Stéphanie Conzelmann

Monocyte chemoattractant protein‐1 secreted by adipose tissue induces direct lipid accumulation in hepatocytes

For many years, adipose tissue has been mainly considered as an inert reservoir for storing triglycerides. Since the discovery that adipocytes may secrete a variety of bioactive molecules (hormones, chemokines, and cytokines), an endocrine and paracrine role for white adipose tissue (WAT) in the regulation of energy balance and other physiological processes has been established, particularly with regard to brain and muscle. In contrast, little is known about the interactions of WAT with liver. Hence, we examined the effect of the secretory products of WAT on hepatocytes. Conditioned medium of human WAT explants induced significant steatosis in hepatocyte cell lines. Factor(s) responsible for the conditioned medium‐induced steatosis were screened by a battery of blocking antibodies against different cytokines/chemokines shown to be secreted by WAT. In contrast to interleukin‐8 and interleukin‐6, the monocyte chemoattractant protein‐1 was capable of inducing steatosis in hepatocytes in a time‐dependent manner at concentrations similar to those found in conditioned medium. Incubation of conditioned medium with antimonocyte chemoattractant protein‐1 antibodies prevented triglyceride accumulation. Investigation of the mechanism leading to the triglyceride accumulation showed that both a diminution of apolipoprotein B secretion and an increase in phosphoenolpyruvate carboxykinase messenger RNA may be involved. Conclusion: The monocyte chemoattractant protein‐1 secreted by adipose tissue may induce steatosis not only recruiting macrophages but also acting directly on hepatocytes. (HEPATOLOGY 2008.)

The hepatitis C virus core protein indirectly induces alpha-smooth muscle actin expression in hepatic stellate cells via interleukin-8

BACKGROUND & AIMS Progressive deposition of liver fibrosis is a common feature of chronic hepatitis associated with hepatitis C virus (HCV) infection, and it may eventually lead to cirrhosis and liver failure. Although this fibrogenic process appears to be linked to HCV protein expression and replication via indirect mechanisms, i.e., to be mediated by virally-driven inflammation, a direct role of HCV in inducing fibrosis deposition has never been entirely excluded. METHODS We established an in vitro system in which the human hepatic stellate cell line LX-2 was cultured in the presence of conditioned medium from human hepatoma Huh-7 cells transduced with a lentiviral vector expressing HCV core proteins of different genotypes. RESULTS Treatment of LX-2 cells, with conditioned medium from Huh-7 cells expressing HCV core protein, led to the activation of alpha-smooth muscle actin expression. Among the chemokines secreted by cells transduced with HCV core, interleukin-8 was identified as the strongest inducer of alpha-smooth muscle actin expression in LX-2 and primary hepatic stellate cells. This effect was accompanied by a decrease in cell migration and increased focal contact organisation. CONCLUSIONS The expression of the HCV core in hepatocytes may contribute to the establishment of a profibrogenic microenvironment.

Gene expression profile of Huh-7 cells expressing hepatitis C virus genotype 1b or 3a core proteins.

Background: The liver disease expression in chronic hepatitis C patients is variable and may partially depend on the sequence of the infecting viral genotype.

PTEN protein phosphatase activity regulates hepatitis C virus secretion through modulation of cholesterol metabolism

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is dependent on lipid metabolism. Hepatocyte steatosis occurs frequently in HCV infection, but the relationship between steatosis and HCV life cycle is unclear. We showed that HCV induces steatosis via the downregulation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). We here investigated how PTEN may affect HCV production. METHODS The effect of overexpression or silencing of PTEN on HCV secretion was assessed in genomic-length Jc1 infected HuH7 cells. The role of PTEN protein and lipid phosphatase activities on lipid metabolism and infectious viral particle secretion was investigated using dominant-negative PTEN mutants. The importance of cholesterol metabolism for PTEN-dependent lipid droplet biogenesis and viral particle secretion was examined using statins. RESULTS PTEN silencing in Jc1 infected HuH7 cells stimulated HCV particle secretion, while PTEN overexpression decreased virus egress. Viral secretion was also increased by overexpression of protein phosphatase-deleted (PTENY138L), but not lipid phosphatase-deleted (PTENG129E), PTEN mutant, thus indicating that the protein phosphatase activity of PTEN controls viral secretion. Similarly, PTENY138L, but not PTENG129E mutant induced the formation of large lipid droplets. PTENY138L mutant did not affect biosynthesis of triglycerides, but promoted the biosynthesis of cholesterol esters. Consistently, statins prevented the increased cholesterol ester production, large lipid droplet formation, and viral secretion in cells expressing the PTENY138L mutant. CONCLUSIONS Downregulation of PTEN protein phosphatase activity by HCV affects cholesterol metabolism, thereby inducing the appearance of large lipid droplets and increasing virion egress.

Effects of hepatitis C virus on suppressor of cytokine signaling mRNA levels: comparison between different genotypes and core protein sequence analysis

Glucose metabolism disturbances, including insulin resistance and type 2 diabetes, are frequent and important cofactors of hepatitis C. Increasing epidemiological and experimental data suggest that all major genotypes of hepatitis C virus (HCV), albeit to a different extent, cause insulin resistance. The HCV core protein has been shown to be sufficient to impair insulin signaling in vitro through several post‐receptorial mechanisms, mostly via the activation of suppressor of cytokine signaling (SOCS) family members and the consequent decrease of insulin receptor substrate‐1 (IRS‐1). The levels of IRS‐1 and SOCS were investigated upon expression of the core protein of HCV genotypes 1–4. Furthermore, the core protein sequences were analyzed to identify the amino acid residues responsible for IRS‐1 decrease, with particular regard to SOCS mRNA deregulation. The results suggest that the activation of SOCS family members is a general mechanism associated with the common HCV genotypes. A rare genotype 1b variant, however, failed to activate any of the SOCS tested: this allowed to analyze in detail the distinct amino acid sequences responsible for SOCS deregulation. By combining approaches using intergenotypic chimeras and site‐directed mutagenesis, genetic evidence was provided in favor of a role of amino acids 49 and 131 of the HCV core‐encoding sequence in mediating SOCS transactivation. J. Med. Virol. 83:1005–1015, 2011.

HCV 3a Core Protein Increases Lipid Droplet Cholesteryl Ester Content via a Mechanism Dependent on Sphingolipid Biosynthesis

Hepatitis C virus (HCV) infected patients often develop steatosis and the HCV core protein alone can induce this phenomenon. To gain new insights into the pathways leading to steatosis, we performed lipidomic profiling of HCV core protein expressing-Huh-7 cells and also assessed the lipid profile of purified lipid droplets isolated from HCV 3a core expressing cells. Cholesteryl esters, ceramides and glycosylceramides, but not triglycerides, increased specifically in cells expressing the steatogenic HCV 3a core protein. Accordingly, inhibitors of cholesteryl ester biosynthesis such as statins and acyl-CoA cholesterol acyl transferase inhibitors prevented the increase of cholesteryl ester production and the formation of large lipid droplets in HCV core 3a-expressing cells. Furthermore, inhibition of de novo sphingolipid biosynthesis by myriocin - but not of glycosphingolipid biosynthesis by miglustat - affected both lipid droplet size and cholesteryl ester level. The lipid profile of purified lipid droplets, isolated from HCV 3a core-expressing cells, confirmed the particular increase of cholesteryl ester. Thus, both sphingolipid and cholesteryl ester biosynthesis are affected by the steatogenic core protein of HCV genotype 3a. These results may explain the peculiar lipid profile of HCV-infected patients with steatosis.

Role of seipin in lipid droplet morphology and hepatitis C virus life cycle.

Infectious hepatitis C virus (HCV) particle assembly starts at the surface of lipid droplets, cytoplasmic organelles responsible for neutral fat storage. We analysed the relationship between HCV and seipin, a protein involved in lipid droplet maturation. Although seipin overexpression did not affect the total mean volume occupied by lipid droplets nor the total triglyceride and cholesterol ester levels per cell, it caused an increase in the mean diameter of lipid droplets by 60 %, while decreasing their total number per cell. The latter two effects combined resulted in a 34 % reduction of the total outer surface area of lipid droplets per cell, with a proportional decrease in infectious viral particle production, probably due to a defect in particle assembly. These results suggest that the available outer surface of lipid droplets is a critical factor for HCV release, independent of the neutral lipid content of the cell.

Hepatitis C Virus Increases Occludin Expression via the Upregulation of Adipose Differentiation-Related Protein

The hepatitis C virus (HCV) life cycle is closely associated with lipid metabolism. In particular, HCV assembly initiates at the surface of lipid droplets. To further understand the role of lipid droplets in HCV life cycle, we assessed the relationship between HCV and the adipose differentiation-related protein (ADRP), a lipid droplet-associated protein. Different steps of HCV life cycle were assessed in HCV-infected human Huh-7 hepatoma cells overexpressing ADRP upon transduction with a lentiviral vector. HCV infection increased ADRP mRNA and protein expression levels by 2- and 1.5-fold, respectively. The overexpression of ADRP led to an increase of (i) the surface of lipid droplets, (ii) the total cellular neutral lipid content (2.5- and 5-fold increase of triglycerides and cholesterol esters, respectively), (iii) the cellular free cholesterol level (5-fold) and (iv) the HCV particle production and infectivity (by 2- and 3.5-fold, respectively). The investigation of different steps of the HCV life cycle indicated that the ADRP overexpression, while not affecting the viral replication, promoted both virion egress and entry (~12-fold), the latter possibly via an increase of its receptor occludin. Moreover, HCV infection induces an increase of both ADRP and occludin expression. In HCV infected cells, the occludin upregulation was fully prevented by the ADRP silencing, suggesting a specific, ADRP-dependent mechanism. Finally, in HCV-infected human livers, occludin and ADRP mRNA expression levels correlated with each other. Alltogether, these findings show that HCV induces ADRP, which in turns appears to confer a favorable environment to viral spread.

Intrahepatic mRNA levels of SOCS1 and SOCS3 are associated with cirrhosis but do not predict virological response to therapy in chronic hepatitis C.

Antiviral treatment of chronic hepatitis C is not invariably successful, costly and associated with serious side‐effects, and therefore should be indicated only when the chances of benefitting patients exceed the potential risks. The suppressor of cytokine signalling (SOCS) family members have been suggested to affect the rate of virological response to therapy, but the published evidence is conflicting.

1140 HEPATITIS C VIRUS AND LIPID DROPLETS: ROLE OF ADIPOSE DIFFERENTIATION-RELATED PROTEIN IN LIPID DROPLET MORPHOLOGY AND VIRAL LIFE CYCLE

Aims and Objectives: We evaluated whether HBV +ve and HCV +ve patients are at high risk for developing drug induced hepatitis than control subjects during treatment for tuberculosis with standard short course regimens. Study design: Observational cohort study. Place and Duration: This study was conducted at department of Medicine, Liaquat University of Medical and Health sciences Jamshoro from May 2008 to May 2011. Material and Methods: All newly diagnosed active tuberculosis patients were included in the study population and they were further screened for hepatitis B surface antigen and HCV antibodies. All patients were divided into three groups. One having no coinfection with hepatitis B and Hepatitis C and was taken as control group, second group was co-infected with hepatitis B and third was co-infected with hepatitis C. short course anti tuberculous regimen was started and patients were followed for six months. Results: One hundred and twenty eight tuberculous patients were divided into three groups. 92 in control groups without any coinfection with hepatitis B and C, 10 were HBV +ve and 26 were HCV +ve. During follow up 24 developed drug induced hepatitis, 8 (38.33%, n = 24) in control group, 2 (8.33%, n = 24) in hepatitis B group and 14 (58.33%, n = 24) in hepatitis C group. Conclusion: These findings suggest that treatment for tuberculosis in HCV seropositive patients is a risk factor for the development of hepatitis exacerbation and HBV seropositive patients shows no any increased risk of hepatitis exacerbation.