Ceramide triggers ER stress is independent of its downstream metabolite S1P. (A) ACC-M and ACC-2 cells were seeded into 60 mm culture dishes and the next day cells were treated with 5–10 μM S1P. The level of phosphorylated eIF2α and ERK was analyzed. Unphosphorylated eIF2α and ERK were measured and actin were used as loading control. The experiment was repeated several times and the representative result is shown. (B) Proposed mechanism of ceramide-mediated activation of ER stress response in ACCs. Ceramide induces SERCA inhibition and ER calcium depletion. This leads to increase GRP78 and activate PERK/eIF2α and IRE1α/XBP1 arm of ER stress. FB1 inhibits ceramide-induced ER stress. Prolonged ER stress eventually induces apoptosis through activates pro-apoptotic proteins CHOP and JNK.