Tu1811 Correlation Between Mutations in the HFE Gene and Alanine Transaminase Levels May Increase the Risk of Cardiovascular Disease Events in Patients With NASH

Abstract

Background and Aim: Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis (NASH). We have previously reported that saturated free fatty acids (FFAs), but not unsaturated FFAs, induces hepatocyte apoptosis (Malhi et al, JBC, 2006). Cellular inhibitor of apoptosis 1 and 2 (cIAP-1 and -2) protein are potent inhibitors of apoptosis. However, the role of the cIAPs in FFA-mediated apoptosis are unexplored. Our aim was to determine if cIAPs are dysregulated during hepatocyte lipoapoptosis. Methods: Huh-7 cells, a human hepatocellular carcinoma cell line, were treated with FFAs (100-800 uM). cIAPs protein and mRNA expression were assessed by immunoblotting and real time PCR, respectively. cIAP1 and 2, caspase 8 were selectively knocked down using shRNA. Apoptosis was assessed by characteristic nuclear staining with DAPI and caspase 3/7 activity assay. Results: cIAP-1 and 2 protein underwent rapid cellular elimination with 4 hours following treatment with the 800 uM saturated FFA palmitate (PA), but not with the non-toxic unsaturated FFA oleate (OA). In contrast, PA did not alter cIAP-1 and -2 mRNA expression levels suggesting cellular elimination occurs by a post-translational process. Consistent with this concept, cellular elimination of cIAP proteins was inhibited by the proteasome inhibitor MG 132 (10 uM). In contrast, cIAP protein degradation was not affected by either caspase 8 shRNA targeted knock down, or by pancaspase inhibition with QVD-OPH. Incubation with the SMAC mimetic JP1584 (500 nM), which also induces degradation of cIAPs, significantly enhanced PA mediated apoptosis. HuH-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced free fatty acid mediated apoptosis and caspase 3/7 activity. cIAP-1 protein expression was also decreased by 48% in liver biopsy specimens from patients with non-alcoholic steatohepatitis as compared with patients without steatosis . Conclusions: Collectively, these results implicate proteasomal degradation of cIAP-1 by FFA as a mechanism promoting hepatocyte lipoapoptosis.