Phytoceramide and sphingoid bases derived from brewer's yeast Saccharomyces pastorianus activate peroxisome proliferator-activated receptors

Abstract

Background: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate lipid and glucose metabolism. PPARα is highly expressed in the liver and controls genes involved in lipid catabolism. We previously reported that synthetic sphingolipid analogs, part of which contains shorter-length fatty acid chains than natural sphingolipids, stimulated the transcriptional activities of PPARs. Sphingosine and dihydrosphingosine (DHS) are abundant sphingoid bases, and ceramide and dihydroceramide are major ceramide species in mammals. In contrast, phytosphingosine (PHS) and DHS are the main sphingoid bases in fungi. PHS and phytoceramide exist in particular tissues such as the epidermis in mammals, and involvement of ceramide species in PPARβ activation in cultured keratinocytes has been reported. The purpose of the present study is to investigate whether natural sphingolipids with C18 fatty acid and yeast-derived sphingoid bases activate PPARs as PPAR agonists.

Method: Lipids of brewer's yeast contain PHS- and DHS-based sphingolipids. To obtain the sphingoid bases, lipids were extracted from brewer's yeast and acid-hydrolyzed. The sphingoid base fraction was purified and quantified. To assess the effects of sphingolipids on PPAR activation, luciferase reporter assay was carried out. NIH/3T3 and human hepatoma (HepG2) cells were transfected with expression vectors for PPARs and retinoid × receptors, and PPAR responsive element reporter vector. When indicated, the PPAR/Gal4 chimera system was performed to enhance the credibility of experiments. Sphingolipids were added to the cells and the dual luciferase reporter assay was performed to determine the transcriptional activity of PPARs.

Results: We observed that phytoceramide increased the transcriptional activities of PPARs significantly, whereas ceramide and dihydroceramide did not change PPAR activities. Phytoceramide also increased transactivation of PPAR/Gal4 chimera receptors. Yeast-derived sphingoid base fraction, which contained PHS and DHS, or authentic PHS or DHS increased PPAR-dependent transcription. Additionally, phytoceramide stimulated PPARα activity in HepG2 hepatocytes, suggesting that phytoceramide activates genes regulated by PPARα.

Conclusions: Phytoceramide and yeast-derived sphingoid bases activate PPARs, whereas ceramide and dihydroceramide do not change the PPAR activity. The present findings suggest that phytoceramide acts as a PPAR ligand that would regulate PPAR-targeted genes.

Figure 1

Chemical structure of ceramide species (ceramide, phytoceramide and dihydroceramide).

Figure 2

Effects of phytoceramide on PPAR transcriptional activity. (A) NIH/3T3 cells were transfected with 4 × PPRE firefly luciferase reporter plasmid along with expression plasmids for PPARs (PPARα, PPARβ or PPARγ) and RXR and an internal control TK Renilla luciferase vector. After 24 h cultivation, the cells were treated with the indicated concentration of phytoceramide or ceramide for 24 h. As positive control, 10 μM of WY-14643 (WY), L-165,041 (LD) or ciglitazone (CIG) were used. (B) NIH/3T3 cells were transfected as described above, expect for an empty luciferase reporter instead of 4 × PPRE firefly luciferase reporter. Twenty-four hours after transfection, phytoceramide was added to the cells for 24 h. The luciferase activity of the cell was measured, and firefly luciferase activity was normalized to Renilla luciferase activity. The activity of a vehicle control was expressed as 1 and the relative luciferase activities were presented as a fold induction to that of the vehicle control. All results are shown as means ± S.D. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the level of vehicle control (Dunnett's test).

Figure 3

Effect of phytoceramide on PPAR activity in the Gal4/PPAR chimera system. The expression vector for PPAR LBD/Gal4 DBD and Gal4 UAS luciferase reporter were transfected into NIH/3T3 cells. After 24 h, cells were treated with phytoceramide at the indicated concentrations. The luciferase assay was performed as described in the legend to Figure 2A. All results are shown as means ± S.D. (n = 3). *P < 0.05, ***P < 0.001, significantly different from the level of vehicle control.

Figure 4

Comparison between phytoceramide and PHS in PPAR activation. NIH/3T3 cells were transfected as described for Figure 2A. PHS was dissolved in DMSO to carry out the experiments under the same condition with phytoceramide. Transfected cells were treated with phytoceramide or PHS at the indicated concentrations for 24 h, and then the luciferase activity was quantified. All results are shown as means ± S.D. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the level of vehicle control.

Figure 5

Effects of sphingoid bases derived from brewer's yeast in PPAR activation. (A and B) Sphingoid base fraction from brewer's yeast was extracted, treated with o-phthalaldehyde, and analyzed by reversed-phase HPLC. The areas of PHS and DHS were quantified. (C) NIH/3T3 cells were transfected as described for Figure 2A. Cells were stimulated with the indicated concentration of yeast-derived sphingoid base fraction. Cells were also treated with authentic PHS, DHS or the mixture of PHS and DHS. (D) Transfected cells were cultured with phytoceramide or dihydroceramide (1 or 10 μM) for 24 h. Luciferase activity was presented as a fold induction relative to that of the control. All results are shown as means ± S.D. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the level of vehicle control.

Figure 6

Effects of sphingolipid administration on cell viability. NIH/3T3 cells were seeded into a 96-well culture plate. (A) C2 ceramide, phytoceramide and ceramide were dissolved in DMSO, and (B) yeast degradate, PHS and DHS were dissolved in EtOH. These sphingolipids were added to the cells for 24 h. Cell viability was measured using Cell Counting Kit-8. Cell viability is expressed as a relative value to vehicle control. (C) NIH/3T3 cells were transfected with 6 ×AP-1 reporter and TK Renilla luciferase vector for 24 h. Cells were treated with the indicated concentration of phytoceramide for 4 h. The luciferase activity was measured and expressed as fold change of control (Ctl). The results represent the means of triplicate determinations ± S.D. from a representative experiment.

Figure 7

Effect of phytoceramide on transcriptional activity of PPARα in HEPG2 hepatocytes. HEPG2 cells were transfected with 4 × PPRE reporter plasmid and expression vectors for PPARα and RXR. TK Renilla luciferase reporter was cotransfected for normalizing the transfection efficiency. Following transfection, cells were treated for 24 h with phytoceramide (1, 10 μM). Luciferase assays were performed as described elsewhere. All results are shown as means ± S.D. (n = 3). *P < 0.05, significantly different from the level of vehicle control.