Methionine-choline Deficient Diet Diet Research Articles

Fermented field water-dropwort (Oenanthe javanica) alleviates diet-induced non-alcoholic steatohepatitis

ABSTRACT Naturally fermented filed water-dropwort (Oenanthe javanica) extracts by steeping with oligosaccharides (FDE) show a hepatoprotective activity against the liver injury induced by carbon tetrachloride (CCl4) and ethanol. However, the role of FDE in non-alcoholic steatohepatitis (NASH) has not yet been reported. In the present study, we investigated the effect of FDE on NASH using a mouse model with a methionine/choline-deficient diet (MCD). C57BL/6 male mice (9 weeks old) were fed on an MCD diet for 6 weeks with parallel water or FDE orally administration each day. FDE administered mice showed decreasing MCD diet-induced triglyceride (TG) levels, oxidative stress, infiltrating macrophages and elevating inflammatory cytokines in the liver. Our results suggest that FDE suppressed MCD diet-induced liver injury by inhibiting TG synthesis, the blocking of oxidative stress and hepatic inflammation, highlighting FDE as a potential therapeutic agent for the prevention and treatment of NASH.

Mitochondrial Peroxiredoxin III Protects against Non-Alcoholic Fatty Liver Disease Caused by a Methionine-Choline Deficient Diet.

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common chronic liver disease worldwide. In addition, NAFLD may increase the risk of cardiovascular and liver-related diseases, and displays features of metabolic syndrome. In NAFLD, oxidative stress is primarily caused by excessive free fatty acids. The oxidation of fatty acids is usually caused by β-oxidation of mitochondria under normal conditions, resulting in the production of energy. However, when the inflow of fatty acids in NAFLD becomes excessive, the β-oxidation of mitochondria becomes saturated and the oxidation process increases at sites including peroxisomes and microsomes, thereby increasing production of reactive oxygen species (ROS). Thus, hepatic mitochondrial ROS play an important role in the pathogenesis of NAFLD. Eliminating mitochondrial ROS may improve NAFLD, but the underlying mechanism remains unclear. We examined the effect of mitochondrial ROS on NAFLD by focusing on peroxiredoxin (Prx), an antioxidant protein that can remove hydrogen peroxide. The protective effect and pathological phenomenon of mitochondrial peroxiredoxin in methionine-choline deficient diet (MCD)-induced liver injury was assessed in a mouse model of NAFLD. In these mice, mitochondrial peroxiredoxin deficiency significantly increased hepatic steatosis and fibrosis. In addition, ablation of Prx III enhances susceptibility to MCD diet-induced oxidative stress and exacerbates NAFLD progression by promoting inflammation. The binding assay results also showed that Prx III-deficient mice had more severe liver damage than Prx III-abundant mice in MCD diet liver injury models. The present data suggest that mitochondrial peroxiredoxin III could be a therapeutic target for preventing and suppressing diet-induced NAFLD.

Attenuating effects of allantoin on oxidative stress in a mouse model of nonalcoholic steatohepatitis: role of SIRT1/Nrf2 pathway.

Background and purpose:Nonalcoholic steatohepatitis (NASH) is considered a common and serious liver disease, which develops into cirrhosis, fibrosis, and even hepatocellular carcinoma. Oxidative stress is identified as an important factor in the induction and promotion of NASH. Allantoin is a natural and safe compound and has notable effects on lipid metabolism, inflammation, and oxidative stress. Therefore, this study was aimed to assess the role of allantoin on the oxidative stress and SIRT1/Nrf2 pathway in a mouse model of NASH.Experimental approach:C57/BL6 male mice received saline and allantoin (saline as the control and allantoin as the positive control groups). NASH was induced by a methionine-choline deficient diet (MCD). In the NASH-allantoin (NASH-Alla) group, allantoin was injected for 4 weeks in the mice feeding on an MCD diet. Afterward, histopathological, serum, oxidative stress, and western blot evaluations were performed.Findings/Results:We found NASH provided hepatic lipid accumulation and inflammation. Superoxide dismutase (SOD) and glutathione (GSH) levels decreased, lipid peroxidation increased, and the expression of SIRT1 and Nrf2 downregulated. However, allantoin-treatment decreased serum cholesterol, ALT, and AST. Liver steatosis and inflammation were improved. Protein expression of SIRT1 and Nrf2 were upregulated and SOD, CAT, and GSH levels increased and lipid peroxidation decreased.Conclusion and implications:It seems that the antioxidant effects of allantoin might have resulted from the activation of SIRT1/Nrf2 pathway and increase of cellular antioxidant power.

A Mineralocorticoid Receptor Deficiency in Myeloid Cells Reduces Liver Steatosis by Impairing Activation of CD8+ T Cells in a Nonalcoholic Steatohepatitis Mouse Model.

Background and AimsThe mineralocorticoid receptor (MR) and renin-angiotensin-aldosterone system (RAAS) are implicated in non-alcoholic liver fatty disease (NALFD). However, inflammatory mechanisms linking MR and RAAS with disease pathology remain unclear. Here we aimed to evaluate the contribution of myeloid MR to the inflammatory response in an animal model of non-alcoholic steatohepatitis (NASH), induced with a methionine-choline deficient diet (MCD).MethodsMice with a conditional deficiency of MR in myeloid cells (MyMRKO) and their counterpart floxed control mice (FC) were fed for 18 days with MCD or chow diet, respectively. Serum levels of aminotransferases and aldosterone levels were measured and hepatic steatosis, inflammation and fibrosis scored histologically. Hepatic triglyceride content (HTC) and hepatic mRNA levels of pro-inflammatory pro-fibrotic-associated genes were also assessed. Deep flow cytometric analysis was used to dissect the immune response during NASH development.ResultsMyMRKO mice fed with an MCD diet exhibited reduced hepatic inflammation and lower HTC than controls. Absolute number and percentage of liver inflammatory infiltrate cells (except for CD8+ T lymphocytes) were similar in both MyMRKO and control mice fed with an MCD diet but expression of the costimulatory molecule CD86 by dendritic cells and the CD25 activation marker in CD8+ T cells were significantly reduced in MyMRKO.ConclusionsProinflammatory cells are functionally suppressed in the absence of MR. We hypothesized that loss of MR in myeloid cells reduces lipid accumulation in the liver, in part through modulating the adaptive immune response, which is pivotal for the development of steatosis.

IPla2β Deficiency Suppresses Hepatic ER UPR, Fxr, and Phospholipids in Mice Fed with MCD Diet, Resulting in Exacerbated Hepatic Bile Acids and Biliary Cell Proliferation.

Background: Group VIA calcium-independent phospholipase A2 (iPla2β) regulates homeostasis and remodeling of phospholipids (PL). We previously showed that iPla2β−/− mice fed with a methionine-choline-deficient diet (MCD) exhibited exaggerated liver fibrosis. As iPla2β is located in the endoplasmic reticulum (ER), we investigated the mechanisms for this by focusing on hepatic ER unfolded protein response (UPR), ER PL, and enterohepatic bile acids (BA). Methods: Female WT (wild-type) and iPla2β−/− mice were fed with chow or MCD for 5 weeks. PL and BA profiles were measured by liquid chromatography-mass spectrometry. Gene expression analyses were performed. Results: MCD feeding of WT mice caused a decrease of ER PL subclasses, which were further decreased by iPla2β deficiency. This deficiency alone or combined with MCD downregulated the expression of liver ER UPR proteins and farnesoid X-activated receptor. The downregulation under MCD was concomitant with an elevation of BA in the liver and peripheral blood and an increase of biliary epithelial cell proliferation measured by cytokeratin 19. Conclusion: iPla2β deficiency combined with MCD severely disturbed ER PL composition and caused inactivation of UPR, leading to downregulated Fxr, exacerbated BA, and ductular proliferation. Our study provides insights into iPla2β inactivation for injury susceptibility under normal conditions and liver fibrosis and cholangiopathies during MCD feeding.

Allantoin improves methionine-choline deficient diet-induced nonalcoholic steatohepatitis in mice through involvement in endoplasmic reticulum stress and hepatocytes apoptosis-related genes expressions.

Objective(s):Non-alcoholic steatohepatitis (NASH) is defined by steatosis and inflammation in the hepatocytes, which can progress to cirrhosis and possibly hepatocellular carcinoma. However, current treatments are not entirely effective. Allantoin is one of the principal compounds in many plants and an imidazoline I receptor agonist as well. Allantoin has positive effects on glucose metabolism and inflammation. In this study, the effects of allantoin on the NASH induced animals and the pathways involved have been evaluated. Materials and Methods:C57/BL6 male mice received saline and allantoin as the control groups. In the next group, NASH was induced by the methionine-choline-deficient diet (MCD) for eight weeks. In the NASH+allantoin group, allantoin was injected four weeks in the mice feeding on an MCD diet. Histopathological evaluations, serum analysis, ELISA assay, and real-time RT-PCR were performed. Results:Allantoin administration decreased serum alanine aminotransferase (ALT), cholesterol, low-density lipoprotein (LDL), hepatic lipid accumulation, and liver tumor necrosis factor (TNFα) level. Also, treatment with allantoin down-regulated the gene expression of glucose-regulated protein 78 (GRP78), activating transcription factor 6 (AFT6), TNFα, sterol regulatory element binding proteins 1c (SREBP1c), fatty acid synthase (FAS), Bax/Bcl2 ratio, caspase3, and P53. On the other hand, peroxisome proliferator-activated receptor alpha (PPARα), apolipoprotein B (Apo B), and acetyl-coenzyme acetyltransferase 1 (ACAT1) gene expression increased after allantoin injection. Conclusion:This study indicated that allantoin could improve animal induced NASH by changes in the expression of endoplasmic reticulum stress-related genes and apoptotic pathways.

Oral Hydrogen water – reduce hepatic inflammatory cells in mouse

Introduction: Nonalcoholic fatty liver disease (NAFLD) is characterized by a wide spectrum of liver damages spanning from steatosis, nonalcoholic steatohepatitis (NASH), cryptogenic liver cirrhosis, even to hepatocellular carcinoma. Oxidative stress is a strong contributor to the progression from simple fatty liver to nonalcoholic steatohepatitis (NASH). In this study we investigated the effect of hydrogen in the prevention of NASH in C57BL6 mouse, known as the NASH-related model. Our aim was to investigate the effects of hydrogen water on liver NASH and the mechanism underling these effects. Method: A methionine choline deficient diet (MCD) was prepared for the mouse models. In our investigation we made two experimental groups as follows: (1) MCD diet + normal water group(n=12); (2) MCD diet + hydrogen water group(n=12). Both groups were fed for three different periods, which are 8 weeks, 12 weeks, and 16 weeks. The livers were histologically examined using H&E, CD68 and Masson's trichrome staining methods. Hydrogen water was produced by placing a metallic magnesium stick into drinking water (final hydrogen concentration; 200-300 ppm). Result: The control group and hydrogen water group average concentration of ROS at 8 weeks (588±24 FORT units vs 164±8; P=0.029), at 12 weeks (587±26 FORT units vs 163±5; P=0.029) and at 16 weeks (439±176 FORT units vs 160±0; P=0.029). From H&E staining the inflammation cells was vicissitudinous in control group and HW group. At 8, 12, and 16 weeks demonstrated that (control group vs HW group; P=0.420)(control group vs HW group; P=0.729)(control group vs HW; P=0.017). Immunohistochemical identification of CD68-positive cells in control and HW group were counted that at 8 weeks P=0.151, at 12 weeks P=0.006 and 16 weeks P=0.000. Conclusion: Daily consumption of hydrogen water reduces nonalcoholic fatty liver disease and may be an effective treatment for NASH by reducing hepatic oxidative stress, apoptosis, and inflammation.

Differential Effects of a Glucagon-Like Peptide 1 Receptor Agonist in Non-Alcoholic Fatty Liver Disease and in Response to Hepatectomy

Non-alcoholic fatty liver disease (NAFLD) is associated with post-operative liver failure (PLF) and impaired liver regeneration. We investigated the effects of a glucagon-like peptide-1 (GLP-1) receptor agonist on NAFLD, PLF and liver regeneration in mice fed chow diet or methionine/choline-deficient diet (MCD) or high fat diet (HFD). Fc-GLP-1 decreased transaminases, reduced intrahepatic triglycerides (TG) and improved MCD-induced liver dysfuction. Macrophage/Kupffer cell-related markers were also reduced although Fc-GLP-1 increased expression of genes related to natural killer (NK), cytotoxic T lymphocytes and hepatic stellate cell (HSC) activation. After partial hepatectomy (PH), survival rates increased in mice receiving Fc-GLP-1 on chow or MCD diet. However, the benefit of Fc-GLP-1 on NASH-like features was attenuated 2 weeks post-PH and liver mass restoration was not improved. At this time-period, markers of NK cells and cytotoxic T lymphocytes were further elevated in Fc-GLP-1 treated mice. Increased HSC related gene expression in livers was observed together with decreased retinyl ester content and increased retinal and retinoic acid, reflecting HSC activation. Similar effects were found in mice fed HFD receiving Fc-GLP-1. Our results shed light on the differential effects of a long-acting GLP-1R agonist in improving NAFLD and PLF, but not enhancing liver regeneration in mice.

Circulating microRNA 122 in the methionine and choline-deficient mouse model of non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) and is a major cause of liver cirrhosis and hepatic failure. The methionine choline-deficient diet (MCD) is a frequently used hepatotoxicity animal model of NASH that induces hepatic transaminase (ALT, AST) elevations and hepatobiliary histological changes similar to those observed in human NASH. Liver-specific microRNA-122 (miR-122) has been shown as a key regulator of cholesterol and fatty acid metabolism in adult liver, and has recently been proposed as a sensitive and specific circulating biomarker of hepatic injury. The purpose of this study was to assess miR-122 serum levels in mice receiving an MCD diet for 0, 3, 7, 14, 28 and 56 days and compare the performance vs. routine clinical chemistry when benchmarked against the histopathological liver findings. MiR-122 levels were quantified in serum using RT-qPCR. Both miR-122 and ALT/AST levels were significantly elevated in serum at all timepoints. MiR-122 levels increased on average by 40-fold after 3 days of initiating the MCD diet, whereas ALT and AST changes were 4.8- and 3.3-fold, respectively. In general, miR-122 levels remained elevated across all time points, whereas the ALT/AST increases were less robust but correlated with the progressive severity of NASH as assessed by histopathology. In conclusion, serum levels of miR-122 can potentially be used as a sensitive biomarker for the early detection of hepatotoxicity and can aid in monitoring the extent of NAFLD-associated liver injury in mouse efficacy models.

Dysregulation of the unfolded protein response in db/db mice with diet-induced steatohepatitis

In humans with nonalcoholic fatty liver, diabetes is associated with more advanced disease. We have previously shown that diabetic db/db mice are highly susceptible to methionine choline-deficient diet (MCD)-induced hepatic injury. Because activation of the unfolded protein response (UPR) is an important adaptive cellular mechanism in diabetes, obesity, and fatty liver, we hypothesized that dysregulation of the UPR may partially explain how diabetes could promote liver injury. Db/db and db/m mice were fed the MCD or control diet for 4 weeks to characterize differences in UPR activation and downstream injury. Wildtype mice (C57BLKS/J) fed the MCD or control diet were treated with SP600125; a c-Jun N-terminal kinase (JNK) inhibitor and its effect on liver injury and UPR activation was measured. The MCD diet resulted in global up-regulation of the UPR in both diabetic db/db and nondiabetic db/m mice. db/db mice had an inadequate activation of recovery pathways (GADD34, XBP-1(s)) and accentuated activation of injury pathways related to persistent eif2-α phosphorylation (activating transcription factor 4 [ATF-4], C/EBP homologous transcription factor [CHOP], oxireductase endoplasmic reticulum oxidoreductin-1 [ERO-1α], JNK, nuclear factor kappaB [NF-κB]) compared to db/m mice. This led to increased expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), ICAM-1, and MCP-1 compared to db/m mice. Interestingly, whereas pharmacologic JNK inhibition did not prevent the development of MCD diet-induced steatohepatitis, it did attenuate UPR and downstream inflammatory signaling. MCD-fed db/db mice develop a more proinflammatory milieu than db/m mice associated with an impaired ability to dephosphorylate eif2-α through GADD34, impairing cellular recovery. These data may enhance our understanding of why diabetics with nonalcoholic steatohepatitis are prone to develop more severe liver injury than nondiabetic patients.

ALR expression indicates liver regeneration upon distinct tissue damage

Hepatocellular regeneration requires various factors promoting cellular proliferation. Among them, ALR (augmenter of liver regeneration), was discovered to be highly expressed in vivo under circumstances of liver regeneration and in liver diseases such as cirrhosis and carcinoma. Interestingly, ALR was shown to exhibit its action exclusively on regenerating livers and hepatoma cells. Therefore ALR is an interesting target to analyze liver regeneration. The aim of this study was to investigate the molecular regulation and expression of ALR in murine models of liver damage caused by tissue loss through 70% liver resection, by bile duct ligation, CCl4 intoxication, by fatty liver through a methionine choline deficient diet (MCD) and a high fat diet (lard, cholesterol, Na-cholate) model. ALR mRNA Expression was analyzed by quantitative RT-PCR in liver tissue samples at different time points. ALR mRNA levels were significantly enhanced in liver samples after 2/3 partiel hepatectomy in short (up to 72h) and long term oberservation (12d). Additionally, carbon tetrachloride treatment resulted in a slightly increase of ALR expression within 24 to 72 hours. Bile duct ligation as a model for acute fibrosis displayed intense ALR mRNA levels after 6 weeks. As models for non alcoholic steatohepatits (NASH), MCD and Paigen diet were used representing fatty livers with high and mild fibrotic scores, respectively. In both models liver sample demonstrated high ALR mRNA levels after 3 and 12 weeks with higher expression rates within Paigen mice. In all models a profound inflammation reaction was described and therefore it is likely, that enhanced levels of proflammatory cytokines such as IL-1 are abundant. The latter was shown to activate ALR promoter and ALR expression and might explain our observations. Analysis of ALR expression in different mouse liver disease models might be a useful indicator of an activated process leading to liver regeneration and repair.

Inhibition of JNK along with activation of ERK1/2 MAPK pathways improve steatohepatitis among the rats

Oxidative stress-induced hepatocyte apoptosis is implicated in the onset of non-alcoholic steatohepatitis (NASH). Regarding the pronounced roles of mitogen activated protein kinases (MAPKs) in oxidative stress-induced cellular damages and the development of NASH, we evaluated the effect of Teucrium polium ethyl acetate (EtoAc) extract on rats with NASH and on the modulation of MAPKs activities. To induce NASH, a methionine choline deficient diet (MCD) was given to N-Mary rats. These animals were then compared to the control group receiving normal diet and/or MCD diet plus EtoAc extract (0.5g/kg/day). After 8 weeks, liver histopathology, malondialdehyde (MDA) and immunoblot analyses of caspase-3 cleavage, ERK/pERK and JNK/pJNK were examined. Simultaneous treatments with MCD diet and the EtoAc extract resulted in pronounced improvements in liver steatosis, ballooning degeneration and inflammation. Elevated MDA and caspase-3 levels among MCD-fed rats were also markedly decreased. In addition, the EtoAc extract treatments caused significant reduction in the phosphorylated form of JNK along with an increase in the phosphorylated level of ERK1/2. These results indicate that the anti-apoptotic effect of T. polium extract is mediated through inhibition of ROS generation. Moreover, the inhibitory effect of T. polium on the development and progression of NASH is apparently governed by the attenuation of JNK activation and augmentation of ERK1/2 activity through phosphorylation.

Modulation of hepatic microsomal triglyceride transfer protein (MTP) induced by S-nitroso- N-acetylcysteine in ob/ob mice

We evaluated the effects of a potent NO donor, S-nitroso- N-acetylcysteine (SNAC), on microsomal triglyceride transfer protein (MTP) expression in ob/ob mice. NAFLD was induced in male ob/ob mice using a methionine–choline deficient diet (MCD) concomitantly with oral SNAC fed solution ( n = 5) or vehicle (control; n = 5) by gavage daily for 4 weeks. Livers were collected for histology and for assessing MTP by RT-qPCR, Western blot, immunohistochemistry and immunogold electron microscopy analyses. Histological analysis showed diffuse macro and microvesicular steatosis, moderate hepatocellular ballooning and moderate inflammatory infiltrate in ob/ob mice fed the MCD diet. With SNAC, mice showed a marked reduction in liver steatosis ( p < 0.01), in parenchymal inflammation ( p = 0.02) and in MTP protein immunoexpression in zone III ( p = 0.05). Moreover, SNAC caused reduction of MTP protein in Western blot analysis ( p < 0.05). In contrast, MTP mRNA content was significantly higher ( p < 0.05) in mice receiving SNAC. Immuno-electron microscopy showed MTP localized in the rough endoplasmic reticulum of hepatocytes in both treated and untreated groups. However with SNAC treatment, MTP was also observed surrounding fat globules. Histological improvement mediated by a nitric oxide donor is associated with significantly altered expression and distribution of MTP in this animal model of fatty liver disease. Further studies are in progress to examine possible mechanisms and to develop SNAC as a possible therapy for human fatty liver disease.

Effect of insulin-sensitizing agents in combination with ezetimibe, and valsartan in rats with non-alcoholic fatty liver disease

To assess whether treatment with insulin-sensitizing agents (ISAs) in combination with ezetimibe and valsartan have greater effect on hepatic fat content and lipid peroxidation compared to monotherapy in the methionine choline-deficient diet (MCDD) rat model of non-alcoholic fatty liver disease (NAFLD). Rats (n = 6 per group) were treated with different drugs, including MCDD only, MCDD diet with either metformin (200 mg/kg), rosiglitazone (3 mg/kg), metformin plus rosiglitazone (M+R), ezetimibe (2 mg/kg), valsartan (2 mg/kg), or combination of all drugs for a total of 15 wk. Liver histology, lipids, parameters of oxidative stress and TNF-alpha were measured. Fatty liver (FL) rats demonstrated severe hepatic fatty infiltration (> 91% fat), with an increase in hepatic TG (+ 1263%, P < 0.001), hepatic cholesterol (+ 245%, P < 0.03), hepatic MDA levels (+ 225%, P < 0.001), serum TNF-alpha (17.8 +/- 10 vs 7.8 +/- 0.0, P < 0.001), but a decrease in hepatic alpha tocopherol (-74%, P<0.001) as compared to the control rats. Combination therapy with all drugs produced a significant decrease in liver steatosis (-54%), hepatic TG (-64%), hepatic cholesterol (-31%) and hepatic MDA (-70%), but increased hepatic alpha tocopherol (+ 443%) as compared to FL rats. Combination therapy with ISA alone produced a smaller decrease in liver steatosis (-32% vs -54%, P < 0.001) and in hepatic MDA levels (-55% vs -70%, P < 0.01), but a similar decrease in hepatic lipids when compared with the all drugs combination. TNF-alpha levels decreased significantly in all treatment groups except in ISA group. Combination therapies have a greater effect on liver fat content as compared to monotherapy. Rosiglitazone appears to improve hepatic steatosis to a greater extent than metformin.