Structure-dependent absorption of atypical sphingoid long-chain bases from digestive tract into lymph.

BACKGROUND: Dietary sphingolipids have various biofunctions, including skin barrier improvement and anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are expected to be important for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear. METHODS: In this study, five structurally different LCBs were prepared from glucosylceramides (GlcCers) with LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH moieties derived from konjac tuber (Amorphophallus konjac), from GlcCers with an LCB 18(9Me):2(4E,8E);2OH moiety derived from Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and from ceramide 2-aminoethyphosphonate with LCB 18:3(4E,8E,10E);2OH moiety and LCB 18(9Me):3(4E,8E,10E);2OH moiety derived from giant scallop (Mizuhopecten yessoensis), and their absorption percentages and metabolite levels were analyzed using a lymph-duct-cannulated rat model via liquid chromatography tandem mass spectrometry (LC/MS/MS) with a multistage fragmentation method. RESULTS: The five orally administered LCBs were absorbed and detected in chyle (lipid-containing lymph) as LCBs and several metabolites including ceramides, hexosylceramides, and sphingomyelins. The absorption percentages of LCBs were 0.10-1.17%, depending on their structure. The absorption percentage of LCB 18:2(4E,8Z);2OH was the highest (1.17%), whereas that of LCB 18:3(4E,8E,10E);2OH was the lowest (0.10%). The amount of sphingomyelin with an LCB 18:2(4E,8Z);2OH moiety in chyle was particularly higher than sphingomyelins with other LCB moieties. CONCLUSIONS: Structural differences among LCBs, particularly geometric isomerism at the C8-C9 position, significantly affected the absorption percentages and ratio of metabolites. This is the first report to elucidate that the absorption and metabolism of sphingolipids are dependent on their LCB structure. These results could be used to develop functional foods that are more readily absorbed.

Plant sphingolipids promote extracellular vesicle release and alleviate amyloid-ß pathologies in a mouse model of Alzheimer's disease.

The accumulation of amyloid-ß protein (Aß) in brain is linked to the early pathogenesis of Alzheimer's disease (AD). We previously reported that neuron-derived exosomes promote Aß clearance in the brains of amyloid precursor protein transgenic mice and that exosome production is modulated by ceramide metabolism. Here, we demonstrate that plant ceramides derived from Amorphophallus konjac, as well as animal-derived ceramides, enhanced production of extracellular vesicles (EVs) in neuronal cultures. Oral administration of plant glucosylceramide (GlcCer) to APP overexpressing mice markedly reduced Aß levels and plaque burdens and improved cognition in a Y-maze learning task. Moreover, there were substantial increases in the neuronal marker NCAM-1, L1CAM, and Aß in EVs isolated from serum and brain tissues of the GlcCer-treated AD model mice. Our data showing that plant ceramides prevent Aß accumulation by promoting EVs-dependent Aß clearance in vitro and in vivo provide evidence for a protective role of plant ceramides in AD. Plant ceramides might thus be used as functional food materials to ameliorate AD pathology.

Rescue of cell growth by sphingosine with disruption of lipid microdomain formation in Saccharomyces cerevisiae deficient in sphingolipid biosynthesis.

In the yeast Saccharomyces cerevisiae, sphingolipids are essential for cell growth. Inactivation of sphingolipid biosynthesis, such as by disrupting the serine palmitoyltransferase gene (LCB2), is lethal, but cells can be rescued by supplying an exogenous LCB (long-chain base) like PHS (phytosphingosine) or DHS (dihydrosphingosine). In the present study, supplying SPH (sphingosine), an unnatural LCB for yeast, similarly rescued the Deltalcb2 cells, but only when SPH 1-phosphate production was inhibited by deleting the LCB kinase gene LCB4. Exogenously added SPH was adequately converted into phosphoinositol-containing complex sphingolipids. Interestingly, cells carrying SPH-based sphingolipids exhibited a defect in the association of Pma1p with Triton X-100-insoluble membrane fractions, and displayed sensitivities to both Ca2+ and hygromycin B. These results suggest that the SPH-based sphingolipids in these cells have properties that differ from those of the PHS- or DHS-based sphingolipids in regard to lipid microdomain formation, leading to abnormal sensitivities towards certain environmental stresses. The present paper is the first report showing that in sphingolipid-deficient S. cerevisiae, the requirement for LCB can be fulfilled by exogenous SPH, although this supplement results in failure of lipid microdomain formation.

Suppression of mast cell degranulation by a novel ceramide kinase inhibitor, the F-12509A olefin isomer K1.

Antigen-induced degranulation of mast cells plays a pivotal role in allergic and inflammatory responses. Recently, ceramide kinase (CERK) and its phosphorylated product ceramide 1-phosphate (C1P) have emerged as important players in mast cell degranulation. Here, we describe the synthesis of a novel F-12509A olefin isomer, K1, as an effective CERK inhibitor. In vitro kinase assays demonstrated that K1 effectively inhibits CERK without inhibiting sphingosine kinase and diacylglycerol kinase. Treating RBL-2H3 cells with K1 reduced cellular C1P levels to 40% yet had no effect on cell growth. Furthermore, treatment with K1 significantly suppressed both calcium ionophore- and IgE/antigen-induced degranulation, indicating that K1 interferes with signals that happen downstream of Ca(2+) mobilization. Finally, we show that K1 affects neither IgE/antigen-induced global tyrosine phosphorylation nor subsequent Ca(2+) elevation, suggesting a specificity for CERK-mediated signals. Our novel CERK inhibitor provides a useful tool for studying the biological functions of CERK and C1P. Moreover, to our knowledge, this is the first report demonstrating that inhibition of CERK suppresses IgE/antigen-induced mast cell degranulation. This finding suggests that CERK inhibitors might be a potential therapeutic tool in the treatment of allergic diseases.

Thalidomide-induced antiangiogenic action is mediated by ceramide through depletion of VEGF receptors, and is antagonized by sphingosine-1-phosphate.

Thalidomide, which is clinically recognized as an efficient therapeutic agent for multiple myeloma, has been thought to exert antiangiogenic action through an unknown mechanism. We here show a novel mechanism of thalidomide-induced antiangiogenesis in zebrafish embryos. Thalidomide induces the defect of major blood vessels, which is demonstrated by their morphologic loss and confirmed by the depletion of vascular endothelial growth factor (VEGF) receptors such as neuropilin-1 and Flk-1. Transient increase of ceramide content through activation of neutral sphingomyelinase (nSMase) precedes thalidomide-induced vascular defect in the embryos. Synthetic cell permeable ceramide, N-acetylsphingosine (C2-ceramide) inhibits embryonic angiogenesis as well as thalidomide. The blockade of ceramide generation by antisense morpholino oligonucleotides for nSMase prevents thalidomide-induced ceramide generation and vascular defect. In contrast to ceramide, sphingosine-1-phosphate (S1P) inhibits nSMase-dependent ceramide generation and restores thalidomide-induced embryonic vascular defect with an increase of expression of VEGF receptors. In human umbilical vein endothelial cells (HUVECs), thalidomide-induced inhibition of cell growth, generation of ceramide through nSMase, and depletion of VEGF receptors are restored to the control levels by pretreatment with S1P. These results suggest that thalidomide-induced antiangiogenic action is regulated by the balance between ceramide and S1P signal.

Sphingosine-1-phosphate lyase is involved in the differentiation of F9 embryonal carcinoma cells to primitive endoderm.

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased approximately 2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.