An Experimental DUAL Model of Advanced Liver Damage

1051 Hepatology CommuniCations, Vol. 5, no. 6, 2021 An Experimental DUAL Model of Advanced Liver Damage Raquel BenedéUbieto,1,2 Olga EstévezVázquez,1,2* Feifei Guo,2* Chaobo Chen ,2 Youvika Singh,3 Helder I. Nakaya,3,4 Manuel Gómez del Moral ,5 Arantza LamasPaz,2 Laura Morán,2 Nuria LópezAlcántara,2,6 Johanna Reissing,7 Tony Bruns,7 Matías A. Avila ,8-10 Eva Santamaría,8,9 Marina S. Mazariegos,2 Marius Maximilian Woitok,7 Ute Haas,7 Kang Zheng,2,11,12 Ignacio Juárez,2 José Manuel MartínVilla,2,13 Iris Asensio,9,13,14 Javier Vaquero ,9,13,14 Maria Isabel Peligros,15 Josepmaria Argemi,16-18 Ramón Bataller ,16,19 Javier Ampuero,9,20 Manuel Romero Gómez ,9,20 Christian Trautwein,7 Christian Liedtke ,7 Rafael Bañares,2,9,13,14 Francisco Javier Cubero ,2,11** and Yulia A. Nevzorova 2,7,11** Individuals exhibiting an intermediate alcohol drinking pattern in conjunction with signs of metabolic risk present clinical features of both alcoholassociated and metabolicassociated fatty liver diseases. However, such combination remains an unexplored area of great interest, given the increasing number of patients affected. In the present study, we aimed to develop a preclinical DUAL (alcoholassociated liver disease plus metabolicassociated fatty liver disease) model in mice. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for 10, 23, and 52 weeks (DUAL model). Animals fed with DUAL diet elicited a significant increase in body mass index accompanied by a pronounced hypertrophy of adipocytes, hypercholesterolemia, and hyperglycemia. Significant liver damage was characterized by elevated plasma alanine aminotransferase and lactate dehydrogenase levels, extensive hepatomegaly, hepatocyte enlargement, ballooning, steatosis, hepatic cell death, and compensatory proliferation. Notably, DUAL animals developed lobular inflammation and advanced hepatic fibrosis. Sequentially, bridging cirrhotic changes were frequently observed after 12 months. Bulk RNAsequencing analysis indicated that dysregulated molecular pathways in DUAL mice were similar to those of patients with steatohepatitis. Conclusion: Our DUAL model is characterized by obesity, glucose intolerance, liver damage, prominent steatohepatitis and fibrosis, as well as inflammation and fibrosis in white adipose tissue. Altogether, the DUAL model mimics all histological, metabolic, and transcriptomic gene signatures of human advanced steatohepatitis, and therefore serves as a preclinical tool for the development of therapeutic targets. (Hepatology Communications 2021;5:1051-1068). Excessive alcohol drinking is a leading cause of chronic liver disease and accounts for up to 60%- 80% of liverrelated mortality in Europe.(1) These data become even more relevant considering that alcoholassociated liver disease (ALD) receives only about 5% of the attention in the field of hepatology.(2) The principal fact that only about 6%- 30% of heavy drinkers develop cirrhosis indicates that additional factors modulate the risk of ALD progression.(1) Clinical observations commonly suggest a wide individual susceptibility, and indicate several risk factors for ALD including drinking patterns, female gender, genetic background, cigarette smoking, occupational hazards, and hepatotropic viruses. Obesity and metabolic syndrome (MS) represent another important group of risk factors that accelerate fibrosis Abbreviations: ALD, alcoholassociated liver disease; ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; Bcl2, B cell lymphoma 2; BMI, body mass index; BW, body weight; CPT1c, carnitine palmitoyltransferase 1c; DEN, diethylnitrosamine; DUAL, ALD plus MAFLD; ECM, extracellular matrix; EtOH, ethanol; FFA, free fatty acid; H&E, hematoxylin and eosin; HSC, hepatic stellate cell; IF, immunofluorescence; IHC, immunohistochemistry; LDH, lactate dehydrogenase; MAFLD, metabolic associated fatty liver disease; mRNA, messenger RNA; MS, metabolic syndrome; NAFLD, nonalcoholic fatty liver disease; NFκB, nuclear factor kappa B; ORO, Oil Red O; PCNA, proliferating cell nuclear antigen; Pi3K, phosphoinositide 3kinase; RNAseq, RNA sequencing; SR, sirius red; TEM, transmission electron microscopy; TG, triglycerides; TLR, tolllike receptor; TNFα, tumor necrosis factorα; WAT, white adipose tissue; WD, Western diet; αSMA, αsmooth muscle actin. Received November 3, 2020; accepted February 7, 2021. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep4.1698/suppinfo. *These authors contributed equally to this work. **These authors contributed equally as senior authors. Supported by EXOHEPCM (S2017/BMD3727), Ramón y Cajal (RYC201415242 and RYC201517438), NanoLiverCM (Y2018/ NMT4949), COST Action (CA17112), AMMF (2018/117), ERAB (EA 18/14), MINECO Retos (SAF201678711 and SAF201787919R), and German Research Foundation (DFG NE 2128/21, SFB 1382403224013/A02, and SFB/TRR57/P04). FJC is a Gilead Research Liver Hepatology CommuniCations, June 2021BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1052 progression, hepatic carcinogenesis, and mortality in ALD.(3) Epidemiological studies using a large cohort of patients(46) clearly showed that obese patients with alcoholism have 23times 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 liverrelated death of 18.9 compared with 3.16 in their lean counterparts.(5) However, the patients with intermediate levels of ethanol use plus the presence of metabolic risks (i.e., dual clinical features of ALD and metabolic associated fatty liver disease [MAFLD]) represent a large understudied area in hepatology with a huge unmet need in preclinical and clinical studies.(7) Herein we report a physiological, innovative experimental DUAL (ALD plus MAFLD) model that synergistically combines the effects of alcohol and Western diet (WD). The taste of alcohol was camouflaged by adding glucose to the drinking water, consequently increasing the daily ethanol (EtOH) consumption and remarkably intensifying liver damage. Within 23weeks, this preclinical DUAL model reproduced all of the key metabolic and histological features of human steatohepatitis, with consistent development of hepatic fibrosis, enhanced obesity, glucose intolerance, as well as inflammation of white adipose tissue (WAT). Altogether, this preclinical model might be wellconsidered as a useful experimental model to study the dangerous combination of ALD plus MAFLD in human, and therefore be further used for the development of therapeutic options. Materials And Methods animal HusBanDRy All animal procedures were carried out according to Spanish legal requirements and animal protection Scholar. The research group belongs to the validated Research group Ref. 970935 “Liver Pathophysiology”, 920631 “Lymphocyte immunology”, 920361 “Immunogenética e inmunología de las mucosas” and IBL6 (imas12associated). FG and KZ are Chinese Scholarship Council (CSC) fellows. O.E.- V is supported by Beca FPI (associated to MINECO SAF201787919R) and R.B.- U. by Contratos predoctorales de personal investigador en formación UCMBanco Santander (CT63/19). © 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases. This is an open access article under the terms of the Creative Commons AttributionNonCommercialNoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep4.1698 Potential conflict of interest: Dr. Bruns consults for Intercept. He received grants from Gilead, Falk, Abbvie, Norgine, and Merck. aRtiCle inFoRmation: From the 1 Department of Physiology, Genetics and Microbiology, Faculty of Biology, Complutense University Madrid, Madrid, Spain; 2 Department of Immunology, Ophthalmology and ENT, Complutense University School of Medicine, Madrid, Spain; 3 Depart ment of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil; 4 Scientific P latform Pasteur, University of São Paulo, São Paulo, Brazil; 5 Department of Cell Biology, Complutense University School of Medicine, Madrid, Spain; 6 Institute for Endocrinology and Diabetes, Center of Brain Behavior & Metabolism, University of Lübeck, Lübeck, Germany; 7 Department of Internal Medicine III, University Hospital RWTH, Aachen, Germany; 8 Hepatology Program, CIMA, University of Navarra, Pamplona, Spain; 9 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid, Spain; 10 Instituto de Investigaciones Sanitarias de Navarra, Pamplona, Spain; 11 12 de Octubre Health Research Institute, Madrid, Spain; 12 Department of Anesthesiology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China; 13 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; 14 Servicio de Aparato Digestivo, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 15 Servicio de Anatomía Patológica, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 16 Division of Gastroenterology, Hepatology and Nutrition, Center for Liver Diseases, University of Pittsburgh, Pittsburgh, PA, USA; 17 Liver Unit, Clinica Universidad de Navarra, University of Navarra, Pamplona, Spain; 18 Hepatology Program, Centro de Investigación Médica Aplicada, Universidad de Navarra, Pamplona, Spain; 19 P ittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA; 20 Biomedical Research Networking Center in Hepatic and Digestive Diseases, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío de Sevilla, University of Sevilla, Sevilla, Spain. aDDRess CoRResponDenCe anD RepRint ReQuests to: Yulia A. Nevzorova, M.D., Ph.D. Department of Immunology, Ophthalmology and ENT Complutense University School of Medicine Madrid, Spain Email: [email protected] Tel.: +49- (0)2418080662 Hepatology CommuniCations, Vol. 5, no. 6, 2021 BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1053 law, and approved by the authority of environment conservation and consumer protection of the Regional Government of Madrid (PROEX210/18). All animals were maintained in the Animal Facility at the Faculty of Biology, Complutense University Madrid, in a temperaturecontrolled room with 12hour light/ dark cycle with free access to food and water according to the guidelines of the Federation for Laboratory Animal Science Associations. animal eXpeRimentation Female 10weekold C57BL6/J mice were randomly assigned to four different experimental groups. The total number of mice per group was five to seven. Mice were treated with a DUAL diet consisting of WD (D09100301; Research Diets, Inc., New Brunswick, NJ) and 10% vol/vol EtOH absolute in sweetened drinking water containing 6.75% Dglucose (SigmaAldrich, St. Louis, MO). Controls were fed with either normal diet only (Altomin, Lage, Germany), WD only, or EtOH in sweetened drinking water only (Supporting TablesS1S3). statistiCal analysis Data are expressed as mean ± SD. GraphPad Prism version 8.0 (https://www.graph pad.com/ scien tific - softw are/prism/) (San Diego, CA) was used for statistical analysis and graph design. Statistical significance was determined by oneway analysis of variance ANOVA followed by a Tukey post hoc test. Oneway paired ANOVA followed by Bonferroni’s post hoc test was used to evaluate the differences between 10week and 23week feeding groups. Values with different superscripts are significantly different from each other (P < 0.05), as assessed by oneway ANOVA. Differences (P<0.05) between time points (10weeks vs. 23weeks) for each pairing group were assessed by pairing oneway ANOVA and denoted by “#.” Results CHaRaCteRiZation oF a pReCliniCal Dual moDel Because low EtOH intake due to natural aversion in mice is the main limiting factor for ALD development,(8) we masked the taste of alcohol by adding 6.75% Dglucose to the drinking water and gradually increased EtOH concentration from 1% to 10% vol/ vol. Mice usually consume alcohol up to 25% vol/vol.(9) Thus, initially we performed a pilot study to assess the optimal concentration of EtOH in the drinking water. Surprisingly we found that the concentration of EtOH significantly affected the volume, and, consequently the quantity of the daily consumed ethanol. As the concentration of EtOH increased (20%), the mice drank less volume, and intake significantly decreased. At a 5% concentration, the quantity of consumed EtOH remained relatively low (Supporting Fig. S1A). Only a concentration of 10% vol/vol of EtOH resulted in severe pathophysiologic changes (Supporting Fig. S1B,C). Consequently, for all of the following experiments, animals were randomly assigned to four groups: (1) control group; (2) WD plus sweetened water group; (3) 10% vol/vol EtOH in sweetened water plus chow diet group; and (4) DUAL group (Supporting Fig.S2A). oBesity, DyslipiDemia, anD glyCemia: FeatuRes oF tHe Dual Diet Body weight (BW) increased steadily in all treated groups throughout the experimental period (Fig. 1A [left] and Supporting Table S4). Previous studies in both humans and rodents showed significantly elevated EtOH intake in subjects consuming fat.(10) Consistently, our model nicely reflected this positive correlation, as DUALfed animals demonstrated greater EtOH intake compared with the EtOHfed group (Supporting Fig.S3A). In turn, alcohol stimulated the ingestion of a fatrich diet, thus creating a positive feedback loop (Supporting Fig.S3B). This finally resulted in higher daily caloric consumption (Fig. 1A [middle panel]) and robust increase in the body mass index (BMI) in DUALfed mice (Fig.1A [right panel]). Obesity and ALD are associated with profound changes in the function of adipose tissue, which has important systemic and hepatic consequences.(11) The morphometric evaluation of adipocytes in WAT from visceral fat pad showed that DUAL diet led to an increase in adipocyte size by 93.2% after 23weeks of feeding (Fig. 1B and Supporting Fig. S4A). Additionally, many crownlike structures, formed by macrophages aggregated around dying adipocytes, were observed in the DUAL group (Fig.1B). These Hepatology CommuniCations, June 2021BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1054 Hepatology CommuniCations, Vol. 5, no. 6, 2021 BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1055 results were confirmed by a trend toward increased F4/80 and CD45 positive cell infiltration of WAT in DUALfed animals (Fig.1C). Excessive amounts of extracellular matrix (ECM) and fibrosis in WAT accelerates adipose tissue dysfunction in obese individuals.(12) Consistently, sirius red (SR) staining revealed that DUAL diet dramatically increased collagen deposition in WAT compared with the rest of the experimental groups (Fig.1D and Supporting Fig.S4B). Obesity is strongly associated with other features of MS, including glycemia and dyslipidemia.(13) In fact, the circulating levels of total cholesterol were constantly higher in DUALfed animals (10weeks and 23 weeks) compared with control groups (Fig. 1E). Additionally, DUALfed mice developed hyperglycemia at 10 weeks that was sustained up to 23 weeks of treatment (Fig. 1F). Hence, no significant difference between control animals and treated ones was detected by glucose tolerance test. Insulin tolerance test (ITT) after 6hours of fasting showed the impaired insulin sensitivity only in WD and EtOH groups (Supporting Fig.S5A,B). Dual FeeDing tRiggeRs Hepatomegaly anD Fatty liVeR Disease Obesity and MS predispose to the development of fatty liver disease.(14) After 23 weeks of DUAL diet, animals exhibited enlarged livers, which were pale and yellowish in color, indicating lipid accumulation (Fig.2A [left]). Accordingly, the hepatic mass was increased, and the hepatosomatic ratio of DUAL mice reached almost 8.5% (Fig.2A [middle and right panels]). Hepatocytes of DUALfed animals lost their typical hexagonal shape and became enlarged round cells, as demonstrated by phalloidin staining (Fig.2B). Blinded quantitative analysis performed by an experienced pathologist revealed that animals treated with DUAL diet exhibited microvesicular and macrovesicular steatosis grade 2 associated with hepatocyte ballooning, reaching in most of the animals a S2A4 NAFLD score (moderate steatosis, severe activity according to Bedossa system(15)) (Fig.2C and Supporting Fig.S6AC). All experimental groups after 23 weeks of treatment displayed positive Oil Red O (ORO) staining compared with control mice. However, numerous macrolipid and microlipid droplets were more profuse in DUALfed mice (Fig. 2D). Consistently, hepatic triglycerides (TG) were increased in all mice, with the highest levels found in those treated with DUAL diet (Supporting FigS6D). Dietary free fatty acids (FFAs) are the main source of TG in the liver. Interestingly, while the level of nonesterified fatty acids (NEFAs) in the serum of WDfed mice was significantly increased, the NEFAs in the DUAL animals were not upregulated (Supporting Fig. S6E). This indicates the massive FFA flux into the liver. Consistently we found significant upregulation of CD36 expression (FA translocase) in the livers of DUAL animals (Supporting Fig. S6F). At the same time, the level of TG in serum of DUAL mice was not proportionally increased, reflecting the possible impairment in hepatic secretion of very lowdensity lipoproteins(16) (Supporting Fig.S6G). Expectedly, the lipid load induced an increase in lipid oxidation, as observed by raised carnitine palmitoyltransferase 1c (CPT1c) levels in WD. However, protein expression of CPT1c in the DUAL group was not as high as in WD mice. Altogether, high FFA flux into the liver, poor TG secretion, in combination with reduced lipid oxidation contributed to accumulation of a remarkable amount of fat in the liver parenchyma of DUAL mice (Fig.2E). Fig. 1. Metabolic profile of mice treated with DUAL diet and control groups. (A) Left: BW curve during the feeding period. Statistical differences between DUAL and control groups are shown (n=57). Differences (P<0.05) between control and DUAL group are denoted by “*.” Middle: Calorie intake per day including calories in food and in drinking water (Dglucose and/or EtOH) (n=46). Right: BMI was calculated after 10 and 23weeks of feeding (body surface area [m2/BW]; n=59). (B) Representative WAT H&E. Scale bar=100µm (n=3). (C) Representative CD45 and F4/80 IF staining of WAT. Positive immune cells are stained in green. Nuclei are stained in blue using DAPI as a counterstain. Scale bar=100µm (n=3). (D) SR staining performed in WAT. Scale bar=100µm (n=3). (E) Levels of cholesterol in serum (n=57). (F) Basal glucose levels in blood after 12hours of fasting (n=3). Values with different superscripts are significantly different from each other (P<0.05), assessed by oneway ANOVA. Differences (P<0.05) between time points (10 weeks vs. 23 weeks) for each pairing group were assessed by pairing oneway ANOVA and denoted as “#.” Abbreviation: DAPI, 4′,6diamidino2phenylindole. Hepatology CommuniCations, June 2021BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1056 Hepatology CommuniCations, Vol. 5, no. 6, 2021 BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1057 oXiDatiVe stRess anD HepatoCyte Cell DeatH in DualFeD miCe Extensive fat accumulation in the liver tissue together with alcohol consumption generate oxidative stress.(17) Immunoblotting showed increased expression of cytochrome P450 in the DUAL group after 23 weeks feeding (Fig. 3A). Consequently, we performed 4hydroxinonenal staining in all groups of mice. Overall, DUAL diet led to significant induction of lipid peroxides in the liver (Fig.3B). Oxidative stress led to significant ultrastructural morphological changes in DUAL animals identified by transmission electron microscopy (TEM).(18) After 23 weeks, DUAL diet promoted important morphological changes in mitochondria, which exhibited irregular circled shapes, accumulation of cristae, and multiple electron dense particles (Fig. 3C). Importantly, enlargement of the rough endoplasmic reticulum and cisternae dilation were specifically found in DUAL mice (Fig.3C and Supporting Fig.S7A,B). Oxidative and mitochondrial stress likely contributed to liver damage and caused modest but significant increases of the plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), all major clinical indicators of cellular liver injury (Fig. 3D and Supporting Fig. S7C). Consistently, the cell death by TUNEL (terminal deoxynucleotidyl transferase– mediated deoxyuridine triphosphate nickend labelling) staining in DUALfed livers was significantly higher compared with other experimental groups (Fig.3E and Supporting Fig.S7D). 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 with the rest of experimental groups (Fig.3F and Supporting Fig.S7E). Dual FeeDing inDuCes eXtensiVe HepatiC inFlammation Fat accumulation and cell death in the liver further caused immune cell infiltration and hepatic inflammation.(19) All treated groups showed an increased accumulation of CD45 and F4/80 positive Kupffer cells/macrophages, as assessed by immunofluorescence (IF) staining, but such infiltration was clearly more pronounced in DUAL animals (Fig.4AC and Supporting Fig.S8A,B). Neutrophil infiltration is an important hallmark of alcoholic hepatitis and correlates with the severity of disease.(20) Immunohistochemistry (IHC) staining revealed that neutrophil infiltration in the liver was significantly higher in DUAL mice (Supporting Fig. S8C,D). Infiltrating cells actively produce different cytokines and further contribute to create a proinflammatory microenvironment. Consistently, messenger RNA (mRNA) expression of tumor necrosis factorα (TNFα) was significantly increased in mice fed with DUAL diet, especially after 23weeks of feeding (Fig.4D). Dual Diet leaDs to inCReaseD HepatiC stellate Cell aCtiVation anD HepatiC FiBRogenesis TNFα overproduction induces activation of hepatic stellate cells (HSCs) in the liver.(21) We found strong expression of αsmooth muscle actin (αSMA), a marker of HSCs activation, using western blot analysis and IHC staining in DUALfed animals (Fig. 5A,B [upper panel]). Activated HSCs are the major source of ECM during progression of fibrosis.(22) Hence, SR staining clearly demonstrated that feeding a WD or EtOH alone induced only minor collagen expression in the liver, whereas rapid and Fig. 2. DUAL mice develop hepatomegaly and advanced steatosis. (A) Left: Liver macroscopic images after 23weeks of feeding. Middle: Liver weight (g) (n=57). Right: Liver weight– toBW ratio (%) (n=57). (B) Representative phalloidinstained liver images and size of hepatocytes in phalloidinstained liver pictures quantified by ImageJ software. Scale bar=100µm (n=3). (C) H&E representative images after 10 weeks or 23weeks of feeding. Scale bar=100µm. Steatosis score assigned after 23weeks of treatment (n=37). (D) Illustrative OROstained liver sections from each group and timepoint feeding. Scale bar=100µm. Quantification of OROstained area (n=3). (E) CPT1c immunoblot using GAPDH as loading control. Ratio between CPT1c and GAPDH was calculated. Values with different superscripts are significantly different from each other (P<0.05), assessed by oneway ANOVA. Differences (P<0.05) between time points (10 weeks vs. 23 weeks) for each pairing group were assessed by pairing oneway ANOVA and denoted as “#.” Abbreviation: GAPDH, glyceraldehyde 3phosphate dehydrogenase. Hepatology CommuniCations, June 2021BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1058 Hepatology CommuniCations, Vol. 5, no. 6, 2021 BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1059 severe progression of fibrosis was a notable histological feature of DUALfed mice. The first signs of fibrosis were detectable as soon as 10weeks of treatment (Supporting Fig. S9A,B), with remarkable escalation of fibrogenesis at 23weeks (Fig.5B,C [middle panel]). These findings were additionally confirmed by IF staining for collagen I (Fig.5B,D [lower panel]). Histopathological evaluation of fibrosis revealed F1a stage after 10 weeks and F1b in all DUAL animals after 23weeks of feeding (Fig.5E). Additionally, collagen fibers were further identified though TEM analysis in DUAL mice (Supporting Fig. S10). Finally, Fig. 3. DUAL diet stimulates early oxidative stress and hepatocyte cell death. (A) CYP2E1 western blot, βactin used as a loading control. Ratio between CYP2E1 and βactin was calculated. (B) Illustrative 4HNEstained liver sections from each group after 23weeks of feeding and 4HNE quantification (n=3). Scale bar=100µm. (C) Representative TEM pictures of control and DUAL groups. Mitochondria and endoplasmic reticulum are shown. Arrows mark cristae inclusions in mitochondrial matrix. (D) ALT and LDH measurements in serum after 12hours of fasting (n=57). (E) Representative TUNELstained photomicrographs at 23weeks. Scale bar=100µm. (F) Ki67 liver IHC staining after 23weeks of feeding. Scale bar=100µm. Values with different superscripts are significantly different from each other (P<0.05), assessed by oneway ANOVA. Abbreviations: 4HNE, 4hydroxinonenal; CYP2E1, cytochrome P450; ER, endoplasmic reticulum; TUNEL, terminal deoxynucleotidyl transferase– mediated deoxyuridine triphosphate nickend labeling. Fig. 4. DUAL mice manifest enhanced hepatic inflammation. (A) Illustrative CD45 and F4/80 IF staining in liver sections of mice fed for 23 weeks. Positive immune cells are stained in green. Nuclei are stained in blue using DAPI as a counterstain. Arrows indicate CD45 or F4/80 positive cells, respectively. Scale=100µm. (B,C) Quantification of %CD45 and F4/80 positive cells, respectively, using ImageJ software (n=3). (D) TNFα mRNA relative expression to GAPDH after 10 weeks and 23 weeks on diet (n=36). Values with different superscripts are significantly different from each other (P<0.05), assessed by oneway ANOVA. Abbreviations: DAPI, 4′,6diamidino2phenylindole; GAPDH, glyceraldehyde 3phosphate dehydrogenase. Hepatology CommuniCations, June 2021BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1066 We used WD from the Amylin liver NASH study(16) as a combination of 40% fat, 22% fructose, and 2% cholesterol, resulting in relatively strong steatohepatitis. Moreover, glucose in the drinking water potentiates absorption of fructose from the diet, whereas fructose catalyzes glucose uptake and storage in the liver, leading together to stronger harm.(34) More importantly, by incorporating Dglucose in the water we overcome mouse aversion to alcohol. Sweetened water successfully masked the taste of alcohol, thus increasing alcohol intake. DUALfed animals consume 32.2g/kg of alcohol daily. Hence, the relative risk of alcoholassociated cirrhosis increases in humans who drink more than 25g/day.(35) Additionally, the consumption of fatty WD further elevated EtOH intake. Previous reports suggested the existence of a positive correlation between EtOH and fat, whereby each nutrient stimulates the intake of the other. Within this synergistic vicious cycle, calorie intake can dramatically increase.(10) Consequently, following initiation of the DUAL diet, mice started to gain weight and developed obesity, characterized by an increase in BMI. Over a period of 23weeks, the DUAL group became 19% heavier than the control group. Abdominal obesity is a predominant underlying risk factor for MS.(13) Simultaneously with obesity, DUAL animals developed dyslipidemia and hyperglycemia: altogether three important medical conditions of MS.(36) Hence, it is essential to mention that impaired fasting glycemia was not accompanied either by glucose intolerance or by insulin resistance in DUAL mice. In fact, the ITT area under the curve was significantly lower in animals treated with DUAL diet compared with controls. Nevertheless, the explanation for this phenomenon by enhanced insulin action is an obvious misinterpretation of the results. We believe that DUAL mice exhibited a defect in the counterregulatory response to insulin in— particularly— gluconeogenesis.(37) In fact, RNAseq data demonstrated that the level of glucose6 phosphatase was remarkably lower in the DUAL group. Another key feature of the DUAL obesity is the remarkable damage of WAT: Hypertrophic adipocytes develop an inflammatory phenotype and crownlike structures, consisting of necrotic adipocytes. Adipocyte death and subsequent inflammation increase lipolysis, flux of lipids to the liver, production of proinflammatory cytokines, and significantly contribute to disease progression.(3,11) Previous animal studies showed that highfat diet in combination with EtOH binge feeding synergistically induce liver injury by stimulating hepatocytes to produce chemokine (CXC motif) ligand 1 (CXCL1), which subsequently promoted hepatic neutrophil recruitment.(38) RNAseq data showed upregulation of CXCL1 (up to 30 times) and concomitant pathways related to neutrophilmediated immunity in the liver of DUAL animals. Hence, hepatic steatosis induced by DUAL diet takes a short period of time. Due to the massive flux of FFAs into the liver, relatively poor ßoxidation and reduced TG secretion, lipids accumulate in large amounts already after 10weeks of feeding and gradually increase over time. DUAL mice developed microvesicular and macrovesicular steatosis with hepatocytes ballooning. Consequently, the increased accumulation of FFAs in the liver led to metabolism deregulation and the generation of oxidative stress, which triggered cell death, inflammation, and immune cell infiltration into the hepatic parenchyma. Inflammatory cytokines, produced by immune cells (e.g., TNFα), further activated HSCs and stimulated the production of collagen fibers and ECM deposition in the liver, leading to fibrogenesis.(39) Mice fed a DUAL diet exhibited steatohepatitis already after 10 weeks, whereas feeding for 23weeks resulted in more fibrotic stage, particularly affecting the portal and bridging areas. Notably, transcriptomic changes relevant to chronic liver diseases in humans were also demonstrated in DUAL mice, including (1) Btk, Csf1, Sfpi1, Irf8 and Syk, which play critical roles in the development and function of myeloid and lymphoid cells, associated with chronic inflammation(4043); (2) Bcl2, Ptgs2, Plcg2 and Fyn, which promote apoptosis and proliferation(25,44,45); and (3) Pi3k and Foxo1, which are master regulators of glucose/lipid homeostasis.(46,47) The upregulation of TNF and NFκB(24)in DUAL animals entirely recapitulated the pathogenesis of humanlike steatohepatitis and correlated with disease progression to advanced fibrosis. Importantly, the activation of TLR4/9 indicated that besides the direct toxic effect of alcohol and WD on the liver, the abnormal microbiome and the loss of intestinal barrier function may potentially contribute to the pathogenesis in DUAL mice.(26,48) DUAL mice were similar to human disease, not only in terms of initiation of steatohepatitis, but also in development of cirrhosis and tumorigenesis. Thus, after 1year of DUAL diet, animals demonstrated extensive collagen accumulation and micronodular cirrhotic changes. Hepatology CommuniCations, Vol. 5, no. 6, 2021 BENEDÉUBIETO, ESTÉVEZVÁZQUEZ, ET AL. 1067 The reported DUAL diet additionally functions as an excellent tumor promoter for DENinduced liver tumorigenesis in mice. With this diet, preneoplastic nodules develop within only 26weeks following DEN injection. This observation supports the importance of identifying patients with excess alcohol consumption and MS, as they are at a higher risk of liverrelated cancer. Altogether, our preclinical model induces the development of liver damage in the context of key risk factors for the human condition (e.g., alcohol consumption, obesity, MS), naturally mimicking human pathology and the progression to advanced liver fibrosis, cirrhosis, and endstage tumorigenesis. Importantly, it is an easy, affordable, highly reproducible, timeefficient diet that does not require any special skill or expensive equipment. Moreover, it is associated with no mortality even using longterm feeding, and as a mild procedure, it should be easily approved by ethical committees. However, there are some limitations and methodological difficulties in our DUAL model. First, according to our observations, as typical nocturnal animals, mice consumed significant amounts of EtOH in the drinking water primarily during the dark cycle. Hence, given the fast metabolic capacity of mice, on the following morning their blood alcohol level levels drop significantly. Therefore, we calculated the daily amount of consumed alcohol based on the volume of the water drunk from the bottle. Second, we tested our model on male and female mice. Independently of gender, all mice developed steatohepatitis and fibrosis, and advanced cirrhotic changes. Nonetheless, the phenotype in male mice was slightly stronger, including a more elevated BMI, more pronounced steatosis, and moderately increased ALT (data not showed). However, we dealt with some difficulties with the housing of male mice. Food intake was negatively affected by the subordination, raising concerns about welfare, alcohol consumption, and negatively impacted research validity. Hence, according to our experience, the only possibility for male mice is individual housing (for DUAL diet and the corresponding control groups). Finally, lack of clinical studies with tissue of patients with DUAL alcoholic and metabolicassociated steatohepatitis was a limitation of our study. Moreover, there were major methodological concerns about the existing studies, as many reports failed to consider the pattern and type of alcohol use and/or did not separate between lifetime and current abstainers. In addition, underreporting of alcohol use is a major concern, particularly when assessing patients who are aware of their liver disease.(49,50) Altogether, DUAL alcoholic and MAFLD remains as not wellexplored area of great interest, despite the increased number of affected patients. Our innovative preclinical DUAL model can be a valuable toolbox, as it mimics all histological, metabolic, transcriptomic gene signatures of human disease, thereby contributing to the development of very much needed therapeutic targets (Supporting Fig.S13). ReFeRenCes 1) Pimpin L, CortezPinto H, Negro F, Corbould E, Lazarus JV, Webber L, et al. Burden of liver disease in Europe: epidemiology and analysis of risk factors to identify prevention policies. 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