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;48:799-807.)

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Background & aims: miR-21-5p is a potent oncogenic microRNA targeting many key tumour suppressors including phosphatase and tensin homolog (PTEN). We recently identified PTEN as a key factor modulated by hepatitis C virus (HCV) to promote virion egress. In hepatocytes, expression of HCV-3a core protein was sufficient to downregulate PTEN and to trigger lipid droplet accumulation. Here, we investigated whether HCV controls PTEN expression through miR-21-5p-dependent mechanisms to trigger steatosis in hepatocytes and to promote HCV life cycle.Methods: MiR-21-5p expression in HCV-infected patients was evaluated by transcriptome meta-analysis. HCV replication and viral particle production were investigated in Jc1-infected Huh-7 cells after miR-21-5p inhibition. PTEN expression and steatosis were assessed in HCV-3a core protein-expressing Huh-7 cells and in mouse primary hepatocytes having miR-21-5p inhibited or genetically deleted respectively.HCV-3a core-induced steatosis was assessed in vivo in Mir21a knockout mice. Results: MiR-21-5p expression was significantly increased in hepatic tissues from HCV-infected patients. Infection by HCV-Jc1, or transduction with HCV-3a core, upregulated miR-21-5p expression and/or activity in Huh-7 cells. miR-21-5p inhibition decreased HCV replication and release of infectious virions by Huh-7 cells. HCV-3a core-induced PTEN downregulation and steatosis were further prevented in Huh-7 cells following miR-21-5p inhibition or in Mir21a knockout mouse primary hepatocytes. Finally, steatosis induction by AAV8-mediated HCV-3a core expression was reduced in vivo in Mir21a knockout mice. Conclusion: MiR-21-5p activation by HCV is a key molecular step, promoting both HCV life cycle and HCV-3a core-induced steatosis and may be among the molecular changes induced by HCV-3a to promote carcinogenesis. K E Y W O R D S hepatitis C, lipid metabolism, microRNA, phosphatase and tensin homolog | 1227 CLÉMENT ET aL.

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.

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.

The expression of HCV core proteins of different genotypes leads to a specific gene expression profile. This may account for the variable disease expression associated with HCV infection.

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.