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PubMed1995

Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells.

A series of radiolabelled ceramides (D-erythro and L-threo) and dihydroceramides (DL-erythro and DL-threo) with 2, 4 or 6 carbon N-acyl groups were synthesized. These analogues were incubated with cultured CHO cells and radioactive products isolated and analyzed. In addition to synthesis of short-chain sphingomyelin and glucosylceramide, radiolabelled sphingosine and sphinganine were released from short-chain ceramides and dihydroceramides and subsequently utilized for synthesis of long-chain ceramide and sphingolipids. Substrate preference for short-chain sphingomyelin synthesis in cells was D-erythro-ceramides > L-threo-ceramides > DL-erythro-dihydroceramides > DL-threo-dihydroceramides, and C4- and C6-analogues were preferred over the C2-analogue. Kinetic constants for conversion of short-chain (dihydro)ceramides to short-chain sphingomyelin were determined using CHO cell membranes and found to correlate with substrate preference in cultured cells. D-erythro-C6-Ceramide was the preferred substrate for short-chain glucosylceramide synthesis. D-erythro-C2-ceramide inhibited incorporation of [3H]serine into sphingomyelin, glucosylceramide and ceramide rapidly (2 h) and in a dose-dependent manner. Over a similar time period, [3H]choline-labelling of sphingomyelin was not affected. Inhibition of [3H]serine-labelling of sphingolipids appeared to correlate with release of [3H]long-chain bases from short-chain ceramides and dihydroceramides and synthesis of long-chain sphingolipids. However, some discrepancies between DL-erythro-C4- and C6-dihydroceramides, and D-erythro-C2-ceramide suggested that short-chain dihydroceramides were less efficient in suppressing de novo synthesis from [3H]serine, while contributing substantially to endogenous sphingolipid synthesis. Inhibition of de novo sphingolipid synthesis by short-chain ceramides and dihydroceramides could not be related to inhibition of serine palmitoyltransferase activity in vitro.

Animals↗
PubMed1982

A change in the cerebrosides and sulfatides in a demyelinating nervous system. Development of the methodology and study of multiple sclerosis and Wallerian degeneration.

This report described a new method for the microanalysis of sphingolipids and its application for the characterization of cerebrosides and sulfatides in multiple sclerosis brain and rat sciatic nerves undergoing Wallerian degeneration. Tissue was extracted with isopropanol/hexane (20:78), and the total lipids obtained were subjected to benzoylation-desulfation. A portion of this was directly analyzed by silica-column high performance liquid chromatography for the determination of nonhydroxycerebroside, hydroxycerebroside, nonhydroxysulfatide, and hydroxysulfatide. Another portion was fractionated by thin-layer chromatography, and the spots corresponding to the sphingolipid derivatives were eluted. The material from each spot was analyzed by reverse phase high performance liquid chromatography for its homolog composition. With this new procedure the concentrations and homolog compositions of cerebrosides and sulfatides were measured in plaque, periplaque, and normal-appearing white matter from brains of multiple sclerosis patients and Wallerian degenerated rat sciatic nerves distal to the nerve transection. One piece of plaque studied contained only 1.86, 2.76, 0.60, and 0.45 nmol of nonhydroxycerebroside, hydroxycerebroside, nonhydroxysulfatide and hydroxysulfatide/mg of protein, respectively. These concentrations are less than 1% of those found in normal white matter. Periplaques were found to contain concentrations of these sphingolipids between those of plaque and normal white matter. The levels of these sphingolipids in degenerative nerves were 10-20% below normal the third day after the nerve was severed and about 70% below normal after 10 days. The rate of decrease lessened from ten days to 55 days. The homolog compositions of these sphingolipids in both multiple sclerosis brain and degenerating nerves were similar to those in the control. The implications of these findings and the advantages of this new analytical method are discussed.

Adult↗
PubMed1990

Fatty acid and sterol metabolism of cultured Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus grown in a fetal calf serum-based culture medium, contained as major lipids (i.e., greater than 10% of total) cholesterol, phosphatidylethanolamine and phosphatidylcholine. T. vaginalis also contained sphingomyelin and T. foetus glycophosphosphingolipids. The culture medium contained (greater than 10%) cholesterol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine. The fatty acyl groups of these major lipids of the trichomonads and the culture medium were similar. Those present in amounts greater than 5% of the total fatty acyl groups for a given lipid were myristic, palmitic, hexadecaenoic, stearic, oleic, linoleic, arachidonic and docosahexaenoic. When the trichomonads were exposed to radiolabeled lipids and lipid precursors, [14C]-labeled acetate and potential acetate precursors (glucose, threonine) were poorly incorporated and failed to label the fatty acyl groups of the trichomonad lipids. [14C]-labeled, C12-C22 saturated and unsaturated fatty acids were incorporated, unaltered, into phosphoglycerides and sphingolipids (sphingomyelin and glycophosphosphingolipids), but not into cholesteryl esters or triacylglycerols. Phosphoglycerides were preferentially labeled with unsaturated fatty acids and sphingolipids with saturated ones. This information inferred that the trichomonads: 1) were unable to biosynthesize fatty acids de novo, 2) took up unesterified fatty acids from the culture medium and used them in phosphoglyceride and sphingolipid biosynthesis and/or turnover, 4) did not use unesterified fatty acids in the biosynthesis or turnover of cholesteryl esters or triacylglycerols. Phosphatidylcholine and phosphatidylethanolamine, with [14C]labeled fatty acyl groups, and sphingomyelin, with 14C-labeled choline, were incorporated by the trichomonads. The phospholipids strongly labeled phosphoglycerides and sphingolipids, but not triacylglycerols, while the radioactivity of sphingomyelin [14C]choline remained associated solely with trichomonad sphingomyelin. Triacylglycerol, with 14C-labeled fatty acyl groups, was also incorporated, and labeled phosphoglycerides and sphingolipids. The results of those experiments suggested that trichomonads: (1) could take up culture medium phospholipids and triacylglycerols; (2) actively deacylated and reacylated phospholipids, but not triacylglycerols; (3) hydrolyzed exogenous triacylglycerols and used their fatty acyl groups for phospholipid acylations. Radiolabeled acetate, mevalonate and squalene were not incorporated into trichomonad cholesterol or cholesteryl esters. [14C]Cholesterol was incorporated unaltered, but was not esterified.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Sphingoid bases and their phosphates: transient activation and delayed repression of protein kinase C isoforms and their possible involvement in fumonisin B1 cytotoxicity.

Fumonisin B(1), a potent inhibitor of ceramide synthase, leads to accumulation of sphinganine, and later sphingosine, in vivo and in vitro. Fumonisin B(1) modulates the activity of protein kinase C (PKC), however, which metabolite of disrupted sphingolipid metabolism is involved, has not been ascertained. In the present study, we evaluated the modulation of PKC by sphingolipid bases and their metabolites using exogenous sphingolipid analogues in porcine renal epithelial (LLC-PK(1)) cells. In preliminary studies we found that fumonisin B(1) (1 microM) selectively and transiently activated PKCalpha, whereas fumonisin B(1) concentrations of 1-50 microM at 48 h repressed PKC-alpha, -delta, - epsilon and -zeta isoforms in a concentration-dependent manner. Addition of exogenous sphinganine-1-phosphate (1 microM for 5 min) alone stimulated cytosolic to membrane translocation of PKCalpha. Co-exposure of fumonisin B(1) with N,N-dimethylsphingosine, an inhibitor of sphingosine/sphinganine kinase, prevented the effects of fumonisin B(1) on PKCalpha. Sphinganine, sphingosine, sphingosine-1-phosphate and ceramide (all at 1 microM) added exogenously, did not alter PKCalpha cytosolic to membrane translocation at 5 min. Fumonisin B(1) (10 microM), sphinganine, sphingosine and ceramide (1 microM each) significantly repressed PKC-alpha and -delta isoforms at 48 h, whereas all the exogenously added sphingolipids significantly repressed PKC- epsilon and zeta similar to fumonisin B(1). Co-exposure of myriocin with fumonisin B(1) prevented the delayed inhibitory effects of fumonisin B(1) on PKC isoforms in LLC-PK(1) cells. This study demonstrated that selective and transient activation of PKCalpha may be due to the fumonisin B(1)-induced accumulation of the bioactive sphinganine-1-phosphate, whereas the long-term repression of PKC isoforms may be predominantly due to the accumulation of sphinganine or its metabolite, and to a lesser extent sphingosine or its metabolite in LLC-PK(1) cells. These findings suggest that the direct or indirect modulation of PKC by these sphingolipids is involved at least in part in the action of fumonisin B(1).

Animals↗
PubMed2002

Sphingosine kinase, sphingosine-1-phosphate, and apoptosis.

The sphingolipid metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play an important role in the regulation of cell proliferation, survival, and cell death. Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S1P stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph. Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both. Anticancer therapeutics targeting SphK are potentially clinically relevant. Indeed, inhibition of SphK has been shown to suppress gastric tumor growth [Cancer Res. 51 (1991) 1613] and conversely, overexpression of SphK increases tumorigenicity [Curr. Biol. 10 (2000) 1527]. Moreover, S1P has also been shown to regulate angiogenesis, or new blood vessel formation [Cell 99 (1999) 301], which is critical for tumor progression. Furthermore, there is intriguing new evidence that S1P can act in an autocrine and/or paracrine fashion [Science 291 (2001) 1800] to regulate blood vessel formation [J. Clin. Invest. 106 (2000) 951]. Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth. The cytoprotective effects of SphK/S1P may also be important for clinical benefit, as S1P has been shown to protect oocytes from radiation-induced cell death in vivo [Nat. Med. 6 (2000) 1109]. Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S1P, in apoptosis.

Animals↗
PubMed2000

Sphingosine-1-phosphate and sphingosylphosphorylcholine constrict renal and mesenteric microvessels in vitro.

Sphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act both intracellularly and at G-protein-coupled receptors, some of which were cloned and designated as Edg-receptors. Sphingolipid-induced vascular effects were determined in isolated rat mesenteric and intrarenal microvessels. Additionally, sphingolipid-induced elevations in intracellular Ca(2+) concentration were measured in cultured rat aortic smooth muscle cells. SPPC and SPP (0.1-100 micromol l(-1)) caused concentration-dependent contraction of mesenteric and intrarenal microvessels (e.g. SPPC in mesenteric microvessels pEC(50) 5.63+/-0.17 and E(max) 49+/-3% of noradrenaline), with other sphingolipids being less active. The vasoconstrictor effect of SPPC in mesenteric microvessels was stereospecific (pEC(50) D-erythro-SPPC 5.69+/-0.08, L-threo-SPPC 5.31+/-0.06) and inhibited by pretreatment with pertussis toxin (E(max) from 44+/-5 to 19+/-4%), by chelation of extracellular Ca(2+) with EGTA and by nitrendipine (E(max) from 40+/-6 to 6+/-1 and 29+/-6%, respectively). Mechanical endothelial denudation or NO synthase inhibition did not alter the SPPC effects, while indomethacin reduced them (E(max) from 87+/-3 to 70+/-4%). SPP and SPPC caused transient increases in intracellular Ca(2+) concentrations in rat aortic smooth muscle cells in a pertussis toxin-sensitive manner. Our data demonstrate that SPP and SPPC cause vasoconstriction of isolated rat microvessels and increase intracellular Ca(2+) concentrations in cultured rat aortic smooth muscle cells. These effects appear to occur via receptors coupled to pertussis toxin-sensitive G-proteins. This is the first demonstration of effects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone.

Animals↗
PubMed2000

Sphingosine-1-phosphate reduces rat renal and mesenteric blood flow in vivo in a pertussis toxin-sensitive manner.

Sphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine constrict isolated rat intrarenal and mesenteric microvessels in vitro. The present study investigates their effects on the cardiovascular system in vivo in anaesthetized rats. The animals were given intravenous or intrarenal arterial bolus injections of sphingolipids (0.1-100 microg kg(-1)) with subsequent measurements of mean arterial pressure, heart rate and renal and mesenteric blood flows (RBF, MBF) using a pressure transducer and electromagnetic flow probes, respectively. Intravenous injection of SPP rapidly (within 30 s), transiently and dose-dependently reduced RBF (maximally -4.0+/-0.3 ml min(-1)) and MBF (maximally -1.4+/-0.2 ml min(-1)), without affecting mean arterial pressure or heart rate. Other sphingolipids had no significant effect. Intrarenal arterial SPP administration caused greater blood flow reductions (maximally -6.4+/-0.3 ml min(-1)) than systemic administration. Upon intrarenal administration, sphingosylphos- phorylcholine also lowered RBF (maximally -2.8+/-0.6 ml min(-1)), while the other sphingolipids remained without effect. Pretreatment with pertussis toxin (PTX, 10 microg kg(-1)) 3 days before the acute experiment abolished the SPP-induced reductions of RBF and MBF. These data demonstrate, that SPP is a potent vasoconstrictor in vivo, particularly in the renal vasculature, while the other structurally related sphingolipids had little if any effects. The PTX-sensitivity strongly suggests that the effects of SPP on renal and mesenteric blood flow are mediated by receptors coupled to G(i)-type G-proteins.

Animals↗
PubMed1998

Ceramide and its interconvertible metabolite sphingosine function as indispensable lipid factors involved in survival and dendritic differentiation of cerebellar Purkinje cells.

Ceramide generated from sphingomyelin has emerged as a new but conserved type of biologically active lipid. We previously found that endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and that sphingomyelin is present abundantly in the somatodendritic region of these cells. To gain further insight into a potential role of the sphingomyelin/ceramide pathway, we investigated the effects of depletion of sphingolipids on the phenotypic growth and survival of immature Purkinje cells and the ability of ceramide or other sphingolipids to antagonize these effects. Inhibition of ceramide synthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, decreased cellular levels of sphingolipids. This treatment resulted in a decrease in cell survival accompanied by an induction of apoptotic cell death and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons. Cell-permeable ceramides, sphingosine, or sphingomyelin overcame these abnormalities more effectively than other sphingolipids when added simultaneously with ISP-1. Exposure to bacterial sphingomyelinase in turn enhanced cell survival and dendritic branching complexity of Purkinje cells at different optimal concentrations. Furthermore, cell-permeable ceramide acted synergistically with the neurotrophin family, which has been previously shown to support Purkinje cell survival. These observations suggest that ceramide is a requisite for the survival and the dendritic differentiation of Purkinje cells.

Animals↗
PubMed1998

Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase.

Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, has a high affinity for sphingomyelin. Chinese hamster ovary (CHO) cells exhibited a high cytolytic sensitivity to lysenin, but treatment with sphingomyelinase rendered the cells resistant to lysenin. Temperature-sensitive CHO mutant cells defective in sphingolipid synthesis were resistant to lysenin, and this lysenin resistance was suppressed by metabolic complementation of sphingolipids. Selection of lysenin-resistant variants from mutagenized CHO cells yielded two types of sphingomyelin-deficient mutants, both of which showed less lysenin binding capability than wild-type cells. One mutant strain was severely defective in sphingomyelin synthesis but not glycosphingolipid synthesis, and another strain (designated LY-B) was incapable of de novo synthesis of any sphingolipid species and had no activity of serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzing the first step of sphingolipid biosynthesis. LY-B cells lacked the LCB1 protein, a component of SPT, and transfection of LY-B cells with the hamster LCB1 cDNA restored both SPT activity and sphingolipid synthesis to the cells. Expression of an affinity peptide-tagged LCB1 protein in LY-B cells caused the endogenous LCB2 protein to adsorb to a tag affinity matrix. In addition, an anti-hamster LCB2 protein antibody co-immunoprecipitated both SPT activity and the wild-type LCB1 protein with the LCB2 protein. Thus, cell surface sphingomyelin is essential for lysenin-induced cytolysis, and lysenin is a useful tool for isolation of sphingomyelin-deficient mutants. Moreover, these results demonstrate that the SPT enzyme comprises both the LCB1 and LCB2 proteins.

Acyltransferases↗
PubMed2001

Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids in the heat stress response through inhibition of nutrient import (Chung, N., Jenkins, G. M., Hannun, Y. A., Heitman, J., and Obeid, L. M. (2000) J. Biol. Chem. 275, 17229-17232). In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous phytosphingosine (PHS, 20 microm) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, dihydrosphingosine, C(2)-phytoceramide (PHC), and stearylamine, only PHS inhibited growth. Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS. Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself. By using mutant yeast strains defective in various steps in sphingolipid metabolism, we further determined the specificity of PHS. The elo2Delta strain, which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of ceramides and to be hypersensitive to PHS (5 microm), suggesting that PHS does not need to be converted to PHC. The lcb4Delta lcb5Delta strain is defective in the conversion of PHS to PHS 1-phosphate, and it was as sensitive to PHS as the wild-type strain. The syr2Delta mutant strain was defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high concentrations of DHS (40 microm) that inhibited the growth of an isogenic wild-type strain, demonstrating that DHS needs to be converted to PHS to inhibit growth. Together, these data demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely related and yet unidentified metabolite.

Biological Transport↗
PubMed2006

Effects of sphingosine-1-phosphate and ceramide-1-phosphate on rat intestinal smooth muscle cells: implications for postoperative ileus.

Postoperative ileus, a major cause of morbidity after abdominal surgery, is characterized by intestinal dysmotility and inflammation. The aim was to investigate the involvement of sphingolipids in postoperative intestinal inflammation using a standardized rat model of intestinal surgical manipulation. Sphingolipid analysis (ESI-MS) of intestinal muscularis after manipulation revealed a time-dependent increase of sphingosine 1-phosphate (S1P) and of ceramide 1-phosphate (C1P). We therefore established a culture system of primary rat intestinal smooth muscle cells and examined the potential role of these sphingolipids in intestinal inflammation. Incubation of cells with either of the two sphingolipid-phosphates resulted in an elevated production of PGE(2). Further analysis revealed that S1P enhances cyclooxygenase 2 (COX-2) expression whereas C1P increases release of arachidonic acid, indicating an enhanced phospholipase A(2) activity. S1P-induced COX-2 expression was pertussis toxin sensitive, suggesting the involvement of Gi/o protein-coupled S1P receptors. Further downstream mediators of S1P induced COX-2 expression appear to be extracellular regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Collectively, our results demonstrate that intestinal smooth muscle cells represent a major target for both C1P and S1P activity. Thus, the sustained elevated concentration of the two bioactive sphingolipids in this tissue could at least in part explain postoperative intestinal dysmotility.

Animals↗
PubMed2005

A novel method for the measurement of in vitro fatty acid 2-hydroxylase activity by gas chromatography-mass spectrometry.

Fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is an enzyme responsible for the de novo synthesis of sphingolipids containing 2-hydroxy fatty acids. 2-Hydroxy sphingolipids are highly abundant in the brain, as major myelin galactolipids (galactosylceramide and sulfatide) contain a uniquely high proportion ( approximately 50%) of 2-hydroxy fatty acids. Other tissues, such as epidermis, epithelia of the digestive tract, and certain cancers, also contain 2-hydroxy sphingolipids. The physiological significance of the 2-hydroxylation on N-acyl chains of subsets of sphingolipids is poorly understood. To study the roles of FA2H and 2-hydroxy sphingolipids in various tissues, we developed a highly sensitive in vitro FA2H assay. FA2H-dependent fatty acid 2-hydroxylation requires an electron transfer system, which was reconstituted in vitro with an NADPH regeneration system and purified NADPH:cytochrome P-450 reductase. A substrate [3,3,5,5-D(4)]tetracosanoic acid was solubilized in alpha-cyclodextrin solution, and the 2-hydroxylated product was quantified by gas chromatography-mass spectrometry after conversion to a trimethylsilyl ether derivative. When the microsomes of FA2H-transfected COS7 cells were incubated with the electron transfer system and deuterated tetracosanoic acid, deuterated 2-hydroxy tetracosanoic acid was formed in a time- and protein-dependent manner. With this method, FA2H activities were reproducibly measured in murine brains and tissue culture cell lines.

Animals↗
PubMed2001

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane.

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

Androstenes↗
PubMed2005

Maternal fumonisin exposure and risk for neural tube defects: mechanisms in an in vivo mouse model.

BACKGROUND: Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides, a common contaminant of corn worldwide. FB1 disrupts sphingolipid biosynthesis by inhibiting the enzyme ceramide synthase, resulting in an elevation of free sphingoid bases and depletion of downstream glycosphingolipids. A relationship between maternal ingestion of FB1-contaminated corn during early pregnancy and increased risk for neural tube defects (NTDs) has recently been proposed in human populations around the world where corn is a dietary staple. The current studies provide an in vivo mouse model of FB1 teratogenicity. METHODS: Pregnant LM/Bc mice were injected with increasing doses of FB1 on GD 7.5 and 8.5, and exposed fetuses were examined for malformations. Sphingolipid profiles and (3)H-folate concentrations were measured in maternal and fetal tissues. Immunohistochemical expression of the GPI-anchored folate receptor (Folbp1) and its association with the lipid raft component, ganglioside GM1, were characterized. Rescue experiments were performed with maternal folate supplementation or administration of gangliosides. RESULTS: Maternal FB1 administration (20 mg/kg of body weight) during early gestation resulted in 79% NTDs in exposed fetuses. Sphingolipid profiles were significantly altered in maternal and embryonic tissues following exposure, and (3)H-folate levels and immunohistochemical expression of Folbp1 were reduced. Maternal folate supplementation partially rescued the NTD phenotype, whereas GM1 significantly restored folate concentrations and afforded almost complete protection against FB1-induced NTDs. CONCLUSIONS: Maternal FB1 exposure altered sphingolipid metabolism and folate concentrations in LM/Bc mice, resulting in a dose-dependent increase in NTDs that could be prevented when adequate folate levels were maintained.

Animals↗
PubMed1995

Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin.

ISP-1/myriocin is a new type of remarkably potent immunosuppressant, the structure of which is homologous to sphingosine. ISP-1/myriocin inhibited the proliferation of an IL-2-dependent mouse cytotoxic T cell line, CTLL-2, at nanomole concentrations. ISP-1/myriocin inhibits serine palmitoyltransferase activity at picomole concentrations. This enzyme catalyzes the first step of sphingolipid biosynthesis and reduces the intracellular pool of sphingolipid intermediates. The growth inhibition induced by ISP-1/myriocin was completely abolished by the addition of sphingosines or sphingosine-1-phosphate, but not by sphingomyelin or glycosphingolipids. These results suggest that sphingosines or sphingosine-1-phosphate are associated with CTLL-2 proliferation, and ISP-1/myriocin suppresses T cell proliferation by the modulation of sphingolipid metabolism. ISP-1/myriocin should be a useful tool for the study of the sphingolipid pathway, which has been associated with various kinds of signal transduction.

Acyltransferases↗
PubMed1996

Sphingoid bases and phospholipase D activation.

There is increased interest in physiological functions and mechanisms of action of sphingolipids metabolites, ceramide, sphingosine, and sphingosine-1-phosphate (SPP), members of a new class of lipid second messengers. This review summarizes current knowledge regarding the role of these sphingolipids metabolites in the actions of growth factors and focuses on the second messenger roles of sphingosine and its metabolite, SPP, in the regulation of cell growth. We also discuss possible interactions with intermediates of the well known glycerophospholipid cycle. Sphingosine and SPP generally provide positive mitogenic signals whereas ceramide has been reported to induce apoptosis and cell arrest in several mammalian cell lines. Stimulation of phospholipase D leading to an increase in phosphatidic acid, a positive regulator of cell growth, by sphingosine and SPP, and its inhibition by ceramide, might be related to their opposite effects on cell growth. This also indicates that sphingolipid turnover could regulate the diacylglycerol cycle. Cross-talk between sphingolipid turnover pathways and the diacylglycerol cycle increases complexity of signaling pathways leading to cellular proliferation and adds additional sites of regulation.

Animals↗
PubMed2005

Sphingosine kinases, sphingosine-1-phosphate and sphingolipidomics.

It has become abundantly clear over the past decade that sphingolipids and their metabolites are key signaling molecules. Ceramide, the backbone of all sphingolipids, predominantly inhibits cell growth and induces apoptosis, while its metabolite, sphingosine-1-phosphate promotes growth and survival. Given the interconvertibility of these two opposing signaling molecules, it is essential that any study that examines the effects of one also look at the other. The newly available technology of liquid chromatography-tandem mass spectroscopy (LC-MS/MS) is increasingly being applied for this purpose, as it can quickly identify and measure many different sphingolipids simultaneously. An added benefit of LC-MS/MS is that it is several orders of magnitude more sensitive than enzymatic methods or more traditional chromatographic techniques, allowing smaller sample sizes and increased throughput. Here, we briefly discuss the importance of LC-MS/MS for measuring sphingolipid metabolites and some future directions researchers may take given the increasingly accessibility to this technology.

Animals↗
PubMed2000

Sphingomyelin metabolites in vascular cell signaling and atherogenesis.

The atherosclerotic lesion most probably develops through a number of cellular events which implicate all vascular cell types and include synthesis of extracellular proteins, cell proliferation, differentiation and death. Sphingolipids and sphingolipid metabolizing enzymes may play important roles in atherogenesis, not only because of lipoprotein alterations but also by mediating a number of cellular events which are believed to be crucial in the development of the vascular lesions such as proliferation or cell death. Exogenous sphingolipids may mediate various biological effects such as apoptosis, mitogenesis or differentiation depending on the cell type. Moreover, several molecules present in the atherogenic lesion, such as oxidized LDL, growth factors or cytokines, which activate intracellular signaling pathways leading to vascular cell modifications, can stimulate sphingomyelin hydrolysis and generation of ceramide (and other metabolites as sphingosine-1-phosphate). Here we review the potential implication of the sphingomyelin/ceramide cycle in vascular cell signaling related to atherosclerosis, and more generally the role of sphingolipids in the events observed during the atherosclerotic process as cell differentiation, migration, adhesion, retraction, proliferation and death.

Arteriosclerosis↗