Reduced HNF3b (FOXA2) levels in steatosis leads to diminished expression of anti-lipoapoptotic protein ALR

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of hepatic pathologies, ranging from steatosis in its most benign form to cirrhosis in its most advanced form with non-alcoholic steatohepatitis (NASH) in an intermediate level. NASH has been thought to be a liver disease triggered by various factors such as triglyceride accumulation in hepatocytes and followed by oxidant stress and lipid peroxidation causing inflammation and fibrosis. The progression of Steatosis to NASH is facilitated when adaptive mechanisms to protect hepatocytes from fatty acid-mediated lipotoxicity are failing. The winged Helix Forkhead Box A2, FOXA2 (HNF3β) transcription factor plays an important role in controlling both metabolism and homeostasis of lipids through regulation of multiple target genes in the liver. ALR (augmenter of liver regeneration), a protein known to augment liver regeneration was further shown to reveal hepatoprotective properties against liver toxins. We have previously shown that gene expression of ALR is regulated by FOXA2 in liver cells. In addition, we have demonstrated that ALR attenuates the ER stress-mediated apoptosis induced by palmitic acid in hepatic cells. The aim of our study was to analyze the expression of FOXA2 and ALR and to investigate the potential protective role of ALR under conditions of NASH/steatosis. Steatotic liver tissues obtained from high fat diet (HFD) mouse model (n= 6) and patients with NAFLD (n= 27) were analyzed to determine the expression of both FOXA2 and ALR demonstrating reduced FOXA2 and ALR levels compared to control groups. Furthermore we cultured HepG2 cells treated with palmitic pcid (PA) followed by FOXA2 immunofluorescence staining. PA-treated cells revealed significant translocation of FOXA2 from nucleus into cytosol compared to non-treated cells. In addition, we found diminished ALR mRNA expression levels in PA-loaded cells compared to non-treated cells. To overcome the functional redundancy, we aimed to investigate the role of ALR in PA treated hepatoma cells by gain-of-function experiments. Therefore, we generated stable-transfected HepG2 cells expressing human 15 kDa (short form) ALR protein (HepG2-ALR). Interestingly, culturing HepG2 and HepG2-ALR cells treated with increasing concentrations of PA showed a significant lower lipotoxicity for HepG2-ALR cells accompanied by reduced triglycerides storage compared to mock HepG2 cells.

This study provides evidence that ALR, mediated Foxa2 (HNF3β), plays an important role in NAFLD by alleviating the lipid-mediated ER stress and reducing lipid accumulation in liver cells.

Corresponding author: Dayoub, Rania

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