The JNK signaling pathway mediates the proapoptotic effects of palmitate, is activated by the ceramide biosynthetic pathway, and is inhibited by exendin-4. (A, B) Effects of JNK inhibition on palmitate-induced c-Jun phosphorylation and apoptosis. Human CPCs were treated with 20 µmol/L SP600125 for 1 hour and then exposed to palmitate for 16 hours. (A) Phosphorylation of c-Jun was evaluated by immunoblotting and (B) apoptosis by enzyme-linked immunosorbent assay for cytoplasmic oligonucleosomes. *P < 0.05 vs basal (no palmitate); †P < 0.05 vs cells not treated with SP600125. (C, D) Effects of ceramide synthase inhibition on palmitate-induced c-Jun phosphorylation. CPCs were pretreated with (C) 20 µmol/L FB1 for 8 hours or transfected with (D) 20 µmol/L siRNA specific to CERS5 for 24 hours, or left untreated, and then exposed to 0.25 mmol/L palmitate for 16 hours. Representative immunoblots and quantitation of c-Jun protein content and phosphorylation from at least three independent experiments are shown. *P < 0.05 vs basal (no palmitate); †P < 0.05 vs cells not treated with FB1; ‡P < 0.05 vs cells not transfected with siCERS5. (E) Effects of exendin-4 on the activation of the JNK pathway by palmitate. Human CPCs were incubated in the presence of 20 nmol/L exendin-4 for 8 hours, or left untreated, and then exposed to 0.25 mmol/L palmitate for 16 hours. The panels show the quantitation of JNK1, JNK2, and c-Jun phosphorylation. All data are presented as mean ± standard error of the mean of at least three experiments, which were carried out using cells from different human donors; *P < 0.05 vs basal (no palmitate); †P < 0.05 vs cells not treated with exendin-4. Ex-4, exendin4; p-JNK, phospho-JNK; Palm, palmitate;siCERS5, siRNA to CERS5.