The estimated prevalence of non‐alcoholic fatty liver disease (NAFLD) worldwide is approximately 25%. However, the real prevalence of NAFLD and the associated disorders is unknown mainly because reliable and applicable diagnostic tests are lacking. This is further complicated by the lack of consensus on the terminology of different entities such as NAFLD or nonalcoholic steatohepatitis (NASH). Although assessing fatty infiltration in the liver is simple by ultrasound, the gold standard for the assessment of fibrosis, the only marker of progression towards more severe liver disease is still liver biopsy. Although other non‐invasive tests have been proposed, they must still be validated in large series. Because NAFL/NAFLD/NASH and related metabolic diseases represent an economic burden, finding an inexpensive method to diagnose and stage fatty liver is a priority. A translational approach with the use of cell and/or animal models could help to reach this goal.

BackgroundIn vitro exposure of liver cells to high concentrations of free fatty acids (FFA) results in fat overload which promotes inflammatory and fibrogenic response similar to those observed in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH). Since the mechanisms of this event have not been fully characterized, we aimed to analyze the fibrogenic stimuli in a new in vitro model of NASH.MethodsHuH7 cells were cultured for 24 h in an enriched medium containing bovine serum albumin and increasing concentrations of palmitic and oleic acid at a molar ratio of 1:2 (palmitic and oleic acid, respectively). Cytotoxic effect, apoptosis, oxidative stress, and production of inflammatory and fibrogenic cytokines were measured.ResultsFFA induces a significant increment in the intracellular content of lipid droplets. The gene expression of interleukin-6, interleukin-8 and tumor necrosis factor alpha was significantly increased. The protein level of interleukin-8 was also increased. Intracellular lipid accumulation was associated to a significant up-regulation in the gene expression of transforming growth factor beta 1, alpha 2 macroglobulin, vascular endothelial growth factor A, connective tissue growth factor, insulin-like growth factor 2, thrombospondin 1. Flow cytometry analysis demonstrated a significant increment of early apoptosis and production of reactive oxygen species.ConclusionsThe exposure of hepatocytes to fatty acids elicits inflammation, increase of oxidative stress, apoptosis and production of fibrogenic cytokines. These data support a primary role of FFA in the pathogenesis of NAFLD and NASH.

Background & aims Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that contributes to the inflammatory response to injury. MIF is expressed by multiple cell types; however, the cellular source and actions of MIF in alcoholic liver disease (ALD) are not well known. Here we tested the hypothesis that non-myeloid cells, specifically hepatocytes, are an important cellular source of MIF in ALD. Methods MIF expression was measured in HuH7 and differentiated THP-1 cells in response to ethanol. Ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif−/− bone marrow chimeras. MIF was measured in peripheral and suprahepatic serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH). Results HuH7 hepatocytes, but not THP-1 macrophages, released MIF in response to challenge with ethanol in culture. In chimeric mice expressing MIF in non-myeloid cells (Mif−/− →WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and expression of cytokine/chemokine mRNA. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→ Mif−/−) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH. Conclusions Taken together, these data provide evidence that hepatocyte-derived MIF is critical to the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH.

Non Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) are the hepatic manifestations of the metabolic syndrome; worrisome is the booming increase in pediatric age. To recreate the full spectrum of juvenile liver pathology and investigate the gender impact, male and female C57Bl/6 mice were fed with high fat diet plus fructose in the drinking water (HFHC) immediately after weaning (equal to 3-years old human), and disease progression followed for 16 weeks, until adults (equal to 30-years old human). 100% of subjects of both genders on HFHC diet developed steatosis in 4weeks, and some degree of fibrosis in 8weeks, with the 86% of males and 15% of females presenting a stage 2 fibrosis at 16weeks. Despite a similar final liver damage both groups, a sex difference in the pathology progression was observed. Alterations in glucose homeostasis, dyslipidemia, hepatomegaly and obese phenotype were evident from the very beginning in males with an increased hepatic inflammatory activity. Conversely, such alterations were present in females only at the end of the HFHC diet (with the exception of insulin resistance and the hepatic inflammatory state). Interestingly, only females showed an altered hepatic redox state. This juvenile model appears a good platform to unravel the underlying gender dependent mechanisms in the progression from NAFLD to NASH, and to characterize novel therapeutic approaches.

Hepatocellular carcinoma is one of the most common cancers worldwide. Despite several efforts to elucidate hepatocellular carcinoma molecular pathogenesis, it is still not fully understood. To acquire further insights into the molecular mechanisms of hepatocellular carcinoma, we performed a systematic functional genomic approach on human HuH-7 and JHH-6 cells. The subsequent analysis of the differentially expressed genes in human specimens revealed a molecular signature of 11 genes from which we selected the LGALS1 gene, which was overexpressed in hepatocellular carcinoma. The expression analysis in humans of Galectin-1 (Gal-1), the protein encoded by LGALS1, showed a Gal-1 preferential accumulation in the stromal tissue around hepatocellular carcinoma tumors. Moreover, a significant association between increased expression of Gal-1 in hepatocellular carcinoma and the presence of metastasis was observed. Interestingly, Gal-1 overexpression resulted in an increase of cell migration and invasion. In conclusion, this study provides a portfolio of targets useful for future investigations into molecular marker-discovery studies on a large number of patients and functional assays. In addition, our data provide evidence that Gal-1 plays a role in hepatocellular carcinoma cell migration and invasion, and we suggest that further studies should be conducted to fully establish the role of Gal-1 in hepatocellular carcinoma pathogenesis and evaluate Gal-1 as a potential molecular therapeutic target.