Figure 4.

GPRC5B Deficiency Suppresses Palmitate-Induced Accumulation of Cer and DAG

(A and B) Lipid compositions of MEFs in the indicated culture conditions were analyzed by high-performance thin-layer chromatography (HPTLC). Lipids were extracted from cultured cells in serum-free medium supplemented with BSA or palmitate-BSA complex for 16 hr. (A) Total lipid extracts (left) and saponified lipids (right) were developed on an HPTLC plate and visualized with copper sulfate/phosphoric acid. (B) Quantification of SM, Cer, and DAG contents in MEFs.

(C and D) Lipid compositions in MEFs analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (C) LC-MS/MS analyses revealed that palmitate exposure induces de novo (sphinganine and dihydroceramides) and recycling (sphingosine and Cer) pathways for Cer synthesis in WT MEFs. (D) LC-MS/MS quantification of different molecular species of Cer and dihydroceramide.

(E and F) Lipid compositions of SMS2−/− MEFs transfected with an expression plasmid for Super-SMS2. After 24 hr of transfection, cells were cultured in serum-free DMEM containing BSA or palmitate-BSA complex for 16 hr. Image of HPTLC plate (E) and quantification of the indicated lipids (F) are shown.

(B–D) and (F) Data are means ± SEM (n = 3; ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). ns, Not significant.