FATP4 stimulates JNK activation via ER stress and Rac1/MLK3/p-MKK7 signaling leading to enhanced lipoapoptosis

In non-alcoholic steatohepatitis (NASH), saturated fatty acids injure hepatocytes by inducing apoptosis. It has been reported that inhibitors of fatty acyl-CoA synthetases inhibit palmitate-induced apoptosis. While the functions of FATP4 in the liver are not known, we aim to study the mechanisms of FATP4 on lipoapoptosis in liver cells, and whether FATP4 directs hepatic lipid metabolism to specific pathways. We generated stable cell lines by transfection of HUH-7 cells with GFP or FATP4-plasmids. Compared to GFP^+/+, FATP4^+/+ cells were more susceptible towards apoptosis induced by palmitate by 3-folds. For both GFP^+/+ and FATP4^+/+ cells, the extent of palmitate-induced apoptosis was further increased when cultured in methionine- and choline-deficient medium. This indicates the dependence of lipoapoptosis on phosphatidylcholine synthesis which requires choline and methionine. It is known that lipoapoptosis occurs via JNK activation. With greater response in FATP4^+/+ compared to GFP^+/+ cells, palmitate treatment enhanced phosphorylation of JNK and its downstream ATF-2 and c-Jun molecules. These activities were inhibitable by FATP4 knockdown. Two mechanisms responsible for FATP4-mediated JNK activation during lipoapoptosis were delineated: one involves activation of ER stress (via CHOP and PUMA), and the other involves an induction of cytoskeletal reorganization via MLK-3/p-MKK-7 signalling pathway. The latter was evidenced by Rac1/Cdc42 activation and increased expression of cytoskeletal proteins upon palmitate treatment of FATP4^+/+ cells. Detailed ESI/MS/MS analysis revealed that FATP4^+/+ cells contained decreased intracellular levels of plasmalogens which are important structural phospholipids. FATP4 may contribute to increased apoptosis sensitivity during NASH; as serum plasmalogens in NASH patients have been shown to be low. Taken together, our data demonstrated the effects of FATP4 on lipoapoptosis via JNK activation which leads to activation of apoptotic ER stress and cdc42/Rac1/MLK-3/p-MKK-7 pathways. Our data implicate the role of FATP4 on hepatocellular apoptosis during NASH.