Methionine- And Choline-deficient Diet Research Articles

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH-related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild-type (WT) mice, Patched-deficient (Ptc(+/-) ) (overly active Hh signaling) mice, and OPN-deficient mice before and after feeding methionine and choline-deficient (MCD) diets to induce NASH-related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc(+/-) mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN-deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up-regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up-regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte-derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). OPN is Hh-regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH.

Treatment with Ω-3 fatty acids but not exendin-4 improves hepatic steatosis

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in the Western world. The aim of this study was to evaluate the biochemical and histological effects of omega-3 fatty acid and exendin-4 treatment on NAFLD in an animal model. Sixty-three 8-week-old outbred Sprague-Dawley male rats were used for this study. Three animals were used as procedure controls, and 30 rats were fed a methionine and choline deficient (MCD) diet and 30 were fed a regular chow diet. In each group of 30 animals, 10 served as controls, 10 received exendin-4, and 10 received omega-3 fatty acids. After 75 days of treatment, the animals were euthanized, the tissues and serum were harvested, and the livers were formalin-fixed for histology. The MCD diet was exceptionally efficient at producing fatty livers. The MCD control animals had a liver steatosis score of 38+/-6.7 (of 50 possible); treatment with exendin-4 was not associated with a significant reduction of steatosis (44+/-5.16, P=0.07) and the omega-3 fatty acid treatment was associated with a significant decrease in the liver steatosis score (15.6+/-13.46, P<0.001) compared with both the controls and the exendin-4 groups. The omega-3 fatty acid treatment increased serum aspartate aminotransferase significantly, whereas exendin-4 had no effect. In an animal model of NAFLD, the omega-3 fatty acid therapy was associated with significant improvement in hepatic steatosis compared with exendin-4. These data suggest that omega-3 fatty acid supplements may have a potential therapeutic role in patients with NAFLD.

The expression of chemerin and chemerin receptor in rats with methionine-and choline-deficient diet induced nonalcoholic fatty liver disease

Objective To explore the expression of chemerin and chemerin receptor ( chemokine-like receptor 1, CMKLR1) during different periods of non-alcoholic fatty liver disease ( NAFLD) rat model induced by methionine- and choline-deficient ( MCD) diet. Methods Thirty-six Wistar rats were divided into control group and MCD group in random. After one week quarantine and acclimation period, these two groups were fed either normal chow or MCD diet. The animals were respectively sacrificed at the first week, the forth week, and the tenth week. The levels of alanine transaminase (ALT), blood lipid profile, liver function, and the content of triglyceride in liver were detected. HE staining was done to observe the morphologic change of liver. The mRNA expression changes of chemerin and CMKLR1 in liver were measured using real-time PCR, and the change in chemerin mRNA level was further confirmed in liver by Northern blot. Finally, the concentration of chemerin in serum was measured by Western blot. Results The mRNA level of chemerin decreased significantly after four and ten weeks MCD feeding, although no obvious changes were found at first week, similar changes were found in serum chemerin (1.00±0.11 vs 0.96±0.39; 1.00±0.12 vs 0.21 ±0.77; 1.00±0.42 vs 0.21 ±0. 11). Contrasting with the change of chemerin(1.00±0.08 vs 0.72±0.10;1.00±0.24 vs 0.63±0. 31 ;1.00±0.05 vs 0.50±0.13), the mRNA level of CMKLR1 increased after MCD feeding( 1.00±0. 14 vs 0. 84±0. 26; 1.00±0. 38 vs 1. 51 ±0. 33; 1. 01 ±0. 13 vs 1. 84 ± 0. 39 ). Conclusion The change of chemerin and its receptor may participate in the process of the nonalcoholic fatty liver disease. Key words: Methionine- and choline-deficient diet; Chemerin; CMKLR1; Nonalcoholic fatty liver disease

Anti-apoptotic and anti-inflammatory effects of Silybum marianum in treatment of experimental steatohepatitis

In this study, we were aimed to evaluate the probable effect of the crud extract of Silybum marianum, with high polyphenolic content, on experimental nonalcoholic steatohepatitis (NASH). To induce NASH, a methionine and choline deficient (MCD) diet was given to N-Mary rats for 8 weeks. After NASH development, MCD-fed rats were divided into two groups: MCD groups received MCD diet and MCD + S group was fed MCD diet plus crude extract of S. marianum orally for 3 weeks. Control group was fed a normal diet for 11 weeks. Finally, all rats were sacrificed. Plasma alanine amino transferase (ALT) and aspartate amino transferase (AST) levels were evaluated. In addition, the following hepatic factors were also evaluated: liver histology, malondialdehyde (MDA) and reduced glutathione (GSH) contents, gene expressions of TNF-α and TGF-β and immunoblot evaluations of caspase-3, ERK/p-ERK, JNK/pJNK and p38/pp38. Histopathological evaluations of the liver samples revealed that treatment with the S. marianum extract has abated the severity of NASH among the MCD-fed rats. Also, a significant reduction was observed in the sera ALT and AST activities. In addition, the extract caused dramatic reduction in the elevated hepatic TNF-α and TGF-β mRNA and MDA levels along with an increase in the GSH content. Moreover, the plant extract treatments significantly lowered activation of procaspase-3 to active caspase-3 and also lowered the phosphorylated form of JNK among the same group of rats. These results suggest that the S. marianum crude extract beneficial effects on NASH are mainly due to its antioxidant and anti-inflammatory activities.

Chymase inhibitor prevents the nonalcoholic steatohepatitis in hamsters fed a methionine‐ and choline‐deficient diet

Mast cells may be involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). The mast cell protease chymase contributes to the formation of angiotensin II and matrix metalloproteinase (MMP)-9, both of which are intimately involved in liver fibrosis. Therefore, we hypothesized that chymase plays an important role in the development of NASH. Hamsters were fed a methionine- and choline-deficient (MCD) diet for 8 weeks. These animals were divided into two groups and received either TY-51469 (1 mg/kg per day) or placebo. A third group was fed a normal diet as a control. Total plasma bilirubin, triglycerides, and hyaluronic acid levels were significantly higher in the MCD diet-fed hamsters than in the normal diet-fed hamsters, but the levels were significantly lower in chymase inhibitor-treated MCD diet-fed hamsters than in placebo-treated MCD diet-fed hamsters. Using histological analysis, marked steatosis and fibrosis were observed in MCD diet-fed hamsters, but these changes were significantly attenuated by treatment with the chymase inhibitor. Increases in mast cells and chymase-positive cells were observed in the liver after the MCD diet, but the increases disappeared in the chymase inhibitor-treated group. The significant increase observed in chymase activity in liver tissue extract from the MCD diet-fed group was also reduced by treatment with the chymase inhibitor. Chymase inhibition significantly reduced not only angiotensin II expression but also matrix metallopeptidase 9 activity in MCD diet-fed hamsters. These findings demonstrate that the mast cell protease chymase may play a crucial role in the development of NASH in hamsters.

Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionine- and choline-deficient diet

Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2–Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gstα1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities.

Liver-Specific β-Catenin Knockout Mice Exhibit Defective Bile Acid and Cholesterol Homeostasis and Increased Susceptibility to Diet-Induced Steatohepatitis

Although the role of Wnt/beta-catenin signaling in liver growth and development is well established, its contribution in non-neoplastic hepatic pathologies has not been investigated. Here, we examine the role of beta-catenin in a murine model of diet-induced liver injury. Mice with hepatocyte-specific beta-catenin deletion (KO) and littermate controls were fed the steatogenic methionine and choline-deficient (MCD) diet or the corresponding control diet for 2 weeks and characterized for histological, biochemical, and molecular changes. KO mice developed significantly higher steatohepatitis and fibrosis on the MCD diet compared with wild-type mice. Both wild-type and KO livers accumulated triglyceride on the MCD diet but, unexpectedly, higher hepatic cholesterol levels were observed in KO livers on both control and MCD diets. Gene expression analysis showed that hepatic cholesterol accumulation in KO livers was not attributable to increased synthesis or uptake. KO mice had lower expression of bile acid synthetic enzymes but exhibited higher hepatic bile acid and serum bilirubin levels, suggesting defects in bile export. Therefore, loss of beta-catenin in the liver leads to defective cholesterol and bile acid metabolism in the liver and increased susceptibility to developing steatohepatitis in the face of metabolic stress.

A Silybin-Phospholipid Complex Prevents Mitochondrial Dysfunction in a Rodent Model of Nonalcoholic Steatohepatitis

Mitochondrial dysfunction and oxidative stress are determinant events in the pathogenesis of nonalcoholic steatohepatitis. Silybin has shown antioxidant, anti-inflammatory, and antifibrotic effects in chronic liver disease. We aimed to study the effect of the silybin-phospholipid complex (SILIPHOS) on liver redox balance and mitochondrial function in a dietary model of nonalcoholic steatohepatitis. To accomplish this, glutathione oxidation, mitochondrial oxygen uptake, proton leak, ATP homeostasis, and H(2)O(2) production rate were evaluated in isolated liver mitochondria from rats fed a methionine- and choline-deficient (MCD) diet and the MCD diet plus SILIPHOS for 7 and 14 weeks. Oxidative proteins, hydroxynonenal (HNE)- and malondialdehyde (MDA)-protein adducts, and mitochondrial membrane lipid composition were also measured. Treatment with SILIPHOS limited glutathione depletion and mitochondrial H(2)O(2) production. Moreover, SILIPHOS preserved mitochondrial bioenergetics and prevented mitochondrial proton leak and ATP reduction. Finally, SILIPHOS limited the formation of HNE- and MDA-protein adducts. In conclusion, SILIPHOS is effective in preventing severe oxidative stress and preserving hepatic mitochondrial bioenergetics in nonalcoholic steatohepatitis induced by the MCD diet. The modifications of mitochondrial membrane fatty acid composition induced by the MCD diet are partially prevented by SILIPHOS, conferring anti-inflammatory and antifibrotic effects. The increased vulnerability of lipid membranes to oxidative damage is limited by SILIPHOS through preserved mitochondrial function.

IL-6 Deficiency Attenuates Murine Diet-Induced Non-Alcoholic Steatohepatitis

BackgroundThe role of inflammation in the pathogenesis of non-alcoholic steatohepatitis (NASH), a common cause of liver disease, is still poorly understood. This study aimed at assessing the involvement of a major inflammatory cytokine, IL-6, in NASH.Materials and MethodsSteatohepatitis was induced by feeding wild-type or IL-6−/− mice for 5 weeks with a methionine and choline-deficient (MCD) diet.ResultsWhereas MCD diet-induced weight loss and decreases in serum glucose, cholesterol and triglyceride levels were similar in both genotypes, serum alanine aminotransferase was less elevated in IL-6−/− mice than in wild-type animals. Despite having a comparable liver steatosis score, IL-6-deficient mice exhibited less lobular inflammation than their wild-type littermates. Liver gene expression of TGF-β and MCP-1 was also strongly attenuated in mutant mice; a more modest reduction was observed for PPAR-γ and F4/80 transcripts as well as proteins. Chromatographic analysis of liver lipids demonstrated that MCD diet induced in normal and mutant mice a similar decrease in the ratio of phosphatidylcholine to phosphatidylethanolamine. However, the diet-induced increase in the levels of sphingomyelin and ceramide was less important in IL-6−/− mice.ConclusionAltogether, these results indicate that IL-6 deficiency does not block the development of NASH; yet, IL-6 plays a critical role in the accompanying liver inflammation.

Deletion of nuclear factor-E2-related factor-2 leads to rapid onset and progression of nutritional steatohepatitis in mice

Oxidative stress is a critical mediator in liver injury of steatohepatitis. The transcription factor Nrf2 serves as a cellular stress sensor and is a key regulator for induction of hepatic detoxification and antioxidative stress systems. The involvement of Nrf2 in defense against the development of steatohepatitis remains unknown. We aimed to investigate the protective roles of Nrf2 in nutritional steatohepatitis using wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. WT and Nrf2-null mice were fed a methionine- and choline-deficient (MCD) diet for 3 and 6 wk, and the liver tissues were analyzed for pathology and for expression levels of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. In WT mice fed an MCD diet, Nrf2 was potently activated in the livers, and steatohepatitis did not develop over the observation periods. However, in Nrf2-null mice fed an MCD diet, the pathological state of the steatohepatitis was aggravated in terms of fatty changes, inflammation, fibrosis, and iron accumulation. In the livers of the Nrf2-null mice, oxidative stress was significantly increased compared with that of WT mice based on the increased levels of 4-hydroxy-2-nonenal and malondialdehyde. This change was associated with the decreased levels of glutathione, detoxifying enzymes, catalase, and superoxide dismutase activity. Correlating well with the liver pathology, the mRNA levels of factors involved in fatty acid metabolism, inflammatory cytokines, and fibrogenesis-related genes were significantly increased in the livers of the Nrf2-null mice. These findings demonstrate that Nrf2 deletion in mice leads to rapid onset and progression of nutritional steatohepatitis induced by an MCD diet. Activation of Nrf2 could be a promising target toward developing new options for prevention and treatment of steatohepatitis.

Loss of Nrf2 markedly exacerbates nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) arises from nonalcoholic fatty liver disease (NAFLD) as a consequence of oxidative stress. Herein we report that the development of NASH is greatly accelerated in mice lacking transcription factor Nrf2 when they are challenged with a methionine- and choline-deficient (MCD) diet. After 14 days of feeding on an MCD diet, livers from Nrf2 −/− mice showed a substantial increase in macro- and microvesicular steatosis and a massive increase in the number of neutrophil polymorphs, compared to livers from wild-type mice treated similarly. Livers of Nrf2 −/− mice on the MCD diet suffered more oxidative stress than their wild-type counterparts as assessed by a significant depletion of reduced glutathione that was coupled with increases in oxidized glutathione and malondialdehyde. Furthermore, livers from Nrf2 −/− mice on the MCD diet suffered heightened inflammation as judged by an ∼10-fold increase in the amount of nuclear NF-κB p65 protein and ∼5-fold increases in the levels of mRNA for interleukin-1β, tumor necrosis factor α, cyclooxygenase 2, and inducible nitric oxide synthase compared with livers from similarly treated wild-type mice. Thus, impairment of Nrf2 activity may represent a major risk factor for the evolution of NAFLD to NASH.

Homocysteine Supplementation Attenuates the Unfolded Protein Response in a Murine Nutritional Model of Steatohepatitis

Hyperhomocysteinemia has been correlated with hepatic steatosis and activation of the unfolded protein response (UPR), yet a causal relationship has not been established. Although methionine and choline are essential components of homocysteine metabolism, the role of homocysteine in the pathogenesis of a methionine- and choline-deficient (MCD) diet remains unknown. We explored the effects of homocysteine supplementation on hepatic steatosis and the UPR in mice fed a control or MCD diet. Mice fed the MCD diet developed severe hyperhomocysteinemia and activation of the hepatic UPR. Supplementing the MCD diet with homocysteine attenuated the MCD diet-induced hepatic UPR activation and other injurious effects of the MCD diet including hepatic cholesterol accumulation, weight loss, and plasma ALT elevation. Homocysteine supplementation replenished the MCD diet-induced depletion of hepatic S-adenosylmethionine (SAM). Depleting SAM in HepG2 cells using MAT1alpha siRNA or cycloleucine resulted in enhanced activation of the UPR upon exposure to thapsigargin. Mice fed a control diet supplemented with homocysteine had a 3-fold elevation in plasma homocysteine level by 2 weeks and 6-fold elevation by 6 weeks but demonstrated no other pathophysiologic change. In summary, we found that homocysteine attenuates MCD diet-induced hepatic UPR activation, likely via repletion of hepatic SAM. Furthermore, homocysteine supplementation alone does not cause hepatic steatosis or UPR activation despite inducing hyperhomocysteinemia. These studies indicate that although hyperhomocysteinemia is often associated with hepatic steatosis and UPR activation, these effects may be a secondary response rather than a direct effect of homocysteine.

Highly Purified Eicosapentaenoic Acid Ethyl Ester Prevents Development of Steatosis and Hepatic Fibrosis in Rats

Pathogenesis of nonalcoholic steatohepatitis (NASH) is considered to be involved in fat accumulation, oxidative stress, inflammation, and fibrosis in liver, but no drug therapy has been established as yet. Eicosapentaenoic acid (EPA) is an agent used clinically to treat hypertriglyceridemia, and has been reported to suppress reactive oxygen species and inflammation. Here, we aimed to assess the effect of EPA on progression of hepatic fibrosis in an animal model of NASH. Wistar rats were fed a methionine- and choline-deficient (MCD) diet and given EPA ethyl ester (EPA-E) (1,000 mg/kg/day) or vehicle by gavage for 8 or 20 weeks. The MCD diet caused development of hepatic fibrosis and nodule formation at 20 weeks. EPA-E treatment significantly suppressed MCD-induced increase in fibrosis and hepatic hydroxyproline, and inhibited nodule formation. EPA-E treatment also decreased hepatic transforming growth factor (TGF)-beta1, and messenger RNA (mRNA) levels of connective tissue growth factor. EPA-E suppressed MCD-induced elevation of serum levels of ferritin, 8-isoprostane, soluble tumor necrosis factor receptor 1 (sTNFR1), and sTNFR2 at 20 weeks, and hepatic triglyceride accumulation at 8 weeks. EPA-E prevents progression of hepatic fibrosis in an MCD-induced NASH model with reduction of oxidative stress, inflammation, and initial hepatic steatosis. Thus, EPA-E treatment may be a potential therapy to treat NASH.

Heme Oxygenase-1 Protects Against Steatohepatitis in Both Cultured Hepatocytes and Mice

Heme oxygenase-1 (HO-1), an antioxidant defense enzyme, has been shown to protect against oxidant-induced tissue injury. We investigated the role of HO-1 in nutritional steatohepatitis in vitro and in vivo. AML-12 hepatocytes were cultured in methionine- and choline-deficient (MCD) medium. Cells were transfected with an adenovirus vector that expressed HO-1 (Ad-HO-1) or incubated with the HO-1 inducer hemin or the HO-1 inhibitor stannic mesoporphyrin for 24 hours. C57BL6 mice and db/db mice were fed MCD or control diets, with or without hemin, for up to 4 weeks. AML-12 cells exposed to MCD medium developed significant steatosis, increased release of alanine aminotransferase, and showed signs of oxidative injury. Incubation with hemin induced HO-1 protein, suppressed steatosis, and reduced levels of alanine aminotransferase and lipid peroxidation. A comparable effect was observed in cells transfected with Ad-HO-1, whereas incubation of these cells with stannic mesoporphyrin completely abolished the Ad-HO-1- or hemin-mediated protection of hepatocytes. Mice injected with hemin significantly attenuated MCD-induced steatohepatitis and increased HO-1 protein and activity. This effect was associated with up-regulation of antioxidant chaperones and enzymes, down-regulation of proinflammatory cytokines, and up-regulation of the anti-inflammatory interleukin-22. Moreover, the reduction in steatosis caused by hemin was affected by up-regulation of peroxisome proliferator-activated receptor-alpha and by down-regulation of sterol regulatory element binding protein-1c. HO-1 can interrupt progression of nutritional steatohepatitis by inducing an antioxidant pathway, suppressing production of cytokines, and modifying fatty acid turnover. Induction of HO-1 might provide a new approach for treatment of steatohepatitis.

Pro-oxidant-mediated hepatic fibrosis and effects of antioxidant intervention in murine dietary steatohepatitis

The mechanistic significance of oxidative stress to fibrogenesis in the methionine and choline-deficient (MCD) diet-induced model of steatohepatitis was evaluated by antioxidant intervention, using either vitamin E or L-2-oxothiazolidine-4-carboxylate (OTC), a cysteine precursor that promotes glutathione synthesis. Significant depletion of hepatic reduced glutathione (GSH) and elevation of thiobarbituric acid reactive substances (TBARS) occurred from week 3 in association with hepatic injury in mice fed the MCD diet. Hepatic stellate cell (HSC) activation and increased collagen alpha1(I) mRNA expression, together with morphologic fibrosis were evident from week 5. Vitamin E repleted GSH, reduced TBARS, steatosis, inflammation, HSC activation and collagen alpha1(I) mRNA expression, and ameliorated fibrosis. Vitamin E did not effect the expression of either profibrogenic cytokines (transforming growth factor-beta 1, connective tissue growth factor) or matrix remodeling enzymes (tissue inhibitor of metalloproteinase-1 and -2, matrix metalloproteinase-2 and -13). Despite repletion of hepatic GSH in OTC-supplemented mice, the initial benefit in the reduction of hepatic TBARS and inhibition of collagen alpha1(I) mRNA expression at week 5, failed to protect these mice from hepatic injury or fibrosis at later time points. Oxidative stress or products of lipid peroxidation mediate HSC activation and collagen gene expression directly in the MCD model of steatohepatitis. Vitamin E but not glutathione augmentation can interrupt this pathogenic process.

Farnesoid X receptor agonist WAY-362450 attenuates liver inflammation and fibrosis in murine model of non-alcoholic steatohepatitis

The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily, which plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. Here we investigated whether WAY-362450, a synthetic potent FXR agonist, could protect against non-alcoholic steatohepatitis (NASH) in mice fed a methionine and choline-deficient (MCD) diet. Male C57BL/6 mice on the MCD diet were treated with or without WAY-362450 (30 mg/kg) for 4 weeks. The elevations of serum ALT and AST activities induced by the MCD diet were decreased with WAY-362450 treatment. In terms of liver histology, while WAY-362450 treatment showed no impact on hepatic triglyceride accumulation, it significantly reduced inflammatory cell infiltration and hepatic fibrosis. The reduction in inflammatory cell infiltration correlated with deceased serum levels of keratinocyte derived chemokine (mKC) and MCP 1 and decreased hepatic gene expression of MCP-1 and VCAM-1. The reduction of hepatic fibrosis by WAY-362450 treatment corresponded to a reduction in hepatic gene expression of fibrosis markers. The positive effects of WAY-362450 were FXR-dependent since no protection was observed in MCD diet-fed FXR deficient mice. These findings demonstrate that FXR agonists may be useful for the treatment of non-alcoholic steatohepatitis.

Melatonin ameliorates methionine‐ and choline‐deficient diet‐induced nonalcoholic steatohepatitis in rats

Nonalcoholic steatohepatitis (NASH) may progress to advanced fibrosis and cirrhosis. Mainly, oxidative stress and excessive hepatocyte apoptosis are implicated in the pathogenesis of progressive NASH. Melatonin is not only a powerful antioxidant but also an anti-inflammatory and anti-apoptotic agent. We aimed to evaluate the effects of melatonin on methionine- and choline-deficient diet (MCDD)-induced NASH in rats. Thirty-two male Wistar rats were divided into four groups. Two groups were fed with MCDD while the other two groups were fed a control diet, pair-fed. One of the MCDD groups and one of the control diet groups were administered melatonin 50 mg/kg/day intraperitoneally, and the controls were given a vehicle. After 1 month the liver tissue oxidative stress markers, proinflammatory cytokines and hepatocyte apoptosis were studied by commercially available kits. For grading and staging histological lesions, Brunt et al.'s system was used. Melatonin decreased oxidative stress, proinflammatory cytokines and hepatocyte apoptosis. The drug ameliorated the grade of NASH. The present study suggests that melatonin functions as a potent antioxidant, anti-inflammatory and antiapoptotic agent in NASH and may be a therapeutic option.

The role of cyclooxygenase‐2 in the development of steatohepatitis

This study was designed to investigate the role of cyclooxygenase‐2 (COX‐2) in the pathogenesis of steatohepatitis by using COX‐2 transgenic and knockout mice fed methionine‐and choline‐deficient (MCD) diet. We generated transgenic (Tg) mice with targeted expression of COX‐2 in the liver by using the albumin promoter‐enhancer driven vector. The COX‐2 transgenic (Tg), COX‐2 knockout (KO) and wild type mice were fed with MCD diet or normal diet for 3 weeks. Liver injury was assessed by histopathological examination and measurement of serum ALT and AST levels. Liver tissues were obtained for Western blot analysis. Hepatic triglycerides and lipid peroxide levels were determined. Our data showed that with the MCD diet, the COX‐2 Tg mice exhibited significantly elevated serum ALT and AST, higher levels of hepatic triglycerides and lipid peroxide, and more prominent hepatic steatosis and inflammation when compared to wild type mice (p&lt;0.01). The COX‐2 KO mice showed significantly less liver injury than the COX‐2 Tg or wild type mice when these animals were placed on MCD diet. Western blot analysis revealed that transgenic expression of COX‐2 increases the accumulation of PPAR (peroxisome proliferator‐activated receptor)‐γ and cannabinoid CB1 receptor in the liver tissue. These findings suggest that hepatocyte COX‐2 facilitates the development of steatohepatitis and this effect may involve PPARγ and CB1.

Polaprezinc attenuates liver fibrosis in a mouse model of non‐alcoholic steatohepatitis

The effect of polaprezinc, a zinc-carnosine chelate compound, on the development of non-alcoholic steatohepatitis (NASH) was investigated in dietary methionine and choline deficient (MCD) mice. Mice were fed the MCD diet with or without polaprezinc (2.2 g/kg diet) for 10 weeks. Liver histopathology, triglyceride and lipid peroxide levels, and the expression of genes linked to fibrosis were then assessed. MCD mice developed steatohepatitis accompanied by mild fibrosis with an increase in lipid peroxidation, hepatic stellate cell (HSC) activation, and the augmented mRNA expression of tumor necrosis factor-alpha, transforming growth factor-beta1 and procollagen alpha1(I). The mRNA expression levels of matrix metalloproteinase (MMP)-2 and tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also enhanced. Histopathologically, polaprezinc supplementation did not influence the development of steatosis but it apparently attenuated fibrosis. Polaprezinc slightly reduced lipid peroxidation and suppressed HSC activation as well as the mRNA expression of pro-inflammatory cytokines. Polaprezinc affected the MCD diet-enhanced expression of TIMP-1 even when administered relatively late. These results suggest that polaprezinc attenuates fibrosis in NASH by reducing inflammation and lipid peroxidation and, during a later phase, promoting fibrolysis via the inhibition of TIMP expression in the liver. Further investigation is required to clarify the clinical efficacy of polaprezinc in patients with NASH.

Hepatic effects of a methionine–choline-deficient diet in hepatocyte RXRα-null mice

Retinoid X receptor-α (RXRα) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXRα deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXRα-null (H-RXRα-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid β-oxidation were not altered in WT mice, but were decreased in the MCD diet-fed H-RXRα-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXRα-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXRα-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXRα-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXRα-null mice. In conclusion, these data suggest a critical role for RXRα in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.