transforming-growth-factor-beta and Fatty-Liver

Chymase plays a crucial role in angiotensin II formation in various tissues. Angiotensin II induces gene expressions of transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9, and chymase also converts precursors of TGF-β and MMP-9 to their active forms. All of angiotensin II, TGF-β and MMP-9 are considered to be closely involved in the development and progression of metabolic syndrome and its complications. In a diabetic animal model, chymase induced pancreatic disorganization via attack of oxidative stress induced by augmentation of chymase-forming angiotensin II. In atherosclerotic lesions in patients, accumulation of chymase-positive cells was observed, and chymase inhibition prevented the development of atherosclerosis in an animal model. In Apo E-deficient mice, chymase inhibition prevents the development of angiotensin II-induced abdominal aneurysmal aorta (AAA). In this model, the AAA development on an increase in MMP-9 activities induced by angiotensin II, but the inhibition of MMP-9 activation by chymase inhibitor resulted in attenuation of the AAA development. Cardiac dysfunction after myocardial infarction was also attenuated by chymase inhibition. Steatosis and fiblosis in liver were strongly prevented by chymase inhibition in an animal model with nonalcoholic steatohepatitis which is involved in metabolic syndrome. Therefore, chymase inhibition may be useful for attenuating MMP-9 and TGF-β levels, in addition to reducing angiotensin II formation, and this function may provide powerful preventions of organ damages. In this review, we propose the significance of chymase as a target to prevent complications of metabolic syndrome.

Topics: Angiotensin II; Animals; Atherosclerosis; Chymases; Diabetes Complications; Diabetic Retinopathy; Fatty Liver; Humans; Hypertension; Matrix Metalloproteinase 9; Metabolic Syndrome; Mice; Transforming Growth Factor beta

Diabetes mellitus is characterized by a lack of insulin causing elevated blood glucose, often with associated insulin resistance. Over time, especially in genetically susceptible individuals, such chronic hyperglycemia can cause tissue injury. One pathological response to tissue injury is the development of fibrosis, which involves predominant extracellular matrix (ECM) accumulation. The main factors that regulate ECM in diabetes are thought to be pro-sclerotic cytokines and protease/anti-protease systems. This review will examine the key markers and regulators of tissue fibrosis in diabetes and whether their levels in biological fluids may have clinical utility.

Topics: Animals; Basement Membrane; Biomarkers; Cardiomyopathies; Connective Tissue Growth Factor; Diabetes Complications; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Retinopathy; Endothelium, Vascular; Extracellular Matrix; Fatty Liver; Fibrosis; Glycation End Products, Advanced; Heart Diseases; Heart Failure; Humans; Hyperglycemia; Immediate-Early Proteins; Insulin Resistance; Intercellular Signaling Peptides and Proteins; Liver Cirrhosis; Metalloproteases; Peptide Fragments; Procollagen; Renin-Angiotensin System; Transforming Growth Factor beta; Tunica Intima; Up-Regulation

Topics: Adult; Age Factors; Aged; Alcoholism; alpha-Tocopherol; Antiviral Agents; Biopsy; Child; Cicatrix; Enzyme-Linked Immunosorbent Assay; Fatty Liver; Female; Hemochromatosis; Hepacivirus; Hepatitis C, Chronic; HIV Infections; Humans; Immunohistochemistry; Inflammation; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Necrosis; Phenotype; Risk Factors; Sex Factors; Transforming Growth Factor beta

Non-alcoholic steatohepatitis is a distinct entity, characterized by fatty change, lobular inflammation and fibrosis of the liver. Some cases of non-alcoholic steatohepatitis progress to cirrhosis, but it is not easy to distinguish this disease from non-alcoholic fatty liver by non-invasive examinations. No proven therapy for non-alcoholic steatohepatitis exists. Transforming growth factor-beta1 is implicated in the development of liver fibrosis, and is inhibited by alpha-tocopherol (vitamin E) in the liver. Therefore, in this study, the significance of the measurement of the level of plasma transforming growth factor-beta1 and the effect of alpha-tocopherol on the clinical course of non-alcoholic steatohepatitis were investigated.. Twelve patients with non-alcoholic steatohepatitis and 10 patients with non-alcoholic fatty liver, with a diagnosis confirmed by liver biopsy, were studied. None of the patients had a history of alcohol abuse, habitual medicine or malignant or inflammatory diseases. All patients were negative for hepatitis B, C and G virus. Patients were given dietary instruction for 6 months, and then alpha-tocopherol (300 mg/day) was given for 1 year. Blood chemistries, measurement of plasma transforming growth factor-beta1 level and liver biopsies were undertaken before and after the 1-year alpha-tocopherol treatment.. The serum alanine transaminase level decreased in non-alcoholic fatty liver patients, but not in non-alcoholic steatohepatitis patients, after 6 months of dietary therapy. Although the serum alanine transaminase level in non-alcoholic steatohepatitis patients was reduced during the 1-year alpha-tocopherol treatment, alpha-tocopherol had no effect on the serum alanine transaminase level in non-alcoholic fatty liver patients. The histological findings, such as steatosis, inflammation and fibrosis, of the non-alcoholic steatohepatitis patients were improved after alpha-tocopherol treatment. The plasma transforming growth factor-beta1 level in non-alcoholic steatohepatitis patients was significantly elevated compared with that in non-alcoholic fatty liver patients and healthy controls, and decreased, accompanied by an improvement in serum alanine transaminase level, with alpha-tocopherol treatment.. Our data suggest that the measurement of the level of plasma transforming growth factor-beta1 represents a possible method of distinguishing between non-alcoholic steatohepatitis and non-alcoholic fatty liver. Long-term alpha-tocopherol treatment may be safe and effective for non-alcoholic steatohepatitis. A randomized, controlled, double-blind trial is needed to confirm the full potential of alpha-tocopherol in the management of non-alcoholic steatohepatitis.

Topics: Adult; Alanine Transaminase; Alkaline Phosphatase; alpha-Tocopherol; Aspartate Aminotransferases; Body Weight; Cholesterol; Fatty Liver; Female; gamma-Glutamyltransferase; Hepatitis; Humans; Liver Cirrhosis; Male; Pilot Projects; Transforming Growth Factor beta; Transforming Growth Factor beta1; Triglycerides

Hepatic fibrosis is the primary determinant of mortality in patients with metabolic dysfunction-associated steatohepatitis (MASH). Transforming growth factor-β (TGFβ), a master profibrogenic cytokine, is a promising therapeutic target that has not yet been translated into an effective therapy in part because of liabilities associated with systemic TGFβ antagonism. We have identified that soluble folate receptor γ (FOLR3), which is expressed in humans but not in rodents, is a secreted protein that is elevated in the livers of patients with MASH but not in those with metabolic dysfunction-associated steatotic liver disease, those with type II diabetes, or healthy individuals. Global proteomics showed that FOLR3 was the most highly significant MASH-specific protein and was positively correlated with increasing fibrosis stage, consistent with stimulation of activated hepatic stellate cells (HSCs), which are the key fibrogenic cells in the liver. Exposure of HSCs to exogenous FOLR3 led to elevated extracellular matrix (ECM) protein production, an effect synergistically potentiated by TGFβ1. We found that FOLR3 interacts with the serine protease HTRA1, a known regulator of TGFBR, and activates TGFβ signaling. Administration of human FOLR3 to mice induced severe bridging fibrosis and an ECM pattern resembling human MASH. Our study thus uncovers a role of FOLR3 in enhancing fibrosis.

Topics: Animals; Diabetes Mellitus, Type 2; Fatty Liver; Folic Acid; Hepatic Stellate Cells; Humans; Mice; Transforming Growth Factor beta

Type-2 diabetes mellitus and NAFLD are considered as one of the greatest worldwide metabolic disorders with growing incidence. It was found that patients with T2DM have two-fold increase to develop NAFLD. Evidence that some antidiabetic agents improve NAFLD/NASH in patients with T2DM is evolving. However, there are no certain pharmacologic therapies. The current study aimed to investigate the underlying mechanisms for the hepatoprotective effect of dapagliflozin against steatohepatitis in diabetic rats. Type-2 diabetes was induced by HFD followed by a single dose of STZ (30 mg/kg I.P). Fifty rats were randomly divided into 5 groups: Group1; normal control, Group 2; diabetic control, Groups (3-5); diabetic rats received daily dapagliflozin (0.75, 1.5, 3 mg/kg, p.o.) respectively for 6 weeks. At the end of the experiment, blood glucose level and serum insulin were measured. Hepatic tissue homogenization was performed for measuring inflammatory and oxidative stress markers. In addition, histopathological investigation of the hepatic tissue was done. Diabetic rats exhibited remarkable increase in liver weight and liver enzymes, along with histopathological changes, significant elevation in MDA, IL-1 β, TGFβ levels and, NF-κB, alpha-SMA expressions. Dapagliflozin treatment decreased liver weight, liver enzymes, together with marked improvement in histopathological changes. Furthermore, dapagliflozin increased antioxidant enzymes, GSH levels. Interestingly, Dapagliflozin reduced IL-1 β, TGFβ levels and, NF-κB, alpha-SMA expressions. Present data show that dapagliflozin represent a viable approach to protect the liver against diabetes-encouraged steatohepatitis through inhibiting oxidative stress, inflammation and fibrosis progression thus conserving liver function.

Topics: Actins; Alanine Transaminase; Animals; Anti-Inflammatory Agents; Aspartate Aminotransferases; Benzhydryl Compounds; Blood Glucose; Diabetes Mellitus, Experimental; Fatty Liver; Glucosides; Insulin; Interleukin-1beta; Liver; Male; Malondialdehyde; NF-kappa B; Oxidative Stress; Rats; Sodium-Glucose Transporter 2 Inhibitors; Transforming Growth Factor beta

Dysregulated transforming growth factor-beta (TGF-β) signaling contributes to fibrotic liver disease and hepatocellular cancer (HCC), both of which are associated with fatty liver disease. SIRT6 limits fibrosis by inhibiting TGF-β signaling through deacetylating SMAD2 and SMAD3 and limits lipogenesis by inhibiting SREBP1 and SREBP2 activity. Here, we showed that, compared to wild-type mice, high-fat diet-induced fatty liver is worse in TGF-β signaling-deficient mice (SPTBN1

Topics: Animals; Carcinoma, Hepatocellular; Fatty Liver; Fibrosis; Liver Neoplasms; Mice; Sirtuins; Sterol Regulatory Element Binding Protein 1; Transforming Growth Factor beta

Liver fibrosis risk is a heritable trait, the outcome of which is the net deposition of extracellular matrix by hepatic stellate cell-derived myofibroblasts. Whereas nucleotide sequence variations have been extensively studied in liver fibrosis, the role of copy number variations (CNV) in which genes exist in abnormal numbers of copies (mostly due to duplication or deletion) has had limited exploration.. The impact of the XPO4 CNV on histological liver damage was examined in a cohort comprised 646 Caucasian patients with biopsy-proven MAFLD and 170 healthy controls. XPO4 expression was modulated and function was examined in human and animal models.. Here we demonstrate in a cohort of 816 subjects, 646 with biopsy-proven metabolic associated liver disease (MAFLD) and 170 controls, that duplication in the exportin 4 (XPO4) CNV is associated with the severity of liver fibrosis. Functionally, this occurs via reduced expression of hepatic XPO4 that maintains sustained activation of SMAD3/SMAD4 and promotes TGF-β1-mediated HSC activation and fibrosis. This effect was mediated through termination of nuclear SMAD3 signalling. XPO4 demonstrated preferential binding to SMAD3 compared to other SMADs and led to reduced SMAD3-mediated responses as shown by attenuation of TGFβ1 induced SMAD transcriptional activity, reductions in the recruitment of SMAD3 to target gene promoters following TGF-β1, as well as attenuation of SMAD3 phosphorylation and disturbed SMAD3/SMAD4 complex formation.. We conclude that a CNV in XPO4 is a critical mediator of fibrosis severity and can be exploited as a therapeutic target for liver fibrosis.. ME and JG are supported by the Robert W. Storr Bequest to the Sydney Medical Foundation, University of Sydney; a National Health and Medical Research Council of Australia (NHMRC) Program Grant (APP1053206) and Project and ideas grants (APP2001692, APP1107178 and APP1108422). AB is supported by an Australian Government Research Training Program (RTP) scholarship. EB is supported by Horizon 2020 under grant 634413 for the project EPoS.

Topics: Adult; Animals; Cell Line; DNA Copy Number Variations; Fatty Liver; Female; Humans; Karyopherins; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Middle Aged; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta

Nonalcoholic Fatty Liver Disease (NAFLD) is a common cause of chronic liver disease in childhood and strongly associated with obesity. Routine biochemical non-invasive tests remain with low accuracy for diagnosis of NAFLD. We performed a cross-sectional study to examine potential associations between anthropometric and biochemical parameters, specially TGF-β, a prognosis marker for hepatic steatosis (HS). Between May and October 2019, seventy-two overweight adolescents were enrolled, of which 36 had hepatic steatosis. Hepatic, lipidic and glycemic profiles, and levels of vitamin D, ferritin and TGF-β were analyzed. Hierarchical cluster and a discriminant model using canonical correlations were employed to depict the overall expression profile of biochemical markers and the biochemical degree of perturbation. Median values of alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), and TGF-β were higher in the adolescents with HS. Values of body mass index (BMI)/age and ALT, but not of TGF-β, were gradually increased proportionally to augmentation of steatosis severity. In a multivariate analysis, TGF-β plasma concentrations were associated with occurrence of hepatic steatosis independent of other covariates. Discriminant analysis confirmed that TGF-β concentrations can identify HS cases. Our data reveal that HS patients exhibit a distinct biosignature of biochemical parameters and imply TGF-β as an important biomarker to evaluate risk of steatosis development.

Topics: Adolescent; Alanine Transaminase; Biomarkers; Child; Cross-Sectional Studies; Fatty Liver; Female; gamma-Glutamyltransferase; Humans; Male; Pediatric Obesity; Risk; Severity of Illness Index; Transforming Growth Factor beta

Hepatitis C virus (HCV) infection promotes metabolic disorders, and the severity of lipogenic disease depends upon the infecting virus genotype. Here, we have examined HCV genotype 1-, 2-, or 3-specific regulation of lipid metabolism, involving transforming growth factor β (TGF-β)-regulated phospho-Akt (p-Akt) and peroxisome proliferator-activated receptor alpha (PPARα) axes. Since HCV core protein is one of the key players in metabolic regulation, we also examined its contribution in lipid metabolic pathways. The expression of regulatory molecules, TGF-β1/2, phospho-Akt (Ser473), PPARα, sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FASN), hormone-sensitive lipase (HSL), and acyl dehydrogenases was analyzed in virus-infected hepatocytes. Interestingly, HCV genotype 3a exhibited much higher activation of TGF-β and p-Akt, with a concurrent decrease in PPARα expression and fatty acid oxidation. A significant and similar decrease in HSL, unlike in HCV genotype 1a, was observed with both genotypes 2a and 3a. Similar observations were made from ectopic expression of the core genomic region from each genotype. The key role of TGF-β was further verified using specific small interfering RNA (siRNA). Together, our results highlight a significant difference in TGF-β-induced activity for the HCV genotype 2a- or 3a-induced lipogenic pathway, exhibiting higher triglyceride synthesis and a decreased lipolytic mechanism. These results may help in therapeutic modalities for early treatment of HCV genotype-associated lipid metabolic disorders.

Topics: Cell Line; Fatty Acid Synthase, Type I; Fatty Liver; Genotype; Hep G2 Cells; Hepacivirus; Hepatitis C; Hepatitis C, Chronic; Hepatocytes; Humans; Lipid Metabolism; Lipids; Lipogenesis; Liver Cirrhosis; PPAR alpha; Proto-Oncogene Proteins c-akt; Sterol Esterase; Sterol Regulatory Element Binding Protein 1; Transforming Growth Factor beta

Adipose tissue fibrosis is associated with inflammation and insulin resistance in human obesity. In particular, visceral fat fibrosis is correlated with hyperlipidemia and ectopic fat accumulation. Myocardin-related transcription factor A (MRTFA) is an important coactivator that mediates the transcription of extracellular matrix and other fibrogenic genes. Here, we examine the role of MRTFA in the development of adipose tissue fibrosis and identify a signaling pathway that regulates the fate of vascular progenitors. We demonstrate that obesity induces the formation of Sca1

Topics: Adipocytes; Adipose Tissue; Animals; Cell Differentiation; Chronic Disease; Energy Metabolism; Fatty Liver; Fibrosis; Hypertrophy; Inflammation; Insulin Resistance; Integrin alpha5; Mice, Inbred C57BL; Models, Biological; Myofibroblasts; Obesity; Stem Cells; Trans-Activators; Transforming Growth Factor beta

Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor β1 (TGFβ1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFβ1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding specificity is a promising candidate for a potentially simple therapeutic approach for the prevention and treatment of obesity and associated complications.

Topics: Adiposity; Animals; Azo Compounds; Benzoflavones; Diet, Western; Fatty Liver; Hepatocytes; Indoleamine-Pyrrole 2,3,-Dioxygenase; Intra-Abdominal Fat; Kynurenine; Lipids; Lipoproteins, LDL; Male; Mice; Mice, Inbred C57BL; Obesity; Pyrazoles; Receptors, Aryl Hydrocarbon; Signal Transduction; Toll-Like Receptor 2; Transforming Growth Factor beta

Obesity is one of the major risk factors for nonalcoholic fatty liver disease (NAFLD), and NAFLD is highly associated with an increased risk of cardiovascular disease (CVD). Scholars have suggested that certain probiotics may significantly impact cardiovascular health, particularly certain Lactobacillus species, such as Lactobacillus reuteri GMNL-263 (Lr263) probiotics, which have been shown to reduce obesity and arteriosclerosis in vivo. In the present study, we examined the potential of heat-killed bacteria to attenuate high fat diet (HFD)-induced hepatic and cardiac damages and the possible underlying mechanism of the positive effects of heat-killed Lr263 oral supplements. Heat-killed Lr263 treatments (625 and 3125 mg/kg-hamster/day) were provided as a daily supplement by oral gavage to HFD-fed hamsters for eight weeks. The results show that heat-killed Lr263 treatments reduce fatty liver syndrome. Moreover, heat-killed Lactobacillus reuteri GMNL-263 supplementation in HFD hamsters also reduced fibrosis in the liver and heart by reducing transforming growth factor β (TGF-β) expression levels. In conclusion, heat-killed Lr263 can reduce lipid metabolic stress in HFD hamsters and decrease the risk of fatty liver and cardiovascular disease.

Topics: Animals; Cricetinae; Diet, High-Fat; Fatty Liver; Fibrosis; Heart Diseases; Limosilactobacillus reuteri; Male; Obesity; Probiotics; Transforming Growth Factor beta

Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high-cholesterol (HC), methionine-choline-deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high-fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll-like receptor 4 protein (TLR4) levels through suppression of the endosomal-lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)β-induced activation by down-regulating the expression of bone morphogenetic protein and activin membrane-bound inhibitor. Mammalian-cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element-binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage-activating protein (Scap) to insulin-induced gene (Insig) disrupted the SREBP2-mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig-1 down-regulation. In addition, the suppression of peroxisome proliferator-activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA-33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFβ-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH.. These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH.

Topics: Animals; Cholesterol; Disease Models, Animal; Down-Regulation; Fatty Liver; Hepatic Stellate Cells; Liver Cirrhosis; Macrophage Activation; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; MicroRNAs; PPAR gamma; Receptors, LDL; RNA, Messenger; Signal Transduction; Sterol Regulatory Element Binding Protein 2; Toll-Like Receptor 4; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor beta (TGF-β) signaling activates Smad- and TGF-β-activated kinase 1 (TAK1)-dependent signaling to regulate cell survival, proliferation, fibrosis, and tumorigenesis. The effects of TGF-β signaling on metabolic syndrome, including nonalcoholic fatty liver disease, remain elusive. Wild-type (WT) and hepatocyte-specific TGF-β receptor type II-deficient (Tgfbr2ΔHEP) mice were fed a choline-deficient amino acid (CDAA)-defined diet for 22 weeks to induce NASH. WT mice fed a CDAA diet displayed increased activation of Smad2/3 and had marked lipid accumulation, inflammatory cell infiltration, hepatocyte death, and fibrosis; in comparison, Tgfbr2ΔHEP mice fed a CDAA diet had suppressed liver steatosis, inflammation, and fibrosis. Both palmitate-induced steatotic hepatocytes and hepatocytes isolated from WT mice fed a CDAA diet had increased susceptibility to TGF-β-mediated death. TGF-β-mediated death in steatotic hepatocytes was inhibited by silencing Smad2 or blocking reactive oxygen species (ROS) production and was enhanced by inhibiting TAK1 or nuclear factor kappa B. Increased hepatic steatosis in WT mice fed a CDAA diet was associated with the increased expression of lipogenesis genes (Dgat1 and Srebp1c), whereas the decreased steatosis in Tgfbr2ΔHEP mice was accompanied by the increased expression of genes involved in β-oxidation (Cpt1 and Acox1). In combination with palmitate treatment, TGF-β signaling promoted lipid accumulation with induction of lipogenesis-related genes and suppression of β-oxidation-related genes in hepatocytes. Silencing Smad2 decreased TGF-β-mediated lipid accumulation and corrected altered gene expression related to lipid metabolism in hepatocytes. Finally, we confirmed that livers from patients with nonalcoholic steatohepatitis (NASH) displayed phosphorylation and nuclear translocation of Smad2/3.. TGF-β signaling in hepatocytes contributes to hepatocyte death and lipid accumulation through Smad signaling and ROS production that promote the development of NASH.

Topics: Animals; Apoptosis; Cells, Cultured; Choline Deficiency; Disease Models, Animal; Disease Progression; Fatty Liver; Gene Deletion; Hepatocytes; Lipid Metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Topics: Animals; Apoptosis; Fatty Liver; Hepatocytes; Lipid Metabolism; Male; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Apoptosis; Fatty Liver; Hepatocytes; Lipid Metabolism; Male; Signal Transduction; Transforming Growth Factor beta

The aim of the present study was to establish a progressive steatohepatitis mouse model because few reported animal models of non-alcoholic steatohepatitis (NASH) show the progression from fatty liver to steatohepatitis. C57BL/6N mice were fed a high-fat diet (HFD) to develop obesity and were either administered carbon tetrachloride (CCl4 ) eight times (0.05 mL/kg, s.c., once, followed by 0.1 mL/kg, s.c., seven times) or not. Serum parameters and hepatic histopathology were examined. In a separate experiment, CCl4 was administered subcutaneously from 0 to eight times to HFD-fed obese mice to investigate progressive changes. Markers of oxidative stress, inflammation and apoptosis, as well as histopathological changes in the liver, were analysed. The HFD-fed obese mice showed fatty liver but not steatohepatitis. In contrast, HFD-fed mice administered CCl4 eight times showed histopathological features of steatohepatitis (fatty liver, inflammation, hepatocellular ballooning and fibrosis) and increased serum alanine aminotransferase levels. However, the multiple administration of CCl4 to obese mice reduced the ratio of reduced glutathione to oxidized glutathione, superoxide dismutase activity and mitochondrial DNA copy number, leading to the development of chronic oxidative stress, increased numbers of apoptotic cells and increased levels of both tumour necrosis factor-α and transforming growth factor-β mRNA. The resulting inflammation led to increased hydroxyproline content in the liver and fibrosis. The present study demonstrates that multiple administration of CCl4 to HFD-fed obese mice induces chronic oxidative stress that triggers inflammation and apoptosis and leads to the development of fibrosis in the liver, resulting in progression from fatty liver to steatohepatitis. This murine model will be useful in the research of hepatic disorders.

Topics: Alanine Transaminase; Animals; Apoptosis; Carbon Tetrachloride; Diet, High-Fat; Disease Models, Animal; DNA Copy Number Variations; DNA, Mitochondrial; Fatty Liver; Fibrosis; Glutathione; Glutathione Disulfide; Inflammation; Liver; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Obesity; Oxidative Stress; RNA, Messenger; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Bovine pancreatic trypsin inhibitor (BPTI) has been reported to relieve liver ischemia-reperfusion-induced injury in rats.. This study was designed to determine whether the recombinant BPTI (rBPTI) can prevent the chronic liver injury induced by CCl4 in rats.. Fifty male Wistar rats were divided into five groups. Rats were treated with 40% CCl4 at a dose of 2 ml/kg body weight twice a week subcutaneously for 12 weeks. In the 8th week, they were administered intraperitoneally with rBPTI (80 MU/kg), BPTI (80 MU/kg) or hepatocyte growth-promoting factor (pHGF; 100 mg/kg) daily for the next 4 weeks.. rBPTI significantly prevented the disruption of liver function of alanine aminotransferase (ALT; 172.7 ± 18.16 versus 141.2 ± 15.28, p=0.003), aspartate aminotransferase (AST; 225.10 ± 36.54 versus 170.06 ± 27.14, p=0.007) and hydroxyproline (Hyp; 1.14 ± 0.27 versus 0.62 ± 0.17, p=0.001). rBPTI significantly decreased the level of thiobarbituric acid reactive substances (TBARS; 1.15 ± 0.16 versus 0.87 ± 0.15, p=0.003) and increased the activities of superoxide dismutase (SOD; 6.07 ± 0.95 versus 7.75 ± 1.12, p=0.007). rBPTI reduced the production of cytokines of IL-1β and TGF-β. The hepatocyte necrosis, fibrosis, fatty degeneration and inflammatory cell infiltration were ameliorated by rBPTI administration.. This study demonstrated that rBPTI exerted a hepatoprotective effect on chronic liver fibrosis induced by CCl4, which suggests that rBPTI may have the potential application for chronic liver injury induced by drugs metabolism and toxic substances.

Topics: Alanine Transaminase; Animals; Aprotinin; Aspartate Aminotransferases; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury, Chronic; Cytoprotection; Disease Models, Animal; Fatty Liver; Hydroxyproline; Inflammation Mediators; Interleukin-1beta; Liver; Liver Cirrhosis, Experimental; Male; Necrosis; Protective Agents; Rats; Rats, Wistar; Recombinant Proteins; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Transforming Growth Factor beta

Sympathetic nervous system (SNS) signalling regulates murine hepatic fibrogenesis through effects on hepatic stellate cells (HSC), and obesity-related hypertension with SNS activation accelerates progression of non-alcoholic fatty liver disease (NAFLD), the commonest cause of chronic liver disease. NAFLD may lead to cirrhosis. The effects of the SNS neurotransmitters norepinephrine (NE), epinephrine (EPI) and neuropeptide Y (NPY) on human primary HSC (hHSC) function and in NAFLD pathogenesis are poorly understood.. to determine the mechanistic effects of NE/EPI/NPY on phenotypic changes in cultured hHSC, and to study SNS signalling in human NAFLD livers.. Freshly isolated hHSC were assessed for expression of cathecholamine/neuropeptide Y receptors and for the synthesis of NE/EPI. The effects of NE/EPI/NPY and adrenoceptor antagonists prazosin (PRZ)/propranolol (PRL) on hHSC fibrogenic functions and the involved kinases and interleukin pathways were examined. Human livers with proven NAFLD were then assessed for upregulation of SNS signalling components.. Activated hHSC express functional α/β-adrenoceptors and NPY receptors, which are upregulated in the livers of patients with cirrhotic NAFLD. hHSC in culture synthesize and release NE/EPI, required for their optimal basal growth and survival. Exogenous NE/EPI and NPY dose-dependently induced hHSC proliferation, mediated via p38 MAP, PI3K and MEK signalling. NE and EPI but not NPY increased expression of collagen-1α2 via TGF-β without involvement of the pro-fibrogenic cytokines leptin, IL-4 and IL-13 or the anti-fibrotic cytokine IL-10.. hHSC synthesize and require cathecholamines for optimal survival and fibrogenic functionality. Activated hHSC express directly fibrogenic α/β-adrenoceptors and NPY receptors, upregulated in human cirrhotic NAFLD. Adrenoceptor and NPY antagonists may be novel anti-fibrotic agents in human NAFLD.

Topics: Base Sequence; Catecholamines; Cells, Cultured; Chromatography, High Pressure Liquid; Collagen; DNA Primers; Fatty Liver; Hepatic Stellate Cells; Humans; Interleukins; Liver Cirrhosis; Neuropeptide Y; Non-alcoholic Fatty Liver Disease; Norepinephrine; Receptors, Adrenergic; Reverse Transcriptase Polymerase Chain Reaction; Sympathetic Nervous System; Transforming Growth Factor beta; Up-Regulation

Cytokines play important roles in all stages of steatohepatitis, including hepatocyte injury, the inflammatory response, and the altered function of sinusoidal cells. This study examined the involvement of a major inflammatory cytokine, interferon-γ (IFN-γ), in the progression of steatohepatitis. In a steatohepatitis model by feeding a methionine- and choline-deficient high-fat (MCDHF) diet to both wild-type and IFN-γ-deficient mice, the liver histology, expression of genes encoding inflammatory cytokines, and fibrosis-related markers were examined. To analyze the effects of IFN-γ on Kupffer cells in vitro, we examined the tumor necrosis factor-α (TNF-α) production by a mouse macrophage cell line. Forty two days of MCDHF diet resulted in weight loss, elevated aminotransferases, liver steatosis, and inflammation in wild-type mice. However, the IFN-γ-deficient mice exhibited less extensive changes. RT-PCR revealed that the expression of tumor necrosis factor-α (TNF-α), transforming growth factor-β, inducible nitric oxide synthase, interleukin-4 and osteopontin were increased in wild-type mice, although they were suppressed in IFN-γ-deficient mice. Seventy days of MCDHF diet induced much more liver fibrosis in wild-type mice than in IFN-γ-deficient mice. The expression levels of fibrosis-related genes, α-smooth muscle actin, type I collagen, tissue inhibitor of matrix metalloproteinase-1, and matrix metalloproteinase-2, were dramatically increased in wild-type mice, whereas they were significantly suppressed in IFN-γ-deficient mice. Moreover, in vitro experiments showed that, when RAW 264.7 macrophages were treated with IFN-γ, they produced TNF-α in a dose-dependent manner. The present study showed that IFN-γ deficiency might inhibit the inflammatory response of macrophages cells and subsequently suppress stellate cell activation and liver fibrosis. These findings highlight the critical role of IFN-γ in the progression of steatohepatitis.

Topics: Animals; Cell Line; Choline Deficiency; Diet, High-Fat; Disease Models, Animal; Fatty Liver; Gene Expression Profiling; Hepatic Stellate Cells; Inflammation; Interferon-gamma; Kupffer Cells; Liver; Liver Cirrhosis; Macrophages; Male; Methionine; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent, chronic liver diseases, worldwide. It is a multifactorial disease caused by complex interactions between genetic, epigenetic and environmental factors. Recently, several microRNAs, some of which epigenetically regulated, have been found to be up- and/or down-regulated during NAFLD development. However, in NAFLD, the essential role of the Polycomb Group protein Enhancer of Zeste Homolog 2 (EZH2), which controls the epigenetic silencing of specific genes and/or microRNAs by trimethylating Lys27 on histone H3, still remains unknown. In this study, we demonstrate that the nuclear expression/activity of the EZH2 protein is down-regulated both in livers from NAFLD rats and in the free fatty acid-treated HepG2. The drop in EZH2 is inversely correlated with: (i) lipid accumulation; (ii) the expression of pro-inflammatory markers including TNF-α and TGF-β; and (iii) the expression of miR-200b and miR-155. Consistently, the pharmacological inhibition of EZH2 by 3-Deazaneplanocin A (DZNep) significantly reduces EZH2 expression/activity, while it increases lipid accumulation, inflammatory molecules and microRNAs. In conclusion, the results of this study suggest that the defective activity of EZH2 can enhance the NAFLD development by favouring steatosis and the de-repression of the inflammatory genes and that of specific microRNAs.

Topics: Adenosine; Animals; Disease Models, Animal; Down-Regulation; Enhancer of Zeste Homolog 2 Protein; Fatty Liver; Hep G2 Cells; Histones; Humans; MicroRNAs; Non-alcoholic Fatty Liver Disease; Oleic Acid; Palmitic Acid; Polycomb Repressive Complex 2; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The innate immune system, including toll-like receptor-4 (TLR4) signaling cascade and angiotensin-II (AT-II) play important roles in the progression of liver fibrosis development; the cross talk between TLR4 and AT-II has not been elucidated yet. The aim of the current study was to elucidate the effect of AT-II type 1 receptor blocker (ARB), on the liver fibrosis development, especially in conjunction with the interaction of TLR4 and AT-II in the rat model of non-alcoholic steatohepatitis.. Fischer 344 rats were fed a choline-deficient, L-amino-acid-defined diet for 8 weeks and the effects of losartan were elucidated in conjunction with activated hepatic stellate cells (Ac-HSC) activation, TLR4, nuclear factor-κB (NF-κB), and transforming growth factor-β (TGF-β) expressions. In vitro study was carried out to elucidate the effect of AT-II on several indices including TLR4, myeloid differentiation factor 88, NF-κB, and TGF-β expressions in the rat HSC.. ARB markedly inhibited liver fibrosis development along with suppression of the number of Ac-HSC and TGF-β. These inhibitory effects of ARB were almost in parallel with suppression of the hepatic TLR4 and NF-κB expressions. This in vitro study showed that AT-II significantly augmented the TLR4 expression in a dose- and time-dependent manner via AT-II type 1 receptor in the Ac-HSC. AT-II also augmented the lipopolysaccharide-induced myeloid differentiation factor 88 (MyD88), NF-κB, and TGF-β and these increments were attenuated by treatment with ARB.. These studies indicated that the cross talk between TLR4 signaling cascade and AT-II plays a pivotal role in liver fibrosis development in non-alcoholic steatohepatitis.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Cell Communication; Disease Models, Animal; Disease Progression; DNA Primers; Dose-Response Relationship, Drug; Fatty Liver; Gene Expression; Hepatic Stellate Cells; Liver Cirrhosis; Losartan; Male; NF-kappa B; Non-alcoholic Fatty Liver Disease; Rats; Rats, Inbred F344; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Toll-Like Receptor 4; Transforming Growth Factor beta

Chemokine-like receptor 1 (CMKLR1) ligands chemerin and resolvin E1 are suggested to have a role in non-alcoholic fatty liver disease (NAFLD). Here, expression of CMKLR1 in liver cells and NAFLD was studied. CMKLR1 was detected in primary human hepatocytes (PHH), Kupffer cells, bile-duct cells and hepatic stellate cells. In human and rodent fatty liver and in fibrotic liver of mice fed a methionine-choline deficient diet CMKLR1 was reduced. Hepatocytes are the major cells in the liver and effects of adipokines, cytokines and lipids on CMKLR1 in PHH were analyzed. Increased cellular triglyceride or cholesterol content, lipopolysaccharide, IL-6, TNF and leptin did not influence CMKLR1 levels in PHH whereas profibrotic TGFβ tended to reduce CMKLR1. Adiponectin strongly upregulated CMKLR1 mRNA and protein in PHH and hepatic CMKLR1 when injected into wild type mice. Further, CMKLR1 was suppressed in the liver of adiponectin deficient mice. These data indicate that low CMKLR1 in NAFLD may partly result from reduced adiponectin activity.

Topics: Adiponectin; Aged; Animals; Bile Ducts; Choline; Diet; Fatty Liver; Female; Hepatic Stellate Cells; Hepatocytes; Humans; Kupffer Cells; Liver; Male; Methionine; Mice; Middle Aged; Non-alcoholic Fatty Liver Disease; Primary Cell Culture; Receptors, Chemokine; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in tumor metastasis and carcinogenesis. Although the expression of GnT-V is induced in chronic liver diseases, the biological meaning of GnT-V in the diseases remains unknown. The aim of this study was to investigate the effects of GnT-V on the progression of chronic hepatitis, using GnT-V transgenic (Tg) mice fed a high fat and high cholesterol (HFHC) diet, an experimental model of murine steatohepatitis. Although enhanced hepatic lymphocytes infiltration and fibrosis were observed in wild-type (WT) mice fed the HFHC diet, they were dramatically prevented in Tg mice. In addition, the gene expression of inflammatory Th1 cytokines in the liver was significantly decreased in Tg mice than WT mice. Inhibition of liver fibrosis was due to the dysfunction of hepatic stellate cells (HSCs), which play pivotal roles in liver fibrosis through the production of transforming growth factor (TGF)-β1. Although TGF-β1 signaling was enhanced in Tg mouse-derived HSCs (Tg-HSCs) compared with WT mouse-derived HSCs (WT-HSCs), collagen expression was significantly reduced in Tg-HSCs. As a result from DNA microarray, cyclooxygenase-2 (COX2) expression, known as a negative feedback signal for TGF-β1, was significantly elevated in Tg-HSCs compared with WT-HSCs. Prostaglandin E2 (PGE2), the product of COX2, production was also significantly elevated in Tg-HSCs. COX2 inhibition by celecoxib decreased PGE2 and increased collagen expression in Tg-HSCs. In conclusion, GnT-V prevented steatohepatitis progression through modulating lymphocyte and HSC functions.

Topics: Animals; Celecoxib; Cyclooxygenase 2; Fatty Liver; Hepatic Stellate Cells; Male; Mice; Mice, Transgenic; N-Acetylgalactosaminyltransferases; Pyrazoles; Sulfonamides; Transforming Growth Factor beta

Non-alcoholic steatohepatitis (NASH) is typically associated with pro-apoptotic caspase activation. A potential role for pro-inflammatory caspases remains incompletely understood. Our aims were to examine a potential role of caspase-1 in the development of liver damage and fibrosis in NASH. C57BL/6 wild type (WT) developed marked steatohepatitis, activation, fibrosis and increased hepatic caspase-1 and interleukin-1β expression when placed on the methionine- and choline-deficient (MCD) diet. Marked caspase-1 activation was detected in the liver of MCD-fed mice. Hepatocyte and non-parenchymal fractionation of the livers further demonstrated that caspase-1 activation after MCD feeding was mainly localized to non-parenchymal cells. Caspase-1-knockout (Casp1(-/-)) mice on the MCD diet showed marked reduction in mRNA expression of genes involved in inflammation and fibrogenesis (tumor necrosis factor-α was 7.6-fold greater in WT vs Casp1(-/-) MCD-fed mice; F4/80 was 1.5-fold greater in WT vs Casp1(-/-) MCD-fed mice; α-smooth muscle actin was 3.2-fold greater in WT vs Casp1(-/-) MCD-fed mice; collagen 1-α was 7.6-fold greater in WT vs Casp1(-/-) MCD-fed mice; transforming growth factor-β was 2.4-fold greater in WT vs Casp1(-/-) MCD-fed mice; cysteine- and glycine-rich protein 2 was 3.2-fold greater in WT vs Casp1(-/-) MCD-fed mice). Furthermore, Sirius red staining for hepatic collagen deposition was significantly reduced in Casp1(-/-) MCD-fed mice compared with WT MCD-fed animals. However, serum alanine aminotransferase levels, caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells were similar in Casp1(-/-) and WT mice on the MCD diet. Selective Kupffer cell depletion by clodronate injection markedly suppressed MCD-induced caspase-1 activation and protected mice from fibrogenesis and fibrosis associated with this diet. The conclusion of this study is that it uncovers a novel role for caspase-1 in inflammation and fibrosis during NASH development.

Topics: Actins; Animals; Antigens, Differentiation; Caspase 1; Caspase 3; Choline Deficiency; Clodronic Acid; Collagen Type I; Fatty Liver; Hepatic Stellate Cells; Hepatocytes; Inflammation; Interleukin-1beta; Kupffer Cells; LIM Domain Proteins; Liver; Liver Cirrhosis; Methionine; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Proteins; Nuclear Proteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

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.

Topics: Animals; Chemokine CCL2; Collagen Type I; Dietary Fats; Disease Models, Animal; Fatty Liver; Hepatic Stellate Cells; Liver Cirrhosis; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Species Specificity; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

The tumor suppressor p53 is a primary sensor of stressful stimuli, controlling a number of biologic processes. The aim of our study was to examine the roles of p53 in non-alcoholic steatohepatitis (NASH).. Male wild type and p53-deficient mice were fed a methionine- and choline-deficient diet for 8 weeks to induce nutritional steatohepatitis. mRNA expression profiles in normal liver samples and liver samples from patients with non-alcoholic liver disease (NAFLD) were also evaluated.. Hepatic p53 and p66Shc signaling was enhanced in the mouse NASH model. p53 deficiency suppressed the enhanced p66Shc signaling, decreased hepatic lipid peroxidation and the number of apoptotic hepatocytes, and ameliorated progression of nutritional steatohepatitis. In primary cultured hepatocytes, transforming growth factor (TGF)-β treatment increased p53 and p66Shc signaling, leading to exaggerated reactive oxygen species (ROS) accumulation and apoptosis. Deficient p53 signaling inhibited TGF-β-induced p66Shc signaling, ROS accumulation, and hepatocyte apoptosis. Furthermore, expression levels of p53, p21, and p66Shc were significantly elevated in human NAFLD liver samples, compared with results obtained with normal liver samples. Among NAFLD patients, those with NASH had significantly higher hepatic expression levels of p53, p21, and p66Shc compared with the group with simple steatosis. A significant correlation between expression levels of p53 and p66Shc was observed.. p53 in hepatocytes regulates steatohepatitis progression by controlling p66Shc signaling, ROS levels, and apoptosis, all of which may be regulated by TGF-β. Moreover, p53/p66Shc signaling in the liver appears to be a promising target for the treatment of NASH.

Topics: Animals; Apoptosis; Caspase 3; Choline Deficiency; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Disease Progression; Fatty Liver; Hepatocytes; Humans; Male; Methionine; Mice; Mice, Inbred C57BL; Mice, Knockout; Non-alcoholic Fatty Liver Disease; Primary Cell Culture; Proto-Oncogene Proteins p21(ras); Reactive Oxygen Species; RNA, Messenger; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Up-Regulation

Loss of signal transducer and activator of transcription 5 (STAT5) from liver tissue results in steatosis and enhanced cell proliferation. This study demonstrates that liver-specific Stat5-null mice develop severe hepatic steatosis as well as hepatocellular carcinomas at 17 months of age, even in the absence of chemical insults. To understand STAT5's role as a tumor suppressor, we identified and investigated new STAT5 target genes. Expression of Nox4, the gene encoding the reactive oxygen species (ROS)-generating enzyme NOX4, was induced by growth hormone through STAT5. In addition, the genes encoding the proapoptotic proteins PUMA and BIM were induced by growth hormone through STAT5, which bound to GAS motifs in the promoter regions of all three genes. We further show that STAT5-induced activation of Puma and Bim was dependent on NOX4. Treatment of mice with transforming growth factor-β, an inducer of apoptosis, resulted in cleaved caspase-3 in control but not in liver-specific Stat5-null mice. This study demonstrates for the first time that cytokines through STAT5 regulate the expression of the ROS-generating enzyme NOX4 and key proapoptotic proteins.. STAT5 harnesses several distinct signaling pathways in the liver and thereby functions as a tumor suppressor. Besides suppressing the activation of STAT3, STAT5 induces the expression of proapoptotic genes and the production of ROS.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carbon Tetrachloride; Carcinoma, Hepatocellular; Caspase 3; Cells, Cultured; Fatty Liver; Fibroblasts; Gene Expression Regulation, Neoplastic; Growth Hormone; Hepatocytes; Liver; Liver Neoplasms; Membrane Proteins; Mice; NADPH Oxidase 4; NADPH Oxidases; Promoter Regions, Genetic; Proto-Oncogene Proteins; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; STAT5 Transcription Factor; Transforming Growth Factor beta; Tumor Suppressor Proteins

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is among the most potent environmentally toxic compounds. Serum metabolomics identified azelaic acid monoesters as significantly increased metabolites after TCDD treatment, due to downregulation of hepatic carboxylesterase 3 (CES3, also known as triglyceride hydrolase) expression in an arylhydrocarbon receptor (AhR)-dependent manner in mice. The decreased CES3 expression was accomplished by TCDD-stimulated TGFβ-SMAD3 and IL6-STAT3 signaling, but not by direct AhR signaling. Methionine- and choline-deficient (MCD) diet-treated mice also showed enhanced serum azelaic acid monoester levels after attenuation of hepatic CES3 expression, while db/db mice did not, thus suggesting an association with steatohepatitis. Forced expression of CES3 reversed serum azelaic acid monoester/azelaic acid ratios and hepatic TGFβ mRNA levels in TCDD- and MCD diet-treated mice and ameliorated steatohepatitis induced by MCD diet. These results support the view that azelaic acid monoesters are possible indicators of TCDD exposure and steatohepatitis and suggest a link between CES3, TGFβ, and steatohepatitis.

Topics: Animals; Carboxylic Ester Hydrolases; Cells, Cultured; Dicarboxylic Acids; Diet; Fatty Liver; Hepatocytes; Inflammation; Interleukin-6; Male; Metabolomics; Mice; Mice, Inbred C57BL; Mice, Obese; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta

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.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Fatty Liver; Lipid Peroxidation; Liver; Liver Function Tests; Male; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Plant Extracts; Rats; Rats, Inbred Strains; Real-Time Polymerase Chain Reaction; Seeds; Silybum marianum; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

In the present study, we evaluated the ability of Teucrium polium ethyl acetate fraction, with high antioxidant activity, in the treatment of nonalcoholic steatohepatitis (NASH) in rats and its possible effect on factors involved in pathogenesis of the disease. 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 2 groups: NASH group that received MCD diet and NASH + T group which was fed MCD diet plus ethyl acetate fraction of T. polium orally for 3 weeks. Histopathological evaluations revealed that treatment with the extract has abated the severity of NASH among the MCD-fed rats. In addition, the fraction reduced the elevated levels of hepatic tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) gene expression and also the elevated level of malondialdehyde (MDA). In addition, the extract increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and enhanced the level of hepatic glutathione (GSH). Moreover, the fraction treatments lowered caspase-3 level and the phosphorylated form of C-Jun N-terminal kinase (JNK) and augmented the phosphorylated level of extracellular regulated kinase1/2 (ERK1/2). These results indicate that the ethyl acetate fraction of T. poium effectively reversed NASH, mainly due to its strong antioxidant and anti-inflammatory properties.

Topics: Animals; Antioxidants; Caspase 3; Choline Deficiency; Fatty Liver; Gene Expression; Glutathione Peroxidase; Liver; Male; Malondialdehyde; MAP Kinase Kinase 4; Methionine; Plant Extracts; Rats; Rats, Inbred Strains; Superoxide Dismutase; Teucrium; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The identification of the cellular and molecular pathways that mediate the development of non-alcoholic steatohepatitis is of crucial importance. Cytokines produced by liver-resident and infiltrating inflammatory cells, play a pivotal role in liver inflammation. The role of the proinflammatory cytokines IL-1α and IL-1β in steatohepatitis remains elusive.. We employed IL-1α and IL-1β-deficient mice and transplanted marrow cells to study the role of liver-resident and bone marrow-derived IL-1 in steatosis and its progression to steatohepatitis.. Atherogenic diet-induced steatohepatitis in wild-type mice was associated with 16 and 4.6 fold-elevations in mRNA levels of hepatic IL-1α and IL-1β, respectively. In mice deficient in either IL-1α or IL-1β the transformation of steatosis to steatohepatitis and liver fibrosis was markedly reduced. This protective effect in IL-1α-deficient mice was noted despite increased liver cholesterol levels. Deficiency of IL-1α markedly reduced plasma serum amyloid A and steady-state levels of mRNA coding for inflammatory genes (P-selectin, CXCL1, IL-6, and TNFα) as well as pro-fibrotic genes (MMP-9 and Collagen) and particularly a 50% decrease in TGFβ levels (p = 0.004). IL-1α mRNA levels were two-folds lower in IL-1β-deficient mice, and IL-1β transcripts were three-folds lower in IL-1α-deficient compared to wild-type mice. Hepatic cell derived IL-1α rather than from recruited bone marrow-derived cells was required for steatohepatitis development.. These data demonstrate the critical role of IL-1α and IL-1β in the transformation of steatosis to steatohepatitis and liver fibrosis in hypercholesterolemic mice. Therefore, the potential of neutralizing IL-1α and/or IL-1β to inhibit the development of steatohepatitis should be explored.

Topics: Analysis of Variance; Animals; Chemokine CXCL1; Collagen; Diet, Atherogenic; Disease Progression; Fatty Liver; Gene Expression; Hepatitis; Hypercholesterolemia; Interleukin-1; Interleukin-1alpha; Interleukin-1beta; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; P-Selectin; RNA, Messenger; Serum Amyloid A Protein; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Connective tissue growth factor (CTGF) is induced in liver fibrosis and enhances the activity of transforming growth factor β (TGFβ). Recently we have shown that the hepatoprotective adipokine adiponectin downregulates CTGF in primary human hepatocytes (PHH). In the current study, the mechanisms mediating suppression of CTGF by adiponectin and the well described downstream effector of adiponectin receptor 2 (AdipoR2), peroxisome proliferator activated receptor α (PPARα), were analyzed in more detail. Adiponectin downregulated CTGF mRNA and protein in primary human hepatocytes (PHH) and suppression was blocked by a PPARα antagonist indicating that AdipoR2 is involved. The PPARα agonists fenofibrate and WY14643 also reduced CTGF protein in these cells. Adiponectin further impaired TGFβ-mediated upregulation of CTGF. Phosphorylation of the TGFβ downstream effectors SMAD2 and -3 was reduced in PHH incubated with adiponectin or PPARα agonists suggesting that early steps in TGFβ signal transduction are impaired. CTGF and TGFβ mRNA levels were increased in human non-fibrotic non-alcoholic steatohepatitis (NASH), and here AdipoR2 expression was significantly reduced. Current data show that CTGF and TGFβ are already induced in non-fibrotic NASH and this may be partly explained by low adiponectin bioactivity which interferes with TGFβ signaling by reducing phosphorylation of SMAD2/3 and by downregulating CTGF.

Topics: Adiponectin; Anticholesteremic Agents; Connective Tissue Growth Factor; Down-Regulation; Fatty Liver; Female; Fenofibrate; Hepatocytes; Humans; Male; Non-alcoholic Fatty Liver Disease; Phosphorylation; PPAR alpha; Primary Cell Culture; Pyrimidines; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Mechanisms associated with the progression of simple steatosis to non-alcoholic fatty liver disease (NAFLD) remain undefined. Regulatory T cells (T(regs)) play a critical role in regulating inflammatory processes in non-alcoholic steatohepatitis (NASH) and because T helper type 17 (Th17) functionally oppose T(reg)-mediated responses, this study focused on characterizing the role of Th17 cells using a NAFLD mouse model. C57BL/6 mice were fed either a normal diet (ND) or high fat (HF) diet for 8 weeks. Mice in the HF group had a significantly higher frequency of liver Th17 cells compared to ND-fed mice. Neutralization of interleukin (IL)-17 in HF mice ameliorated lipopolysaccharide (LPS)-induced liver injury reflected by decreased serum alanine aminotransferase (ALT) levels and reduced inflammatory cell infiltrates in the liver. In vitro, HepG2 cells cultured in the presence of free fatty acids (FFA; oleic acid and palmitic acid) for 24 h and IL-17 developed steatosis via insulin-signalling pathway interference. IL-17 and FFAs synergized to induce IL-6 production by HepG2 cells and murine primary hepatocytes which, in combination with transforming growth factor (TGF-β), expanded Th17 cells. It is likely that a similar process occurs in NASH patients, as there were significant levels of IL-17(+) cell infiltrates in NASH patient livers. The hepatic expression of Th17 cell-related genes [retinoid-related orphan receptor gamma (ROR)γt, IL-17, IL-21 and IL-23] was also increased significantly in NASH patients compared to healthy controls. Th17 cells and IL-17 were associated with hepatic steatosis and proinflammatory response in NAFLD and facilitated the transition from simple steatosis to steatohepatitis. Strategies designed to alter the balance between Th17 cells and T(regs) should be explored as a means of preventing progression to NASH and advanced liver diseases in NAFLD patients.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cells, Cultured; Diet, High-Fat; Electrophoretic Mobility Shift Assay; Fatty Acids; Fatty Liver; Gene Expression Regulation; Hep G2 Cells; Hepatocytes; Humans; Insulin; Interleukin-17; Interleukin-6; Lipopolysaccharides; Liver; Male; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Signal Transduction; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta

c-Jun N-terminal kinase (JNK) is activated by multiple profibrogenic mediators; JNK activation occurs during toxic, metabolic, and autoimmune liver injury. However, its role in hepatic fibrogenesis is unknown.. JNK phosphorylation was detected by immunoblot analysis and confocal immunofluorescent microscopy in fibrotic livers from mice after bile duct ligation (BDL) or CCl(4) administration and in liver samples from patients with chronic hepatitis C and non-alcoholic steatohepatitis. Fibrogenesis was investigated in mice given the JNK inhibitor SP600125 and in JNK1- and JNK2-deficient mice following BDL or CCl(4) administration. Hepatic stellate cell (HSC) activation was determined in primary mouse HSCs incubated with pan-JNK inhibitors SP600125 and VIII.. JNK phosphorylation was strongly increased in livers of mice following BDL or CCl(4) administration as well as in human fibrotic livers, occurring predominantly in myofibroblasts. In vitro, pan-JNK inhibitors prevented transforming growth factor (TGF) beta-, platelet-derived growth factor-, and angiotensin II-induced murine HSC activation and decreased platelet-derived growth factor and TGF-beta signaling in human HSCs. In vivo, pan-JNK inhibition did not affect liver injury but significantly reduced fibrosis after BDL or CCl(4). JNK1-deficient mice had decreased fibrosis after BDL or CCl(4), whereas JNK2-deficient mice displayed increased fibrosis after BDL but fibrosis was not changed after CCl(4). Moreover, patients with chronic hepatitis C who displayed decreased fibrosis in response to the angiotensin receptor type 1 blocker losartan showed decreased JNK phosphorylation.. JNK is involved in HSC activation and fibrogenesis and represents a potential target for antifibrotic treatment approaches.

Topics: Angiotensin II; Animals; Anthracenes; Carrier Proteins; Cell Division; Cells, Cultured; Disease Models, Animal; Fatty Liver; Fibroblasts; Hepatic Stellate Cells; Hepatitis C, Chronic; Humans; Liver Cirrhosis; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Mitogen-Activated Protein Kinase 9; Phosphorylation; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Transforming Growth Factor beta

Non-alcoholic steatohepatitis is associated with the deposition of lipid droplets in the liver, and is characterised histologically by the infiltration of inflammatory cells, hepatocellular degeneration and liver fibrosis. Oxidative stress may play an important role in the onset and deterioration of non-alcoholic steatohepatitis. We previously reported that an Eriobotrya japonica seed extract, extracted in 70% ethanol, exhibited antioxidant actions in vitro and in vivo. In this study, we examined the effect of this extract in a rat model of non-alcoholic steatohepatitis.. The seed extract was given in the drinking water to fats being fed a methionine-choline-deficient diet for 15 weeks.. Increases in alanine aminotransferase and aspartate aminotransferase levels were significantly inhibited in rats fed the seed extract compared with the group on the diet alone. Formation of fatty droplets in the liver was also inhibited. Antioxidant enzyme activity in liver tissue was higher than in the diet-only group and lipid peroxidation was reduced compared with rats that also received the extract. Expression of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal was lower in the rats given the seed extract than in the diet-only group. In the former, liver tissue levels of transforming growth factor-beta and collagen were also decreased.. Thus, the E. japonica seed extract inhibited fatty liver, inflammation and fibrosis, suggesting its usefulness in the treatment of non-alcoholic steatohepatitis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Antioxidants; Body Weight; Deoxyguanosine; Disease Models, Animal; Eriobotrya; Fatty Liver; Liver; Liver Cirrhosis, Experimental; Liver Function Tests; Male; Organ Size; Oxidative Stress; Plant Extracts; Rats; Rats, Wistar; Seeds; Transforming Growth Factor beta

Diets high in saturated fat and fructose have been implicated in the development of obesity and nonalcoholic steatohepatitis (NASH) in humans. We hypothesized that mice exposed to a similar diet would develop NASH with fibrosis associated with increased hepatic oxidative stress that would be further reflected by increased plasma levels of the respiratory chain component, oxidized coenzyme Q9 ((ox)CoQ9). Adult male C57Bl/6 mice were randomly assigned to chow, high-fat (HF), or high-fat high-carbohydrate (HFHC) diets for 16 weeks. The chow and HF mice had free access to pure water, whereas the HFHC group received water with 55% fructose and 45% sucrose (wt/vol). The HFHC and HF groups had increased body weight, body fat mass, fasting glucose, and were insulin-resistant compared with chow mice. HF and HFHC consumed similar calories. Hepatic triglyceride content, plasma alanine aminotransferase, and liver weight were significantly increased in HF and HFHC mice compared with chow mice. Plasma cholesterol (P < 0.001), histological hepatic fibrosis, liver hydroxyproline content (P = 0.006), collagen 1 messenger RNA (P = 0.003), CD11b-F4/80+Gr1+ monocytes (P < 0.0001), transforming growth factor beta1 mRNA (P = 0.04), and alpha-smooth muscle actin messenger RNA (P = 0.001) levels were significantly increased in HFHC mice. Hepatic oxidative stress, as indicated by liver superoxide expression (P = 0.002), 4-hydroxynonenal, and plasma (ox)CoQ9 (P < 0.001) levels, was highest in HFHC mice.. These findings demonstrate that nongenetically modified mice maintained on an HFHC diet in addition to developing obesity have increased hepatic ROS and a NASH-like phenotype with significant fibrosis. Plasma (ox)CoQ9 correlated with fibrosis progression. The mechanism of fibrosis may involve fructose inducing increased ROS associated with CD11b+F4/80+Gr1+ hepatic macrophage aggregation, resulting in transforming growth factor beta1-signaled collagen deposition and histologically visible hepatic fibrosis.

Topics: Animals; Body Composition; Collagen; Dietary Carbohydrates; Dietary Fats; Disease Models, Animal; Fatty Liver; Fructose; Insulin Resistance; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Obesity; Reactive Oxygen Species; Trans Fatty Acids; Transforming Growth Factor beta; Ubiquinone

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.

Topics: Animals; Chemokine CCL2; Choline Deficiency; Diet; Fatty Liver; Inflammation; Liver; Male; Methionine; Mice; Mice, Inbred C57BL; Mice, Knockout; Transforming Growth Factor beta

Nonalcoholic fatty liver disease (NAFLD) and its advanced stage, nonalcoholic steatohepatitis (NASH), are the most common causes of chronic liver disease in the United States. NASH features the metabolic syndrome, inflammation, and fibrosis. Probiotics exhibit immunoregulatory and anti-inflammatory activity. We tested the hypothesis that probiotic VSL#3 may ameliorate the methionine-choline-deficient (MCD) diet-induced mouse model of NASH. MCD diet resulted in NASH in C57BL/6 mice compared to methionine-choline-supplemented (MCS) diet feeding evidenced by liver steatosis, increased triglycerides, inflammatory cell accumulation, increased tumor necrosis factor alpha levels, and fibrosis. VSL#3 failed to prevent MCD-induced liver steatosis or inflammation. MCD diet, even in the presence of VSL#3, induced up-regulation of serum endotoxin and expression of the Toll-like receptor 4 signaling components, including CD14 and MD2, MyD88 adaptor, and nuclear factor kappaB activation. In contrast, VSL#3 treatment ameliorated MCD diet-induced liver fibrosis resulting in diminished accumulation of collagen and alpha-smooth muscle actin. We identified increased expression of liver peroxisome proliferator-activated receptors and decreased expression of procollagen and matrix metalloproteinases in mice fed MCD+VSL#3 compared to MCD diet alone. MCD diet triggered up-regulation of transforming growth factor beta (TGFbeta), a known profibrotic agent. In the presence of VSL#3, the MCD diet-induced expression of TGFbeta was maintained; however, the expression of Bambi, a TGFbeta pseudoreceptor with negative regulatory function, was increased. In summary, our data indicate that VSL#3 modulates liver fibrosis but does not protect from inflammation and steatosis in NASH. The mechanisms of VSL#3-mediated protection from MCD diet-induced liver fibrosis likely include modulation of collagen expression and impaired TGFbeta signaling.

Topics: Actins; Animals; Choline Deficiency; Collagen Type I; Diet; Disease Models, Animal; Fatty Liver; Female; Liver Cirrhosis; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Methionine; Mice; Mice, Inbred C57BL; NF-kappa B; Peroxisome Proliferator-Activated Receptors; Probiotics; Signal Transduction; Transforming Growth Factor beta

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease and is one of the most common liver diseases in the developed world. The histological findings of NASH are characterized by hepatic steatosis, inflammation, and fibrosis. However, an optimal treatment for NASH has not been established. Tranilast, N-(3',4'-dimethoxycinnamoyl)-anthranilic acid, is an antifibrogenic agent that inhibits the action of transforming growth factor beta (TGF-beta). This drug is used clinically for fibrogenesis-associated skin disorders including hypertrophic scars and scleroderma. TGF-beta plays a central role in the development of hepatic fibrosis, and tranilast may thus ameliorate the pathogenesis of NASH. We investigated the effects of tranilast using an established dietary animal model of NASH, obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and nondiabetic control Long-Evans Tokushima Otsuka (LETO) rats fed a methionine-deficient and choline-deficient diet. Treatment with 2% tranilast (420 mg/kg/day) for 8 weeks prevented the development of hepatic fibrosis and the activation of stellate cells, and down-regulated the expression of genes for TGF-beta and TGF-beta-target molecules, including alpha1 procollagen and plasminogen activator-1. In addition, tranilast attenuated hepatic inflammation and Kupffer cell recruitment, and down-regulated the expression of tumor necrosis factor alpha. Unexpectedly, tranilast ameliorated hepatic steatosis and up-regulated the expression of genes involved in beta-oxidation, such as peroxisome proliferator-activated receptor alpha and carnitine O-palmitoyltransferase-1. Most of these effects were observed in LETO rats and OLETF rats, which suggest that the action of tranilast is mediated through the insulin resistance-independent pathway.. Our findings suggest that targeting TGF-beta with tranilast represents a new mode of therapy for NASH.

Topics: Animals; Carnitine O-Palmitoyltransferase; Cell Line; Choline Deficiency; Diet; Fatty Acids; Fatty Liver; Interleukin-6; Kupffer Cells; Lipopolysaccharides; Liver; Liver Cirrhosis; Macrophages; Male; Methionine; ortho-Aminobenzoates; Oxidation-Reduction; Oxidative Stress; PPAR alpha; Rats; Rats, Inbred OLETF; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Non-alcoholic steatohepatitis (NASH), which is a common liver disease in industrialized countries, is associated with obesity, hypertension, and type-2 diabetes (metabolic syndrome). Since angiotensin II (ANG II) has been suggested to play an important role in liver inflammation and fibrosis, the purpose of this study was to investigate whether therapy against renin-angiotensin system (RAS) may provide some beneficial effect in liver of an animal model of metabolic syndrome.. For 6 months, obese Zucker rats (OZRs) were treated as follows: OZR-group, OZR + Perindopril (P) group, OZR + Irbesartan (IRB) group, OZR + Amlodipine (AML) group, and lean Zucker rats (LZRs) group as a control. Livers were evaluated by immunohistochemistry techniques using corresponding antibodies.. All treated groups showed a similar reduction in blood pressure compared to untreated OZR. Therapy either with IRB or P improves insulin sensitivity and reduces hepatic enzyme level with respect to untreated OZR. Conversely, AML failed to modify both parameters. Untreated OZR displayed higher hepatic ANG II levels and steatosis together with a marked increase in tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-beta1) level compared to LZR. Following RAS inhibition either by P or IRB, a significant reduction (P < 0.01) in the immunostaining of TNF-alpha, IL-6 and TGF-beta1 compared to untreated OZR was observed.. These results indicate that ANG II expression is increased in the liver of these animals with steatohepatitis. Furthermore, RAS control by either angiotensin-converting enzyme inhibition or AT1 receptor blockade seems to provide a beneficial modulation concerning the inflammatory response to liver injury in this model. Consequently, blockade of RAS could be a new approach to prevent or to treat patients with NASH.

Topics: Amlodipine; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Biphenyl Compounds; Disease Models, Animal; Fatty Liver; Interleukin-6; Irbesartan; Liver; Liver Cirrhosis; Male; Metabolic Syndrome; Obesity; Perindopril; Rats; Rats, Zucker; Renin-Angiotensin System; Tetrazoles; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

All major cell types in pancreatic islets express the transforming growth factor (TGF)-beta superfamily receptor ALK7, but the physiological function of this receptor has been unknown. Mutant mice lacking ALK7 showed normal pancreas organogenesis but developed an age-dependent syndrome involving progressive hyperinsulinemia, reduced insulin sensitivity, liver steatosis, impaired glucose tolerance, and islet enlargement. Hyperinsulinemia preceded the development of any other defect, indicating that this may be one primary consequence of the lack of ALK7. In agreement with this, mutant islets showed enhanced insulin secretion under sustained glucose stimulation, indicating that ALK7 negatively regulates glucose-stimulated insulin release in beta-cells. Glucose increased expression of ALK7 and its ligand activin B in islets, but decreased that of activin A, which does not signal through ALK7. The two activins had opposite effects on Ca(2+) signaling in islet cells, with activin A increasing, but activin B decreasing, glucose-stimulated Ca(2+) influx. On its own, activin B had no effect on WT cells, but stimulated Ca(2+) influx in cells lacking ALK7. In accordance with this, mutant mice lacking activin B showed hyperinsulinemia comparable with that of Alk7(-/-) mice, but double mutants showed no additive effects, suggesting that ALK7 and activin B function in a common pathway to regulate insulin secretion. These findings uncover an unexpected antagonism between activins A and B in the control of Ca(2+) signaling in beta-cells. We propose that ALK7 plays an important role in regulating the functional plasticity of pancreatic islets, negatively affecting beta-cell function by mediating the effects of activin B on Ca(2+) signaling.

Topics: Activin Receptors, Type I; Activins; Animals; Calcium; Fatty Liver; Gene Expression Regulation; Hyperinsulinism; Insulin; Insulin-Secreting Cells; Mice; Mice, Transgenic; Models, Biological; Pancreas; Signal Transduction; Transforming Growth Factor beta

Insulin resistance is a major pathological condition associated with obesity and metabolic syndrome. Insulin resistance and the renin-angiotensin system are intimately linked. We evaluated the role of the renin-angiotensin system in the pathogenesis of insulin resistance-associated, non-alcoholic steatohepatitis by using the angiotensin II type 1 receptor blocker olmesartan medoxomil in a diabetic rat model. The effects of olmesartan on methionine- and choline-deficient (MCD) diet-induced steatohepatitis were investigated in obese, diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and control Long-Evans Tokushima Otsuka (LETO) rats. Components of the renin-angiotensin system were up-regulated in the livers of OLETF rats, compared with LETO rats. In OLETF, but not LETO, rats, oral administration of olmesartan for 8 weeks ameliorated insulin resistance. Moreover, olmesartan suppressed MCD diet-induced hepatic steatosis and the hepatic expression of lipogenic genes (sterol regulatory element-binding protein-1c and fatty acid synthase) in OLETF, but not LETO, rats. In both OLETF and LETO rats, olmesartan inhibited hepatic oxidative stress (4-hydroxy-2-nonenal-modified protein) and expression of NADPH oxidase. Olmesartan also inhibited hepatic fibrosis, stellate cell activation, and expression of fibrogenic genes (transforming growth factor-beta, alpha 1 [I] procollagen, plasminogen activator inhibitor-1) in both OLETF and LETO rats. In conclusion, pharmacological blockade of the angiotensin II type 1 receptor slows the development of steatohepatitis in the OLETF rat model. This angiotensin II type 1 receptor blocker may exert insulin resistance-associated effects against hepatic steatosis and inflammation as well as direct effects against the generation of reactive oxygen species and fibrogenesis.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Choline Deficiency; Disease Models, Animal; Fatty Acids; Fatty Liver; Imidazoles; Insulin Resistance; Liver Cirrhosis, Experimental; Male; Methionine; Oxidative Stress; Rats; Rats, Long-Evans; RNA, Messenger; Sterol Regulatory Element Binding Protein 1; Tetrazoles; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accumulates and remains stable in the fatty tissues and liver of rodents for a long time. Considering the pronounced difference between species, long-term, low dose hepatic effects of TCDD were investigated after subcutaneous administration of TCDD into rhesus monkeys during pregnancy. Macroscopic and histopathological examination of the liver carried out 4 y after TCDD administration demonstrated intrahepatic focal fatty changes, infarction, hemorrhage, microthrombi-formation, sinusoidal ectasia, small hepatocyte hyperplasia, and increased number of alpha-smooth muscle actin (alpha-SMA)-positive cells. An electron microscopic study disclosed sinusoidal endothelial cell degeneration and injury in the liver of TCDD-treated monkeys. Western blot analysis showed downregulation of aryl hydrocarbon receptor (AhR) protein expression and decreased level of vascular endothelial (VE) cadherin but increased expression levels of CYP1A1 and transforming growth factor beta (TGF-beta) protein in the liver tissues. These changes observed in TCDD-exposed monkeys indicated sinusoidal endothelial cell injury and impairment in intrasinusoidal microcirculation. Infarction, focal fatty change, and microthrombi-formation are considered to be closely associated with intrahepatic circulatory impairment. Increased number of alpha-SMA-positive cells and decreased level of VE cadherin expression in the liver tissues might also be associated with sinusoidal endothelial cell injury. In addition, downregulation of AhR expression and increased CYP1A1 protein levels in the liver were consistent with persistent effects of TCDD. Although it has been reported that TCDD induced endothelial cell injury, this is the first report to describe vascular disorders and protein expression in the liver after injection with TCDD in a primate model.

Topics: Animals; Antigens, CD; Blotting, Northern; Cadherins; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP1A1; Endothelial Cells; Fatty Liver; Female; Hemorrhage; Infarction; Injections, Subcutaneous; Liver Diseases; Macaca mulatta; Microscopy, Electron; Muscle, Smooth; Polychlorinated Dibenzodioxins; Pregnancy; Receptors, Aryl Hydrocarbon; Thrombosis; Time Factors; Transforming Growth Factor beta

The increasing prevalence of nonalcoholic steatohepatitis (NASH) is due to the epidemic of obesity and type 2 diabetes, both of which are associated with insulin resistance.. To clarify the causal relationship between insulin resistance and the development of NASH, steatohepatitis was induced in obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) and nondiabetic control Long-Evans Tokushima Otsuka (LETO) rats by feeding them a methionine and choline-deficient (MCD) diet. Insulin sensitivity of the rats was altered by adding a high-fat (HF) diet or the peroxisomal-proliferator activated receptor-gamma agonist pioglitazone to the MCD diet.. The MCD diet-induced steatohepatitis was accelerated in OLETF rats after 8 weeks. Steatosis preceded inflammation, which led to fibrosis and the development of steatohepatitis. The hepatic gene expression for transforming growth factor-beta, alpha1 procollagen and plasminogen activator inhibitor-1 was up-regulated in OLETF rats compared with LETO rats. The MCD + HF diet further enhanced insulin resistance and led to rapid development of pre-cirrhosis in OLETF rats by increasing the triglyceride pool, activating stellate cells, and up-regulating gene expression for sterol regulatory element-binding protein-1c and fatty acid synthase in the liver. In contrast, pioglitazone attenuated the MCD diet-induced steatohepatitis in OLETF rats but not in LETO rats by reversing the underlying pathogenesis involved in this model through improvement of insulin resistance. These results confirm a link between insulin resistance and the development/progression of steatohepatitis, at least partly via up-regulation of genes for lipogenesis, inflammation, and fibrogenesis, in animal models.. Insulin resistance and/or diabetes may accelerate the entire pathologic spectrum of NASH.

Topics: Animal Feed; Animals; Choline; Collagen Type I; Collagen Type I, alpha 1 Chain; Diabetes Mellitus, Type 2; Dietary Fats; Disease Models, Animal; Fatty Liver; Hyperinsulinism; Hypoglycemic Agents; Insulin Resistance; Liver; Male; Methionine; Obesity; Pioglitazone; Plasminogen Activator Inhibitor 1; PPAR gamma; Rats; Rats, Inbred OLETF; Rats, Long-Evans; RNA, Messenger; Sterol Regulatory Element Binding Protein 1; Thiazolidinediones; Transforming Growth Factor beta; Up-Regulation

The aim of the present study was to evaluate the preventive role of genistein, a phytoestrogen with a wide variety of pharmacological effects, in an experimental non-alcoholic steatohepatitis (NASH) model.. Thirty-six Sprague-Dawley rats were divided into three groups. Group 1 (control) received only a standard rat diet, group 2 (placebo) was given a high fat diet (HFD) plus 0.5 mL/day saline subcutaneously, and group 3 (genistein group) a HFD plus subcutaneous genistein injection at dose of 0.2 mg/kg/day for 6 weeks. All rats were killed after 6 weeks. Serum aminotransferases, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and plasma and liver malondialdehyde (MDA) levels were measured. Additionally, steatosis, ballooning degeneration and inflammation of the liver were examined histopathologically.. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (P < 0.001 for each), plasma and liver tissue MDA and plasma TNF-alpha levels (P < 0.001, <0.001, <0.01, respectively) were found to be higher in the placebo group than in the control group. TGF-beta levels, however, were comparable in the placebo and control groups (P > 0.05). Histopathologically, steatosis, inflammatory cells per mm(2) and ballooning degeneration were significantly higher in the placebo group than in the control group (P < 0.001 for each). Nevertheless, AST and ALT (P < 0.05 for each), plasma and liver tissue MDA (P < 0.05 for each) and plasma TNF-alpha levels (P < 0.001) were significantly decreased in the genistein group compared to the placebo group. Histopathologically, steatosis (P < 0.05), inflammatory cells per mm(2) and ballooning degeneration (P < 0.01 for each) in the genistein group were also significantly lower than in the placebo group.. Genistein, a strong antioxidant agent, significantly decreased the plasma TNF-alpha level and remarkably prevented the emergence of NASH by improving the biochemical and histopathological abnormalities via attenuating oxidative stress.

Topics: Alanine Transaminase; Animals; Anti-Inflammatory Agents; Antioxidants; Aspartate Aminotransferases; Dietary Fats; Disease Models, Animal; Fatty Liver; Genistein; Hepatitis; Lipid Peroxidation; Liver; Male; Malondialdehyde; Organ Size; Oxidative Stress; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To investigate the efficacy of angiotensin II receptor antagonist on hepatic stellate cells (HSCs) activation in the patients with non-alcoholic steatohepatitis (NASH).. Seven patients with NASH were prescribed losartan, a selective angiotensin II type 1 receptor antagonist (50 mg/d) for 48 wk. Liver biopsies were performed both at the entry and end of the study in all patients. Quiescent and activated HSCs were identified by double immunostaining using anti-p75 and -smooth muscle actin antibodies, and the number of each phenotype was counted. Similarly, the liver specimens obtained from the eight patients with non-alcoholic fatty liver (NAFL) were also examined as controls.. In NASH hepatic tissues, activated HSCs were dominantly distributed as compared with those in NAFL. The 48-wk losartan treatment induced a remarkable decrease in activated HSCs and a mild increase in quiescent phenotypes.. Our data suggest the crucial involvement of HSCs in anti-fibrotic effect of angiotensin II receptor antagonist on patients with NASH.

Topics: Adult; Aged; Angiotensin II Type 1 Receptor Blockers; Fatty Liver; Female; Humans; Liver; Liver Cirrhosis; Losartan; Male; Middle Aged; Transforming Growth Factor beta; Transforming Growth Factor beta1

We evaluated the effects of bezafibrate, a peroxisome proliferator-activated receptor (PPAR) pan-agonist, and GW501516, a PPARdelta agonist, on mice fed a methionine- and choline-deficient (MCD) diet, a model of non-alcholic steatohepatitis (NASH), to investigate (a) the efficacy of bezafibrate against non-alcholic steatohepatitis and (b) the relation between non-alcholic steatohepatitis and the functional role of PPARdelta. Bezafibrate (50 or 100 mg/kg/day) and GW501516 (10 mg/kg/day) were administered by gavage once a day for 5 weeks. Hepatic lipid contents, plasma triglyceride, high density lipoprotein (HDL)-cholesterol and alanine aminotransferase (ALT) concentrations were evaluated, as were histopathological changes in the liver and hepatic mRNA expression levels. Bezafibrate and GW501516 inhibited the MCD-diet-induced elevations of hepatic triglyceride and thiobarbituric acid-reactants contents and the histopathological increases in fatty droplets within hepatocytes, liver inflammation and number of activated hepatic stellate cells. In this model, bezafibrate and GW501516 increased the levels of hepatic mRNAs associated with fatty acid beta-oxidation [acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-1 (CPT-1), liver-fatty acid binding protein (L-FABP) and peroxisomal ketothiolase], and reduced the levels of those associated with inflammatory cytokines or chemokine [transforming growth factor (TGF)-beta1, interleukin (IL)-6, IL-1beta, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) alpha and nuclear factor (NF)-kappaB1]. In addition, bezafibrate characteristically reduced the elevation in the level of plasma ALT, but enhanced that in plasma adiponectin and increased the mRNA expression levels of its receptors (adiponectin receptors 1 and 2). These results suggest that (a) bezafibrate (especially) and GW501516 might improve hepatic steatosis via an improvement in fatty acid beta-oxidation and a direct prevention of inflammation, (b) treatment with a PPARdelta agonist might improve non-alcholic steatohepatitis, (c) bezafibrate may improve non-alcholic steatohepatitis via activation not only of PPARalpha but also of PPARdelta, because bezafibrate is a PPAR pan-agonist.

Topics: Acyl-CoA Oxidase; Alanine Transaminase; Animals; Bezafibrate; Carnitine O-Palmitoyltransferase; Cholesterol, HDL; Choline; Diet; Dose-Response Relationship, Drug; Fatty Acid-Binding Proteins; Fatty Liver; Gene Expression; Interleukin-6; Liver; Male; Methionine; Mice; Mice, Inbred C57BL; Peroxisome Proliferator-Activated Receptors; PPAR delta; RNA, Messenger; Thiazoles; Thiobarbituric Acid Reactive Substances; Transforming Growth Factor beta; Transforming Growth Factor beta1; Triglycerides

The number of patients suffering from metabolic syndrome is increasing rapidly. Metabolic syndrome causes severe pathological changes in various organs, including the liver, and its main phenotype is nonalcoholic fatty liver disease (NAFLD). NAFLD has a broad spectrum ranging from simple fatty change to severe steatohepatitis with marked fibrosis. Recently, several experimental animal models for NAFLD have been proposed. However, most were established by rather artificial conditions such as genetic alteration. In the present study, we tried to establish a unique animal model mimicking some of the physiopathological features of NAFLD using high-cholesterol-fed rabbits.. Male rabbits fed with standard rabbit food containing 1% cholesterol for 8 weeks and 12 weeks were compared to controls (six rabbits/group). The weight of food was strictly restricted to 100 g/rabbit per day.. Body weights and fasting plasma insulin levels showed no significant differences among the groups. In contrast, characteristic fine fibrosis was extended from perivenular to pericellular areas, and microvesicular fatty change with ballooning degeneration was observed in perivenular areas in livers of the cholesterol-fed rabbits. Increase of serum cholesterol level, activation of hepatic stellate cells, and exposure to oxidative stress were also recognized.. Cholesterol-fed rabbits share several physiopathological features of NAFLD. Because this model did not show insulin resistance or obesity, it may be useful for elucidating the mechanism of NAFLD related mainly to hyperlipidemia.

Topics: Animals; Cholesterol, Dietary; Enzyme-Linked Immunosorbent Assay; Fatty Liver; Immunohistochemistry; Insulin; Insulin Resistance; Lipid Peroxides; Lipids; Liver Cirrhosis, Experimental; Male; Rabbits; Severity of Illness Index; Transforming Growth Factor beta

Cystathionine beta-synthase (CBS) deficiency causes severe hyperhomocysteinemia, which confers diverse clinical manifestations, notably liver disease. To investigate this aspect of hyperhomocysteinemia, we performed a thorough investigation of liver pathology in CBS-deficient mice, a murine model of severe hyperhomocysteinemia.. The degree of liver injury and inflammation was assessed by histologic examination, by measurements of products of lipid peroxidation, and by formation of carbonyl groups on protein as a measure for the occurrence of protein oxidation. Analysis of profibrogenic, proinflammatory factors and cell apoptosis was performed by Western blots, real-time quantitative reverse-transcription polymerase chain reaction, caspase-3 activity, DNA laddering, and TUNEL assay.. Histologic evaluation of liver specimens of 8- to 32-week-old CBS-deficient mice showed that CBS-deficient mice develop inflammation, fibrosis, and hepatic steatosis, concomitant with an enhanced expression of tissue inhibitor of metalloproteinase-1, alpha-smooth muscle actin, pro(alpha)1 collagen type I, transforming growth factor-beta1, and proinflammatory cytokines. Moreover, even if the proapoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated, caspase-3 was not activated, DNA laddering was not detected, and number of positive TUNEL cells was not increased in liver of CBS-deficient mice compared with wild-type mice.. The results show that hyperhomocysteinemia in liver of CBS-deficient mice promotes oxidative stress, which may cause mitochondrial damage in association with activation of hepatic stellate cells, leading to liver injury. The absence of caspase-3 activation, DNA fragmentation, and TUNEL-positive cells shows that protective signals may counteract apoptotic signals in liver of CBS-deficient mice.

Topics: Animals; Apoptosis; Collagen Type I; Cystathionine beta-Synthase; Disease Models, Animal; Fatty Liver; Homocysteine; Homocystinuria; Hyperhomocysteinemia; Liver; Liver Cirrhosis; Mice; Mice, Mutant Strains; Oxidative Stress; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Administration of a methionine and choline deficient (MCD) diet to rodents causes progressive fibrosing steatohepatitis pathologically similar to human metabolic steatohepatitis. We have previously shown that the peroxisome proliferator-activated receptor-alpha (PPARalpha) agonist, Wy-14,643, prevented the development of MCD diet-induced steatohepatitis. We have now tested whether Wy-14,643 ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice were fed the MCD diet for 51 days to induce severe steatohepatitis. They were then treated with Wy-14,643 together with the MCD diet for 5 or 12 days; positive controls continued on the MCD diet for 5 or 12 days. After 5 days of Wy-14,643 treatment, alanine aminotransferase (ALT) levels were significantly decreased, steatohepatitis less severe, and hepatic lipoperoxides significantly reduced. After 12 days, hepatic triglycerides were normalized and there was near resolution of histological changes. MCD dietary feeding was associated with increased expression of vascular cell adhesion molecule (VCAM)-1, and increased numbers of activated macrophages in the liver. Treatment with Wy-14,643 reduced VCAM-1 expression and macrophage numbers. MCD diet-fed mice developed hepatic fibrosis with increased hepatic collagen alpha1(I), tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and matrix metalloproteinase (MMP)-13 mRNA levels. After treatment with Wy-14,643, expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells and near resolution of liver fibrosis. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by treatment with Wy-14,643. It is likely that activation of PPARalpha reverses fibrosis indirectly by reducing stimuli, such as lipid peroxides, and activation of cells responsible for promoting hepatic fibrosis.

Topics: Animals; Cell Adhesion Molecules; Connective Tissue Growth Factor; Dietary Fats; Fatty Liver; Gene Expression; Hepatocytes; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lipid Peroxides; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Peroxisome Proliferators; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Triglycerides

The therapeutic efficacy of angiotensin II receptor antagonist, losartan, was studied in patients with nonalcoholic steatohepatitis (NASH). Seven patients with both NASH and hypertension were treated with losartan (50 mg/d) for 48 weeks. Treatment with losartan resulted in a significant decrease in blood markers of hepatic fibrosis, plasma TGF-beta1 and serum ferritin concentration concurrently with an improvement in serum aminotransferase levels. Histological assessment showed improvement of hepatic necroinflammation in five patients, reduction of hepatic fibrosis in four patients, and disappearance of iron deposition in two patients. No side effect of treatment was noted at any time during the study. In conclusion, the present data raise the possibility that an angiotensin II receptor antagonist may be therapeutically efficacious for NASH.

Topics: Adult; Angiotensin Receptor Antagonists; Biomarkers; Fatty Liver; Female; Ferritins; Hepatitis; Humans; Hypertension; Iron; Liver; Liver Cirrhosis; Losartan; Male; Middle Aged; Necrosis; Osmolar Concentration; Transaminases; Transforming Growth Factor beta; Transforming Growth Factor beta1

Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV.. Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation.. Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry.. Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV.

Topics: Adult; Antineoplastic Agents; Collagen Type I; Cytochrome P-450 CYP2E1; Fatty Liver; Female; Gene Expression; Hepatitis C, Chronic; Humans; Lipopolysaccharide Receptors; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Pathogenesis of non-alcoholic steatohepatitis (NASH) remains poorly understood. Cytochrome P450 2E1 (CYP 2E1), cytokines, oxidative stress and activation of hepatic stellate cells seem to play a role in this process. The aim was to determine the potential implication of these factors in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis.. Animals were fed a standard diet, a 5% orotic acid-diet (OA) developing hepatic steatosis, or the methionine-choline deficient (MCD) diet inducing steatohepatitis for 2 and 6 weeks. Lipid peroxidation, CYP 2E1 expression and activity, expression of UCP-2, interleukin (IL)-6, transforming growth factor (TGF)beta1, KLF6 mRNAs, and activation of hepatic stellate cells were examined by gas chromatography, high-performance liquid chromatography, Western blotting, quantitative polymerase chain reaction and immunohistochemistry.. Lipid peroxidation increased in the MCD model whereas only minor changes occurred in the OA model. KLF6 and TGFbeta1 mRNAs were selectively up-regulated in MCD animals. Stellate cell activation, inflammation and collagen deposition only occurred in the MCD group. CYP 2E1 expression and activity increased in the OA group while both decreased in MCD animals. UCP-2 and IL-6 mRNA increased in both groups.. In the context of steatosis, lipid peroxidation is associated with inflammation and stellate cell activation with concomitant increase in TGFbeta1 production, possibly through up-regulation of KLF6.

Topics: Actins; Animals; Cytochrome P-450 Enzyme System; Diagnosis, Differential; Disease Progression; Fatty Liver; Immunohistochemistry; Interleukin-6; Ion Channels; Kruppel-Like Factor 6; Kruppel-Like Transcription Factors; Lipid Metabolism; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Membrane Transport Proteins; Microsomes, Liver; Mitochondrial Proteins; Muscle, Smooth; Oxidative Stress; Proto-Oncogene Proteins; Rats; Rats, Wistar; RNA, Messenger; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Uncoupling Protein 2; Up-Regulation

We explored the involvement of cell types, cytokines and lipid peroxidation in a rat dietary model of fibrosing steatohepatitis.. Male rats were fed a high fat diet deficient in methionine and choline (MCD) for up to 17 weeks. Whole liver, hepatocytes and non-parenchymal cells were analysed for reduced glutathione (GSH) levels, products of lipid peroxidation (thiobarbituric acid reactive substances, TBARS), liver injury, and fibrosis.. MCD diet-fed rats developed hepatic steatosis at week 2 and focal necroinflammatory change by week 5, while pericellular fibrosis evolved and progressed between weeks 12 and 17. Collagen alpha(1)(1) gene expression was upregulated by week 5 and increased fivefold by week 17. Stellate cells were the unique source of collagen gene expression. TIMP-1 and -2 were increased at week 12. Livers of MCD diet-fed rats exhibited lowered levels of GSH and elevated TBARS. Hepatocytes were the source of lipid peroxidation, and mRNA levels for TGFbeta1 were increased only in this cell type.. The MCD model of 'fibrosing steatohepatitis' replicates the histologic features of human steatohepatitis, and the sequence of steatosis, inflammatory cell injury and fibrogenesis. The temporal sequence is consistent with a concept for involvement of oxidative injury in inflammatory recruitment and pathogenesis of hepatic fibrogenesis.

Topics: Animals; Choline; Diet; Fatty Liver; Gene Expression; Hepatocytes; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Methionine; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Insulin resistance and systemic hypertension are predictors of advanced fibrosis in obese patients with non-alcoholic fatty liver disease (NAFLD). Genetic factors may also be important. We hypothesize that high angiotensinogen (AT) and transforming growth factor-beta1 (TGF-beta1) producing genotypes increase the risk of liver fibrosis in obese subjects with NAFLD.. One hundred and five of 130 consecutive severely obese patients having a liver biopsy at the time of laparoscopic obesity surgery agreed to have genotype analysis. Influence of specific genotype or combination of genotypes on the stage of hepatic fibrosis was assessed after controlling for known risk factors.. There was no fibrosis in 70 (67%), stages 1-2 in 21 (20%) and stages 3-4 fibrosis in 14 (13%) of subjects. There was no relationship between either high AT or TGF-beta1 producing genotypes alone and hepatic fibrosis after controlling for confounding factors. However, advanced hepatic fibrosis occurred in five of 13 subjects (odds ratio 5.7, 95% confidence interval 1.5-21.2, P=0.005) who inherited both high AT and TGF-beta1 producing polymorphisms.. The combination of high AT and TGF-beta1 producing polymorphisms is associated with advanced hepatic fibrosis in obese patients with NAFLD. These findings support the hypothesis that angiotensin II stimulated TGF-beta1 production may promote hepatic fibrosis.

Topics: Adult; Fatty Liver; Female; Gene Frequency; Genotype; Humans; Liver Cirrhosis; Male; Middle Aged; Obesity, Morbid; Polymorphism, Genetic; Predictive Value of Tests; Risk Factors; Severity of Illness Index; Transforming Growth Factor beta; Transforming Growth Factor beta1

Alcohol consumption accelerates the appearance of liver fibrosis and hepatocellular carcinoma in patients with chronic hepatitis C virus (HCV) infection, but the mechanisms of these interactions are unknown. We therefore investigated the effects of chronic ethanol consumption in HCV core protein-expressing transgenic mice.. Ethanol was progressively added (up to 20%) to the drinking water that was given ad libidum.. In vivo fatty acid oxidation was not inhibited by ethanol consumption and/or HCV core expression. Both chronic ethanol consumption and HCV core expression decreased hepatic lipoprotein secretion and caused steatosis, but had no additive effects on lipoprotein secretion or steatosis. However, chronic ethanol consumption and HCV core protein additively increased lipid peroxidation and acted synergistically to increase the hepatic expression of transforming growth factor-beta (TGF-beta) and, to a less extent, tumor necrosis factor-alpha (TNF-alpha).. HCV core protein expression and chronic alcohol consumption have no effects on in vivo fatty acid oxidation and do not additively impair hepatic lipoprotein secretion, but additively increase hepatic lipid peroxidation and synergistically increase hepatic TNF-alpha and TGF-beta expression. These effects may be involved in the activation of fibrogenesis and the development of hepatocellular carcinoma in patients cumulating alcohol abuse and HCV infection.

Topics: Animals; Central Nervous System Depressants; Disease Models, Animal; Ethanol; Fatty Acids; Fatty Liver; Gene Expression; Hepacivirus; Hepatitis B Core Antigens; Lipid Peroxidation; Lipoproteins; Liver; Liver Diseases, Alcoholic; Mice; Mice, Inbred C57BL; Mice, Transgenic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Obesity is associated with hyperleptinemia and is also a risk factor for fibrosis and severity of fibrosis in several chronic liver diseases. The correlation between increased leptin, obesity and hepatic fibrosis prompted us to hypothesise that leptin has profibrogenic effects on the liver.. We analysed the role of leptin in liver fibrosis in leptin-deficient mice fed a diet which generates steatohepatitis, and in chronic carbon tetrachloride-induced hepatic injury.. Leptin-deficient mice failed to develop fibrosis during steatohepatitis or in response to chronic toxic liver injury, and failed to up-regulate collagen-I while developing similar hepatic injury as their genetic controls. Restitution of physiological levels of circulating leptin by injection of exogenous leptin, but not correction of the obese phenotype by dietary manipulation, restored liver fibrosis in leptin-deficient mice during chronic liver injury. These results confirmed the absolute requirement of leptin for hepatic fibrosis. We showed that leptin deficiency did not alter hepatic TNF regulation but that leptin is necessary for induction of bioactive transforming growth factor beta 1 (TGFbeta1) protein in the context of chronic liver injury.. These data establish that leptin is an essential mediator of hepatic fibrosis in response to chronic liver injury, whether metabolic or toxic in aetiology.

Topics: Animals; Carbon Tetrachloride; Chronic Disease; Disease Progression; Fatty Liver; Female; Leptin; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Oxidative Stress; Phenotype; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non-alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade.. Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic stellate cell activation (alpha-smooth muscle actin and TGF-beta expression) and collagen type I synthesis (procollagen alpha1 (I) mRNA).. Lipid peroxidation occurred in and adjacent to fat-laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). alpha-Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them.. These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes.

Topics: Actins; Adult; Fatty Liver; Female; Hemochromatosis; Hemochromatosis Protein; Hepatocytes; Histocompatibility Antigens Class I; HLA Antigens; Humans; Immunohistochemistry; Iron; Iron Overload; Lipid Peroxidation; Liver Cirrhosis; Male; Malondialdehyde; Membrane Proteins; Middle Aged; Procollagen; Transforming Growth Factor beta

Cirrhosis is a diffuse process of hepatic fibrosis and regenerative nodule formation of unknown pathogenesis. Transforming growth factor (TGF) beta 1 induces the production of extracellular matrix proteins by liver cells and has been implicated in the pathogenesis of hepatic fibrosis in laboratory animals. TGF alpha is a hepatocyte mitogen that participates in liver regeneration.. Using Northern blot analysis, we studied the expression of TGF beta 1 messenger RNA (mRNA) in liver specimens from 42 patients with chronic hepatitis and cirrhosis and 12 subjects with either normal or fatty livers. The results were correlated with measurements of procollagen Type I mRNA in liver tissue, procollagen Type III peptide in serum, and the degree of histologic injury. We also investigated whether TGF alpha mRNA would be detectable in biopsy specimens of livers with proliferative activity.. TGF beta 1 mRNA expression correlated closely with the expression of procollagen Type I mRNA (r = 0.94) and serum procollagen Type III peptide (r = 0.89) and with the histologic activity index (r = 0.73). All patients with increased fibrogenic activity (serum procollagen Type III peptide level, greater than 11.9 micrograms per liter) had increased levels of TGF beta 1 mRNA (2 to 14 times the levels in the control group or in patients with normal fibrogenic activity), and both TGF alpha and H3 histone (a marker of DNA synthesis) mRNAs were detectable in patients with regenerative nodules. Six of eight patients with hepatitis C treated with interferon alfa for one year had sustained clinical responses with normalization of serum procollagen Type III peptide and aminotransferase activity. All these patients had normal levels of TGF beta 1 mRNA in liver specimens obtained at the end of the year.. TGF beta 1 may have an important role in the pathogenesis of fibrosis in patients with chronic liver disease, and TGF alpha expression may be associated with liver regeneration in these patients.

Topics: Adult; Blotting, Northern; Extracellular Matrix Proteins; Fatty Liver; Female; Glyceraldehyde-3-Phosphate Dehydrogenases; Hepatitis B; Hepatitis C; Histones; Humans; Interferon Type I; Liver; Liver Cirrhosis; Liver Regeneration; Male; Middle Aged; Procollagen; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

Topics: Biomarkers; Chronic Disease; Fatty Liver; Hepatitis; Humans; Liver; Liver Cirrhosis; Liver Regeneration; Peptide Fragments; Procollagen; RNA, Messenger; Transforming Growth Factor beta