696 XBP-1 Inhibitor Toyocamycin Attenuates Palmitate-Induced Hepatocyte Apoptosis and Steatosis

Abstract

Background and Aim: Free fatty acid-induced hepatocyte cytotoxicity is deeply associated withmorbidity of Non-alcoholic fatty liver disease (NAFLD) (Malhi et al, 2008, Gastroenterology). Saturated free fatty acids induce hepatocyte lipoapoptosis via endoplasmic reticulum stress (ER) response, which leads to translational activation of DR5 and BH3 only protein Bim (Cazanave et al, JBC, 2011). We previously reported that X-box binding protein-1 (XBP-1) is activated downstream of IRE-1 arm of ER stress by free fatty acid treatment upon its splicing (Akazawa et al, 2009, J Hepatol). However, role of XBP-1 during hepatocyte lipoapoptosis remains relatively unexplored. We examined the impact of inhibiting XBP-1 on hepatocyte lipoapoptosis and steatosis employing Toyocamycin, which was recently reported to inhibit ER stress-mediated XBP-1 splicing (Ri M et al, Blood Cancer J. 2012). Methods: We employed hepatocellular carcinoma cell line, Huh-7 cells, for these studies. Cells were incubated with palmitate (200~800μM) in presence or absence of Toyocamycin (0.1~3μM). Apoptosis was assessed by DAPI staining and caspase 3/7 activity assay. Steatosis was assessed by Nile red staining. mRNA expression of Bim and DR5 was examined by real time PCR. Expression of Bim protein and phosphorylation of JNK was assessed by immunoblotting. Results: As expected, Toyocamycin inhibited palmitate-mediated XBP-1 splicing without affecting amount of total RNA. Toyocamycin significantly reduced palmitateinduced hepatocyte lipoapoptosis (p<0.05) and caspase 3/7 activity (p<0.05) in dose dependent manner. Interestingly, Toyocamycin also attenuated free fatty acid-induced steatosis. In addition, Toyocamycin attenuated expression of Bim protein andmRNA (p<0.05) following palmitate treatment. Furthermore, PAmediated-transcriptional activation of DR5was reduced by Toyocamycin (p<0.05). In contrast, palmitate-induced JNK phosphorylation, which also occurs downstream of IRE-1 arm of ER stress, was not inhibited by Toyocamycin. Conclusions: These data imply that Toyocamycin attenuates hepatocyte lipotoxicity as well as steatosis, likely through inhibition of XBP-1 splicing. Toyocamycin might be beneficial for NASH treatment.