MCD Diet Research Articles

Effect of miR-146 targeted HDMCP up-regulation in the pathogenesis of nonalcoholic steatohepatitis.

Backgrounds/AimsMitochondrial dysfunction plays an important role inthe pathogenesis of nonalcoholic steatohepatitis (NASH), where uncoupling protein (UCP) is actively involved. We previously reported the uncoupling activity of HDMCP and its role in liver steatosis. We now aim to investigate the degree and therapeutic effect of HDMCP in NASH and the regulatory role of miR-146 on HDMCP.MethodsNASH animal model was established by feeding BALB/c mice with MCD diet while L02 cell was cultured with high concentration of fatty acid (HFFA) for 72h to mimic the steatosis and inflammation of NASH in-vitro appearance. The steatosis level was assessed by H-E/oil-red staining and serum/supernatant marker detection. The inflammation activity was evaluated by levels of Hepatic activity index, transwell, apoptosis degree (TUNEL/flow cytometry) and serum/supernatant marker. HDMCP level was detected by western blot and miRNA expression was tested by qRT-PCR. NASH severity change was recorded after RNA interference while the regulatory role of miR-146 on HDMCP was confirmed by dual luciferase report system. The H2O2 and ATP levels were measured for mechanism exploration.ResultsIncreased HDMCP expression was identified in NASH animal model and HFFA-72h cultured L02 cell. Moreover, under regulation of miR-146, NASH alleviation was achieved after HDMCP downregulation in both in vivo and in vitro, according to the declination of steatosis and inflammation related markers. Though H2O2 and ATP levels were increased and decreased in NASH models, HDMCP down regulation both increased their levels.ConclusionsThe miR-146-HDMCP-ATP/H2O2 pathway may provide novel mechanism and treatment option for NASH.

Role of ADAMTS13 in diet-induced liver steatosis.

Previous studies, predominantly based on increased or decreased plasma levels, have reported conflicting data on a potential functional role of ADAMTS13 in the pathogenesis of liver diseases, including non‑alcoholic steatohepatitis (NASH). The aim of the current study was to evaluate whether ADAMTS13 deficiency affects development of NASH. Therefore, male wild‑type (WT) and Adamts13 deficient (Adamts13‑/‑) mice were kept on a steatosis‑inducing diet devoid of methionine and choline (MCD) or a control diet (MCC) for 4weeks. Induction of NASH did not affect plasma ADAMTS13 antigen levels of WT mice. MCD as compared with MCC feeding resulted in reduced body and liver weight with no differences between the genotypes. Plasma levels of the liver enzymes AST and ALT were significantly higher for MCD vs. MCC fed Adamts13‑/‑ and WT mice, however were not different between the genotypes. Liver triglyceride levels were also higher after MCD feeding, but were not different between WT and Adamts13‑/‑ mice. Adamts13‑/‑ mice on the two diets exhibited higher insulin sensitivity when compared with WT mice. On the MCC diet, the genotype did not show clear histological abnormalities in the liver, whereas severe steatosis and fibrosis were observed on MCD diet, however were comparable for both genotypes. This was supported by comparably enhanced hepatic expression in the two genotypes on MCD diet of the steatosis marker CD36 and of the fibrosis marker tissue inhibitor of metalloproteinase 1. Thus, the results of the current study do not support a functional role of ADAMTS13 in this murine model of NASH.

PS-028 - A2a receptor stimulation prevents the multiple processes that lead to hepatic “immuno-lipotoxicity” in mice fed with MCD diet and blocks non-alcoholic steatohepatitis development

PS-028 - A2a receptor stimulation prevents the multiple processes that lead to hepatic “immuno-lipotoxicity” in mice fed with MCD diet and blocks non-alcoholic steatohepatitis development

FRI-358 - Aramchol reduces established fibrosis in MCD diet animal model

FRI-358 - Aramchol reduces established fibrosis in MCD diet animal model

Fluorescing fatty acids in rat fatty liver models.

The autofluorescence (AF) of NAD(P)H and flavins has been at the basis of many in-situ studies of liver energy metabolism and functionality. Conversely, few data have been so far reported on fluorescing lipids. In this work we investigated the AF of liver lipid extracts from two fatty liver models, Wistar rats fed with MCD diet for 12 days (Wi-MCD), and obese (fa/fa) Zucker rats. Among the most abundant fatty acids in the lipid extracts, indicated by mass spectrometry, arachidonic acid (AA) exhibited higher quantum yield than the other fluorescing fatty acids (FLFA), and red shifted AF spectrum. This allowed to estimate the AA contribution to the overall emission of lipid extracts by curve fitting analysis. AA prevailed in obese Zucker livers, accounting for the different AF spectral profiles between the two models. AF and mass spectrometry indicated also a different balance between the fluorescing fraction and the overall amount of AA in the two models. The ability of AF to detect directly AA and FLFA was demonstrated, suggesting its supportive role as tool in wide-ranging applications, from the control of animal origin food, to experimental investigations on liver fat accumulation, lipotoxicity and disease progression, with potential translation to the clinics.

Transcriptional repression of SIRT1 by protein inhibitor of activated STAT 4 (PIAS4) in hepatic stellate cells contributes to liver fibrosis.

Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression in vivo. PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis.

Hepatocyte-specific Keap1 deletion reduces liver steatosis but not inflammation during non-alcoholic steatohepatitis development

Generation of reactive oxygen species (ROS) in response to fatty acids accumulation has been classically proposed as a possible “second hit” triggering progression from simple steatosis to non-alcoholic steatohepatitis (NASH). In this study we challenged hepatocyte-specific Keap1 knockout mice (Keap1Δhepa) and littermate Cre- controls (Keap1fx/fx) with two different diet models of NASH in order to evaluate the effects of the anti-oxidant transcription factor Nrf2 over-activation on hepatic metabolism and disease progression. After 4 weeks of MCD diet the liver/body weight ratio of Keap1Δhepa mice was significantly higher compared to littermate controls with no differences in total body weight. Strikingly, liver histology revealed a dramatic reduction of lipid droplets confirmed by a decreased content of intra-hepatic triglycerides in Keap1Δhepa compared to controls. In parallel to reduced expression of genes involved in lipid droplet formation, protein expression of Liver X Receptor (LXRα/β) and Peroxisome proliferator-activated receptor α (PPARα) was significantly decreased. In contrast, genes involved in mitochondrial lipid catabolism were markedly up-regulated in Keap1Δhepa livers. A similar phenotype characterized by inhibition of lipogenesis in favor of increased mitochondrial catabolic activity was also observed after 13 weeks of western diet administration. MCD-induced apoptosis was significantly dampened in Keap1Δhepa compared to Keap1fx/fx as detected by TUNEL, cleaved caspase-3 and Bcl-2 protein expression analyses. However, no differences in inflammatory F4/80- and CD11b-positive cells and pro-fibrogenic genes were detected between the two groups. Although hepatic lack of Keap1 did not ameliorate inflammation, the resulting constitutive Nrf2 over-activation in hepatocytes strongly reduced hepatic steatosis via enhanced lipid catabolism and repressed de novo lipogenesis during murine NASH development.

Glucotoxicity Caused by Change in Hepatic Metabolic Pathways Leading to Type 2 Diabetes

Recent reviews emphasize type 2 diabetes initiation by chronic liver exposure to lipotoxicity and glucotoxicity by adding to the deterioration of glucose maintenance which characterizes type 2 diabetes. To investigate the interaction of these contributing factors, we utilized 4 groups (10 each) of OB/OB mice: Grp 1 received a normal diet (Harlan Teklad); Grp 2 received the same diet with all methionine and choline lacking (MCD diet, same % CHO, protein, fat). Grp 3 was the normal diet with a high clinical dose of Ω3-fatty acids, grp 4 received the MCD diet with the same dose of Ω3-fatty acids as group 3. Hypothesis The MCD diet for 2 months would change liver lipid and fatty acid composition, while Ω3-fatty acid feeding would change steatosis. The OB/OB mice (leptin k/o) have high food intake which aids glucose desensitization. Together the metabolic picture emulates type 2 diabetes. At 2 weeks, half of group 3 died suddenly , Leptin receptor analysis was normal. At 2 months, necropsies on all others showed >600 blood glucose in most grp 4 survivors with significantly depressed hepatic glycogen. Image J analysis was performed to quantitatively estimate the percentage of glycogen in the study groups. Grp 1 had a glycogen content of 17.55 % (±3.724), grp 2 20.53 (±6.25), grp 3 11.8 (±2.95) and grp 4 19.809 (±7.75). Analysis of insulin showed elevated levels in the hyperglycemic mice in grp 3 as compared to the normoglycemic mice in grp 4. Beta cells of pancreas in these animals showed minimal inflammation. These data demonstrate an important input of hepatic metabolic pathways on the glucotoxity and lipotoxicity that may define type 2 diabetes.

Representation of human non-alcoholic fatty liver disease in murine models

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries. Murine models are the key mechanistic platform for the disease. We systematically evaluated genome-wide mRNA expression data from five commonly used murine models of NAFLD (high-fat diet, MCD diet, streptozotocin diet, PTEN (Fox) KO) in comparison to human data from 39 controls and 77 NAFLD samples spanning the range of the phenotype. First, profound differences between human and murine expression patterns exist, that override any disease or phenotype signature. In fact, from the significantly regulated human genes in NASH/NAFLD only between 1 and 16 are significantly and concordantly regulated in mice. The dataset, depending on mouse model and human phenotype, shows correct reflection of key pathways, lack of regulation, or reverse effects thereby providing a guide for murine model selection in future experiments to improve applicability to human NAFLD.

MIF shows profibrotic properties after MCD diet via NKT cell population

Background: Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine. It has recently been recognized to function as a chemokine-like molecule in chronic inflammatory diseases. In a model of hepatotoxic liver injury, MIF shows anti-fibrotic properties via CD74 and AMPK. In a model of non-alcoholic steatohepatitis, MIF has anti-steatotic properties. Because NASH is also associated with fibrosis within the liver, we compared Mif -/- with wild-type mice in an experimental liver fibrosis and metabolic disease model.

Increased expression of the Glucose-transporter GLUT1 during hepatic stellate cell activation promotes hepatic fibrosis

Normal liver reveals only low expression of the facilitative glucose transporter (GLUT) 1. Recently, we have shown that GLUT1 expression is increased in hepatocellular carcinoma (HCC), where GLUT1 acts as a tumor promotor. Hyperglycemia is one of the factors known to induce and promote hepatic fibrogenesis, and the activation of hepatic stellate cells (HSCs) is the key event of hepatic fibrosis. Furthermore, these cells form and transduce the HCC stroma. The aim of this study was to assess the expression and functional role of GLUT1 in hepatic fibrosis. Methods and Results: Hepatic GLUT1 expression was significantly increased in different murine models of hepatic fibrosis, namely chronic TAA or CCl4-application, bile-duct-ligation and dietary models of non-alcoholic steatohepatitis (NASH). Also in cirrhotic human livers and human NASH GLUT1 expression was increased. Interestingly, hepatic GLUT1 expression revealed a significant correlation with collagen I and alpha-sma levels indicating activated HSCs as cellular source of GLUT1, which was confirmed by immunohistochemical analysis. In line with this, we found that GLUT1 expression increased during in vitro activation of primary murine and human HSCs. To gain insight in the functional role of GLUT1 in activated HSCs we inhibited GLUT1 expression with siRNA or the specific GLUT1 inhibitor WZB117 and found that GLUT1 suppression impaired glucose uptake and lactate secretion of HSCs indicative for reduced anaerobic glycolysis. Functional analysis demonstrated that reduced GLUT1 expression led to lower apoptosis resistance of HSCs. Transgenic GLUT1 knockdown mice subjected to TAA induced liver fibrosis model revealed less pronounced liver damage than WT animals confirmed by hepatic sirius red staining and alpha-sma protein expression. In line with this, treatment with GLUT1 inhibitor WZB117 in combination with the NASH inducing MCD diet resulted in less hepatic alpha-sma and collagen I expression than animals treated with vehicle. Conclusions: Increased GLUT1 expression during HSC activation functionally affects HSCs, and herewith, likely promotes the development and progression of hepatic fibrosis. Therefore and based on its known role as tumor-promotor, GLUT1 appears as attractive novel target to inhibit the progression of chronic liver disease. Corresponding author: Czech, Barbara E-Mail:barbara.czech@ukr.de

Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal Permeability.

Background & AimsEmerging data suggest that changes in intestinal permeability and increased gut microbial translocation contribute to the inflammatory pathway involved in nonalcoholic steatohepatitis (NASH) development. Numerous studies have investigated the association between increased intestinal permeability and NASH. Our meta-analysis of this association investigates the underlying mechanism.MethodsA meta-analysis was performed to compare the rates of increased intestinal permeability in patients with NASH and healthy controls. To further address the underlying mechanism of action, we studied changes in intestinal permeability in a diet-induced (methionine-and-choline-deficient; MCD) murine model of NASH. In vitro studies were also performed to investigate the effect of MCD culture medium at the cellular level on hepatocytes, Kupffer cells, and intestinal epithelial cells.ResultsNonalcoholic fatty liver disease (NAFLD) patients, and in particular those with NASH, are more likely to have increased intestinal permeability compared with healthy controls. We correlate this clinical observation with in vivo data showing mice fed an MCD diet develop intestinal permeability changes after an initial phase of liver injury and tumor necrosis factor-α (TNFα) induction. In vitro studies reveal that MCD medium induces hepatic injury and TNFα production yet has no direct effect on intestinal epithelial cells. Although these data suggest a role for hepatic TNFα in altering intestinal permeability, we found that mice genetically resistant to TNFα-myosin light chain kinase (MLCK)–induced intestinal permeability changes fed an MCD diet still develop increased permeability and liver injury.ConclusionsOur clinical and experimental results strengthen the association between intestinal permeability increases and NASH and also suggest that an early phase of hepatic injury and inflammation contributes to altered intestinal permeability in a fashion independent of TNFα and MLCK.

Apolipoprotein A‐I and adenosine triphosphate‐binding cassette transporter A1 expression alleviates lipid accumulation in hepatocytes

Abnormal lipid metabolism may contribute to the pathogenesis of non-alcoholic steatohepatitis. ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to high density lipoprotein apolipoproteins. The lipidation of apolipoprotein A-I (apoA-I) by ABCA1 is the rate-limiting step in reverse cholesterol transport and the generation of plasma high density lipoprotein. Here, we examined the effect of apoA-I or ABCA1 overexpression on hepatic lipid levels in BEL-7402 cells. Human ABCA1 or apoA-I was overexpressed in BEL-7402 hepatocytes by transfection and human apoA-I was overexpressed via adenoviral vector in C57BL/6J mice with MCD diet. Overexpression of either apoA-I or ABCA1 resulted in an increase in cholesterol efflux and a decrease in cellular fatty acids and triglycerides. However, after repression of ABCA1 by its siRNA, overexpression of apoA-I failed to decrease both cellular fatty acids and triglycerides. ApoA-I or ABCA1 overexpression also resulted in a decrease in the expression of the endoplasmic reticulum stress-related proteins GRP78 and SREBP-1. Overexpression of apoA-I in mice also reduced hepatic lipid levels. Expression of apoA-I or ABCA1 can reduce steatosis by decreasing lipid storage in hepatocytes through lipid transport and may also reduce endoplasmic reticulum stress, further lessening hepatic steatosis.

Enhancer of Zeste Homolog 2 (EZH2) expression and activation in in vivo and in vitro non-alcoholic fatty liver disease (NAFLD)

via dimethylarginine-dimethylaminohydrolase (DDAH). This study investigated whether changes in serum levels of ADMA occur in a rat model of non-alcoholic steatohepatitis (NASH) and what is the mechanism involved. Materials and methods: NASH was induced in Male Wistar rats by 8 weeks of feeding with an MCD diet (methionine/cholinedeficient diet). Blood samples and hepatic biopsies were collected after 1, 2, 3, 4 and 8 weeks. Serum hepatic enzymes (AST, ALT and g-GT) and ADMA were evaluated. Hepatic biopsies were used for in situ NAD(P)H autofluorescence detection and for mRNA expression of DDAH and ADMA transporters (CAT-1) by RT-PCR. Tissue DDAH activity and content of lipid peroxides, glutathione and ATP were also quantified. Results: NASH injury was confirmed by altered serum levels of hepatic enzymes. A time dependent decrease in serum ADMA levels andan increase inmRNAexpressionofDDAHandCAT-1were found. The hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high lipid peroxide levels and low GSH content. A decrease in ATP levels and in the NAD(P)Hbound/free ratio reflecting the mitochondria alterations were detected. Conclusions: These results indicate that the oxidative stress observed can contribute to the reduction of DDAH activity. This enzyme is a cysteine hydrolase that may be inhibited by increased reactive oxygen species associated to mitochondria dysfunction. The observed decrease in serum ADMAmay be due to the increase in ADMA transporter, CAT-1. These data confirm and support the crucial role of the liver in the control of ADMA levels by taking up large amounts of ADMA from the systemic circulation. (Supported by Fondazione Cariplo, grant no. 2011-0439.)

β7-Integrins and MAdCAM-1 play an opposing role during the development of Non-alcoholic steatohepatitis (NASH)

Introduction: Non-alcoholic steatohepatitis (NASH), the hepatic correlate of the metabolic syndrome, is the third most common cause of chronic liver disease in western countries. Its global incidence is on the rise and reflects one of the fastest growing medical problems. Aim: The significance of leukocyte infiltration during the development of NASH remains unclear until now. We therefore investigated the role of β7-Integrin and one of its receptors MAdCAM-1 in a mouse model of NASH development. Methods: Constitutive MAdCAM-1 and β7-Integrin knockout (KO) mice were fed a MCD diet (methionine and choline deficient) for 4 weeks. Results: Interestingly β7-deficient mice displayed an earlier and faster progressing steatohepatitis during MCD treatment, while MAdCAM-1 KO showed less histomorphological changes in contrast to wildtype mice. Oxidative stress analysis in β7 KO mice showed a stronger response as indicated by DHE (dihydroethidium) staining and a down regulation of transcription factors involved in anti-oxidative stress and fatty acid metabolism (e.g. FAS and SREBP). MAdCAM-1 KO mice on the other hand had an upregulation of the anti-oxidative stress response. Moreover, we detected a stronger hepatic infiltration of inflammatory cells (CD4+ and CD11b+) in the β7 KO group, reflecting an earlier onset of NASH. Those changes finally resulted in an earlier and stronger collagen accumulation (Sirius red) in the β7-Integrin deficient group, while MAdCAM-1 KO mice were protected. Conclusion: MAdCAM-1 and β7 Integrins mediate opposing effects in the MCD model, with protection in MAdCAM-1 KO animals and a more severe phenotype and significantly stronger fibrosis progression in β7-Integrin KO mice. Therefore, the interaction of β7 Integrins and their receptor MAdCAM-1 provide a novel interesting target for therapeutic interventions during NASH development.

Osteopontin: Versatile modulator of liver diseases

Osteopontin (OPN) is a multifunctional protein, involved in pathological conditions including inflammation, immunity, angiogenesis, fibrosis and cancer progression in various tissues. Hepatic inflammation and fibrosis induced by feeding with a diet deficient in methionine and choline (MCD diet) were markedly attenuated in OPN knockout mice when compared with wild-type mice in the model of non-alcoholic steatohepatitis (NASH). Hepatic cholangiocytes, myofibroblastic stellate cells and natural killer T cells were suggested to secret OPN in mice fed an MCD diet. Plasma and hepatic OPN levels were significantly higher in patients with NASH with advanced fibrosis than in those with early fibrosis. Hepatic OPN mRNA level was correlated with hepatic neutrophil infiltration and fibrosis in patients with alcoholic liver diseases. In those with hepatocellular carcinoma (HCC), OPN levels in plasma and HCC were prognostic factors after liver resection or transplantation. Downregulation of OPN inhibited tumor growth and lung metastasis in nude mice implanted with HCC cells. The single nucleotide polymorphism in the promoter region of the OPN gene was shown to be associated with activity of hepatitis in chronic hepatitis C patients, prognosis in patients with HCC, and growth and lung metastasis of HCC xenografts in nude mice. OPN was reported to be a downstream effecter of Hedgehog pathway, which modulates hepatic fibrosis and carcinogenesis. This review focuses on the roles of OPN in hepatic inflammation, fibrosis and cancer progression. Further elucidation of cellular interactions and molecular mechanisms associated with OPN actions may contribute to development of novel strategies for treatment of the liver diseases.

Abstract 399: Increased Gene Transcription via Ire1a/cJun Enhances Plasma ALT/AST in MTP Inhibited and MCD Diet Fed Mice

Microsomal triglyceride transfer protein (MTP) is a target to reduce plasma lipids because of its indispensable role in triglyceride-rich lipoprotein biosynthesis. MTP inhibition in western diet fed mice decreased plasma triglycerides/cholesterol while increasing plasma alanine/aspartate aminotransferases (ALT/AST) and hepatic triglycerides/free cholesterol. Free cholesterol accumulated in the endoplasmic reticulum (ER) and mitochondria resulting in ER and oxidative stresses. Mechanistic studies revealed that MTP inhibition increased transcription of the GPT/GOT1 genes through up-regulation of the IRE1α/cJun pathway leading to increased synthesis and release of ALT1/AST1. We also observed that IRE1α/cJun pathway is involved in augmenting plasma ALT/AST in mice fed methionine-choline deficient diet. Thus, transcriptional up-regulation of GPT/GOT1 genes is a major mechanism elevating plasma transaminases under different conditions.

Retracted: Quercetin Decreases Liver Damage in Mice with Non‐Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a frequent condition in obese patients that may progress to end-stage liver disease. This study was designed to evaluate the modulation of this condition by use of quercetin (Q), a flavonoid largely found in vegetable foods, with known anti-inflammatory and antioxidant properties, in the experimental model of non-alcoholic steatohepatitis (NASH) using a diet deficient in methionine and choline (MCD). Male C57BL6 mice were divided into four groups (n=16): (i) Control plus vehicle (control ration plus carboxymethylcellulose 1% used as vehicle, CO+V); (ii) Control ration plus Q 50mg/kg (CO+Q); (iii) MCD diet plus vehicle (NASH+V); and (iv) MCD diet plus Q (NASH+Q). Diets were administered for 4weeks. At the end of the experimental period, liver alterations, bioindicators of oxidative stress and DNA damage were assessed. NASH was diagnosed in 100% of the mice that were fed the MCD diet. In addition, a significant increase in DNA damage in liver tissue from NASH+V group was observed in comparison with CO+V. The group NASH+Q showed a significant decrease in hepatic damage enzymes, lipoperoxidation, DNA damage and a lower degree of macrovesicular steatosis, ballooning and inflammatory process. These findings suggest that Q may have protective effects by improving liver integrity in NASH.

Amelioration of hepatic inflammation in a mouse model of NASH using a dithiocarbamate derivative.

The process whereby liver inflammation develops in non-alcoholic steatohepatitis (NASH) is not fully understood. While modification of the inflammatory milieu is an attractive target to prevent the development of hepatocellular injury, most antiinflammatory agents have proven ineffective in this setting. Tetraethylthiuram disulfide (TDSF) is able to induce S-glutathionylation of NF-κB along with critical signaling proteins involved with inflammation, especially when complexed with a heavy metal. For this reason, we hypothesized that administration of TDSF would function to ameliorate necroinflammatory activity in a mouse model of NASH. Mice were divided into five groups and received control chow versus a methionine-choline-deficient diet. After 6weeks of TDSF versus sham gavage, animals were necropsied. Using conventional H&E staining, livers were examined using the Brunt scoring system by a hepatopathologist blinded to treatment groups. Validated mouse primer sets were used for quantitative real-time PCR to evaluate changes in mRNA expression. Livers treated with TDSF demonstrated a qualitative reduction in lobular inflammation, lipogranuloma formation, and Kupffer cell accumulation, but not steatosis. Significant reductions in inflammatory transcripts for α-1-collagen, TGF-β, Mmp2, MCP-1, and TNF-1α were also observed. Animals treated with TDSF exhibit a reduction in lobular inflammation that is independent of lipid accumulation when administered MCD diet. Similar reductions are seen in several inflammatory transcripts associated with NASH. Additional work in this area may reveal a therapeutic role for TDSF or similar agents in curtailing inflammatory signaling within the liver.

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.