A mouse model for nonalcoholic steatohepatitis

Background

Nonalcoholic steatohepatitis (NASH) is a hepatic manifestation of the growing metabolic syndrome epidemic that could progress to cirrhosis. Animal models adequately mimicking this condition in humans are scanty.

Aim

The objective of our study was to investigate whether high-fat diets (HFD) with adequate methionine and choline levels can induce pathophysiological features typical of human NASH in C57BL/6J mice.

Methods

Forty C57BL/6J mice, divided into control and high-fat (HF) groups, were fed low-fat diet and HFD, ad libitum respectively for 20 weeks. At the end of 20 weeks, animals were sacrificed and assays were performed for blood biomarkers typical of human NASH. Adipose tissue depots were collected and liver samples were processed for histological examination.

Results

High-fat feeding led to increased triglyceride accumulation in the liver (8.9 μmol/100 mg liver tissue vs. 2.6 μmol/100 mg for control) and induced histopathological features of human NASH including hepatic steatosis, ballooning inflammation and fibrosis. Expressions of proteins and chemokines predominant in NASH including collagens I, III and IV and platelet derived growth factor (PDGF) A and B were significantly higher in animals fed the HFD. Liver enzymes alanine transaminase, aspartate transaminase and alkaline phosphatase were significantly (P<.05) elevated in the HF group compared to controls. Mice on HFD also developed hyperglycemia, hyperinsulinemia, hypoadiponectinemia along with elevated tumor necrosis factor α, resistin, leptin, free fatty acids, transforming growth factor β and malondialdehyde levels that characterize NASH in humans.

Conclusion

Long-term HF feeding with adequate methionine and choline can induce many of the pathophysiological features typical of human NASH in C57BL/6J mice.     

Effects of long-term ethanol administration in a rat total enteral nutrition model of alcoholic liver disease

Male Sprague-Dawley rats were chronically fed a high-unsaturated-fat diet for 130 days by using total enteral nutrition (TEN), or the same diet in which ethanol (EtOH) isocalorically replaced carbohydrate calories. Additional groups were supplemented with the antioxidant N-acetylcysteine (NAC) at 1.7 g·kg(-1)·day(-1). Relative to an ad libitum chow-fed group, the high-fat-fed controls had three- to fourfold greater expression of fatty acid transporter CD36 mRNA and developed mild steatosis but little other hepatic pathology. NAC treatment resulted in increased somatic growth relative to controls (4.0 ± 0.1 vs. 3.1 ± 0.1 g/day) and increased hepatic steatosis score (3.5 ± 0.6 vs. 2.7 ± 1.2), associated with suppression of the triglyceride hydrolyzing protein adiponutrin, but produced no elevation in serum alanine aminotransferase (ALT). Chronic EtOH treatment increased expression of fatty acid transport protein FATP-2 mRNA twofold, resulting in marked hepatic steatosis, oxidative stress, and a twofold elevation in serum ALT. However, no changes in tumor necrosis factor-α or transforming growth factor-β expression were observed. Fibrosis, as measured by Masson's trichrome and picrosirius red staining, and a twofold increase in expression of type I and type III collagen mRNA, was only observed after EtOH treatment. Long-term EtOH treatment increased hepatocyte proliferation but did not modify the hepatic mRNAs for hedgehog pathway ligands or target genes or genes regulating epithelial-to-mesenchymal transition. Although the effects of NAC on EtOH-induced fibrosis could not be fully evaluated, NAC had additive effects on hepatocyte proliferation and prevented EtOH-induced oxidative stress and necrosis, despite a failure to reverse hepatic steatosis.     

Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet

Despite the increasing incidence of nonalcoholic steatohepatitis (NASH) with the rise in lifestyle-related diseases such as the metabolic syndrome, little is known about the changes in the liver proteome that precede the onset of inflammation and fibrosis. Here, we investigated early changes in the liver-soluble proteome of female C57BL/6N mice fed an NASH-inducing diet by 2D-DIGE and nano-HPLC-MS/MS. In parallel, histology and measurements of hepatic content of triglycerides, cholesterol and intermediates of the methionine cycle were performed. Hepatic steatosis manifested itself after 2 days of feeding, albeit significant changes in the liver-soluble proteome were not evident before day 10 in the absence of inflammatory or fibrotic signs. Proteomic alterations affected mainly energy and amino acid metabolism, detoxification processes, urea cycle, and the one-carbon/S-adenosylmethionine pathways. Additionally, intermediates of relevant affected pathways were quantified from liver tissue, confirming the findings from the proteomic analysis.     

Cytokine and chemokine expression associated with steatohepatitis and hepatocyte proliferation in rats fed ethanol via total enteral nutrition

To determine the temporal relationship between alcohol-induced changes in cytokines and chemokines, development of liver pathology and stimulation of hepatocyte proliferation, male Sprague-Dawley rats were intragastrically fed low carbohydrate-containing ethanol (EtOH) diets via total enteral nutrition (TEN) for up to 49 d. Induction of EtOH metabolism and appearance of steatosis preceded development of oxidative stress, inflammation, and cell death. A transitory peak of tumor necrosis factor (TNFalpha) and interferon gamma (IFN gamma) was observed at 14 d followed by reduced expression of TNFalpha, IFN gamma and another Th1 cytokine IL-12 accompanied by reduced expression of the Th1 regulators T-bet and STAT4. After 35-49 d of EtOH, at a time when hepatocyte proliferation was stimulated, IL-12 returned to control values and a second peak of TNFalpha occurred. The Th2 cytokine IL-4 remained suppressed throughout the study and was accompanied by reductions in the Th2 regulator GATA3. There was no temporal effect of EtOH on expression of IL-6 or TGFbeta. IL-5 and IL-13 mRNA were undetectable. Chemokine CXCL-2 expression increased progressively up to 35 d and preceded the appearance of inflammatory infiltrates. These data suggest that steatosis, increased ethanol metabolism, a transient induction of the innate immune response and suppression of Th2 responses were acute consequences of ethanol treatment and were followed by suppression of Th1 responses. However, the majority of necrosis, apoptosis and a late peak of TNFalpha only occurred after 6-7 weeks of ethanol, coincided with the appearance of inflammatory infiltrates and were associated with stimulation of hepatocyte proliferation.     

Bifidobacterium adolescentis protects from the development of nonalcoholic steatohepatitis in a mouse model

To investigate the hypothesis that an oral supplementation of Bifidobacterium adolescentis protects against a diet-induced nonalcoholic steatohepatitis in a mouse model, C57BL/6 mice were fed either a Western-style or a control diet±tap water fortified with B. adolescentis (5×10⁷ cfu/ml) ad libitum for 12 weeks. Mice fed a Western-style diet gained significantly more weight than mice fed a control diet and developed a mild steatohepatitis. Western-style diet fed groups concomitantly treated with B. adolescentis had significantly decreased liver damage, whereas portal endotoxin levels and toll-like receptor-4 protein levels as well as myeloid differentiation factor 88 mRNA were increased in livers of both Western-style diet fed groups. The protective effects of the B. adolescentis were associated with a significant attenuation of the formation of reactive oxygen species, activation of nuclear factor κB (NFκB) and induction of markers of inflammation in the liver. Taken together, our data suggest that an oral supplementation of the B. adolescentis attenuates diet-induced steatohepatitis, and this effect is associated with prevention from lipid peroxidation, NFκB activation and finally inflammation in the liver.     

A novel noninvasive index for nonalcoholic steatohepatitis: a pilot study

Abstract

Objective: The potential development of a noninvasive marker predicting nonalcoholic steatohepatitis (NASH).

Methods: Thirty patients with biopsy-proven nonalcoholic fatty liver disease were evaluated by numerous anthropometric, clinical and biochemical parameters.

Results: Serum glutamic oxaloacetic transaminase (SGOT; p?=?0.027), log (erythrocyte sedimentation rate) (ESR; p?=?0.034) and homocysteine (p?=?0.041) were associated with NASH independently from gender, age and body mass index. When combined, the regression model provided R²?=?0.563 (p?=?0.001) and area under the ROC curve?=?0.873?±?0.066 (p?<?0.001).

Conclusion: This noninvasive marker, named HSENSI (acronym of homocysteine, SGOT, ESR, Nonalcoholic Steatohepatitis Index), consists of three low cost, easily measurable parameters and may accurately predict NASH.     

Genes of the antioxidant response undergo upregulation in a rodent model of nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease encompasses a spectrum of hepatic pathologies ranging from simple fatty liver to an inflammatory state known as nonalcoholic steatohepatitis (NASH). NASH is also characterized by severe hepatic oxidative stress. The goal of this study was to determine whether genes of the antioxidant response are induced in rodent models of nonalcoholic fatty liver disease. To simulate simple fatty liver and NASH, respectively, male Sprague-Dawley rats were fed a high-fat (HF) or a methionine and choline-deficient (MCD) diet for 8 weeks. Key marker genes of the antioxidant response that are known to undergo upregulation via activation of Nuclear Factor Erythroid 2-Related Factor 2 were measured using the branched DNA signal amplification assay. Messenger RNA levels of the antioxidant response, including NAD(P)H:quinone oxidoreductase-1 (Nqo1), Glutamate cysteine ligase catalytic (Gclc), and Heme oxygenase-1 (Ho-1), were significantly induced in MCD rat liver but not in HF rat liver. Furthermore, Nqo1 protein expression and activity underwent significant upregulation in MCD rat liver but not in HF rat liver. These data strongly indicate that the pathology induced by the MCD dietary model of NASH results in upregulation of the antioxidant response in rats.     

High-fat diet decreases activity of the oxidative phosphorylation complexes and causes nonalcoholic steatohepatitis in mice

Nonalcoholic fatty liver disease (NAFLD) is the most frequent histological finding in individuals with abnormal liver-function tests in the Western countries. In previous studies, we have shown that oxidative phosphorylation (OXPHOS) is decreased in individuals with NAFLD, but the cause of this mitochondrial dysfunction remains uncertain. The aims of this study were to determine whether feeding mice a high-fat diet (HFD) induces any change in the activity of OXPHOS, and to investigate the mechanisms involved in the pathogenesis of this defect. To that end, 30 mice were distributed between five groups: control mice fed a standard diet, and mice on a HFD and treated with saline solution, melatonin (an antioxidant), MnTBAP (a superoxide dismutase analog) or uric acid (a scavenger of peroxynitrite) for 28 weeks intraperitoneously. In the liver of these mice, we studied histology, activity and assembly of OXPHOS complexes, levels of subunits of these complexes, gene expression of these subunits, oxidative and nitrosative stress, and oxidative DNA damage. In HFD-fed mice, we found nonalcoholic steatohepatitis, increased gene expression of TNFα, IFNγ, MCP-1, caspase-3, TGFβ1 and collagen α1(I), and increased levels of 3-tyrosine nitrated proteins. The activity and assembly of all OXPHOS complexes was decreased to about 50–60%. The amount of all studied OXPHOS subunits was markedly decreased, particularly the mitochondrial-DNA-encoded subunits. Gene expression of mitochondrial-DNA-encoded subunits was decreased to about 60% of control. There was oxidative damage to mitochondrial DNA but not to genomic DNA. Treatment of HFD-fed mice with melatonin, MnTBAP or uric acid prevented all changes observed in untreated HFD-fed mice. We conclude that a HFD decreased OXPHOS enzymatic activity owing to a decreased amount of fully assembled complexes caused by a reduced synthesis of their subunits. Antioxidants and antiperoxynitrites prevented all of these changes, suggesting that nitro-oxidative stress played a key role in the pathogenesis of these alterations. Treatment with these agents might prevent the development of NAFLD in humans.KEY WORDS: Mitochondrial respiratory chain, Nonalcoholic steatohepatitis, NADPH oxidase, Oxidative phosphorylation, Proteomic, Nitro-oxidative stress     

A porcine animal model to mimic the restart of enteral nutrition (refeeding-model)

With the aim towards establishing an animal model of total parenteral nutrition (TPN), 12 piglets aged 9 weeks (mean body weight 21 kg) were surgically provided with central venous catheters. Six piglets were nourished parenterally with the objective to reach a 14-d period of TPN; the other six piglets served as control and were fed normally. Only one animal from each group could be monitored over the whole period. Nine piglets were euthanised on d 13 and one on d 12. No animal showed fever or signs of septicaemia during the study. The levels of Ca, Mg, Na and P in the blood were within the normal range as were those for blood glucose and plasma creatinine. Symptoms of the TPN included: transient diarrhoea, occasional appearance of faecal blood and occasional absence of defecation. A reduced small intestine length and altered mucosal morphology and function were observed. One animal showed bile stasis at the end of the study. All TPN animals showed a remarkably high level of blood urea early in the morning. The intestinal symptoms observed may resemble the human situation during TPN. However, due to the fast growth rate, pigs aged 9 weeks have higher nutrient requirements per kg body weight. Consequently, the osmolality of the nutrient solution was necessarily high. Whether the significantly higher blood urea observed in the TPN group reflected a catabolic metabolism during the starving period at night-time could not be conclusively shown. Alternatively, it could reflect a slower growth rate and a resulting quantitative excess of amino acids (AA), or could have been the consequence of a suboptimal AA composition. A permanent infusion would be favourable in order not to overcharge the capacity for glucose uptake and amino acid metabolism during the infusion.     

Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model

The pathogenesis of nonalcoholic steatohepatitis (NASH) is poorly defined. Feeding mice a diet deficient in methionine and choline (MCD diet) induces experimental NASH. Osteopontin (OPN) is a Th1 cytokine that plays an important role in several fibroinflammatory diseases. We examined the role of OPN in the development of experimental NASH. A/J mice were fed MCD or control diet for up to 12 wk, and serum alanine aminotransferase (ALT), liver histology, oxidative stress, and the expressions of OPN, TNF-alpha, and collagen I were assessed at various time points. MCD diet-fed mice developed hepatic steatosis starting after 1 wk and inflammation by 2 wk; serum ALT increased from day 3. Hepatic collagen I mRNA expression increased during 1-4 wk, and fibrosis appeared at 8 wk. OPN protein expression was markedly increased on day 1 of MCD diet and persisted up to 8 wk, whereas OPN mRNA expression was increased at week 4. TNF-alpha expression was increased from day 3 to 2 wk, and evidence of oxidative stress did not appear until 8 wk. Increased expression of OPN was predominantly localized in hepatocytes. Hepatocytes in culture also produced OPN, which was stimulated by transforming growth factor-beta and TNF-alpha. Moreover, MCD diet-induced increases in serum ALT levels, hepatic inflammation, and fibrosis were markedly reduced in OPN(-/-) mice when compared with OPN(+/+) mice. In conclusion, our results demonstrate an upregulation of OPN expression early in the development of steatohepatitis and suggest an important role for OPN in signaling the onset of liver injury and fibrosis in experimental NASH.     

Alpha-syntrophin null mice are protected from non-alcoholic steatohepatitis in the methionine-choline-deficient diet model but not the atherogenic diet model

Adipose tissue dysfunction contributes to the pathogenesis of non-alcoholic steatohepatitis (NASH). The adapter protein alpha-syntrophin (SNTA) is expressed in adipocytes. Knock-down of SNTA increases preadipocyte proliferation and formation of small lipid droplets, which are both characteristics of healthy adipose tissue. To elucidate a potential protective role of SNTA in NASH, SNTA null mice were fed a methionine-choline-deficient (MCD) diet or an atherogenic diet which are widely used as preclinical NASH models. MCD diet mediated loss of fat mass was largely improved in SNTA?/? mice compared to the respective wild type animals. Hepatic lipids were mostly unchanged while the oxidative stress marker malondialdehyde was only induced in the wild type mice. The expression of inflammatory markers and macrophage immigration into the liver were reduced in SNTA?/? animals. This protective function of SNTA loss was absent in atherogenic diet induced NASH. Here, hepatic expression of inflammatory and fibrotic genes was similar in both genotypes though mutant mice gained less body fat during feeding. Hepatic cholesterol and ceramide were strongly induced in both strains upon feeding the atherogenic diet, while hepatic sphingomyelin, phosphatidylserine and phosphatidylethanolamine levels were suppressed.SNTA deficient mice are protected from fat loss and NASH in the experimental MCD model. NASH induced by an atherogenic diet is not influenced by loss of SNTA. The present study suggests the use of different experimental NASH models to study the pathophysiological role of proteins like SNTA in NASH.     

Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis

A small percentage of pathologically obese subjects with fatty livers develop histological signs of necroinflammation and fibrosis, suggesting a variety of cofactors in the pathogenesis of obesity-related liver diseases including nonalcoholic steatohepatitis. Since several observations have linked bacterial endotoxins to liver damage, the aim of this study was to determine the effect of obesity on intestinal mucosal integrity and portal blood endotoxemia in two strains of obese mice: leptin-deficient (ob/ob) and hyperleptinemic (db/db) mice. Murine intestinal mucosal barrier function was assessed using a Ussing chamber, whereas ileum tight junction proteins were analyzed by immunocytochemistry and Western blot analysis. Circulating proinflammatory cytokines and portal blood endotoxin levels were measured by ELISA and the limulus test, respectively. The inflammatory and fibrogenic phenotype of murine hepatic stellate cells (HSCs) was determined by ELISA and quantitative RT-PCR. Ob/ob and db/db mice showed lower intestinal resistance, profoundly modified distribution of occludin and zonula occludens-1 in the intestinal mucosa, and higher circulating levels of inflammatory cytokines and portal endotoxemia compared with lean control mice. Moreover, HSCs isolated from ob/ob and db/db mice showed higher membrane CD14 mRNA levels and more pronounced lipopolysaccharide-induced proinflammatory and fibrogenic responses than HSCs from lean animals. In conclusion, genetically obese mice display enhanced intestinal permeability leading to increased portal endotoxemia that makes HSCs more sensitive to bacterial endotoxins. We suggest that in metabolic syndrome, patients may likewise have a greater intestinal mucosa permeability and increased lipopolysaccharide levels in portal blood that can contribute to the liver inflammatory damage.     

Novel role of IL-13 in fibrosis induced by nonalcoholic steatohepatitis and its amelioration by IL-13R-directed cytotoxin in a rat model

Nonalcoholic steatohepatitis (NASH), the most common cause of chronic liver fibrosis, progresses to cirrhosis in up to 20% of patients. We report that hepatic stellate cells (HSC) in sinusoidal lesions of liver of patients with NASH express high levels of high-affinity IL-13R (IL-13Ralpha2), which is colocalized with smooth muscle actin, whereas fatty liver and normal liver specimens do not express IL-13Ralpha2. HSCs engineered to overexpress IL-13Ralpha2 respond to IL-13 and induce TGFB1 promoter activity and TGF-beta1 production. We also developed NASH in rats by feeding a choline-deficient l-amino acid diet. These rats developed liver fibrosis as assessed by H&E staining, Masson's trichrome and Sirius red staining, and hydroxyproline assays. Treatment of these rats with IL-13R-directed cytotoxin caused a substantial decline in fibrosis and liver enzymes without organ toxicity. These studies demonstrate that functional IL-13Ralpha2 are overexpressed in activated HSCs involved in NASH and that IL-13 cytotoxin ameliorates pathological features of NASH in rat liver, indicating a novel role of this cytotoxin in potential therapy.     

A new device for long-term continuous enteral nutrition of rats by elementary diet via gastrostomy, following extensive oesophageal or lower gastrointestinal surgery.

A device for intragastric nutrition of unsedated and minimally restrained rats under complete alimentary abstinence has been developed. The cannulation system consists of an infusion pump, modified glass syringe as flow swivel, rat-harness and a silicone-tube-gastrostomy. The animals were kept in special cages and coprophagy was prevented by an own model of faecal collection cup. Methionine and Ca-glycerophosphate had to be added to a commercial elementary diet. The rats were allowed to move freely during intragastric infusion, which was applied for 14 to 28 days in 118 Wistar-rats (350-400 g). They maintained weight on an energy supply of 86.4 kcal/day.     

A Western-like fat diet is sufficient to induce a gradual enhancement in fat mass over generations

The prevalence of obesity has steadily increased over the last few decades. During this time, populations of industrialized countries have been exposed to diets rich in fat with a high content of linoleic acid and a low content of α-linolenic acid compared with recommended intake. To assess the contribution of dietary fatty acids, male and female mice fed a high-fat diet (35% energy as fat, linoleic acid:α-linolenic acid ratio of 28) were mated randomly and maintained after breeding on the same diet for successive generations. Offspring showed, over four generations, a gradual enhancement in fat mass due to combined hyperplasia and hypertrophy with no change in food intake. Transgenerational alterations in adipokine levels were accompanied by hyperinsulinemia. Gene expression analyses of the stromal vascular fraction of adipose tissue, over generations, revealed discrete and steady changes in certain important players, such as CSF3 and Nocturnin. Thus, under conditions of genome stability and with no change in the regimen over four generations, we show that a Western-like fat diet induces a gradual fat mass enhancement, in accordance with the increasing prevalence of obesity observed in humans.     

Comprehensive characterization of metabolic,inflammatory and fibrotic changes in a mouse model of diet-derived nonalcoholic steatohepatitis

The objective of this study was to develop a well-characterized mouse model of nonalcoholic steatohepatitis (NASH) with a strong manifestation of liver fibrosis. The progression of metabolic, inflammatory and fibrotic features of this mouse model was monitored by performing in vivo time-course study. Male C57BL/6J mice were fed a high-fat/high-sucrose/high-cholesterol diet (34% fat, 34% sucrose and 2.0% cholesterol, by weight) for 2, 4, 6, 8, 10, 12, 14 or 16 weeks to induce obesity-associated metabolic dysfunctions, inflammation and fibrosis in the liver and white adipose tissue (WAT). Body and liver weights were gradually increased with significant hepatic triglyceride accumulation, i.e., liver steatosis, and marked elevation of serum alanine transaminase levels at week 10. While hepatic inflammation was displayed with the highest expression of macrophage markers and M1 markers at week 6, liver fibrosis determined by collagen accumulation was continuously increased to week 16. In epididymal WAT, weights and adipocyte size peaked at week 6–8. The increased expression of fibrogenic genes preceded inflammatory features (week 2 to 6 vs. week 6 to 16), suggesting that early fibrosis may trigger inflammatory events in the WAT. This study established a mouse model of diet-induced NASH with a strong manifestation of liver fibrosis. This mouse model will be a valuable in vivo tool in studying the pathophysiology of NASH and also in testing preventive and therapeutic potentials of dietary components and drugs against NASH with liver fibrosis.