Impact of amino acid substitutions in the hepatitis C virus genotype 1b core region on liver steatosis and hepatic oxidative stress in patients with chronic hepatitis C

Background: Liver steatosis and hepatic oxidative stress are the histopathological features of chronic hepatitis C. Hepatitis C virus (HCV) genotype 1 core protein induces hepatic steatosis and reactive oxygen species production in transgenic mice. The amino acid substitutions in the HCV core region appear to be related to hepatocarcinogenesis. Aims: The aim of this study was to clarify the impact of mutations in the HCV core region on oxidative stress and lipid metabolism in patients with chronic hepatitis C. Methods: Sixty‐seven patients (35 men, 32 women; mean age, 58.4 ± 10.2 years) with chronic hepatitis C with high titres (>5 log IU/ml) were enrolled. Substitutions in amino acids 70, 75 and 91 of the HCV genotype 1b core region, the percentage of hepatic steatosis, and hepatic 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) levels were investigated in all patients. Urinary 8‐OHdG levels were measured in 35 patients. Results: Body mass index, alanine aminotransferase, γ‐glutamyl transferase, and triglyceride levels and substitutions of amino acid 70/Q (glutamine) were significantly associated with the presence of steatosis on univariate analysis. Multivariate analysis showed that substitution of amino acid 70 of glutamine and triglyceride levels were the independent factors related to liver steatosis. Hepatic and urinary 8‐OHdG levels were significantly higher in patients with methionine at amino acid 91 of the HCV core region than in those with leucine. Conclusion: Substitutions in the amino acids of the HCV genotype1b core region are associated with hepatic steatosis and oxidative stress in patients with chronic hepatitis C.     

Alcohol and hepatitis C virus core protein additively increase lipid peroxidation and synergistically trigger hepatic cytokine expression in a transgenic mouse model

BACKGROUND/AIMS: 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. METHODS: Ethanol was progressively added (up to 20%) to the drinking water that was given ad libidum. RESULTS: 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). CONCLUSIONS: 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.     

Peroxisome proliferator-activated receptor α, a potential therapeutic target for alcoholic liver disease

Alcoholic liver injury represents a progressive process with a range of consequences including hepatic steatosis, steatohepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Targeting key molecular regulators involved in the development of alcoholic liver injury may be of great value in the prevention of liver injury. Peroxisome proliferator-activated receptor α (PPARα) plays a pivotal role in modulation of hepatic lipid metabolism, oxidative stress, inflammatory response and fibrogenesis. As such, PPARα may be a potential therapeutic target for the treatment of alcoholic liver disease.     

Adiponectin serum level in chronic hepatitis C infection and therapeutic profile

Hepatic steatosis is commonly seen in the patients with chronic hepatitis C virus(HCV) infection. HCV is closely associated with lipid metabolism,and viral steatosis is more common in genotype 3 infection owing to a direct cytopathic effect of HCV core protein. In non-genotype3 infection,hepatic steatosis is considered largely to be the result of the alterations in host metabolism; metabolic steatosis is primarily linked with HCV genotype 1. A d i p o s e t i s s u e s e c r e t e s d i f f e r e n t h o r m o n e s involved in glucose and lipid metabolisms. It has been demonstrated that adipocytokines are involved in the pathogenesis of non-alcoholic fatty liver disease,as the decreased plasma adiponectin levels,a soluble matrix protein expressed by adipoctyes and hepatocyte,are associated with liver steatosis. Various studies have shown that steatosis is strongly correlated negatively with adiponectin in the patients with HCV infection. The role of adiponectin in hepatitis C virus induced steatosis is still not completely understood,but the relationship between adiponectin low levels and liver steatosis is probably due to the ability of adiponectin to protect hepatocytes from triglyceride accumulation by increasing β-oxidation of free fatty acid and thus decreasing de novo free fatty acid production.     

Ghrelin promotes hepatic lipogenesis by activation of mTOR-PPARγ signaling pathway

Although ghrelin has been demonstrated to stimulate energy intake and storage through a central mechanism, its effect on hepatic lipid metabolism remains largely uncharacterized. Ghrelin receptor antagonism or gene deletion significantly decreased obesity-associated hepatic steatosis by suppression of de novo lipogenesis, whereas exogenous ghrelin stimulated lipogenesis, leading to hepatic lipid accumulation in mice. The effects of ghrelin were mediated by direct activation of its receptor on hepatocytes. Cultured hepatocytes responded to ghrelin with increased lipid content and expression of lipogenesis-related genes. Ghrelin increased phosphorylation of S6, the downstream target of mammalian target of rapamycin (mTOR) signaling in cultured hepatocytes, whereas ghrelin receptor antagonism reduced hepatic phosphorylation of S6 in db/db mice. Inhibition of mTOR signaling by rapamycin markedly attenuated ghrelin-induced up-regulation of lipogenesis in hepatocytes, whereas activation of hepatic mTOR signaling by deletion of TSC1 increased hepatic lipogenesis. By interacting with peroxisome proliferator-activated receptor-γ (PPARγ), mTOR mediates the ghrelin-induced up-regulation of lipogenesis in hepatocytes. The stimulatory effect of ghrelin on hepatic lipogenesis was significantly attenuated by PPARγ antagonism in cultured hepatocytes and in PPARγ gene-deficient mice. Our study indicates that ghrelin activates its receptor on hepatocytes to promote lipogenesis via a mechanism involving the mTOR-PPARγ signaling pathway.Triglyceride deposition in the liver, which is strongly associated with obesity, is the initial event in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Over time, hepatic steatosis may progress to steatohepatitis, cirrhosis, and primary hepatocellular carcinoma (1). The current therapeutic strategy for NAFLD has been focused on reversal of hepatic steatosis, primarily through weight reduction. Treatment is often ineffective because of the difficulty in achieving sustained weight loss. Alternative approaches are needed but are limited by incomplete understanding of the mechanisms controlling the development of steatosis. Gastric hormones may be involved in regulation of lipid metabolism. Studies in both animals and humans demonstrate that ghrelin, a 28-aa peptide hormone secreted by X/A-like endocrine cells in the gastric fundus (2, 3), stimulates lipid accumulation in adipose tissue (4). Chronic infusion of ghrelin increases both adipose and hepatic lipid storage (5). Genetic disruption of either ghrelin or ghrelin receptor genes renders mice resistant to obesity and to the development of hepatic steatosis (6). Interestingly, the anabolic effect of ghrelin appears to be independent of its hyperphagic action. Chronic third intracerebroventricular infusion of ghrelin in diet-induced obese rats increases adiposity and gene expression of lipogenic enzymes in white adipose tissue while food intake remains unchanged (7). Most studies suggest that ghrelin increases lipid deposition in adipose tissue through an effect on hypothalamic neurons (8). However, the location and anatomical structure of the hypothalamus pose significant hurdles for therapies that target this organ, and peripheral targets would be appealing alternatives. In this study, we demonstrate that ghrelin stimulates lipogenesis and increases triglyceride content in liver by direct activation of its receptor on hepatocytes. This effect is mediated via the mammalian target of rapamycin (mTOR) and peroxisome proliferator-activated receptor-γ (PPARγ) signaling pathway. This study provides direct evidence that both pharmacological and genetic interventions directed at ghrelin receptor ameliorate the development of hepatic steatosis associated with obesity.     

Specific polymorphisms in hepatitis C virus genotype 3 core protein associated with intracellular lipid accumulation

BACKGROUND: Steatosis is a common histological finding and a poor prognostic indicator in patients with hepatitis C virus (HCV) infection. In HCV genotype 3-infected patients, the etiology of steatosis appears to be closely correlated with unknown viral factors that increase intracellular lipid levels. We hypothesize that specific sequence polymorphisms in HCV genotype 3 core protein may be associated with hepatic intracellular lipid accumulation. METHODS: Using selected serum samples from 8 HCV genotype 3-infected patients with or without steatosis, we sequenced the HCV core gene to identify candidate polymorphisms associated with increased intracellular lipid levels. RESULTS: Two polymorphisms at positions 182 and 186 of the core protein correlated with the presence (P= .03) and absence (P= .005) of intrahepatic steatosis. Transfected liver cell lines expressing core protein with steatosis-associated polymorphisms had increased intracellular lipid levels compared with non-steatosis-associated core isolates, as measured by oil red O staining (P= .02). Site-specific mutagenesis performed at positions 182 and 186 in steatosis-associated core genes yielded proteins that had decreased intracellular lipid levels in transfected cells (P= .03). CONCLUSIONS: We have identified polymorphisms in HCV core protein genotype 3 that produce increased intracellular lipid levels and thus may play a significant role in lipid metabolism or trafficking, contributing to steatosis.     

Effective treatment of steatosis and steatohepatitis by fibroblast growth factor 1 in mouse models of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder and is strongly associated with obesity and type 2 diabetes. Currently, there is no approved pharmacological treatment for this disease, but improvement of insulin resistance using peroxisome proliferator-activated receptor-γ (PPARγ) agonists, such as thiazolidinediones (TZDs), has been shown to reduce steatosis and steatohepatitis effectively and to improve liver function in patients with obesity-related NAFLD. However, this approach is limited by adverse effects of TZDs. Recently, we have identified fibroblast growth factor 1 (FGF1) as a target of nuclear receptor PPARγ in visceral adipose tissue and as a critical factor in adipose remodeling. Because FGF1 is situated downstream of PPARγ, it is likely that therapeutic targeting of the FGF1 pathway will eliminate some of the serious adverse effects associated with TZDs. Here we show that pharmacological administration of recombinant FGF1 (rFGF1) effectively improves hepatic inflammation and damage in leptin-deficient ob/ob mice and in choline-deficient mice, two etiologically different models of NAFLD. Hepatic steatosis was effectively reduced only in ob/ob mice, suggesting that rFGF1 stimulates hepatic lipid catabolism. Potentially adverse effects such as fibrosis or proliferation were not observed in these models. Because the anti-inflammatory effects were observed in both the presence and absence of the antisteatotic effects, our findings further suggest that the anti-inflammatory property of rFGF1 is independent of its effect on lipid catabolism. Our current findings indicate that, in addition to its potent glucose-lowering and insulin-sensitizing effects, rFGF1 could be therapeutically effective in the treatment of NAFLD.Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in developed countries and is strongly associated with obesity and type 2 diabetes (1). NAFLD refers to a wide spectrum of liver disorders ranging from simple fatty liver (steatosis) to nonalcoholic steatohepatitis (NASH) with increased risk of developing progressive fibrosis, cirrhosis, and liver cancer (2).Treatment options for NAFLD are limited and are directed mainly at weight loss or pharmacological improvement of insulin resistance (3). Although no pharmacologic therapy has been approved, the thiazolidinedione (TZD) class of insulin sensitizers has been demonstrated to improve steatosis, steatohepatitis, and liver function in mice and patients with NAFLD (1). TZDs improve insulin sensitivity through activation of nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ), which reduces insulin resistance in adipose tissue, liver, and skeletal muscle (4). The exact mechanism by which PPARγ exerts its beneficial effects on NAFLD is not completely understood, but it is believed that improved hepatic insulin sensitivity enhances lipid oxidation and reduces hepatic lipogenesis, thereby reducing steatosis (5). In addition, increased peripheral insulin sensitivity may reduce lipolysis in white adipose tissue and thereby limit ectopic fat accretion.PPARγ and its activators also have broad anti-inflammatory effects. On one hand, PPARγ has been shown to attenuate the expression and secretion of proinflammatory cytokines (including IL-1β and TNF-α) associated with M1 macrophages (6); on the other hand, it reduces macrophage activity via transrepression of NF-κB (7). Despite their efficacy in glycemic control and reduction of steatosis, TZDs are associated with various serious adverse side effects, including weight gain, fluid retention, osteoporosis, and cardiovascular toxicity, which have strongly limited their clinical use (4). These limitations highlight the need for novel approaches such as more selective PPARγ agonists or direct activation of downstream targets.Recently we have identified fibroblast growth factor 1 (FGF1) as a target of PPARγ in visceral adipose tissue and as a critical factor in adipose remodeling (8). Mice with an FGF1 deficiency displayed a severe diabetic phenotype with increased inflammation and fibrosis in adipose tissue. Conversely, pharmacological treatment with recombinant FGF1 (rFGF1) has a potent insulin-sensitizing effect at the systemic level, and in the liver it effectively reduces steatosis in ob/ob mice (9). It remains unclear, however, if and to what extent the hepatic effects of FGF1 are direct or indirect.In this study we used two etiologically different models of NAFLD to determine the mechanism by which rFGF1 improves liver disease: leptin-deficient ob/ob mice, which develop steatosis primarily through excessive food intake, and mice with a dietary choline deficiency, which develop steatosis primarily as a result of a defect in hepatic lipid catabolism (10). Interestingly, we found that rFGF1 effectively reverses steatosis in ob/ob mice but not in mice with a dietary choline deficiency, suggesting that rFGF1 stimulates hepatic lipid catabolism. rFGF1 treatment improved steatohepatitis and plasma alanine transaminase activity (ALT) in both models, indicating that the effects of rFGF1 on hepatic inflammation and liver function are independent of its antisteatotic properties. Together our results provide insight into the mechanism by which rFGF1 improves NAFLD and highlight its potential therapeutic value in the treatment of different aspects of liver disease.     

脂滴自噬在丙型肝炎病毒核心蛋白下调沉默信息调节因子1诱导小鼠肝脂肪变性中的作用

目的研究脂滴自噬在HCV核心蛋白下调沉默信息调节因子1(SIRT1)诱导小鼠肝脂肪变性中的作用。方法小鼠随机分为两组,每组10只。实验组小鼠尾静脉注射HCV core重组表达载体。对照组小鼠尾静脉注射磷酸盐缓冲溶液。1个月后处死小鼠。检测肝功能、脂联素、血清和肝内甘油三酰(TG)、肝脏组织病理学检查肝脂肪变性程度。蛋白质免疫法检测肝脏SIRT1蛋白、脂联素受体(AdipoR)蛋白以及脂滴自噬相关蛋白微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、脂肪分化相关蛋白(ADRP)、尾部作用蛋白(TIP-47)和P62蛋白的表达。计量资料采用t检验。结果与对照组相比,HCV组小鼠出现肝脂肪变性;肝脏TG含量明显增加[(80.9±20.1)比(45.8±10.5)μg/mg,t=4.964,P0.01];血清脂联素[(1.05±0.25)比(1.41±0.45)ng/mL,t=2.211,P0.05]水平下降;SIRT1蛋白水平(0.4±0.1比0.9±0.2,t=7.071,P0.01)和AdipoR2蛋白水平(0.4±0.1比0.8±0.2,t=5.656,P0.01)下降;LC3-Ⅱ蛋白(0.8±0.2比0.4±0.1,t=5.656,P0.01)、TIP-47蛋白(0.9±0.3比0.4±0.1,t=5.000,P0.01)和ADRP蛋白(0.8±0.3比0.4±0.1,t=4.000,P0.01)表达增加;而p62蛋白(0.7±0.2比0.8±0.3,t=0.877,P0.05)表达水平差异无统计学意义。结论 HCV核心蛋白下调SIRT1表达,下调脂联素及受体表达,引起不完全脂滴自噬导致肝脂肪变性。