Escorial, Madrid. Date: 5/05/2023- 6/05/2023. https://www.rcumariacristina.com/ 4. 48th annual congress AEEH- Asociación española para el estudio del hígado. “The beneficial effects of the diet withdrawal and physical exercise on steatohepatitis of dual etiology”. Olga Estévez-Vázquez, Raquel Benedé-Ubieto, Héctor Leal- Lassalle, Jeanne Fabre, Johanna Reissing, Marina Mazariegos León, Carlos Sanz Garcia, Tony Bruns, Javier Vaquero, Rafael Bañares, Francisco Javier Cubero, Yulia A. Nevzorova. Madrid, Spain. Date: 15/03/2023- 17/03/2023. Poster. 5. 48th annual congress AEEH- Asociación española para el estudio del hígado. “Unveiling the Gut-Liver Axis crosstalk in DUAL etiology”. Raquel Benedé-Ubieto, Olga Estévez-Vázquez, Salvador Iborra, Ana Redondo-Urzainqui, Matías A. Avila, José María Herranz, Alexander Tyakht, Viktoria Odintsova, Beatriz Gómez-Santos, Patricia Aspichueta, Johanna Reissing, Oluwatomi Ibidapo-Obe, Tony Bruns, Marina S. Mazariegos, Héctor Leal, Javier Vaquero, Christian Trautwein, Christian Liedtke, Rafael Bañares, Francisco Javier Cubero, Yulia A. Nevzorova. Madrid, Spain. Dates: 15/03/2023- 17/03/2023. Poster. 6. COST action meeting 2023- European Cooperation in Science and Technology. “Closing meeting. WGs meeting, 8th CG & 6th MC Meeting”. Prague, Czech Republic. Date: 09/03/2023- 10/03/2023. 7. 47th annual congress AEEH. “A short-term diet withdrawal ameliorates steatohepatitis in DUAL-fed mice”. Olga Estévez-Vázquez, Raquel Benedé-Ubieto, Johanna Reissing, Tony Bruns, Javier Vaquero, Christian Liedtke, Christian Trautwein, Rafael Bañares Francisco Javier Cubero, Yulia A. Nevzorova. Madrid, Spain. Dates: 25/05/2022- 27/05/2022. Poster. 8. 47th annual congress AEEH. “Papel de p21/CDKN1A en la progresión de la enfermedad del hígado graso no alcohólico (EHGNA)”. Arantza Lamas-Paz, Feifei Guo, Fengjie Hao, Olga Estévez-Vázquez, Raquel Benedé-Ubieto, Elena Vázquez- Ogando, Elena Blázquez- López, Iris Asensio, Javier Vaquero, Rafael Bañares, Carlos Sanz-García, Eduardo Martínez-Naves, Teresa C. Delgado, María Luz Martínez- Appendix 221 Chantar, Pere Puigserver, Yulia A. Nevzorova, Francisco Javier Cubero. Madrid, Spain. Dates: 25/05/2022- 27/05/2022. Oral defense. 9. New insights: Young investigator series PRO-EURO DILI NETWORK. COST ACTION. “Drug-induced liver injury in the context of DUAL model in Zebrafish”. Online defense results obtain in the context of the STSM (Short Term Scientific Mission), internship. Date 19/10/2022. Oral defense. 10. Joint Meeting of ISBRA and ESBRA 2nd World Congress on Alcohol and Alcoholism. “Blood and fecal extracellular vesicles (EVS) as biomarkers of injury in the gut-liver axis during alcohol-induced liver disease”. A Lamas-Paz, L Moran, O Estevez-Vazquez, RL Benede-Ubieto, B Salinas, S Sydor, R Vilchez-Vargas, L Moreno, M Gomez del Moral, LP Bechmann, E Martinez-Naves, J Vaquero, R Banares, YA Nevzorova, FJ Cubero. Krakow, Poland. Dates: 17/10/2022- 20/10/2022. 11. EMBO workshop. Energy balance in metabolic disorders. “A novel role for CDKN1A in metabolic-associated fatty liver disease (MAFLD)”. Arantza Lamas-Paz, Gonzalo Jorquera-Olave, Laura Morán-Blanco, Olga Estévez-Vázquez, Raquel Benedé-Ubieto, Javier Vaquero, Rafael Bañares, Carlos Sanz-García, Eduardo Martinez-Naves, Teresa Cardoso Delgado, María Luz Martinez-Chantar, Pere Puigserver, Yulia Nevzorova, Francisco Javier Cubero. Malaga, Spain. Dates: 03/10/2022- 06/10/2022 12. EMBO workshop. Energy balance in metabolic disorders. “Unveiling the Gut- Liver Axis crosstalk in DUAL etiology”. Benedé-Ubieto, R; Estévez- Vázquez, O; Iborra, S; Redondo- Urzainqui, A; Avila, MA; Herranz, JM; Tyakht, AV; Odintsova, VE; Gómez-Santos, B; Aspichueta, P; Reissing, J; Bruns, T; Mazariegos, MS; Vaquero, J; Trautwein, C; Liedtke, C; Bañares, R; Cubero, FJ; Nevzorova, YA. Malaga, Spain. Dates: 03/10/2022- 06/10/2022. Poster and oral defense. 13. FALK Foundation congress 2022. Pathophysiology and Clinical Management of Alcoholic liver disease. “A short-term diet withdrawal ameliorates steatohepatitis in DUAL-fed mice”. Olga Estévez-Vázquez, Raquel Benedé-Ubieto, Johanna Reissing, Tony Bruns, Javier Vaquero, Christian Liedtke,Christian Trautwein, Rafael Bañares, Francisco Javier Cubero, Yulia A. Nevzorova. Dates: 27/01/2022- 28/01/2022. Mannheim, Germany. Online. Poster. Appendix 222 14. FALK Foundation congress 2022. Pathophysiology and Clinical Management of Alcoholic liver disease. “Gender- related differences in response to DUAL diet in murine model of steatohepatitis”. Benedé Ubieto, R; Estévez-Vázquez, O; Dumartin, M; Reissing, J; Bruns, T; Vaquero, J; Liedtke, C; Trautwein, C; Bañares, R; Cubero, FJ; Nevzorova, YA. Dates: 27/01/2022- 28/01/2022. Mannheim, Germany. Online. Poster. 15. German Association of the Study of the Liver (GASL) virtual annual congress 2022. “Gender-related differences in response to DUAL diet in murine model of steatohepatitis”. Raquel Benedé-Ubieto, Marine Dumartin, Olga Estévez-Vázquez, Johanna Reissing, Tony Bruns, Javier Vaquero, Christian Liedtke, Rafael Bañares, Francisco Javier Cubero, Yulia A. Nevzorova. Hamburg, Germany. Date: 27/01/2022- 28/01/2022. Online. Poster award. 16. 3erd translational hepatology meeting of metabolic hepatic disease AEEH. “Fat: Quality, or Quantity? What Matters Most for the Progression of Metabolic Associated Fatty Liver Disease (MAFLD)”. Olga Estévez-Vázquez, Raquel Benedé- Ubieto, Feifei Guo, Beatriz Gómez-Santos, Patricia Aspichueta, Johanna Reissing, Tony Bruns, Carlos Sanz-García, Svenja Sydor, Lars P. Bechmann, Eva Maranillo, José Ramón Sañudo, María Teresa Vázquez, Arantza Lamas-Paz, Laura Morán, Marina S. Mazariegos, María Isabel Peligros, Andreea Ciudin, Javier Vaquero, Eduardo Martínez-Naves, Christian Liedtke, José R. Regueiro, Christian Trautwein, Rafael Bañares Francisco Javier Cubero, Yulia A. Nevzorova, Juan M Pericàs. Alicante, Spain. Date: 22/10/2021- 23/10/2021. Poster. 17. 3erd translational hepatology meeting of metabolic hepatic disease AEEH. “Gender-related differences in response to DUAL diet in murine model of steatohepatitis”. Raquel Benedé-Ubieto, Marine Dumartin, Olga Estévez-Vázquez, Johanna Reissing, Tony Bruns, Javier Vaquero, Christian Liedtke, Rafael Bañares, Francisco Javier Cubero, Yulia A. Nevzorova. Alicante, Spain. Date: 22/10/2021- 23/10/2021. Poster. 18. 3erd translational hepatology meeting of metabolic hepatic disease AEEH. “Utilidad del sistema Keap1-Nrf2 como marcador de la progresión de la enfermedad hepática”. Laura Morán, Hui Ye, Olga Estévez, Arantza Lamas-Paz, Elena Vázquez, Appendix 223 Elena Blázquez, Iris Asensio Nuria López-Alcántara, Pierluigi Ramadori, Christoph J. Wruck, Mohamed Ramadan Mohamed, Manolo Gómez del Moral, Javier Vaquero, Rafael Bañares, Yulia A. Nevzorova, Francisco Javier Cubero. Alicante, Spain. Date: 22/10/2021- 23/10/2021. Oral defense. 19. Biology PhDay 2021. Biology faculty, Complutense University of Madrid (UCM). “Fat: Quality, or Quantity? What Matters Most for the Progression of Metabolic Associated Fatty Liver Disease (MAFLD)”. Date: 08/10/2021. Poster and oral defense. 20. Digital Liver Cancer Summit meeting by the European Association for the Study of the Liver (EASL). “The combination of alcohol and metabolic syndrome is a fast track to hepatic tumorigenesis”. Benedé-Ubieto R; Chen C; Estévez-Vázquez O; Guo F; Lamas-Paz A; Morán L; Reißing J; Bruns T; Zheng K; Peligros MI; Vaquero J; Trautwein C; Liedtke C; Bañares R; Cubero FJ; Nevzorova YA. Online Dates: 05/02/2021- 06/02/2021. Poster. 21. Translational hepatology sessions from AEEH. Online. Dates: 30/10/2020 and 6/07/2020. 22. The Digital International Liver Congress (DILC) meeting 2020 from EASL. “Shedding light on BASH: A novel experimental model of advanced liver damage”. Benedé-Ubieto R; Guo F; Estévez-Vázquez O; Woitok MM; Zheng K; Asensio I; Juárez Martín-Delgado I; Martín-Villa JM; Vaquero J; Bañares R; Liedtke C; Cubero FJ; Nevzorova YA. Journal of Hepatology, 73(01): S190. DOI: 10.1016/S0168- 8278(20)30887-4. (IF 20.582, Q1). Online. Dates: 27/08/2020- 28/08/2020. Poster. 23. DILC 2020 meeting from EASL. Guo, F., Zheng, K., Benedé-Ubieto, R.*, Woitok, MM., Estévez-Vázquez, O., Kaye, P., Liedtke, C., Cubero, FJ., Nevzorova, YA. “A shortcut from non-alcoholic fatty liver disease to HCC: c-Myc, a promising theranostic target”. Journal of Hepatology, 73(01): S661-S662. DOI: 10.1016/S0168- 8278(20)31785-2. (IF 20.582, Q1). Online. Date: 27/08/2020- 29/08/2020. 24. German Association for the Study of the Liver (GASL) meeting 2020. “Shedding light on BASH: A novel experimental model of advanced liver damage”. Mainz, Alemania. Benedé-Ubieto R; Guo F; Estévez Vázquez O; Woitok MM; Zheng Appendix 224 K; Asensio I; Juárez Martín-Delgado I; Martín-Villa JM; Vaquero J; Bañares R; Liedtke C; Cubero FJ; Nevzorova YA. Online. Dates: 14/02/2020- 15/02/2020. Poster. 25. Annual International Liver Congress (ILC) meeting from EASL. “Cumulative effects of western diet and alcohol abuse: A novel model of ASH/NASH derived liver injury”. Benedé-Ubieto R., Estévez O., Morán L., López-Alcántara N., Guo F., Chaobo C.H., Macias-Rodriguez R.U., Ruíz-Margaín A., Zheng K., Ramadori P., Cubero FJ., Nevzorova YA. Online. Dates: 10/04/2019- 14/04/2019. 26. American Association for the Study of the Liver (AASLD) meeting 2019. “Cumulative effects of western diet and alcohol abuse: a novel experimental mouse model of ASH/NASH-derived liver injury”. Benedé-Ubieto R; Estévez- Vázquez O; Morán L; López-Alcántara N; Guo F; Chaobo CHEN; Macias-Rodriguez RU; Ruíz- Margaín A; Zheng K; Ramadori P; Cubero FJ; Nevzorova YA. Boston, EEUU. Dates: 08/11/2019- 12/11/2019. Poster. 27. NAFLD summit meeting 2019 from EASL. “The genetic background strongly influences the development of steatohepatitis and metabolic syndrome in a novel experimental model of dual ASH/NASH”. Benedé-Ubieto R, Estévez-Vázquez O, Guo F, Zheng K, Woitok MM, Asensio I, Vaquero J, Rafael Bañares, Cubero FJ, Nevzorova YA. Sevilla, Spain. Dates: 26/09/2019- 18/09/2019. Poster. 28. NAFLD summit meeting 2019 from EASL. “Differential effects of palmitic acid on the development of NASH and related metabolic disorders”. Estévez Vázquez O, Guo F, Benedé-Ubieto R, Chaobo Chen, Vaquero J, Bañares R, Cubero FJ, Nevzorova YA. Sevilla, Spain. Dates: 26/09/2019- 18/09/2019. Poster. 29. Paris NASH Meeting 2019. “Differential effects of palmitic acid on the development of NASH and related metabolic disorders”. Estévez-Vázquez O, Benedé- Ubieto R, Guo F, Lamas-Paz A, Vaquero J, Bañares R, Cubero FJ, Nevzorova YA. Paris, France. Dates: 11/07/2019 – 12/07/2019. Poster. 30. The International Liver Congress (ILC) from EASL 2019. “Cumulative effects of western diet and alcohol abuse: A novel model of ASH/NASH derived liver injury”. Benedé-Ubieto R; Estévez- Vázquez O; Morán L; López-Alcántara N; Guo F; Chaobo Appendix 225 CHEN; Macias-Rodriguez RU; Ruíz-Margaín A; Zheng K; Ramadori P; Cubero FJ; Nevzorova YA. Wien, Austria. Dates: 10/04/2019- 14/04/2019. Poster. 31. 44th annual meeting AEEH. “c-MYC overexpression in hepatocytes is associated with spontaneous development of non-alcoholic steatohepatitis (NASH) in mice”. Guo F., Zheng K., Benedé-Ubieto R., Estévez-Vázquez O., Ramadori P., Woitok M.M., Chen C., Trautwein C., Liedtke C., Cubero F.J., Nevzorova Y.A. Madrid, Spain. Dates: 20/02/2019- 22/02/2019. Poster. 32. 44th annual meeting AEEH. “Effects of Western Diet and Palmitic Acid in the development of Non-Alcoholic Fatty Liver Disease (NAFLD) and related metabolic disorders”. Estévez-Vázquez O, Guo F, Benedé-Ubieto R, Chaobo CHEN, Vaquero J, Bañares R, Cubero FJ, Nevzorova YA. Madrid, Spain. Dates: 20/02/2019- 22/02/2019. Poster and oral defense. 33. 44th annual meeting AEEH. “La deleción genética de Keap1 específicamente en hepatocitos mejora el daño hepático, pero desencadena hepatitis por sobreexpresión de Nrf2, en daño hepatotóxico”. Morán L, López-Alcántara N, Estévez O, Ramadori P, Wruck CJ, Asensio I, Vaquero J, Bañares R, Nevzorova YA, Cubero FJ. Madrid, Spain. Dates: 20/02/2019- 22/02/2019. Poster. 34. 1st Translational Hepatology meeting AEEH 2019. “Hepatocyte Keap1 protects against experimental murine hepatic fibrogenesis”. Morán L, López-Alcántara N, Estévez O, Ramadori P, Wruck CJ, Asensio I, Vaquero J, Bañares R, Nevzorova YA, Cubero FJ. San Sebastián, Spain. Date: 2019. Oral defense. 35. 20th International Symposium on Cells of the Hepatic Sinusoid. “Genetic deletion of Keap1 in hepatocytes triggers hepatitis in an Nrf2- dependant manner in an experimental toxic injury model”. Morán L, López-Alcántara N, Estévez O, Ramadori P, Wruck CJ, Asensio I, Vaquero J, Bañares R, Nevzorova YA, Cubero FJ. Sydney, Australia. Dates: 04/09/2019- 07/09/2019. Oral defense. 12.3. Membership of Scientific Societies - Member of the European Association for the Study of the Liver (EASL) Appendix 226 12.4. Most Outstanding Awards - STSM (Short Term Scientific Mission) COST action 2022 scholarship. Stay at Bilkent University (Ankara) and IBG (Izmir Biomedicine and Genome Center) in Izmir, Turkey, in the context of developing a DUAL diet model in zebrafish. - FPI Scholarship (Researcher Staff Training) from MINECO- 2018 call. 12.5. Others - Registration bursary annual EASL congress 2023. Wien, Austria. - Professor of practices of the subject of Immunology, to the students of the degree of Medicine of the Complutense University of Madrid (2019-2023). - BDFACS Celesta flow cytometry course. 2023. - Registration scholarship for the 48th annual congress of the AEEH (Spanish Association for the Study of the Liver) 2023. - Cytometry course at the National Cancer Research Center (CNIO). “Flow Cytometry Induction Course: fundamentals, applications, data analysis and data presentation”. 2022. - Course on the use of the cryostat- UNAM Science and Nanotechnology. Bilkent University Ankara. 2022 - Laboratory safety course Bilkent University, Ankara 2022 - Teaching Innovation Project. Faculty of Medicine of the Complutense University of Madrid (UCM). Department of Immunology, Ophthalmology and ENT. “Internalization of Immunology in the Faculty of Medicine”. 2021-2022. Cubero Francisco Javier, Nevzorova Yulia, Martínez Naves Eduardo, Sanz García Carlos, Estevez Vázquez Olga, Benedé-Ubieto Raquel, Martín Adrados Beatriz, Mazariegos S. Marina. - Registration scholarship for the 47th annual congress of the AEEH 2022. - Teaching Innovation Project. Faculty of Medicine of the Complutense University of Madrid (UCM). Department of Immunology, Ophthalmology and ENT. "Towards a common internalization strategy in the Faculty of Medicine". 2020- 2021. Francisco Javier Cubero Palero, Yulia Nevzorova, Carlos Sanz García, Eduardo Martinez Naves, Beatriz Martín Adrados, Olga Estevez Vázquez, Pedro Roda Navarro, Óscar Aguilar Sopeña. - Organoid Course- The Virtual Human Intestinal Organoid Training Course. Stem Cell 2021 Appendix 227 - Animal experimentation course. Felasa (Federation of European Laboratory animal Science Associations) B. Category B. 2019 - "Young Investigator" registration grant for the EASL NAFLD summit 2019 congress. Seville, Spain. - Registration scholarship 44th annual congress of the AEEH 2018. Appendix 228 Appendix 229 Appendix 230 Appendix 231 Appendix 232 Appendix 233 Tesis Olga Estévez Vázquez Portada Table of Contents Resumen Summary Abbreviations Introduction Open questions Objectives Materials and methods Results Discussion Conclusions References Appendix