Murine Model Of Steatohepatitis Research Articles

Blockade of angiotensin II modulates insulin-like growth factor 1-mediated skeletal muscle homeostasis in experimental steatohepatitis

Sarcopenia is associated with mortality in patients with nonalcoholic steatohepatitis (NASH). Angiotensin II receptor blocker (ARB) has been suggested to prevent sarcopenia, but reports on its effect on NASH-derived skeletal muscle atrophy in conjunction with insulin-like growth factor 1 (IGF-1)-mediated muscle homeostasis are few. Our aim was to examine the combined effect of the ARB losartan and IGF-1 replacement on skeletal muscle atrophy in a methionine–choline deficient (MCD) diet-fed murine steatohepatitis model. The MCD-fed mice developed steatohepatitis and skeletal muscle atrophy, as indicated by the reduction of psoas muscle mass and attenuation of forelimb and hindlimb grip strength. Significantly suppressed steatohepatitis and skeletal muscle atrophy was observed after single treatment with ARB or IGF-1, and these effects were augmented after combination treatment. Treatment with ARB and IGF-1 effectively inhibited ubiquitin proteasome-mediated protein degradation by reducing forkhead box protein O1 (FOXO1) and FOXO3a transcriptional activity in the skeletal muscle. Combined ARB and IGF-1 decreased the intramuscular expression of proinflammatory cytokines (i.e., TNFα, IL6, and IL1β) and increased the Trolox equivalent antioxidant capacity and antioxidant enzymes (CAT, GPX1, SOD2, and CYTB). This antioxidant effect was based on downregulation of NADPH oxidase (NOX) 2, normalization of mitochondrial biogenesis and dynamics. Moreover, ARB increased the hepatic and plasma IGF-1 levels and improved steatohepatitis, leading to enhanced skeletal muscle protein synthesis mediated by IGF-1/ AKT/ mechanistic target of rapamycin signaling. Collectively, combined ARB and IGF-1 replacement could be a promising new therapeutic target for NASH-derived skeletal muscle wasting.

Extraction, characterization, and anti-nonalcoholic steatohepatitis activity of a (1,3) (1,6)-β-D-glucan from the Polyporus umbellatus (Pers.) Fries

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by inflammation and hepatic steatosis that may coincide with fibrotic activity. To date, no pharmacological agents have been approved for NASH treatment. Here, a homogeneous (1,3),(1,6)-β-D-glucan (PUP-W-1, Mw: 41.07 kDa) was successfully purified from Polyporus umbellatus (Pers.) Fries sclerotia and characterized. The analysis showed that the PUP-W-1 backbone consisted of a repeating chain of eight →3)-β-D-Glcp-(1 → units, with branched chains of four β-D-Glcp residues, joined by repeating 1,6-linkage units at the O-6 position of the backbone. The pharmacological effects of PUP-W-1 treatment in the context of NASH pathogenesis were explored using a methionine choline-deficient (MCD) diet-induced murine steatohepatitis model. The MCD model mice exhibited pronounced steatohepatitis, inflammatory activity, steatosis, stellate cell activation, and mild fibrotic activity. Treatment of the mice for three weeks with PUP-W-1 prevented the development of NASH due to the suppression of inflammation, lipid accumulation, and fibrosis. As suggested by these findings, PUP-W-1 may hold promise as a natural drug candidate or precursor for the treatment of NASH.

In Vivo Analysis of Necrosis and Ferroptosis in Nonalcoholic Steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease states such as chronic inflammation and fibrosis. In most liver diseases, immunological responses caused by tissue damages or viral infection contribute to the pathological advances, and various types of cell death have been reported to be implicated in their pathogenesis. However, the conventional detection of necrosis in vivo is not currently available, whereas the detection method for apoptosis has been relatively well-established. We recently reported a method for the in vivo detection of necrotic cells in liver disease models by an intravenous injection of Propidium Iodide (PI) into mice. We also provide standard methods for the evaluation of lipid accumulation and fibrosis characteristic of NASH. In addition, by utilizing these procedures and a murine model of steatohepatitis, we showed that ferroptosis, a type of regulated necrotic cell death, could be involved in the pathogenesis of NASH. These approaches allow us to explore the pathophysiological roles of cell death in liver diseases.

The A3 adenosine receptor agonist, namodenoson, ameliorates non‑alcoholic steatohepatitis in mice

The Wnt/β-catenin pathway confers a chain of molecular events in livers affected by non-alcoholic steato-hepatitis (NASH). Namodenoson, a selective agonist of the A3 adenosine receptor (A3AR), which is highly expressed in pathological liver cells, induces a robust anti-inflammatory effect in the liver, mediated via the de-regulation of the Wnt/β-catenin pathway. Namodenoson also acts as a liver protective agent by inhibiting ischemia/reperfusion injury. Based on these unique characteristics, we investigated the anti-NASH effect of Namodenoson in murine models of steato-hepatitis and in the LX2 human hepatic stellate cell line (HSC). In the STAM model, Namodenoson significantly decreased the non-alcoholic fatty liver disease (NAFLD) activity score, NAS, demonstrating anti-inflammatory and anti-steatotic effects. In the carbon tetrachloride (CCl4) model, Namodenoson reversed alanine aminotransferase (ALT) to normal values and signifi-cantly improved liver inflammation and fibrosis, as well as the adiponectin and leptin levels. Namodenoson de-regulated the Wnt/β-catenin pathway in the liver extracts of the CCl4 model mice and in the LX2 HSCs, manifested by a decrease in the expression of phosphoinositide 3-kinase (PI3K), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), β-catenin, lymphoid enhancer-binding factor 1 (Lef-1) and cyclin D1, and an increase in the expression level of glycogen synthase kinase 3β (GSK-3β). The fibrosis marker, α-smooth muscle actin (α-SMA) was also de-regulated, supporting the anti-fibrotic effect of Namodenoson. On the whole, the findings of this study demonstrate that Namodenoson exerts an anti-NASH effect mediated via the de-regulation of the PI3K/NF-κB/Wnt/β-catenin signaling pathway. Thus, targeting A3AR may prove to be a novel direction in the pharmacotherapy of NAFLD/NASH.

Gpbar1 agonism promotes a Pgc-1\u03b1-dependent browning of white adipose tissue and energy expenditure and reverses diet-induced steatohepatitis in mice

Gpbar1 is a bile acid activated receptor for secondary bile acids. Here we have investigated the mechanistic role of Gpbar1 in the regulation of adipose tissues functionality in a murine model of steatohepatitis (NASH). Feeding wild type and Gpbar1−/− mice with a high fat diet-fructose (HFD-F) lead to development of NASH-like features. Treating HFD-F mice with 6β-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective Gpbar1-ligand, reversed insulin resistance and histologic features of NASH, increased the weight of epWAT and BAT functionality and promoted energy expenditure and the browning of epWAT as assessed by measuring expression of Ucp1 and Pgc-1α. The beneficial effects of BAR501 were lost in Gpbar1−/− mice. In vitro, BAR501 promoted the browning of 3T3-L1 cells a pre-adipocyte cell line and recruitment of CREB to the promoter of Pgc-1α. In conclusion, Gpbar1 agonism ameliorates liver histology in a rodent model of NASH and promotes the browning of white adipose tissue.

A novel diet-induced murine model of steatohepatitis with fibrosis for screening and evaluation of drug candidates for nonalcoholic steatohepatitis.

Many animal models of nonalcoholic steatohepatitis have been reported. While these models exhibit mild onset of hepatitis and fibrosis, induction is often slow. For faster screening of drug candidates, there is a compelling need for convenient animal models of steatohepatitis and nonalcoholic steatohepatitis in which fatty liver and hepatitis are stably induced within a short period. Here, we analyzed the hepatic lipid composition in nonalcoholic steatohepatitis, and used this information to successfully establish a murine model where steatohepatitis is induced within only 1 week using a novel diet (steatohepatitis‐inducing high‐fat diet, STHD‐01) high in saturated fatty acids and cholesterol. After receiving STHD‐01 for 1 week, normal mice (C57BL/6J) showed elevated markers of fatty liver and hepatitis, including hepatic triglycerides and plasma alanine aminotransferase; the administration of angiotensin receptor blockers reduced these symptoms. Furthermore, we confirmed that STHD‐01 administration for 36 weeks induced not only sustained elevation of hepatic triglyceride and plasma alanine aminotransferase levels, but also fibrosis and tumor formation. Pretreatment with the carcinogen diethylnitrosamine accelerated tumor formation, and hepatic lesions were observed within 30 weeks of STHD‐01 feeding following diethylnitrosamine pretreatment. Finally, branched‐chain amino acids, known to reduce the risk for hepatocellular carcinoma in preclinical models, were effective in reducing the progression of liver fibrosis induced by STHD‐01 feeding after diethylnitrosamine pretreatment. We concluded that STHD‐01 administration successfully induces steatohepatitis within a short period of time. The proposed murine model is suitable for studying the long‐term effects of pharmaceutical agents targeting steatohepatitis, fibrosis, and tumor formation.

Abstract 601: Nicotinic Acid Prevents and Restores Steatohepatitis Together with Amelioration of Postprandial Dyslipidemia.

Background and aims: ICR mouse neonatally administered with monosodium glutamate (MSG) shows obesity, insulin resistance, and is a murine model of steatohepatitis. We analysed the effect of nicotinic acid (niacin), an antilipidemic drug and also a potent lipolysis inhibitor. Methods: Neonatal ICR mice were subcutaneously injected with 2 mg/g of MSG or saline at 5 consecutive days after birth and fed a normal chow. Niacin was administered by dietary supplementation (0.5% w/w) since 5 week-old. After 12 weeks, mice were sacrificed and tissue samples were collected. Niacin was also administered to 12 week-old mice already having steatosis for 12 weeks. Results: After 12 weeks of treatment of niacin, MSG-treated mice showed less weight of body (21.5%, p < 0.001) and liver (52.5%, p < 0.001). Histopathological analyses showed markedly prevention of steatohepatitis. NAFLD activity scores (NAS) of niacin and vehicle groups were 0.0 +/- 0.0, 5.2 +/- 0.4, respectively (p < 0.01). Niacin also dramatically ameliorated accomplished steatohepatitis. Niacin treatment ameliorated dyslipidemia; atherogenic LDL fraction was disappeared in fasting plasma, and elevations of postprandial NEFA and triglycerides were suppressed. Hepatic gene expression of lipogenic genes was not decreased, while expression of Fsp27 was slightly (25%, p < 0.05) decreased. On the other hand, niacin-treated group showed postprandial hyperglycemia. Although there were no evidence of insulin resistance by insulin tolerance test and hepatic Akt phosphorylation, postprandial insulin response was impaired by niacin treatment. Conclusion: Niacin prevented and ameliorated steatohepatitis together with amelioration of postprandial dyslipidemia at least partially due to inhibition of NEFA influx into the liver, although niacin impaired postprandial insulin response.

N‐3 polyunsaturated fatty acids worsen inflammation and fibrosis in experimental nonalcoholic steatohepatitis

n-3 polyunsaturated fatty acids (PUFA) ameliorate fatty liver in experimental models, but their effects on inflammation and fibrosis during steatohepatitis are either controversial or lacking. We compared the effects of supplementation with olive oil (OO) alone or OO and n-3 PUFA on the development and progression of experimental steatohepatitis. Balb/C mice (≥5 mice/group) were fed a methionine- and choline-deficient (MCD) diet or a control diet for 4 or 8weeks. At the same time, mice were supplemented with n-3 PUFA (eicosapentaenoic and docosahexahenoic acid, 25mg together with 75mg OO), or OO alone (100mg), two times a week by intragastric gavage. After 8weeks, mice on MCD/n-3 had higher ALT levels compared to MCD/OO and more severe scores of inflammation, including a significant increase in the number of lipogranulomas (26.4±8.4 vs. 5.1±5 per field, P<0.001). Intrahepatic expression of TNF-α and CCL2 was higher in MCD/n-3 mice at both time points. In addition, increased expression of the profibrogenic genes TIMP-1 and TGF-β, and more severe histological scores of fibrosis were evident in MCD/n-3 mice. After 8week of MCD diet, portal pressure was higher in mice receiving n-3 than in those on OO alone (5.1±1.4 vs. 7.0±0.9mmHg, P<0.05). Analysis of hepatic fatty acid profile showed that supplementation resulted in effective incorporation of n-3 PUFA. In a murine model of steatohepatitis, supplementation with n-3 PUFA and OO is associated with more severe necro-inflammation and fibrosis than in mice treated with OO only.

Hepatocyte caspase-8 is an essential modulator of steatohepatitis in rodents

In human and murine models of nonalcoholic steatohepatitis (NASH), increased hepatocyte apoptosis is a critical mechanism contributing to inflammation and fibrogenesis. Caspase 8 (Casp8) is essential for death-receptor-mediated apoptosis activity and therefore its modulation might be critical for the pathogenesis of NASH. The aim was to dissect the role of hepatocyte Casp8 in a murine model of steatohepatitis. We generated hepatocyte-specific Casp8 knockout (Casp8(Δhep) ) mice. Animals were fed with a methionine-choline-deficient (MCD) diet. Liver injury was assessed by histopathological analysis, apoptotic death, serum alanine aminotransferase (ALT), fluorescent-activated cell sorter (FACS), analysis of liver infiltration and inflammation, reactive oxygen species (ROS), and liver fibrosis. MCD feeding triggered steatosis, hepatic lipid storage, and accumulation of free fatty acid (FFA) in wildtype (WT) livers, which were significantly reduced in Casp8(Δhep) animals. Additionally, lack of Casp8 expression in hepatocytes reduced the MCD-dependent increase in apoptosis and decreased expression of proinflammatory cytokines as well as hepatic infiltration. As a consequence, ROS production was lower, leading to a reduction in the progression of liver fibrosis in Casp8(Δhep) livers. Selective ablation of Casp8 in hepatocytes ameliorates development of NASH by modulating liver injury. Casp8-directed therapy might be a plausible treatment for patients with steatohepatitis. (HEPATOLOGY 2013;57:2189-2201).

Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis

Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-β (transforming growth factor-β), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

OC-25 The genetic background differentially affects hepatic necro-inflammation in response to CCL2 in a murine model of steatohepatitis

OC-25 The genetic background differentially affects hepatic necro-inflammation in response to CCL2 in a murine model of steatohepatitis

P87 Vascular Adhesion Protein-1 promotes increases in liver infiltrating CD4+ T cells and NKT cells and induction of fibrogenesis in steatohepatitis

IntroductionVascular Adhesion Protein-1 (VAP-1) is an adhesion molecule and membrane-bound amine oxidase. Our group has previously demonstrated that VAP-1 is able to support lymphocyte recruitment across human hepatic sinusoidal endothelium...

Hepatocyte growth factor overexpression ameliorates liver inflammation and fibrosis in a mouse model of nonalcoholic steatohepatitis

Hepatocyte growth factor (HGF) is a potent growth factor involved in liver regeneration that has various effects on epithelial and nonepithelial cells. Although it has been demonstrated that HGF can reduce liver inflammation or fibrosis caused by pharmaceutical or chemical insult, no examination of its effect on liver injury in nonalcoholic steatohepatitis (NASH) has been reported. To examine the effect of HGF on liver injury in NASH, we generated a murine steatohepatitis model on an HGF overexpression transgenic (Tg) background, and fed the mice a methionine- and choline-deficient diet (MCD). In mice fed the MCD diet, serum ALT levels and the inflammation score for the Tg mice were significantly lower than those for the wild-type (Wt) control mice (P<0.01). The index of lipid peroxidation increased in the liver of the Wt mice as demonstrated by thiobarbituric acid-reactive substances. Furthermore, the liver fibrosis in Tg mice was dramatically suppressed in comparison to that in Wt mice. The gene expression of matrix metalloprotease-13 in the Tg mice was significantly increased in comparison to that of the Wt mice. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay showed the apoptotic cells to significantly decrease in number in the Tg mice in comparison to the Wt mice fed the MCD diet (P<0.01). Hepatocyte growth factor ameliorated liver inflammation and fibrosis in a murine model of NASH as a result of the anti-oxidative and anti-apoptotic effect, and the induction of fibrinolysis.

Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury

ObjectiveMonocyte chemoattractant protein-1 (MCP-1, CCL2), the primary ligand for chemokine receptor C–C chemokine receptor 2 (CCR2), is increased in livers of patients with non-alcoholic steatohepatitis (NASH) and murine models of...

Transcriptional Profile of Genes Involved in Oxidative Stress and Antioxidant Defense in a Dietary Murine Model of Steatohepatitis

Oxidative stress is a core abnormality responsible for disease progression in nonalcoholic steatohepatitis (NASH). However, the relevant pathways that contribute to oxidative damage in vivo remain poorly understood. Here we explore the gene-expression patterns related to oxidative stress, antioxidant defense, and reactive oxygen metabolism in an established dietary murine model of NASH. C57BL/6 mice were placed on either a methionine- and choline-deficient (MCD) or a control (CTL) diet for 6 weeks. Hepatic oxidative damage and the development of NASH were monitored by biochemical and histologic indices. Analysis of 84 oxidative stress-related genes was performed by real-time reverse transcription polymerase chain reaction (PCR) in the livers of the two groups of mice. Mice on the MCD diet showed increased ALT, histologic features of NASH, and oxidative liver damage with increases in 4-hydroxynonenal and 3-nitrotyrosine. Of the genes analyzed, the GPx family were most significantly upregulated, whereas SCD1 was most significantly downregulated. Other genes that were significantly upregulated included Fmo2 and peroxiredoxins, whereas genes downregulated included Catalase and Serpinb1b. Our data demonstrate that oxidative stress-related genes are differentially expressed in the livers of mice with diet-induced NASH. These findings have important implications for NASH pathogenesis and the development of novel therapeutic strategies for patients with this condition.

Monocyte chemoattractant protein-1 deficiency does not affect steatosis or inflammation in livers of mice fed a methionine–choline-deficient diet

Monocyte chemoattractant protein-1 (MCP-1, Ccl2) expression is increased in livers of patients with nonalcoholic steatohepatitis and in murine models of steatohepatitis. Several studies in rodents indicate that MCP-1 contributes to liver steatosis induced by feeding a high-fat diet. However, the extent of MCP-1 involvement in the widely utilized methionine–choline-deficient (MCD) diet model of steatohepatitis has not been determined. We tested the hypothesis that MCP-1 contributes to steatohepatitis in mice fed the MCD diet. MCP-1-deficient mice on a C57Bl/6J background and age-matched C57Bl/6J mice were fed either MCD diet or control diet for 4 weeks. MCP-1 deficiency did not affect steatohepatitis, as indicated by liver histopathology, nor did it affect serum alanine aminotransferase activity, hepatic triglyceride levels, hepatic inflammatory gene induction, or macrophage accumulation in mice fed the MCD diet. MCP-1 deficiency reduced the expression of the profibrogenic genes, pro-collagen 1a1, connective tissue growth factor, and transforming growth factor-β, in mice fed the MCD diet. MCP-1 deficiency significantly reduced collagen deposition and α-smooth muscle actin protein levels in the livers of mice fed the MCD diet. The results indicate that MCP-1 does not contribute to liver steatosis or inflammation in the MCD diet model of steatohepatitis. Rather, the data suggest that MCP-1 contributes to fibrosis in mice fed the MCD diet, independent of effects on steatosis and inflammation.

P46 Vascular adhesion protein-1 promotes inflammation and fibrogenesis in murine steatohepatitis

IntroductionVAP-1 is an adhesion molecule which promotes lymphocyte recruitment to the liver. It is released in a soluble form (sVAP-1) from adipose tissue and the hepatic vascular bed. sVAP-1 has...

Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model, and suppresses fibrogenic response induced by transforming growth factor‐β1 in a cultured stellate cell line

The aim of this study was to investigate the preventive actions of bezafibrate against non-alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model. Male KK-A(y)/TaJcl (KK-A(y)) mice were fed a methionine and choline-deficient (MCD) diet or a MCD diet containing bezafibrate or pioglitazone for 7 weeks, after which biochemical parameters, pathological changes, and hepatic mRNA levels were assessed. An in vitro HSC model was designed by using a previously described RI-T cell line stimulated by transforming growth factor-beta1 (TGF-beta1). MCD diet-fed KK-A(y) mice developed hepatic steatosis, oxidative stress, inflammation, and hepatic fibrosis. Bezafibrate markedly decreased the hepatic content of triglyceride accumulation of fatty droplets within hepatocytes, and increased the expression of hepatic fatty acid beta-oxidative genes in MCD diet-fed KK-A(y) mice. Bezafibrate markedly inhibited the increases in the plasma alanine aminotransferase level and hepatic content of thiobarbituric acid-reactive substances in this model. Moreover, it dramatically reduced hepatic inflammatory changes and fibrosis concomitantly with marked reductions in the mRNA levels for inflammatory cytokine, chemokine, and profibrogenic genes. Importantly, both bezafibrate and pioglitazone markedly reduced the mRNA levels of profibrogenic and fibrogenic genes in TGF-beta1-stimulated cells. Bezafibrate improved hepatic steatosis and potently prevented inflammation, oxidative stress, HSC activation, and fibrogenesis in the liver. Moreover, this study was the first to demonstrate that bezafibrate directly inhibits hepatic fibrogenic response induced by TGF-beta1 in vitro. Hence bezafibrate may be a new therapeutic strategy against NASH and hepatic fibrosis.

Intrahepatic insulin resistance in a murine model of steatohepatitis: effect of PPARγ agonist pioglitazone

Hepatic insulin resistance is associated with hepatic steatosis and is thought to play an important role in the pathogenesis of steatohepatitis. Using a murine model of steatohepatitis (mice fed a diet deficient in methionine and choline—MCD diet), we tested the effects of the insulin-sensitising, PPARγ agonist drug pioglitazone (PGZ) on systemic and intrahepatic insulin sensitivity and on liver pathology. Compared to controls, mice fed the MCD diet develop a significant steatohepatitis, have enhanced glucose tolerance and enhanced systemic response to insulin. PGZ did not affect the systemic insulin sensitivity in control or MCD-fed mice as assessed in vivo by intraperitoneal glucose or insulin dynamic tests. However, PGZ prevented hepatic fat accumulation and steatohepatitis induced by the MCD diet. This effect was associated with an increased mass of adipose tissue and increased expression and release of adiponectin, while hepatic acyl co-enzyme A oxidase and acyl-co-enzyme A carboxylase, regulating hepatic β-oxidation of fatty acid, remained unchanged. Steatohepatitis in MCD-diet-fed mice was associated with intrahepatic insulin resistance as shown by a reduced phosphorylation of hepatic insulin receptor, and Akt in response to an insulin stimulus. PGZ to MCD-fed mice restored the activation of the insulin receptor and of the Akt pathway in response to insulin. In conclusion, PGZ alleviates steatosis and steatohepatitis induced by the MCD diet, an effect associated with correction of intrahepatic insulin resistance.

Longitudinal analysis of murine steatohepatitis model induced by chronic exposure to high‐fat diet

Several lines of epidemiological evidence have suggested that non-alcoholic steatohepatitis (NASH) is closely associated with obesity in humans. However, the precise mechanisms of the progression of NASH and its key metabolic abnormalities remain to be elucidated. We found that long-term high-fat diet (HFD) exposure induces NASH, with excess body weight, hyperinsulinemia and hypercholesteremia in mice. Longitudinal analysis of the model showed that steatohepatitis was induced after onset of metabolic abnormalities. In addition, we found that expression of MCP-1 mRNA was induced in the liver before induction of TNFalpha and type I collagen alpha1 mRNAs, and prior to onset of steatohepatitis. We confirmed that hepatic MCP-1 contents were increased in mice fed HFD for 50 weeks, although the precise role of MCP-1 in the development of NASH remains to be addressed. The mouse model was also characterized by moderate reductions in catalase activity and glutathione content, as well as by overexpression of fatty acid synthase, acetyl-CoA carboxylase 1 and FAT/CD36 mRNAs in the liver. The murine NASH model apparently mimics clinical aspects of the condition and provides insight into NASH.