In the present study, we found that phytoceramide activated PPARs and that PPARβ was more highly stimulated than PPARα and PPARγ. However, ceramide and dihydroceramide did not activate PPARs (Figure 2A and 5D). Analysis of the X-ray structure indicated a hydrogen bond between the ligand and LBD residues of human PPARγ LBD [24, 25]. The hydroxyl group at C4 position in phytoceramide is not present in ceramide and dihydroceramide; therefore, the hydroxyl group at C4 position might be involved in the transcriptional activation of PPARs. Another hypothesis that could explain the superiority of phytoceramide in PPAR activation is that phytoceramide is more easily taken up into the cells than dihydroceramide and ceramide. In that case, the structural difference among ceramide species might have no correlation with PPAR activation.
Phytoceramide is synthesized mainly in the kidney, intestine and skin in mammals, while PHS and DHS are the major sphingoid bases in fungi. Fermented foods contain the PHS-based sphingolipids derived from fungi, and a some of the sphingolipids are hydrolyzed to sphingoid bases in the small intestine . The sphingoid bases are taken up by intestinal cells and reincorporated into complex sphingolipids such as ceramide and sphingomyelin. Some of them reach the lymph, blood and liver [26, 27]. We observed that PHS contained in food was absorbed and reached the mouse liver (unpublished data). We also observed that phytoceramide induced activation of PPARα in HepG2 hepatocytes (Figure 7). Therefore, phytoceramide originating from dietary yeast sphingolipids could be incorporated to the cells, such as hepatocytes and adipocytes, and activate PPARs, regulating the expression of PPARα target genes.
In addition to phytoceramide, yeast-derived sphingoid bases and authentic PHS and DHS treatment activated PPARs (Figure 5C), while ceramide and dihydroceramide treatment did not activate PPARs. There were no distinct differences between yeast-derived sphingoid bases and authentic sphingoid bases in PPAR activation, but their effects were smaller than for the positive controls (WY, LD and CIG) (Figure 5C). These results indicate that activation of PPARs by yeast-derived sphingoid bases does not reach maximal levels, suggesting that unidentified factors included in yeast-derived sphingoid bases do not contribute to PPAR activation. There is a possibility that PHS and DHS interact directly with PPARs and activate PPARs. The incorporation rate of sphingoid bases into the cells is higher than that of ceramide species [28, 29]. If PHS was a true ligand of PPARs, PPAR activation occurring by PHS treatment would be higher than phytoceramide treatment. However, the activation of PPARs by PHS was not more potent than phytoceramide at the same concentration, as shown in Figure 4. Therefore, we considered that PHS-induced activation of PPARs was caused by phytoceramide generated from PHS in the cells. It remains unclear why DHS treatment activates PPARs or whether metabolites of DHS are the active agents. Future studies are needed to elucidate this question.
In this study, PPARβ was most intensively stimulated by phytoceramide in three PPARs. It has been reported that liver-specific PPARβ overexpression decreases glucose production and increases glucose disposal in the liver . Moreover, transgenic mice overexpressing constitutively active PPARβ in white adipocytes exhibit reduced fat mass . Therefore, phytoceramide-induced activation via PPARβ might contribute to the anti-obesity effect.
It has been reported that PPARβ is concerned with keratinocyte differentiation, and the DES2 protein that converts dihydroceramide to phytoceramide is expressed in differentiated keratinocytes. Glucosylceramide in the extracellular spaces of the epidermis is hydrolyzed to free ceramide species , and these lipids are involved in skin barrier function. Alkaline ceramidase is highly expressed in mouse skin and does not digest phytoceramide well . Therefore, there is a possibility that phytoceramide in epidermis is protected from digestion and that phytoceramide might regulate epidermis function as a PPARβ ligand.