Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.