Animal models of NAFLD from a hepatologist's point of view

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disorder closely linked to obesity, hyperlipidemia and type 2 diabetes and is increasingly recognized as a major health problem in many parts of the world. While early stages of NAFLD are characterized by a bland accumulation of fat (steatosis) in hepatocytes, the disease can progress to non-alcoholic steatohepatitis (NASH) which involves chronic liver inflammation, tissue damage and fibrosis and can ultimately lead to end-stage liver disease including cirrhosis and cancer. As no approved pharmacological treatment for NAFLD exists today, there is an urgent need to identify promising pharmacological targets and develop future therapies. For this purpose, basic and translational research in NAFLD animal models is indispensable. While a large number of diverse animal models are currently used in the field, there is an ongoing challenge to identify those models that mirror human pathology the closest to allow good translation of obtained results into further clinical development. This review is meant to provide a concise overview of the most relevant NAFLD animal models currently available and will discuss the strengths and weaknesses of these models with regard to their comparability to human disease conditions.     

The expanding role of fish models in understanding non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a condition in which excessive fat accumulates in the liver of an individual who has not consumed excessive alcohol. Non-alcoholic steatohepatitis (NASH), a severe form of NAFLD, can progress to hepatic cirrhosis and/or hepatocellular carcinoma (HCC). NAFLD is considered to be a hepatic manifestation of metabolic syndrome, and its incidence has risen worldwide in lockstep with the increased global prevalence of obesity. Over the last decade, rodent studies have yielded an impressive list of molecules associated with NAFLD and NASH pathogenesis. However, the identification of currently unknown metabolic factors using mammalian model organisms is inefficient and expensive compared with studies using fish models such as zebrafish (Danio rerio) and medaka (Oryzias latipes). Substantial advances in unraveling the molecular pathogenesis of NAFLD have recently been achieved through unbiased forward genetic screens using small fish models. Furthermore, these easily manipulated organisms have been used to great advantage to evaluate the therapeutic effectiveness of various chemical compounds for the treatment of NAFLD. In this Review, we summarize aspects of NAFLD (specifically focusing on NASH) pathogenesis that have been previously revealed by rodent models, and discuss how small fish are increasingly being used to uncover factors that contribute to normal hepatic lipid metabolism. We describe the various types of fish models in use for this purpose, including those generated by mutation, transgenesis, or dietary or chemical treatment, and contrast them with rodent models. The use of small fish in identifying novel potential therapeutic agents for the treatment of NAFLD and NASH is also addressed.     

Establishment of a General NAFLD Scoring System for Rodent Models and Comparison to Human Liver Pathology

Background and aims

The recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD) by the NASH Clinical Research Network (NASH-CRN) has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models.

Methods

The histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system). For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart.

Results

The criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient) between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p<0.001, respectively) and moderate for the analysis of hypertrophy and inflammation (ICC = 0.685 and 0.650, all p<0.001, respectively). The intra-observer reproducibility between the different observations of one observer was high for the analysis of macrovesicular steatosis, microvesicular steatosis and hypertrophy (ICC = 0.871, 0.871 and 0.896, all p<0.001, respectively) and very high for the analysis of inflammation (ICC = 0.931, p<0.001).

Conclusions

We established a simple NAFLD scoring system with high reproducibility that is applicable for different rodent models and for all stages of NAFLD etiology.     

MicroRNAs in the Pathogenesis of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD), or, more accurately, metabolic associated fatty liver disease, accounts for a large proportion of chronic liver disorders worldwide and is closely associated with other conditions such as cardiovascular disease, obesity, and type 2 diabetes mellitus. NAFLD ranges from simple steatosis to nonalcoholic steatohepatitis (NASH) and can progress to cirrhosis and, eventually, also hepatocellular carcinoma. The morbidity and mortality associated with NAFLD are increasing rapidly year on year. Consequently, there is an urgent need to understand the etiology and pathogenesis of NAFLD and identify effective therapeutic targets. MicroRNAs (miRNAs), important epigenetic factors, have recently been proposed to participate in NAFLD pathogenesis. Here, we review the roles of miRNAs in lipid metabolism, inflammation, apoptosis, fibrosis, hepatic stellate cell activation, insulin resistance, and oxidative stress, key factors that contribute to the occurrence and progression of NAFLD. Additionally, we summarize the role of miRNA-enriched extracellular vesicles in NAFLD. These miRNAs may comprise suitable therapeutic targets for the treatment of this condition.     

Genetics of Nonalcoholic Fatty Liver Disease:An Overview

Nonalcoholic fatty liver disease(NAFLD) is the most common liver disease in the world today.Its incidence in adults and children is rising rapidly due to the ongoing epidemics of obesity and type 2 diabetes.Hence,it has become a global public health issue.Environmental factors have been found to play a major role in the etiology of NAFLD,especially for genetically susceptible populations. Among these,one of the most important factors is junk food,especially the typical "Western-style" diet rich in simple carbohydrates, saturated fat,and highly processed food materials.Genetic predisposition to NAFLD does occur;however,a precise definition of genetic factors responsible for NAFLD is still lacking.Specific variants of different genes have been shown to present a risk for NAFLD.Genetic studies might be helpful in the management of the disease by developing novel treatment strategies based on individual’s genotype.     

A Post-Developmental Genetic Screen for Zebrafish Models of Inherited Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease.     

Advancing the understanding of NAFLD to hepatocellular carcinoma development: From experimental models to humans

Nonalcoholic fatty liver disease (NAFLD) has recently been recognized as an important etiology contributing to the increased incidence of hepatocellular carcinoma (HCC). NAFLD, characterized by fat accumulation in the liver, is affecting at least one-third of the global population. The more aggressive form, nonalcoholic steatohepatitis (NASH), is characterized by hepatocyte necrosis and inflammation. The development of effective approaches for disease prevention and/or treatment heavily relies on deep understanding of the mechanisms underlying NAFLD to HCC development. However, this has been largely hampered by the lack of robust experimental models that recapitulate the full disease spectrum. This review will comprehensively describe the current in vitro and mouse models for studying NAFLD/NASH/HCC, and further emphasize their applications and possible future improvement for better understanding the molecular mechanisms involved in the cascade of NAFLD to HCC progression.     

胆汁酸受体在非酒精性脂肪性肝病中的作用

非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD)作为一种慢性肝病,在全球的发病率逐年递增。胰岛素抵抗和脂质代谢紊乱,以及随后的炎症反应和纤维化的激活,在其发生发展过程中发挥重要作用。但是对其认识仍很欠缺,且临床尚缺乏有效的药物。科研人员正极力探索NAFLD的相关病因及治疗的新的突破口。胆汁酸是在肝中合成的众多代谢产物之一。除帮助脂肪消化吸收外,胆汁酸还作为信号分子激活胆汁酸受体,一种重要的转录调节因子而发挥效应,对维持机体正常生理代谢至关重要。越来越多的证据表明,胆汁酸受体的功能与NAFLD的发生发展关系密切,研究其相关的作用与功能可为治疗NAFLD提供新见解和药物治疗靶点。本文就胆汁酸受体包括核受体,诸如法尼醇X受体 (farnesoid X receptor, FXR)、孕烷X受体 (pregnane X receptor ,PXR)等,和细胞表面受体,诸如跨膜G蛋白偶联胆汁酸受体5(transmembrane G protein-coupled receptor 5, TGR5)、鞘氨醇-1-磷酸受体2(phingosine-1-phosphate receptor 2, S1PR2)和毒蕈碱胆碱受体3 (M3 muscarinic receptor, M3R)通过调节胆汁酸稳态、脂质和糖代谢、能量代谢、肝的炎症和纤维化等参与NAFLD发病机制的研究进展进行总结,并进一步阐述了胆汁酸受体激动剂对NAFLD的治疗现状,以期更全面地了解NAFLD的发病机制以及为治疗找到更有效的途径。     

非酒精性脂肪肝与脂肪性肝炎动物模型研究进展

非酒精性脂肪肝是无酒精滥用的包括单纯性脂肪肝、脂肪性肝炎、脂肪性肝纤维化和肝硬化的肝病综合征,目前已成为广受关注的肝病医学难题。随着抗脂肪肝药物的深入研究,动物模型制作得到很好发展。近年来,在大鼠、沙鼠、小鼠、兔和小猪等动物种属成功地建立了食物、胃肠外营养与蛋氨酸胆碱缺乏等诱导的单纯性脂肪肝和脂肪性肝炎动物模型,这些模型为研究脂肪肝和脂肪性肝炎的发病机理与治疗提供了机会。每种动物模型各有优缺点,合理应用动物模型能更好地开展脂肪肝病的实验和临床研究。本文综述了非酒精性脂肪肝及脂肪性肝炎动物模型制作方法的若干研究进展。     

Pharmacological Inhibition of the Chemokine CXCL16 Diminishes Liver Macrophage Infiltration and Steatohepatitis in Chronic Hepatic Injury

Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality in developed countries, resulting in steatohepatitis (NASH), fibrosis and eventually cirrhosis. Modulating inflammatory mediators such as chemokines may represent a novel therapeutic strategy for NAFLD. We recently demonstrated that the chemokine receptor CXCR6 promotes hepatic NKT cell accumulation, thereby controlling inflammation in experimental NAFLD. In this study, we first investigated human biopsies (n = 20), confirming that accumulation of inflammatory cells such as macrophages is a hallmark of progressive NAFLD. Moreover, CXCR6 gene expression correlated with the inflammatory activity (ALT levels) in human NAFLD. We then tested the hypothesis that pharmacological inhibition of CXCL16 might hold therapeutic potential in NAFLD, using mouse models of acute carbon tetrachloride (CCl₄)- and chronic methionine-choline-deficient (MCD) diet-induced hepatic injury. Neutralizing CXCL16 by i.p. injection of anti-CXCL16 antibody inhibited the early intrahepatic NKT cell accumulation upon acute toxic injury in vivo. Weekly therapeutic anti-CXCL16 administrations during the last 3 weeks of 6 weeks MCD diet significantly decreased the infiltration of inflammatory macrophages into the liver and intrahepatic levels of inflammatory cytokines like TNF or MCP-1. Importantly, anti-CXCL16 treatment significantly reduced fatty liver degeneration upon MCD diet, as assessed by hepatic triglyceride levels, histological steatosis scoring and quantification of lipid droplets. Moreover, injured hepatocytes up-regulated CXCL16 expression, indicating that scavenging functions of CXCL16 might be additionally involved in the pathogenesis of NAFLD. Targeting CXCL16 might therefore represent a promising novel therapeutic approach for liver inflammation and steatohepatitis.     

Imaging Mass Spectrometry Reveals Alterations in N-Linked Glycosylation That Are Associated With Histopathological Changes in Nonalcoholic Steatohepatitis in Mouse and Human

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD) and is characterized by inflammation, hepatocyte injury, and fibrosis. Further, NASH is a risk factor for cirrhosis and hepatocellular carcinoma. Previous research demonstrated that serum N-glycan profiles can be altered in NASH patients. Here, we hypothesized that these N-glycan modifications may be associated with specific liver damage in NAFLD and NASH. To investigate the N-glycome profile in tissue, imaging mass spectrometry was used for a qualitative and quantitative in situ N-linked glycan analysis of mouse and human NAFLD/NASH tissue. A murine model was used to induce NAFLD and NASH through ad libitum feeding with either a high-fat diet or a Western diet, respectively. Mice fed a high-fat diet or Western diet developed inflammation, steatosis, and fibrosis, consistent with NAFLD/NASH phenotypes. Induction of NAFLD/NASH for 18 months using high caloric diets resulted in increased expression of mannose, complex/fucosylated, and hybrid N-glycan structures compared to control mouse livers. To validate the animal results, liver biopsy specimens from 51 human NAFLD/NASH patients representing the full range of NASH Clinical Research Network fibrosis stages were analyzed. Importantly, the same glycan alterations observed in mouse models were observed in human NASH biopsies and correlated with the degree of fibrosis. In addition, spatial glycan alterations were localized specifically to histopathological changes in tissue like fibrotic and fatty areas. We demonstrate that the use of standard staining’s combined with imaging mass spectrometry provide a full profile of the origin of N-glycan modifications within the tissue. These results indicate that the spatial distribution of abundances of released N-glycans correlate with regions of tissue steatosis associated with NAFLD/NASH.     

Calcium channel blockers as potential therapeutics for obesity-associated autophagy defects and fatty liver pathologies

Nonalcoholic fatty liver disease (NAFLD), typically associated with overnutrition and obesity, is one of the most common liver diseases both in the US and worldwide. During obesity and NAFLD, lipotoxic injuries to hepatocytes can provoke formation of protein inclusions consisting of SQSTM1/p62 and ubiquitinated proteins. It has been suggested that autophagy deregulation during obesity contributes to protein inclusion formation and progression of other liver pathologies including insulin resistance, steatohepatitis, and hepatocellular carcinoma. To examine how lipotoxicity and obesity affect autophagy, we established an in vitro system where cultured HepG2 cells exhibit prominent accumulation of SQSTM1 and ubiquitinated proteins in insoluble inclusion bodies upon treatment with saturated fatty acids. Using this system and a mouse model of obesity, we have determined that obesity induces chronic elevation of cytosolic calcium levels in hepatocytes, which interferes with the fusion between autophagosomes and lysosomes. Intriguingly, pharmacological inhibition of calcium channels using the FDA-approved drug verapamil successfully restores autophagic flux and suppresses protein inclusions, not only in HepG2 cells but also in mouse liver. Verapamil also reduces hepatic lipid droplet accumulation, insulin resistance and steatohepatitis, suggesting that calcium channel blockers can be used for correction of general NAFLD pathologies. Indeed, there have been a number of clinical observations in which beneficial effects of calcium channel blockers against obesity-associated metabolic pathologies are observed in humans and animal models.     

实验小鼠在癌症研究中的应用及其进展

小鼠是生物医学研究中使用数量最多的哺乳类实验动物。人类利用小鼠模型进行癌症研究已有100多年的历史,小鼠大量的遗传变异可作为研究人类癌症的借鉴。特别是近年来,培育成功的转基因、基因敲除等遗传工程小鼠模型,使我们对人类癌症发生有了深刻的认识,为评估癌症的诊断方法,革新预防和治疗方案提供了一个很有价值的平台。本文着重介绍了癌症研究中常用的小鼠模型、GEM模型及取得的最新进展等,分析了小鼠肿瘤模型的局限性,并对其发展趋势进行展望。     

Calcium channel blockers as potential therapeutics for obesity-associated autophagy defects and fatty liver pathologies

Nonalcoholic fatty liver disease (NAFLD), typically associated with overnutrition and obesity, is one of the most common liver diseases both in the US and worldwide. During obesity and NAFLD, lipotoxic injuries to hepatocytes can provoke formation of protein inclusions consisting of SQSTM1/p62 and ubiquitinated proteins. It has been suggested that autophagy deregulation during obesity contributes to protein inclusion formation and progression of other liver pathologies including insulin resistance, steatohepatitis, and hepatocellular carcinoma. To examine how lipotoxicity and obesity affect autophagy, we established an in vitro system where cultured HepG2 cells exhibit prominent accumulation of SQSTM1 and ubiquitinated proteins in insoluble inclusion bodies upon treatment with saturated fatty acids. Using this system and a mouse model of obesity, we have determined that obesity induces chronic elevation of cytosolic calcium levels in hepatocytes, which interferes with the fusion between autophagosomes and lysosomes. Intriguingly, pharmacological inhibition of calcium channels using the FDA-approved drug verapamil successfully restores autophagic flux and suppresses protein inclusions, not only in HepG2 cells but also in mouse liver. Verapamil also reduces hepatic lipid droplet accumulation, insulin resistance and steatohepatitis, suggesting that calcium channel blockers can be used for correction of general NAFLD pathologies. Indeed, there have been a number of clinical observations in which beneficial effects of calcium channel blockers against obesity-associated metabolic pathologies are observed in humans and animal models.     

The construction of transgenic and gene knockout/knockin mouse models of human disease

The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.     

Genetic determinants of hepatic steatosis in man

Hepatic steatosis is one of the most common liver disorders in the general population. The main cause of hepatic steatosis is nonalcoholic fatty liver disease (NAFLD), representing the hepatic component of the metabolic syndrome, which is characterized by type 2 diabetes, obesity, and dyslipidemia. Insulin resistance and excess adiposity are considered to play key roles in the pathogenesis of NAFLD. Although the risk factors for NAFLD are well established, the genetic basis of hepatic steatosis is largely unknown. Here we review recent progress on genomic variants and their association with hepatic steatosis and discuss the potential impact of these genetic studies on clinical practice. Identifying the genetic determinants of hepatic steatosis will lead to a better understanding of the pathogenesis and progression of NAFLD.     

The isoxazole based flavonoid derivative 1 ameliorates non-alcoholic fatty liver disease in high-fat diet-induced obese mice by regulating lipid metabolism and inflammatory responses

Globally, non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease, and has attracted considerable research traction in the literature. However, no effective drugs have yet been approved by the Food and Drug Administration. In this study, a new isoxazole derivative (1), which activated AMPK and reduced gluconeogenesis levels, was evaluated in HFD-induced obese mice for NAFLD treatment. Our results showed that 1 reduced body weight gain and attenuated metabolic disorder in these mice. Underlying mechanisms revealed that 1 alleviated hepatic steatosis by activating AMPK, and inflammatory responses by inhibiting NF-κB. Our study suggested that 1 may be clinically valuable as an NAFLD therapeutic candidate.     

肝脏类器官的研究及应用进展

肝脏疾病易感性差异大且个体间的肝脏细胞存在明显的异质性,因此开发体外能够长期存活并具有代谢功能的人体类肝组织细胞模型,对治疗终末期肝病、开展肝脏致病机理研究及药物筛选具有重要意义。过去十年中,体外三维类器官模型发展迅猛,为疾病模拟、精准化治疗领域的研究提供了新的工具,显示出巨大潜力。肝脏类器官具有患者的基因表达与突变特征,在体外能够较长时间地保持肝脏细胞功能,已被应用于疾病模拟及药物有效性研究,并具有进行原位或异位移植发挥治疗作用的应用潜能。就干细胞、肝脏原代细胞等不同来源的肝脏类器官的发展及近年的研究进展作了综述,以期为肝脏类器官在疾病建模、药物发现和器官移植领域的研究和应用提供新的思路。     

Animal models of Huntington's disease

Huntington's disease (HD) is a neurological disorder caused by a genetic mutation in the IT15 gene. Progressive cell death in the striatum and cortex, and accompanying declines in cognitive, motor, and psychiatric functions, are characteristic of the disease. Animal models of HD have provided insight into disease pathology and the outcomes of therapeutic strategies. Earlier studies of HD most often used toxin-induced models to study mitochondrial impairment and excitotoxicity-induced cell death, which are both mechanisms of degeneration seen in the HD brain. These models, based on 3-nitropropionic acid and quinolinic acid, respectively, are still often used in HD studies. The discovery in 1993 of the huntingtin mutation led to the creation of newer models that incorporate a similar genetic defect. These models, which include transgenic and knock-in rodents, are more representative of the HD progression and pathology. An even more recent model that uses a viral vector to encode the gene mutation in specific areas of the brain may be useful in nonhuman primates, as it is difficult to produce genetic models in these species. This article examines the aforementioned models and describes their use in HD research, including aspects of the creation, delivery, pathology, and tested therapies for each model.