Palmitic acid treatment alters expression of arachidonic-acid pathway associated genes in hepatoma cells linking fatty acids to inflammation

Non-alcoholic fatty liver disease (NAFLD) is among the most common liver diseases worldwide and is characterized by abnormal or excessive accumulation of fat in hepatocytes. NAFLD covers a spectrum of liver disorders ranging from simple steatosis (S) to its most aggressive form non-alcoholic steatohepatitis (NASH) and cirrhosis. In contrast to other manifestations of NAFLD NASH is directly linked to inflammatory processes, which are regulated among others by bioactive fatty acids, their derivatives and other types of lipid compounds. To gain more insights gene expression profiling of healthy and diseased tissue is helpful to analyze cluster of dysregulated genes, their associating with cellular pathways and therefore identify potential therapeutic targets. Microarray analysis (Affymetrix GeneChip) on human liver samples was performed with a discovery cohort (normal n = 7, S n = 7, NASH n = 8) followed by a gene set enrichment analysis to identify processes that distinguish between steatosis/NASH and normal groups. Gene Ontology (GO) analysis of differently expressed genes (DEG) identified arachidonic-acid metabolism as one of the main biological processes that is affected in S and NASH samples. Four genes of the DEGs are associated with this metabolic process PTGS2, HPGDS, PLA2G4A and ALOX5AP. The aim of this study was to validate these target genes in a large cohort of human liver samples and to verify these genes as targets of free fatty acids like palmitic acid in vitro.

The target gene expression was validated in a second, independent and large cohort (n = 112) performing quantitative real-time PCR (qRT-PCR). We found that PTGS2 (prostaglandin-endoperoxide synthase 2, COX2) and ALOX5AP (arachidonate 5-lipoxygenase-activating protein) mRNA expression in liver tissue of S (n = 37) and NASH (n = 43) is significantly increased compared to normal (n = 32) liver tissue. HPGDS (hematopoetic prostaglandin synthase) mRNA expression in liver tissue of S significantly decreased compared to normal liver tissue. Furthermore using an in vitro steatosis model we investigated expression of these target genes (mRNA and protein) under conditions of free fatty acid (FFA) treatment. Hepatoma cells (Huh7, HepG2) were incubated time- and dose-dependently with FFAs, and demonstrated enhanced mRNA as well as protein expression of PTGS2 and ALOX5AP in Huh7 cells as well as of ALOX5AP in HepG2 cells treated with palmitic acid (0.4 mM PA). In addition, treatment of HepG2 cells with palmitic (saturated FA, 0.3 mM) and oleic acid (mono unsaturated FA, 0.5 mM), however, had no impact on ALOX5AP mRNA expression. Moreover HPGDS mRNA expression was seen to be reduced in Huh7 cells treated with palmitic acid (0.4 mM).

Results of our in vitro experiments nicely correspond with the findings of the gene expression study using human NAFLD samples. We assume that the toxic FFA, palmitic acid, may be one of the driving forces changing arachidonic acid metabolism and thereby initiating and/or augmenting the inflammatory process in liver tissues of patients with developing NASH. Therefore genes of arachidonic acid metabolism seems to be potential targets to prevent progression from steatosis to NASH.