Our lipidomics analysis showed that several sphingolipid species were significantly upregulated by GW3965. Sphingolipids such as sphinganine, dihydroceramides, ceramides, glucosyl-ceramides, dihydrosphingomyelins, sphingomyelins(Figure 1A-G). There are three major pathways for sphingolipid biosynthesis a shown in Figure 1H: 1) he sphingomyelinase pathway, which uses sphingomyelinase to break down sphingomyelin in the cell membrane and release ceramide; 2) he de novo pathway which synthesizes ceramide from palmitate and serine; and 3) he salvage pathway can generate ceramide through breakdown of complex sphingolipids that are ultimately converted into sphingosine, which then undergoes reacylation to form ceramide. we next looked if the expressions of the key genes regulating sphingolipid synthesis are affected by GW3965. Surprisingly, unlike its effect on the sphingolipid levels, GW3965 had only a modest effect on the expression of sphingolipid biosynthesis genes. Using fatty acid synthase (Fasn) expression as a positive control for LXR transcriptional activity, the relative expression levels of genes involved in the Figure 1H de novo and salvage pathways of ceramide synthesis (e.g., serine palmitoyl transferase SPTLC1-3 , ceramide synthase CerS2-5 , and dihydroceramide desaturase DEGS1-2 ) showed a tendency to increase with LXR activation but did not reach significance (Figure 1I). Similarly, the level of sphingomyelin synthase (SGMS1-2) also did not change. Additionally, GW3965 did not affect the gene expression of phosphatidylinositol-4-phosphate adaptor-2 (FAPP2), a protein needed for intracellular ceramide transport (Figure 1I). These results suggest that LXRs mediated increase in sphingolipid content is possibl independent of the regulation of its biosynthetic genes by LXRs. Therefore, it is possible that LXR agonist may either increase the flux of substrate (i.e., palmitate) via the activity of endogenous Fasn activity or alter the activities of sphingolipid synthesis enzymes. Further studies are needed to determine the specific mechanism(s).
and sphingosine were significantly by GW3965