Non-alcoholic steatohepatitis and animal models: Understanding the human disease

Non-alcoholic fatty liver disease includes a broad spectrum of liver abnormalities ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. Patients with primary NASH have the metabolic (or insulin resistance) syndrome, condition typically associated with obesity, diabetes, hyperlipidemia and hypertension. To understand the mechanisms implicated in development of NASH, animal models of non-alcoholic fatty liver disease have been generated. These have greatly improved our understanding of some of the aspects of this disease. The challenge now is to identify the common mechanisms between the animal models and humans, which could eventually lead to a better prognosis and development of novel therapeutic strategies.     

From Mallory to Mallory-Denk bodies: what, how and why?

Frank B. Mallory described cytoplasmic hyaline inclusions in hepatocytes of patients with alcoholic hepatitis in 1911. These inclusions became known as Mallory bodies (MBs) and have since been associated with a variety of other liver diseases including non-alcoholic fatty liver disease. Helmut Denk and colleagues described the first animal model of MBs in 1975 that involves feeding mice griseofulvin. Since then, mouse models have been instrumental in helping understand the pathogenesis of MBs. Given the tremendous contributions made by Denk to the field, we propose renaming MBs as Mallory-Denk bodies (MDBs). The major constituents of MDBs include keratins 8 and 18 (K8/18), ubiquitin, and p62. The relevant proteins and cellular processes that contribute to MDB formation and accumulation include the type of chronic stress, the extent of stress-induced protein misfolding and consequent proteasome overload, a K8-greater-than-K18 ratio, transamidation of K8 and other proteins, presence of p62 and autophagy. Although it remains unclear whether MDBs serve a bystander, protective or injury promoting function, they do serve an important role as histological and potential progression markers in several liver diseases.     

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。     

Mallory-Denk-bodies: lessons from keratin-containing hepatic inclusion bodies

Inclusion bodies are characteristic morphological features of various neuronal, muscular and other human disorders. They share common molecular constituents such as p62, chaperones and proteasome subunits. The proteins within aggregates are misfolded with increased beta-sheet structure, they are heavily phosphorylated, ubiquitinylated and partially degraded. Furthermore, involvement of proteasomal system represents a common feature of virtually all inclusions. Multiple aggregates contain intermediate filament proteins as their major constituents. Among them, Mallory-Denk bodies (MDBs) are the best studied. MDBs represent hepatic inclusions observed in diverse chronic liver diseases such as alcoholic and non-alcoholic steatohepatitis, chronic cholestasis, metabolic disorders and hepatocellular neoplasms. MDBs are induced in mice fed griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine and resolve after discontinuation of toxin administration. The availability of a drug-induced model makes MDBs a unique tool for studying inclusion formation. Our review summarizes the recent advances gained from this model and shows how they relate to observations in other aggregates. The MDB formation-underlying mechanisms include protein misfolding, chaperone alterations, disproportional protein expression with keratin 8>keratin 18 levels and subsequent keratin 8 crosslinking via transglutaminase. p62 presence is crucial for MDB formation. Proteasome inhibitors precipitate MDB formation, whereas stimulation of autophagy with rapamycin attenuates their formation.     

Lipidomics in pathogenesis,progression and treatment of nonalcoholic steatohepatitis (NASH): Recent advances

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease affecting up to 30% of the general adult population. NAFLD encompasses a histological spectrum ranging from pure steatosis to non-alcoholic steatohepatitis (NASH). NASH can progress to cirrhosis and is becoming the most common indication for liver transplantation, as a result of increasing disease prevalence and of the absence of approved treatments. Lipidomic readouts of liver blood and urine samples from experimental models and from NASH patients disclosed an abnormal lipid composition and metabolism. Collectively, these changes impair organelle function and promote cell damage, necro-inflammation and fibrosis, a condition termed lipotoxicity. We will discuss the lipid species and metabolic pathways leading to NASH development and progression to cirrhosis, as well as and those species that can contribute to inflammation resolution and fibrosis regression. We will also focus on emerging lipid-based therapeutic opportunities, including specialized proresolving lipid molecules and macrovesicles contributing to cell-to-cell communication and NASH pathophysiology.     

Bone Mineral Density in Patients with Nonalcoholic Steatohepatitis among End-Stage Liver Disease Patients Awaiting Liver Transplantation

ObjectiveSeveral studies have shown that patients with end-stage liver disease (ESLD) have lower bone mineral density (BMD) and a higher prevalence of osteoporosis compared to an age-matched population. Hyperinsulinemia and insulin resistance are typically associated with increased BMD. We hypothesized that patients with nonalcoholic steatohepatitis (NASH) and underlying insulin resistance may have higher BMD than patients with cirrhosis from other causes.MethodsWe performed a retrospective chart review of patients with ESLD who underwent liver transplant evaluation at Ochsner Clinic Foundation and had a BMD study as part of initial work up and compared BMD values of patients diagnosed with NASH to patients with cirrhosis due to other causes. Patients were categorized into 3 groups based on the etiology of their liver disease as NASH, alcoholic cirrhosis, or viral hepatitis C or B (HCV/ HBV).ResultsA total of 63 patients met the study inclusion criteria, including 15 with NASH, 17 with alcoholic cirrhosis, and 31 with HCV/HBV. The overall prevalence rates of osteopenia and osteoporosis were 44% and 12%, respectively. BMD values were higher in the NASH group than the HCV/HBV group at lumbar spine, total hip, and femoral neck (P = .01, .03, and .02, respectively). There were no statistical differences in BMD values between NASH and alcoholic cirrhosis groups at any site.ConclusionsWe found a high prevalence of low BMD among patients with ESLD awaiting liver transplantation. NASH patients had higher BMDs than HCV/ HBV patients. The effects of NASH and insulin resistance on bone are complex and should be examined further. (Endocr Pract. 2013;19:414-419)     

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.     

Co-factors in liver disease: the role of HFE-related hereditary hemochromatosis and iron

The severity of liver disease and its presentation is thought to be influenced by many host factors. Prominent among these factors is the level of iron in the body. The liver plays an important role in coordinating the regulation of iron homeostasis and is involved in regulating the level of iron absorption in the duodenum and iron recycling by the macrophages. Iron homeostasis is disturbed by several metabolic and genetic disorders, including various forms of hereditary hemochromatosis. This review will focus on liver disease and how it is affected by disordered iron homeostasis, as observed in hereditary hemochromatosis and due to HFE mutations. The types of liver disease covered herein are chronic hepatitis C virus (HCV) infection, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatocellular carcinoma (HCC) and porphyria cutanea tarda (PCT).     

New insights in the pathogenesis of non-alcoholic fatty liver disease

PURPOSE OF REVIEW: The hallmark of non-alcoholic fatty liver disease is hepatic steatosis. This is mostly a benign condition, but for largely unknown reasons it progresses to liver fibrosis, cirrhosis, and ultimately hepatocellular carcinoma in about 10% of patients. In this review we discuss recent progress in the understanding of the etiology of non-alcoholic fatty liver disease. RECENT FINDINGS: In the last few years many connections between carbohydrate and triglyceride homeostasis, as well as inflammation, have surfaced. These seemingly unrelated metabolic pathways are linked by the action of diverse nuclear receptors. Many intermediates in lipid metabolism were shown to be activating ligands of these receptors, explaining the dysregulation of intermediary metabolism and induction of insulin resistance by a lipid overload. In addition to invoking a derangement in nuclear receptor regulation, excessive hepatic lipid influx may have direct metabolic consequences, particularly on mitochondrial function. SUMMARY: Non-alcoholic fatty liver disease is a multifactorial disease. Many aspects of the disease and the links to inflammation can be understood when the multiple functions of the regulating nuclear receptors are taken into account. Many of these nuclear receptors seem attractive targets to develop therapy for non-alcoholic fatty liver disease and the closely related metabolic syndrome.     

The hepatic circadian clock is preserved in a lipid-induced mouse model of non-alcoholic steatohepatitis

Recent studies have correlated metabolic diseases, such as metabolic syndrome and non-alcoholic fatty liver disease, with the circadian clock. However, whether such metabolic changes per se affect the circadian clock remains controversial. To address this, we investigated the daily mRNA expression profiles of clock genes in the liver of a dietary mouse model of non-alcoholic steatohepatitis (NASH) using a custom-made, high-precision DNA chip. C57BL/6J mice fed an atherogenic diet for 5 weeks developed hypercholesterolemia, oxidative stress, and NASH. DNA chip analyses revealed that the atherogenic diet had a great influence on the mRNA expression of a wide range of genes linked to mitochondrial energy production, redox regulation, and carbohydrate and lipid metabolism. However, the rhythmic mRNA expression of the clock genes in the liver remained intact. Most of the circadianly expressed genes also showed 24-h rhythmicity. These findings suggest that the biological clock is protected against such a metabolic derangement as NASH.     

Genome-Wide Analysis of Copy Number Variation Identifies Candidate Gene Loci Associated with the Progression of Non-Alcoholic Fatty Liver Disease

Between 10 and 25% of individuals with non-alcoholic fatty liver disease (NAFLD) develop hepatic fibrosis leading to cirrhosis and hepatocellular carcinoma (HCC). To investigate the molecular basis of disease progression, we performed a genome-wide analysis of copy number variation (CNV) in a total of 49 patients with NAFLD [10 simple steatosis and 39 non-alcoholic steatohepatitis (NASH)] and 49 matched controls using high-density comparative genomic hybridization (CGH) microarrays. A total of 11 CNVs were found to be unique to individuals with simple steatosis, whilst 22 were common between simple steatosis and NASH, and 224 were unique to NASH. We postulated that these CNVs could be involved in the pathogenesis of NAFLD progression. After stringent filtering, we identified four rare and/or novel CNVs that may influence the pathogenesis of NASH. Two of these CNVs, located at 13q12.11 and 12q13.2 respectively, harbour the exportin 4 (XPO4) and phosphodiesterase 1B (PDE1B) genes which are already known to be involved in the etiology of liver cirrhosis and HCC. Cross-comparison of the genes located at these four CNV loci with genes already known to be associated with NAFLD yielded a set of genes associated with shared biological processes including cell death, the key process involved in ‘second hit’ hepatic injury. To our knowledge, this pilot study is the first to provide CNV information of potential relevance to the NAFLD spectrum. These data could prove invaluable in predicting patients at risk of developing NAFLD and more importantly, those who will subsequently progress to NASH.     

Lipotoxicity and steatohepatitis in an overfed mouse model for non-alcoholic fatty liver disease

The major risk factors for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH.Male C57Bl/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied.Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coincided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor γ (Pparγ), fibroblast growth factor 21 (Fgf21), fatty acid binding protein (Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice.In conclusion: this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established.     

Integrating the contributions of mitochondrial oxidative metabolism to lipotoxicity and inflammation in NAFLD pathogenesis

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing globally. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NASH is the pathological form of the disease characterized by liver steatosis, inflammation, cell injury, and fibrosis. A fundamental contributor to NASH is the imbalance between lipid accretion and disposal. The accumulation of liver lipids precipitates lipotoxicity and the inflammatory contributions to disease progression. This review defines the role of dysregulated of lipid disposal in NAFLD pathophysiology. The characteristic changes in mitochondrial oxidative metabolism pathways and the factors promoting these changes across the spectrum of NAFLD severity are detailed. This includes pathway-specific and integrative perturbations in mitochondrial β-oxidation, citric acid cycle flux, oxidative phosphorylation, and ketogenesis. Moreover, well-recognized and emerging mechanisms through which dysregulated mitochondrial oxidative metabolism mediates inflammation, fibrosis, and disease progression are highlighted.     

S100A9: A Potential Biomarker for the Progression of Non-Alcoholic Fatty Liver Disease and the Diagnosis of Non-Alcoholic Steatohepatitis

Non-alcoholic fatty liver (NAFL) has the potential to progress to non-alcoholic steatohepatitis (NASH) or to promote type 2 diabetes mellitus (T2DM). However, NASH and T2DM do not always develop coordinately. Additionally, there are no definite noninvasive methods for NASH diagnosis currently. We established rat models of NAFL, NASH, and NAFL + T2DM to recapitulate different phenotypes associated with non-alcoholic fatty liver disease (NAFLD) and its progression. Histologic features of rat livers were scored according to criteria established by the Nonalcoholic Steatohepatitis Clinical Research Network. Microarray was performed to assess gene expression changes in rat livers. We find that gene expression of s100a9 was higher in NAFL group compared with control, and was increased in NASH groups and decreased in NAFL + T2DM group compared with NAFL. In contrast, srebf1, tbx21, and gimap4 only showed limited discriminating abilities in different groups. There is a significant positive correlation between serum levels of S100A9 and NAFLD Activity Score (NAS), the severity of hepatic steatosis, and lobular inflammation (r = 0.80, 0.64 and 0.86, P < 0.001). These findings suggest that S100A9 may be extremely useful in the diagnosis of NASH (AUROC: 0.947, CI: 0.845-1.049). Additionally, serum S100A9 levels displayed a strong correlation with ALT, AST and TBil (r = 0.81, 0.89 and 0.91, P < 0.001) but a weak correlation with FBG, HOMA-IR, TG, and TC (r = -0.41, -0.40, 0.47 and 0.49, P < 0.05). Conclusions: The results we provide here suggest that S100A9 may be useful as a biomarker for the hepatic and metabolic progression of NAFLD and the non-invasive diagnosis of NASH.     

Non-alcoholic fatty liver disease (NAFLD)--a novel common aspect of the metabolic syndrome

Non-alcoholic fatty liver disease (NAFLD) is emerging as one of the most common liver disorders claiming the urgent attention of both medical professionals and the public sphere because of the imminent epidemic of advanced liver injury that appendages epidemic of obesity. Recent research reveals simple triglyceride accumulation in hepatocytes (i.e., liver steatosis) frequently becoming complicated by inflammation (i.e., non-alcoholic steatohepatitis, or NASH) that may progress into more advanced stages of the disease including cirrhosis or, eventually, hepatocellular carcinoma. The exact mechanisms of the progression of NAFLD into overt NASH and advanced disease stages are largely unknown. There is urgent need in terms of both intensive research pursuits and effective practical measures to deal with this common threat.     

Mind the gap

The unmet needs of biomedical and clinical research are highlighted by reference to drug -induced liver injury(DILI), non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH). Examples in these areas highlight the major limitations of animal models with respect to predicting, examining and managing these clinically significant forms of liver injury. The way in which these knowledge gaps are being bridged by studies involving the use of human tissues and primary cells are described.     

Oxidized LDL at the crossroads of immunity in non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is viewed as the hepatic manifestation of the metabolic syndrome and is a condition hallmarked by lipid accumulation in the liver (steatosis) along with inflammation (hepatitis). Currently, the etiology and mechanisms leading to obesity-induced hepatic inflammation are not clear and, as a consequence, strategies to diagnose or treat NASH in an accurate manner do not exist. In the current review, we put forward the concept of oxidized lipids as a significant risk factor for NASH. We will focus on the contribution of the different types of oxidized lipids as part of the oxidized low-density lipoprotein (oxLDL) to the hepatic inflammatory response. Furthermore, we will elaborate on the underlying mechanisms linking oxLDL to inflammatory responses in the liver and on how these cascades can be used as therapeutic targets to combat NASH. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.     

Fractures on the chest radiograph in detection of alcoholic liver disease.

The presence of fractures seen on routine chest radiography was assessed in patients with liver disease to see whether it might provide a useful marker of alcoholism. Chest radiographs taken at the time of liver biopsy were examined in 149 patients--72 with alcoholic liver disease (32 (44%) cirrhotic) and 77 with various forms of non-alcoholic liver disease (15 (19 . 5%) cirrhotic)--and in 149 controls. Fractures (85 rib, two clavicular) were much more common in patients with alcoholic liver disease (20 subjects; 28%) than in patients with non-alcoholic liver disease (1; 1 . 3%) or controls 10; 6 . 7%). In alcoholic liver disease rib fractures were significantly more likely to be bilateral or multiple (more than two) or both (p less than 0 . 01). Of patients with alcoholic liver disease, those with fractures were significantly older than those without, but there was no difference in sex, social class, the proportion with cirrhosis, or the proportion known to be alcoholic at the time of the radiograph. In liver disease fractures on the chest radiograph diagnosed alcoholism with 95% specificity and 28% sensitivity. These often overlooked or ignored findings in the chest radiograph may have a wider role in the detection of alcoholism.     

Intermediate filament cytoskeleton of the liver in health and disease

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising approximately 70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.