A diet-induced Sprague–Dawley rat model of nonalcoholic steatohepatitis-related cirrhosis

Certain modified diets containing saturated fatty acids, cholesterol or fructose lead to the development of nonalcoholic steatohepatitis (NASH)-related fibrosis in rodents; however, progression to cirrhosis is rare. Experimental liver cirrhosis models have relied on genetic manipulation or administration of hepatotoxins. This study aimed to establish a reliable dietary model of NASH-related cirrhosis in a relatively short period. Male Sprague–Dawley rats (9 weeks of age) were randomly assigned to normal, high-fat (HF), or two types (1.25% or 2.5% cholesterol) of high-fat and high-cholesterol (HFC) diets for 18 weeks. All HFC diets contained 2% cholic acid by weight. Histopathological analysis revealed that the HFC diets induced obvious hepatic steatosis, inflammation with hepatocyte ballooning and advanced fibrosis (stage 3–4) in all 12 rats at 27 weeks of age. In contrast, all five rats given the HF diet developed mild steatosis and inflammation without fibrosis. The amount of cholesterol in the liver and hepatocellular mitochondrial and microsomal fractions was significantly higher in rats fed the HFC diets than the normal or HF diets. The HFC diets significantly suppressed mRNA levels of microsomal triglyceride transfer protein, adenosine triphosphate binding cassette transporter G5, bile acid CoA: amino acid N-acyltransferase and bile salt export pump, as well as the enzymatic activity of carnitine palmitoyltransferase in the liver. In conclusion, the HFC diets induced liver cirrhosis in conjunction with hepatic features of NASH in Sprague–Dawley rats within 18 weeks, and altered gene expression and enzyme activity to accumulate lipid and bile acid in the liver.     

Mouse Models of Diet-Induced Nonalcoholic Steatohepatitis Reproduce the Heterogeneity of the Human Disease

MethodsMice were fed standard chow, MCD diet for 8 weeks, or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. Liver pathology and metabolic profile were compared.ResultsThe metabolic profile associated with human NASH was better mimicked by Western diet. Although hepatic steatosis (i.e., triglyceride accumulation) was also more severe, liver non-esterified fatty acid content was lower than in the MCD diet group. NASH was also less severe and less reproducible in the Western diet model, as evidenced by less liver cell death/apoptosis, inflammation, ductular reaction, and fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also less robust in the Western diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation.ConclusionFeeding mice a Western diet models metabolic perturbations that are common in humans with mild NASH, whereas administration of a MCD diet better models the pathobiological mechanisms that cause human NAFLD to progress to advanced NASH.     

The Annexin A2-Notch regulatory loop in hepatocytes promotes liver fibrosis in NAFLD by increasing osteopontin expression

BackgroundThe mechanisms underlying the progression of liver disease from simple hepatic steatosis to advanced nonalcoholic steatohepatitis (NASH) and liver fibrosis warrant further investigation. Increased mRNA levels of Annexin A2 protein (Anxa2) have been observed in patients with NASH. However, the role of Anxa2 in NASH remains unclear.MethodsThe protein levels of Anxa2 were analyzed in the livers of mice and patients with NASH. Anxa2-knockout and -knockdown mice were generated, and NASH was induced through a high fructose, palmitate, and cholesterol (FPC) diet or methionine- and choline-deficient (MCD) diet.FindingsWe found elevated expression of Anxa2 in the livers of patients and mice with NASH. Anxa2 knockdown but not knockout ameliorated liver fibrosis in both FPC and MCD diet–fed mice. Liver-specific Anxa2 overexpression increased collagen deposition in mice fed a normal diet. Mechanistically, Anxa2 overexpression in hepatocytes promoted hepatic stellate cell activation in a paracrine manner by increasing osteopontin expression. Notch inhibition suppressed the exogenous overexpression of Anxa2-induced osteopontin and endogenous Anxa2 expression. Additionally, Anxa2 overexpression accelerated the progression of nonalcoholic fatty liver disease (NAFLD) in mice fed a high-fat diet. Moreover, Anxa2 levels were higher in NAFLD patients with advanced liver fibrosis than in those with mild liver fibrosis, as determined using the Gene Expression Omnibus database.InterpretationIn conclusion, we found increased Anxa2 expression in hepatocytes promoted liver fibrosis in NASH mice by increasing osteopontin expression. The Anxa2-Notch positive regulatory loop contributes to this process and represents a novel target for the treatment of NASH-related liver fibrosis.     

Dietary modification dampens liver inflammation and fibrosis in obesity‐related fatty liver disease

Background: Alms1 mutant (foz/foz) mice develop hyperphagic obesity, diabetes, metabolic syndrome, and fatty liver (steatosis). High‐fat (HF) feeding converts pathology from bland steatosis to nonalcoholic steatohepatitis (NASH) with fibrosis, which leads to cirrhosis in humans. Objective: We sought to establish how dietary composition contributes to NASH pathogenesis. Design and Methods: foz/foz mice were fed HF diet or chow 24 weeks, or switched HF to chow after 12 weeks. Serum ALT, NAFLD activity score (NAS), fibrosis severity, neutrophil, macrophage and apoptosis immunohistochemistry, uncoupling protein (UCP)2, ATP, NF‐κB activation/expression of chemokines/adhesion molecules/fibrogenic pathways were determined. Result: HF intake upregulated liver fatty acid and cholesterol transporter, CD36. Dietary switch expanded adipose tissue and decreased hepatomegaly by lowering triglyceride, cholesterol ester, free cholesterol and diacylglyceride content of liver. There was no change in lipogenesis or fatty acid oxidation pathways; instead, CD36 was suppressed. These diet‐induced changes in hepatic lipids improved NAS, reduced neutrophil infiltration, normalized UCP2 and increased ATP; this facilitated apoptosis with a change in macrophage phenotype favoring M2 cells. Dietary switch also abrogated NF‐κB activation and chemokine/adhesion molecule expression, and arrested fibrosis by dampening stellate cell activation. Conclusion: Reversion to a physiological dietary composition after HF feeding in foz/foz mice alters body weight distribution but not obesity. This attenuates NASH severity and fibrotic progression by suppressing NF‐κB activation and reducing neutrophil and macrophage activation. However, adipose inflammation persists and is associated with continuing apoptosis in the residual fatty liver disease. Taken together, these findings indicate that other measures, such as weight reduction, may be required to fully reverse obesity‐related NASH.     

The modulation of hepatic adenosine triphosphate and inflammation by eicosapentaenoic acid during severe fibrotic progression in the SHRSP5/Dmcr rat model

AimsEicosapentaenoic acid (EPA) can ameliorate certain liver lesions involved in non-alcoholic steatohepatitis (NASH). A previous study has found that stroke-prone spontaneously hypertensive 5/Dmcr (SHRSP5/Dmcr) rats fed a high fat-cholesterol (HFC) diet developed fibrotic steatohepatitis with histological similarities to NASH. This study evaluated the potential effects and mechanisms of action of EPA supplementation using this rodent model.Main methodsMale rats were randomly assigned to groups that were fed with either the stroke-prone (SP) diet or HFC diet with or without EPA for 2, 8 and 14 weeks, respectively. The liver histopathology, biochemical features, mRNA and protein levels, and nuclear factor-κB (NF-κB) DNA binding activity were determined.Key findingsThe SP diet-fed rats presented normal livers. Conversely, the HFC diet-fed rats developed microvesicular/macrovesicular steatosis, inflammation, ballooning degeneration and severe fibrosis. At 2 weeks, the administration of EPA inhibited hepatic inflammatory recruitment by blocking the phosphorylation of inhibitor of κB-α (IκBα), which antagonizes the NF-κB activation pathway. The dietary supplementation of EPA for 8 weeks ameliorated hepatic triglyceride accumulation and macrovesicular steatosis by inhibiting the HFC diet-induced decrease in the protein levels of enzymes involved in fatty acid β-oxidation including carnitine palmitoyltransferase 1, very long chain acyl-CoA dehydrogenase and peroxisomal bifunctional protein. Although the administration of EPA elicited no histologically detectable effects on severe fibrosis at 14 weeks, it restored an HFC diet-induced decline in hepatic adenosine triphosphate (ATP) levels and suppressed ballooning degeneration, suggesting that EPA may inhibit HFC diet-induced ATP loss and cell death.SignificanceInitial amelioration of the inflammation and steatosis in the rats after EPA supplementation indicates a possibility to treat steatohepatitis. Additionally, this study provides new insights into the roles of EPA in hepatic ATP depletion and subsequent hepatocellular injury during severe fibrosis.     

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.     

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.     

Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition

Although there are small animal platforms that recapitulate some of the histological features of nonalcoholic fatty liver disease, there are no small animal models of nonalcoholic steatohepatitis (NASH) with consistent hepatocellular ballooning and progressive fibrosis that also exhibit fidelity to the human condition physiologically. We examined the metabolic and histological effects of a diet on the basis of the composition of "fast food" (high saturated fats, cholesterol, and fructose). Mice (n = 8 in each group) were assigned to diets as follows: 1) standard chow (SC), i.e., 13% energy as fat [1% saturated fatty acids (SFA)], 2) high fat (HF), i.e., 60% energy as fat (1% SFA), and 3) fast food (FF), i.e., 40% energy as fat (12% SFA, 2% cholesterol). All three diets were supplemented with high fructose. All diets produced obesity. The HF and FF diets produced insulin resistance. Liver histology was normal in animals fed the SC diet. Steatohepatitis with pronounced ballooning and progressive fibrosis (stage 2) was observed in mice fed the FF diet. Although the HF diet produced obesity, insulin resistance, and some steatosis; inflammation was minimal, and there was no increase in fibrosis. The FF diet produced a gene expression signature of increased fibrosis, inflammation, and endoplasmic reticulum stress and lipoapoptosis. A diet based on high cholesterol, high saturated fat, and high fructose recapitulates features of the metabolic syndrome and NASH with progressive fibrosis. This represents a novel small animal model of fibrosing NASH with high fidelity to the human condition. These results highlight the contribution of dietary composition to the development of nonalcoholic fatty liver disease and NASH.     

A non-mitogenic FGF4 analog alleviates non-alcoholic steatohepatitis through an AMPK-dependent pathway

Background & aimsNon-alcoholic fatty liver disease (NAFLD) has emerged as a major liver disease increasingly in association with non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). However, there are currently no approved therapies for treating NAFLD and NASH. Fibroblast growth factor 4 (FGF4) has recently been shown as a promising drug candidate for several metabolic diseases.MethodsMice fed a high-fat diet with high fructose/glucose drinking water (HF/HFG, Western-like diet) for 21 weeks were intraperitoneally injected with non-mitogenic recombinant FGF4^△NT (rFGF4^△NT, 1.0 mg/kg body weight) every other day for 8 weeks. Primary mouse hepatocytes cultured in medium containing high glucose/palmitic acid (HG/PA) or TNFα/cyclohexane (TNFα/CHX) were treated with 1.0 μg/ml rFGF4^△NT. Changes in parameters for histopathology, lipid metabolism, inflammation, hepatocellular apoptosis and fibrosis were determined. The Caspase6 activity and AMPK pathway were assessed.ResultsAdministration of rFGF4^△NT significantly attenuated the Western-like diet-induced hepatic steatosis, inflammation, liver injury and fibrosis in mice. rFGF4^△NT treatment reduced fatty acid-induced lipid accumulation and lipotoxicity-induced hepatocyte apoptosis, which were associated with inhibition of Caspase6 cleavage and activation. Inhibition of AMP-activated protein kinase (AMPK) by Compound C or deficiency of Ampk abrogated rFGF4^△NT–induced hepatoprotection in primary hepatocytes and in mice with NASH.ConclusionrFGF4^△NT exerts significant protective effects on NASH via an AMPK-dependent signaling pathway. Our study indicates that FGF4 analogs may have therapeutic potential for the Western-like diet induced NASH.     

Lipid Metabolism,Oxidative Stress and Cell Death Are Regulated by PKC Delta in a Dietary Model of Nonalcoholic Steatohepatitis

Steatosis, oxidative stress, and apoptosis underlie the development of nonalcoholic steatohepatitis (NASH). Protein kinase C delta (PKCδ) has been implicated in fatty liver disease and is activated in the methionine and choline-deficient (MCD) diet model of NASH, yet its pathophysiological importance towards steatohepatitis progression is uncertain. We therefore addressed the role of PKCδ in the development of steatosis, inflammation, oxidative stress, apoptosis, and fibrosis in an animal model of NASH. We fed PKCδ^−/− mice and wildtype littermates a control or MCD diet. PKCδ^−/− primary hepatocytes were used to evaluate the direct effects of fatty acids on hepatocyte lipid metabolism gene expression. A reduction in hepatic steatosis and triglyceride levels were observed between wildtype and PKCδ^−/− mice fed the MCD diet. The hepatic expression of key regulators of β-oxidation and plasma triglyceride metabolism was significantly reduced in PKCδ^−/− mice and changes in serum triglyceride were blocked in PKCδ^−/− mice. MCD diet-induced hepatic oxidative stress and hepatocyte apoptosis were reduced in PKCδ^−/− mice. MCD diet-induced NADPH oxidase activity and p47^phox membrane translocation were blunted and blocked, respectively, in PKCδ^−/− mice. Expression of pro-apoptotic genes and caspase 3 and 9 cleavage in the liver of MCD diet fed PKCδ^−/− mice were blunted and blocked, respectively. Surprisingly, no differences in MCD diet-induced fibrosis or pro-fibrotic gene expression were observed in 8 week MCD diet fed PKCδ^−/− mice. Our results suggest that PKCδ plays a role in key pathological features of fatty liver disease but not ultimately in fibrosis in the MCD diet model of NASH.     

The Heme Oxygenase System Rescues Hepatic Deterioration in the Condition of Obesity Co-Morbid with Type-2 Diabetes

The prevalence of non-alcoholic fatty-liver disease (NAFLD) is increasing globally. NAFLD is a spectrum of related liver diseases that progressive from simple steatosis to serious complications like cirrhosis. The major pathophysiological driving of NAFLD includes elevated hepatic adiposity, increased hepatic triglycerides/cholesterol, excessive hepatic inflammation, and hepatocyte ballooning injury is a common histo-pathological denominator. Although heme-oxygenase (HO) is cytoprotective, its effects on hepatocyte ballooning injury have not been reported. We investigated the effects of upregulating HO with hemin or inhibiting it with stannous-mesoporphyrin (SnMP) on hepatocyte ballooning injury, hepatic adiposity and inflammation in Zucker-diabetic-fatty rats (ZDFs), an obese type-2-diabetic model. Hemin administration to ZDFs abated hepatic/plasma triglycerides and cholesterol, and suppressed several pro-inflammatory cytokines and chemokines including, TNF-α, IL-6, IL-1β, macrophage-inflammatory-protein-1α (MIP-1α) and macrophage-chemoattractant-protein-1 (MCP-1), with corresponding reduction of the pro-inflammatory M1-phenotype marker, ED1 and hepatic macrophage infiltration. Correspondingly, hemin concomitantly potentiated the protein expression of several markers of the anti-inflammatory macrophage-M2-phenotype including ED2, IL-10 and CD-206, alongside components of the HO-system including HO-1, HO-activity and cGMP, whereas the HO-inhibitor, SnMP abolished the effects. Furthermore, hemin attenuated liver histo-pathological lesions like hepatocyte ballooning injury and fibrosis, and reduced extracellular-matrix/profibrotic proteins implicated in liver injury such as osteopontin, TGF-β1, fibronectin and collagen-IV. We conclude that hemin restore hepatic morphology by abating hepatic adiposity, suppressing macrophage infiltration, inflammation and fibrosis. The selective enhancement of anti-inflammatory macrophage-M2-phenotype with parallel reduction of pro-inflammatory macrophage-M1-phenotype and related chemokines/cytokines like TNF-α, IL-6, IL-1β, MIP-1α and MCP-1 are among the multifaceted mechanisms by which hemin restore hepatic morphology.     

Amelioration of diet-induced nonalcoholic steatohepatitis in rats by Mn-salen complexes via reduction of oxidative stress

Background

Nonalcoholic steatohepatitis (NASH), a progressive stage of nonalcoholic fatty liver disease (NAFLD), is characterized by steatosis (accumulation of triacylglycerols within hepatocytes) along with inflammation and ballooning degeneration. It has been suggested that oxidative stress may play an important role in the progress of NAFLD to NASH. The aim of present study was to determine whether antioxidant supplementations using EUK-8, EUK-134 and vitamin C could improve the biochemical and histological abnormalities associated with diet-induced NASH in rats.

Methods

NASH was induced in male N-Mary rats by feeding a methionine - choline deficient (MCD) diet. The rats were fed either normal chow or MCD diet for 10 weeks. After NASH development, the MCD-fed rats were randomly divided into four groups of six: the NASH group that received MCD diet, the EUK-8 group which was fed MCD diet plus EUK-8, the EUK-134 group which was fed MCD diet plus EUK-134 and the vitamin C group which received MCD diet plus vitamin C. EUK-8, EUK-134 and vitamin C (30 mg/kg body weight/day) were administered by gavage for eight weeks.

Results

Treatment of MCD-fed rats with salens reduced the sera aminotransferases, cholesterol, low density lipoprotein contents, the extent of lipid peroxidation and protein carbonylation whereas the HDL-C cholesterol levels were significantly increased. In addition, EUK-8 and EUK-134 improved steatosis, ballooning degeneration and inflammation in liver of MCD-fed rats.

Conclusion

Antioxidant (EUK-8, EUK-134 and vitamin C) supplementation reduces NASH-induced biochemical and histological abnormalities, pointing out that antioxidant strategy could be beneficial in treatment of NASH.     

Vismodegib Suppresses TRAIL-mediated Liver Injury in a Mouse Model of Nonalcoholic Steatohepatitis

Hedgehog signaling pathway activation has been implicated in the pathogenesis of NASH. Despite this concept, hedgehog pathway inhibitors have not been explored. Thus, we examined the effect of vismodegib, a hedgehog signaling pathway inhibitor, in a diet-induced model of NASH. C57BL/6 mice were placed on 3-month chow or FFC (high saturated fats, fructose, and cholesterol) diet. One week prior to sacrifice, mice were treated with vismodegib or vehicle. Mice fed the FFC diet developed significant steatosis, which was unchanged by vismodegib therapy. In contrast, vismodegib significantly attenuated FFC-induced liver injury as manifested by reduced serum ALT and hepatic TUNEL-positive cells. In line with the decreased apoptosis, vismodegib prevented FFC-induced strong upregulation of death receptor DR5 and its ligand TRAIL. In addition, FFC-fed mice, but not chow-fed animals, underwent significant liver injury and apoptosis following treatment with a DR5 agonist; however, this injury was prevented by pre-treatment with vismodegib. Consistent with a reduction in liver injury, vismodegib normalized FFC-induced markers of inflammation including mRNA for TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1 and a variety of macrophage markers. Furthermore, vismodegib in FFC-fed mice abrogated indices of hepatic fibrogenesis. In conclusion, inhibition of hedgehog signaling with vismodegib appears to reduce TRAIL-mediated liver injury in a nutrient excess model of NASH, thereby attenuating hepatic inflammation and fibrosis. We speculate that hedgehog signaling inhibition may be salutary in human NASH.     

The Selective SGLT2 Inhibitor Ipragliflozin Has a Therapeutic Effect on Nonalcoholic Steatohepatitis in Mice

Background & Aims

In recent years, nonalcoholic steatohepatitis (NASH) has become a considerable healthcare burden worldwide. Pathogenesis of NASH is associated with type 2 diabetes mellitus (T2DM) and insulin resistance. However, a specific drug to treat NASH is lacking. We investigated the effect of the selective sodium glucose cotransporter 2 inhibitor (SGLT2I) ipragliflozin on NASH in mice.

Methods

We used the Amylin liver NASH model (AMLN), which is a diet-induced model of NASH that results in obesity and T2DM. AMLN mice were fed an AMLN diet for 20 weeks. SGLT2I mice were fed an AMLN diet for 12 weeks and an AMLN diet with 40 mg ipragliflozin/kg for 8 weeks.

Results

AMLN mice showed steatosis, inflammation, and fibrosis in the liver as well as obesity and insulin resistance, features that are recognized in human NASH. Ipragliflozin improved insulin resistance and liver injury. Ipragliflozin decreased serum levels of free fatty acids, hepatic lipid content, the number of apoptotic cells, and areas of fibrosis; it also increased lipid outflow from the liver.

Conclusions

Ipragliflozin improved the pathogenesis of NASH by reducing insulin resistance and lipotoxicity in NASH-model mice. Our results suggest that ipragliflozin has a therapeutic effect on NASH with T2DM.     

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.     

Sugar kelp (Saccharina latissima) inhibits hepatic inflammation and fibrosis in a mouse model of diet-induced nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH), closely associated with obesity, is a health concern worldwide. We investigated whether the consumption of U.S.-grown sugar kelp (Saccharina latissima), an edible brown alga, can prevent obesity-associated metabolic disturbances and NASH in a mouse model of diet-induced NASH. Male C57BL/6J mice were fed a low-fat diet, a high-fat/high-sucrose/high-cholesterol diet (HF), or a HF diet containing sugar kelp (HF-Kelp) for 14 weeks. HF-Kelp group showed lower body weight with increased O₂ consumption, CO₂ production, physical activity, and energy expenditure compared with the HF. In the liver, there were significant decreases in weight, triglycerides, total cholesterol, and steatosis with HF-Kelp. The HF-Kelp group decreased hepatic expression of a macrophage marker adhesion G protein-coupled receptor E1 (Adgre1) and an M1 macrophage marker integrin alpha x (Itgax). HF-Kelp group also exhibited decreased liver fibrosis, as evidenced by less expression of fibrogenic genes and collagen accumulation than those of HF group. In epididymal white adipose tissue (eWAT), HF-Kelp group exhibited decreases in eWAT weight and adipocyte size compared with those of the HF. HF-Kelp group showed decreased expression of collagen type VI alpha 1 chain, Adgre1, Itgax, and tumor necrosis factor α in eWAT. We demonstrated, for the first time, that the consumption of U.S-grown sugar kelp prevented the development of obesity and its associated metabolic disturbances, steatosis, inflammation, and fibrosis in the liver and eWAT of a diet-induced NASH mouse model.     

Components of metabolic syndrome and coronary artery disease in female Ossabaw swine fed excess atherogenic diet

Ossabaw swine have a 'thrifty genotype' (propensity to obesity) that enables them to survive seasonal food shortages in their native environment. Consumption of excess kcal causes animals of the thrifty genotype to manifest components of the metabolic syndrome, including central (intra-abdominal) obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension. We determined whether female Ossabaw swine manifest multiple components of the metabolic syndrome by comparing lean pigs fed a normal maintenance diet (7% kcal from fat; lean, n = 9) or excess chow with 45% kcal from fat and 2% cholesterol (obese, n = 8). After 9 wk, body composition, glucose tolerance, plasma lipids, and intravascular ultrasonography and histopathology of coronary arteries were assessed. Computed tomography (CT) assessed subcutaneous and intra-abdominal fat deposition and was compared with traditional methods, including anatomical measurements, backfat ultrasonography, and proximate chemical composition analysis. Compared with lean animals, obese swine showed 2-fold greater product of the plasma insulin x glucose concentrations, 4.1-fold greater total cholesterol, 1.6-fold greater postprandial triglycerides, 4.6-fold greater low- to high-density lipoprotein cholesterol ratio, hypertension, and neointimal hyperplasia of coronary arteries. The 1.5-fold greater body weight in obese swine was largely accounted for by the 3-fold greater carcass fat mass. High correlation (0.79 to 0.95) of CT, anatomical measurements, and ultrasonography with direct chemical measures of subcutaneous, retroperitoneal, and visceral fat indicates high validity of all indirect methods. We conclude that relatively brief feeding of excess atherogenic diet produces striking features of metabolic syndrome and coronary artery disease in female Ossabaw swine.     

Is Western Diet-Induced Nonalcoholic Steatohepatitis in Ldlr-/- Mice Reversible?

Background

Nonalcoholic fatty liver disease (NAFLD) is a major public health burden in western societies. The progressive form of NAFLD, nonalcoholic steatohepatitis (NASH), is characterized by hepatosteatosis, inflammation, oxidative stress, and hepatic damage that can progress to fibrosis and cirrhosis; risk factors for hepatocellular carcinoma. Given the scope of NASH, validating treatment protocols (i.e., low fat diets and weight loss) is imperative.

Methods

We evaluated the efficacy of two diets, a non-purified chow (NP) and purified (low-fat low-cholesterol, LFLC) diet to reverse western diet (WD)-induced NASH and fibrosis in Ldlr^-/- mice.

Results

Mice fed WD for 22–24 weeks developed robust hepatosteatosis with mild fibrosis, while mice maintained on the WD an additional 7–8 weeks developed NASH with moderate fibrosis. Returning WD-fed mice to the NP or LFLC diets significantly reduced body weight and plasma markers of metabolic syndrome (dyslipidemia, hyperglycemia) and hepatic gene expression markers of inflammation (Mcp1), oxidative stress (Nox2), fibrosis (Col1A, LoxL2, Timp1) and collagen crosslinking (hydroxyproline). Time course analyses established that plasma triglycerides and hepatic Col1A1 mRNA were rapidly reduced following the switch from the WD to the LFLC diet. However, hepatic triglyceride content and fibrosis did not return to normal levels 8 weeks after the change to the LFLC diet. Time course studies further revealed a strong association (r² ≥ 0.52) between plasma markers of inflammation (TLR2 activators) and hepatic fibrosis markers (Col1A, Timp1, LoxL2). Inflammation and fibrosis markers were inversely associated (r² ≥ 0.32) with diet-induced changes in hepatic ω3 and ω6 polyunsaturated fatty acids (PUFA) content.

Conclusion

These studies establish a temporal link between plasma markers of inflammation and hepatic PUFA and fibrosis. Low-fat low-cholesterol diets promote reversal of many, but not all, features associated with WD-induced NASH and fibrosis in Ldlr^-/- mice.     

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1^−/− mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND-fed Cyp7b1^−/− mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1^−/− mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1^−/− mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.