Adiponectin induces the transforming growth factor decoy receptor BAMBI in human hepatocytes

Transforming growth factor (TGF) β is the central cytokine in fibrotic liver diseases. We analyzed whether hepatoprotective adiponectin directly interferes with TGFβ1 signaling in primary human hepatocytes (PHH). Adiponectin induces the TGFβ decoy receptor BMP-and activin-membrane-bound inhibitor (BAMBI) in PHH. Overexpression of BAMBI in hepatoma cells impairs TGFβ-mediated phosphorylation of SMAD2 and induction of connective tissue growth factor. BAMBI is lower in human fatty liver with a higher susceptibility to liver fibrosis and negatively correlates with BMI of the donors. Hepatic BAMBI is reduced in rodent models of liver inflammation and fibrosis. In summary, the current data show that hepatoprotective effects of adiponectin include induction of BAMBI which is reduced in human fatty liver and rodent models of metabolic liver injury.     

Follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis

Activin A, a member of the transforming growth factor-beta superfamily, is constitutively expressed in hepatocytes and regulates liver mass through tonic inhibition of hepatocyte DNA synthesis. Follistatin is the main biological inhibitor of activin bioactivity. These molecules may be involved in hepatic fibrogenesis, although defined roles remain unclear. We studied activin and follistatin gene and protein expression in cultured rat hepatic stellate cells (HSCs) and in rats given CCl4 for 8 wk and examined the effect of follistatin administration on the development of hepatic fibrosis. In activated HSCs, activin mRNA was upregulated with high expression levels, whereas follistatin mRNA expression was unchanged from baseline. Activin A expression in normal lobular hepatocytes redistributed to periseptal hepatocytes and smooth muscle actin-positive HSCs in the fibrotic liver. A 32% reduction in fibrosis, maximal at week 4, occurred in CCl4-exposed rats treated with follistatin. Hepatocyte apoptosis decreased by 87% and was maximal at week 4 during follistatin treatment. In conclusion, activin is produced by activated HSCs in vitro and in vivo. Absence of simultaneous upregulation of follistatin gene expression in HSCs suggests that HSC-derived activin is biologically active and unopposed by follistatin. Our in vivo and in vitro results demonstrate that activin-mediated events contribute to hepatic fibrogenesis and that follistatin attenuates early events in fibrogenesis by constraining HSC proliferation and inhibiting hepatocyte apoptosis.     

The activin axis in liver biology and disease

Activins are a closely related subgroup within the TGFbeta superfamily of growth and differentiation factors. They consist of two disulfide-linked beta subunits. Four mammalian activin beta subunits termed beta(A), beta(B), beta(C), and beta(E), respectively, have been identified. Activin A, the homodimer of two beta(A) subunits, has important regulatory functions in reproductive biology, embryonic development, inflammation, and tissue repair. Several intra- and extracellular antagonists, including the activin-binding proteins follistatin and follistatin-related protein, serve to fine-tune activin A activity. In the liver there is compelling evidence that activin A is involved in the regulation of cell number by inhibition of hepatocyte replication and induction of apoptosis. In addition, activin A stimulates extracellular matrix production in hepatic stellate cells and tubulogenesis of sinusoidal endothelial cells, and thus contributes to restoration of tissue architecture during liver regeneration. Accumulating evidence from animal models and from patient data suggests that deregulation of activin A signaling contributes to pathologic conditions such as hepatic inflammation and fibrosis, acute liver failure, and development of liver cancer. Increased production of activin A was suggested to be a contributing factor to impaired hepatocyte regeneration in acute liver failure and to overproduction of extracellular matrix in liver fibrosis. Recent evidence suggests that escape of (pre)neoplastic hepatocytes from growth control by activin A through overexpression of follistatin and reduced activin production contributes to hepatocarcinogenesis. The role of the activin subunits beta(C) and beta(E), which are both highly expressed in hepatocytes, is still quite incompletely understood. Down-regulation in liver tumors and a growth inhibitory function similar to that of beta(A) has been shown for beta(E). Contradictory results with regard to cell proliferation have been reported for beta(C). The profound involvement of the activin axis in liver biology and in the pathogenesis of severe hepatic diseases suggests activin as potential target for therapeutic interventions.     

Activin-A causes Hepatic stellate cell activation via the induction of TNFα and TGFβ in Kupffer cells

Background & aims

TGFβ superfamily member Activin-A is a multifunctional hormone/cytokine expressed in multiple tissues and cells, where it regulates cellular differentiation, proliferation, inflammation and tissue architecture. High activin-A levels have been reported in alcoholic cirrhosis and non-alcoholic steatohepatitis (NASH). Our aim was to identify the cell types involved in the fibrotic processes induced by activin-A in liver and verify the liver diseases that this molecule can be found increased.

iPla2β deficiency in mice fed with MCD diet does not correct the defect of phospholipid remodeling but attenuates hepatocellular injury via an inhibition of lipid uptake genes

Group VIA calcium-independent phospholipase A2 (iPla2β) is among modifier genes of non-alcoholic fatty liver disease which leads to non-alcoholic steatohepatitis (NASH). Consistently, iPla2β deletion protects hepatic steatosis and obesity in genetic ob/ob and obese mice chronically fed with high-fat diet by replenishing the loss of hepatic phospholipids (PL). As mouse feeding with methionine- and choline-deficient (MCD) diet is a model of lean NASH, we tested whether iPla2β-null mice could still be protected since PL syntheses are disturbed. MCD-diet feeding of female wild-type for 5 weeks induced hepatic steatosis with a severe reduction of body and visceral fat weights concomitant with a decrease of hepatic phosphatidylcholine. These parameters were not altered in MCD-fed iPla2β-null mice. However, iPla2β deficiency attenuated MCD-induced elevation of serum transaminase activities and hepatic expression of fatty-acid translocase Cd36, fatty-acid binding protein-4, peroxisome-proliferator activated receptorγ, and HDL-uptake scavenger receptor B type 1. The reduction of lipid uptake genes was consistent with a decrease of hepatic esterified and unesterified fatty acids and cholesterol esters. On the contrary, iPla2β deficiency under MCD did not have any effects on inflammasomes and pro-inflammatory markers but exacerbated hepatic expression of myofibroblast α-smooth muscle actin and vimentin. Thus, without any rescue of PL loss, iPla2β inactivation attenuated hepatocellular injury in MCD-induced NASH with a novel mechanism of lipid uptake inhibition. Taken together, we have shown that iPla2β mediates hepatic steatosis and lipotoxicity in hepatocytes in both obese and lean NASH, but elicits exacerbated liver fibrosis in lean NASH likely by affecting other cell types.     

脾切除对肝纤维化大鼠肝脏TGFβ 1的表达和血清TGFβ 1水平的影响

目的：研究脾切除对肝纤维化大鼠肝脏TGFβ1的表达和血清TGFβ1水平的影响,探讨脾切除在肝纤维化中的意义。方法：用CCL4建立50例肝纤维化大鼠模型。于建模第3周,6周,及8周分别取大鼠肝脏和脾脏标本。用免疫组化SP方法测定其TGFβ1的表达,HE和姬姆萨染色检测肝纤维化。应用双抗体夹心ELISA方法测定15例模型大鼠行脾脏切除前后的血清TGFβ1水平,以及15例对照组大鼠的血清TGFβ1水平,并于术后4周取两组大鼠的肝脏标本,用免疫组化SP方法测定其TGFβ1的表达。应用CMIAS8彩色图像系统对阳性目标进行分析和处理。结果：随着肝纤维化程度的进展,大鼠肝脏和脾脏TGFβ1的表达也随之增加（P〈0.01）。脾切除组大鼠其血清TGF-β1的水平显著低于对照组大鼠（P〈0.05）,且脾切除组大鼠肝脏TGFβ1的表达低于对照组大鼠（P〈0.05）。结论：脾切除术在一定程度上可延缓肝纤维化的发展。     

Eicosapentaenoic Acid Ameliorates Non-Alcoholic Steatohepatitis in a Novel Mouse Model Using Melanocortin 4 Receptor-Deficient Mice

Many attempts have been made to find novel therapeutic strategies for non-alcoholic steatohepatitis (NASH), while their clinical efficacy is unclear. We have recently reported a novel rodent model of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice, which exhibit the sequence of events that comprise hepatic steatosis, liver fibrosis, and hepatocellular carcinoma with obesity-related phenotypes. In the liver of MC4R-KO mice, there is a unique histological feature termed hepatic crown-like structures (hCLS), where macrophages interact with dead hepatocytes and fibrogenic cells, thereby accelerating inflammation and fibrosis. In this study, we employed MC4R-KO mice to examine the effect of highly purified eicosapentaenoic acid (EPA), a clinically available n-3 polyunsaturated fatty acid, on the development of NASH. EPA treatment markedly prevented the development of hepatocyte injury, hCLS formation and liver fibrosis along with lipid accumulation. EPA treatment was also effective even after MC4R-KO mice developed NASH. Intriguingly, improvement of liver fibrosis was accompanied by the reduction of hCLS formation and plasma kallikrein-mediated transforming growth factor-β activation. Moreover, EPA treatment increased the otherwise reduced serum concentrations of adiponectin, an adipocytokine with anti-inflammatory and anti-fibrotic properties. Collectively, EPA treatment effectively prevents the development and progression of NASH in MC4R-KO mice along with amelioration of hepatic steatosis. This study unravels a novel anti-fibrotic mechanism of EPA, thereby suggesting a clinical implication for the treatment of NASH.     

不同来源肝细胞在体外降脂药物评价中药效反应的对比

目的:通过对比不同来源的人肝癌细胞系HepG2和原代大鼠肝细胞在体外降脂药物评价中药效反应,指导两种肝细胞在体外降脂药物评价中的实际应用。方法:用游离脂肪酸(油酸/棕榈酸,2:1)诱导HepG2细胞、原代大鼠肝细胞脂肪变性,并用100μmol·L-1苯扎贝特干预,检测细胞内甘油三酯(TG)、总胆固醇(TC)、活性氧(ROS)含量,细胞内脂滴数目、并检测细胞上清液中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果:FFA刺激使HepG2细胞和原代大鼠肝细胞脂质沉积(TG、脂滴)和氧化应激(ROS、MDA、SOD)水平上升。苯扎贝特对HepG2细胞1 mmol·L-1FFA造模组和原代大鼠肝细胞0.5 mmol·L-1FFA造模组脂质沉积和氧化应激水平改善显著;而HepG2细胞0.5 mmol·L-1FFA造模组和原代大鼠肝细胞1 mmol·L-1FFA造模组脂质沉积和氧化应激水平在苯扎贝特干预后变化不明显。结论:在相同FFA造模浓度,原代大鼠肝细胞病理特征变化更为明显;苯扎贝特对两种肝细胞在脂质沉积和氧化应激水平的作用也不完全相同。因而HepG2细胞和原代大鼠肝细胞在体外降脂药物评价中药效反应是不完全相同的。     

A human hepatocellular in vitro model to investigate steatosis

The present study was designed to define an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the metabolic and cytotoxic/apoptotic effects could be separated. This was accomplished by defining the experimental conditions of lipid exposure that lead to significant intracellular fat accumulation in the absence of overt cytotoxicity, therefore allowing to differentiate between cytotoxic and apoptotic effects. Palmitic (C16:0) and oleic (C18:1) acids are the most abundant fatty acids (FFAs) in liver triglycerides in both normal subjects and patients with nonalcoholic fatty liver disease (NAFLD). Therefore, human hepatocytes and HepG2 cells were incubated with a mixture of different proportions of saturated (palmitate) and unsaturated (oleate) FFAs to induce fat-overloading. Similar intracellular levels of lipid accumulation as in the human steatotic liver were achieved. Individual FFAs have a distinct inherent toxic potential. Fat accumulation, cytotoxicity and apoptosis in cells exposed to the FFA mixtures were investigated. The FFA mixture containing a low proportion of palmitic acid (oleate/palmitate, 2:1 ratio) is associated with minor toxic and apoptotic effects, thus representing a cellular model of steatosis that mimics benign chronic steatosis. On the other hand, a high proportion of palmitic acid (oleate/palmitate, 0:3 ratio) might represent a cellular model of steatosis in which saturated FFAs promote an acute harmful effect of fat overaccumulation in the liver. These hepatic cellular models are apparently suitable to experimentally investigate the impact of fat overaccumulation in the liver excluding other factors that could influence hepatocyte behaviour.     

Conjugated linoleic acid isomers modulate protein expression profile in rat hepatocytes

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid, which has been recently proven to be effective in reducing body fat mass, but brings as a side effect, the liver enlargement due to an increased lipid content. The in vivo lipogenic activity has been suggested to be due to the reduction in fat mass and to the consequent metabolism of blood glucose to fatty acid in the liver rather than in the adipose tissue. We investigated the ability of CLA to directly induce steatosis by modulating the expression pattern of hepatic proteins involved in lipid metabolism. To avoid interferences derived from CLA metabolism by other tissues, we used the in vitro model of freshly isolated rat hepatocytes incubated in the presence of different CLA isomers. The direct effect of CLA on lipid accumulation in hepatocytes was demonstrated by the altered expression pattern of several proteins involved in lipid metabolism, as assessed by two-dimensional gel electrophoresis and confirmed by Western blotting analysis. The CLA isomer c9,t11 was most effective in modulating the protein expression profile.     

Screening and identification of differentially expressed genes in goose hepatocytes exposed to free fatty acid

The overaccumulation of triglycerides in hepatocytes induces hepatic steatosis; however, little is known about the mechanism of goose hepatic steatosis. The aim of this study was to define an experimental model of hepatocellular steatosis with TG overaccumulation and minimal cytotoxicity, using a mixture of various proportions of oleate and palmitate free fatty acids (FFAs) to induce fat‐overloading, then using suppressive subtractive hybridization and a quantitative PCR approach to identify genes with higher or lower expression levels after the treatment of cells with FFA mixtures. Overall, 502 differentially expressed clones, representing 21 novel genes and 87 known genes, were detected by SSH. Based on functional clustering, up‐ and down‐regulated genes were mostly related to carbohydrate and lipid metabolism, enzyme activity and signal transduction. The expression of 20 selected clones involved with carbohydrate and lipid metabolism pathways was further studied by quantitative PCR. The data indicated that six clones similar to the genes ChREBP, FoxO1, apoB, IHPK2, KIF1B, and FSP27, which participate in de novo synthesis of fatty acid and secretion of very low density lipoproteins, had significantly lower expression levels in the hepatocytes treated with FFA mixtures. Meanwhile, 13 clones similar to the genes DGAT‐1, ACSL1, DHRS7, PPARα, L‐FABP, DGAT‐2, PCK, ACSL3, CPT‐1, A‐FABP, PPARβ, MAT, and ALDOB had significantly higher expression levels in the hepatocytes treated with FFA mixtures. These results suggest that several metabolic pathways are altered in goose hepatocytes, which may be useful for further research into the molecular mechanism of goose hepatic steatosis. J. Cell. Biochem. 111: 1482–1492, 2010. © 2010 Wiley‐Liss, Inc.     

Bromodomain protein 4 is a key molecular driver of TGFβ1-induced hepatic stellate cell activation

Liver fibrosis is characterized by the excessive deposition of extracellular matrix in liver. Chronic liver injury induces the activation of hepatic stellate cell (HSCs), a key step in liver fibrogenesis. The activated HSC is the primary source of ECM and contributes significantly to liver fibrosis. TGFβ1 is the most potent pro-fibrotic cytokine. Bromodomain protein 4 (BrD4), an epigenetic reader of histone acetylation marks, was crucial for profibrotic gene expression in HSCs. The present study aimed to investigate the roles of BRD4 in TGFβ1-dependent HSC activation and liver fibrosis, focusing on TGFβ1-induced alterations of the levels of the fibrotic-related important proteins in HSCs by employing the heterozygous TGFβ1 knockout mice and BrD4 knockdown in vivo and in vitro. Results revealed that BrD4 protein level was significantly upregulated by TGFβ1 and BrD4 knockdown reduced TGFβ1-induced HSC activation and liver fibrosis. BrD4 was required for the influences of TGFβ1 on PDGFβ receptor and on the pathways of Smad3, Stat3, and Akt. BrD4 also mediated TGFβ1-induced increases in histone acetyltransferase p300, the pivotal pro-inflammatory NFkB p65, and tissue inhibitor of metalloproteinase 1 whereas BrD4 reduced Caspase-3 protein levels in HSCs during liver injury, independent of TGFβ1. Further experiments indicated the interaction between TGFβ1-induced BrD4 and NFkB p65 in HSCs and in liver of TAA-induced liver injury. Human cirrhotic livers were demonstrated a parallel increase in the protein levels of BrD4 and NFkB p65 in HSCs. This study revealed that BrD4 was a key molecular driver of TGFβ1-induced HSC activation and liver fibrosis.     

Liver Injury and Fibrosis Induced by Dietary Challenge in the Ossabaw Miniature Swine

BackgroundOssabaw miniature swine when fed a diet high in fructose, saturated fat and cholesterol (NASH diet) develop metabolic syndrome and nonalcoholic steatohepatitis (NASH) characterized by liver injury and fibrosis. This study was conducted to further characterize the development of NASH in this large animal model.MethodsOssabaw swine were fed standard chow (control group; n = 6) or NASH diet (n = 6) for 24 weeks. Blood and liver tissue were collected and liver histology were characterized at 0, 8, 16 and 24 weeks of dietary intervention. Hepatic apoptosis and lipid levels were assessed at week 24.ResultsThe NASH diet group developed metabolic syndrome and progressive histologic features of NASH including: (a) hepatocyte ballooning at 8 weeks which progressed to extensive ballooning (>90% hepatocytes), (b) hepatic fibrosis at week 16, which progressed to moderate fibrosis, and (c) Kupffer cell accumulation with vacuolization at 8 weeks which progressed through week 24. The NASH diet group showed increased hepatocyte apoptosis that correlated with hepatic total and free cholesterol and free fatty acids, but not esterified cholesterol or triglycerides.ConclusionsThis report further characterizes the progression of diet-induced NASH in the Ossabaw swine model. In Ossabaw swine fed the NASH diet: (a) hepatocyte injury and fibrosis can occur without macrovesicular steatosis or excess triglyceride accumulation; (b) hepatocyte ballooning generally precedes the development of fibrosis; (c) there is increased hepatocyte apoptosis, and it is correlated more significantly with hepatic free cholesterol than hepatic free fatty acids and had no correlation with hepatic triglycerides.