FIGURE 4.

The induction of ceramide in response to palmitate and LPS occurs through a NF-κB and MAP kinase-independent mechanism. A and B, macrophages were pretreated with the NF-κB translocation inhibitor SN50 (25 μm) and then with palmitate ± LPS and SN50 for 16 h. TNFα secretion (A) and C16 ceramide levels (B) were quantified by ELISA and LC-MS/MS, respectively. C and D, mRNA expression of IKKβ relative to 36B4 was quantified by quantitative real-time-PCR (C), and ceramide levels were quantified after 16 h of treatment as indicated (D) in macrophages from IKKβ flox mice (FLOX) and from IKKβ flox mice crossed with LysM-Cre mice (KO). E and F, MAP kinase inhibitors for p38 (p38i SB203580, 20 μm), JNK (JNKi SP600125, 20 μm), and ERK (ERKi PD98059,10 μm) were preincubated with pMACs before stimulation with palm and LPS. TNFα secretion (E) and C16 ceramide levels (F) were determined 16 h after treatment. Bar graphs report the mean ± S.E. for a minimum of three experiments, each performed in triplicate. *p < 0.05 for KO/TG versus WT and for inhibitor versus vehicle. ns, not significant.