Synthesis Of Complex Sphingolipids Research Articles

Ceramide sorting into non-vesicular transport is independent of acyl chain length in budding yeast

The transport of ceramide from the endoplasmic reticulum (ER) to the Golgi is a key step in the synthesis of complex sphingolipids, the main building blocks of the plasma membrane. In yeast, ceramide is transported to the Golgi either through ATP-dependent COPII vesicles of the secretory pathway or by ATP-independent non-vesicular transport that involves tethering proteins at ER-Golgi membrane contact sites. Studies in both mammalian and yeast cells reported that vesicular transport mainly carries ceramide containing very long chain fatty acids, while the main mammalian non-vesicular ceramide transport protein CERT only transports ceramides containing short chain fatty acids. However, if non-vesicular ceramide transport in yeast similarly favors short chain ceramides remained unanswered. Here we employed a yeast GhLag1 strain in which the endogenous ceramide synthase is replaced by the cotton-derived GhLag1 gene, resulting in the production of short chain C18 rather than C26 ceramides. We show that block of vesicular transport through ATP-depletion or the use of temperature-sensitive sec mutants caused a reduction in inositolphosphorylceramide (IPC) synthesis to similar extent in WT and GhLag1 backgrounds. Since the remaining IPC synthesis is a readout for non-vesicular ceramide transport, our results indicate that non-vesicular ceramide transport is neither blocked nor facilitated when only short chain ceramides are present. Therefore, we propose that the sorting of ceramide into non-vesicular transport is independent of acyl chain length in budding yeast.

Ceramide Is Involved in Temozolomide Resistance in Human Glioblastoma U87MG Overexpressing EGFR.

Glioblastoma multiforme (GBM) is the most frequent and deadly brain tumor. Many sphingolipids are crucial players in the regulation of glioma cell growth as well as in the response to different chemotherapeutic drugs. In particular, ceramide (Cer) is a tumor suppressor lipid, able to induce antiproliferative and apoptotic responses in different types of tumors including GBM, most of which overexpress the epidermal growth factor receptor variant III (EGFRvIII). In this paper, we investigated whether Cer metabolism is altered in the U87MG human glioma cell line overexpressing EGFRvIII (EGFR+ cells) to elucidate their possible interplay in the mechanisms regulating GBM survival properties and the response to the alkylating agent temozolomide (TMZ). Notably, we demonstrated that a low dose of TMZ significantly increases Cer levels in U87MG cells but slightly in EGFR+ cells (sensitive and resistant to TMZ, respectively). Moreover, the inhibition of the synthesis of complex sphingolipids made EGFR+ cells sensitive to TMZ, thus involving Cer accumulation/removal in TMZ resistance of GBM cells. This suggests that the enhanced resistance of EGFR+ cells to TMZ is dependent on Cer metabolism. Altogether, our results indicate that EGFRvIII expression confers a TMZ-resistance phenotype to U87MG glioma cells by counteracting Cer increase.

Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that serves as a potent extracellular signaling molecule. Metabolic regulation of extracellular S1P levels impacts key cellular activities through altered S1P receptor signaling. Although the pathway through which S1P is degraded within the cell and thereby eliminated from reuse has been previously described, the mechanism used for S1P cellular uptake and the subsequent recycling of its sphingoid base into the sphingolipid synthesis pathway is not completely understood. To identify the genes within this S1P uptake and recycling pathway, we performed a genome-wide CRISPR/Cas9 KO screen using a positive-selection scheme with Shiga toxin, which binds a cell-surface glycosphingolipid receptor, globotriaosylceramide (Gb3), and causes lethality upon internalization. The screen was performed in HeLa cells with their sphingolipid de novo pathway disabled so that Gb3 cell-surface expression was dependent on salvage of the sphingoid base of S1P taken up from the medium. The screen identified a suite of genes necessary for S1P uptake and the recycling of its sphingoid base to synthesize Gb3, including two lipid phosphatases, PLPP3 (phospholipid phosphatase 3) and SGPP1 (S1P phosphatase 1). The results delineate a pathway in which plasma membrane–bound PLPP3 dephosphorylates extracellular S1P to sphingosine, which then enters cells and is rephosphorylated to S1P by the sphingosine kinases. This rephosphorylation step is important to regenerate intracellular S1P as a branch-point substrate that can be routed either for dephosphorylation to salvage sphingosine for recycling into complex sphingolipid synthesis or for degradation to remove it from the sphingolipid synthesis pathway.

TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids.

Plasma membrane lipid composition must be maintained during growth and under environmental insult. In yeast, signaling mediated by TOR Complex 2 (TORC2)-dependent protein kinase Ypk1 controls lipid abundance and distribution in response to membrane stress. Ypk1, among other actions, alleviates negative regulation of L-serine:palmitoyl-CoA acyltransferase, upregulating production of long-chain base precursors to sphingolipids. To explore other roles for TORC2-Ypk1 signaling in membrane homeostasis, we devised a three-tiered genome-wide screen to identify additional Ypk1 substrates, which pinpointed both catalytic subunits of the ceramide synthase complex. Ypk1-dependent phosphorylation of both proteins increased upon either sphingolipid depletion or heat shock and was important for cell survival. Sphingolipidomics, other biochemical measurements and genetic analysis demonstrated that these modifications of ceramide synthase increased its specific activity and stimulated channeling of long-chain base precursors into sphingolipid end-products. Control at this branch point also prevents accumulation of intermediates that could compromise cell growth by stimulating autophagy.

Activation of Protein Kinase C-Mitogen-activated Protein Kinase Signaling in Response to Inositol Starvation Triggers Sir2p-dependent Telomeric Silencing in Yeast

Depriving wild type yeast of inositol, a soluble precursor for phospholipid, phosphoinositide, and complex sphingolipid synthesis, activates the protein kinase C (PKC)-MAPK signaling pathway, which plays a key role in the activation of NAD(+)-dependent telomeric silencing. We now report that triggering PKC-MAPK signaling by inositol deprivation or by blocking inositol-containing sphingolipid synthesis with aureobasidin A results in increased telomeric silencing regulated by the MAPK, Slt2p, and the NAD(+)-dependent deacetylase, Sir2p. Consistent with the dependence on NAD(+) in Sir2p-regulated silencing, we found that inositol depletion induces the expression of BNA2, which is required for the de novo synthesis of NAD(+). Moreover, telomeric silencing is greatly reduced in bna2Δ and npt1Δ mutants, which are defective in de novo and salvage pathways for NAD(+) synthesis, respectively. Surprisingly, however, omitting nicotinic acid from the growth medium, which reduces cellular NAD(+) levels, leads to increased telomeric silencing in the absence of inositol and/or at high temperature. This increase in telomeric silencing in response to inositol starvation is correlated to chronological life span extension but is Sir2p-independent. We conclude that activation of the PKC-MAPK signaling by interruption of inositol sphingolipid synthesis leads to increased Sir2p-dependent silencing and is dependent upon the de novo and salvage pathways for NAD(+) synthesis but is not correlated with cellular NAD(+) levels.

Unperverted synthesis of complex sphingolipids is essential for cell survival under nitrogen starvation

Changes in membrane dynamics are known to occur in cells faced with starvation. However, the functions of the major lipid components of biological membranes, sphingolipids, during the starvation response remain unclear. In this study, we found that yeast cells lacking genes encoding mannosylinositol phosphorylceramide (MIPC) synthases (csg1Δ csh1Δ) underwent rapid cell death upon nitrogen starvation, but not upon carbon starvation or carbon and nitrogen starvation. Addition of NaN3 prevented the nitrogen starvation-induced cell death of the csg1Δ csh1Δ cells, indicating that energy production is required for this rapid cell death. The cell death was caused by an accumulation of inositol phosphorylceramide (IPC) species containing phytosphingosine. Removing Ca(2+) by treating the cells with a calcium chelator or by changing the medium to a Ca(2+) -free medium before nitrogen starvation rescued the cells from death. Approximately half of the cells died shortly after collapse of the vacuole, whereas in the other half, morphological changes in the cytoplasm preceded vacuole disruption. Because the vacuole is the major Ca(2+) storage organelle, we suggest that the vacuole is involved in the cell death either directly or indirectly. We report here that normal synthesis of complex sphingolipids is important for cell survival in nitrogen-starved medium.

TORC1-regulated protein kinase Npr1 phosphorylates Orm to stimulate complex sphingolipid synthesis.

The evolutionarily conserved Orm1 and Orm2 proteins mediate sphingolipid homeostasis. However, the homologous Orm proteins and the signaling pathways modulating their phosphorylation and function are incompletely characterized. Here we demonstrate that inhibition of nutrient-sensitive target of rapamycin complex 1 (TORC1) stimulates Orm phosphorylation and synthesis of complex sphingolipids in Saccharomyces cerevisiae. TORC1 inhibition activates the kinase Npr1 that directly phosphorylates and activates the Orm proteins. Npr1-phosphorylated Orm1 and Orm2 stimulate de novo synthesis of complex sphingolipids downstream of serine palmitoyltransferase. Complex sphingolipids in turn stimulate plasma membrane localization and activity of the nutrient scavenging general amino acid permease 1. Thus activation of Orm and complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to starvation.

Sphingolipid Δ8 unsaturation is important for glucosylceramide biosynthesis and low‐temperature performance in Arabidopsis

Plants contain a large diversity of sphingolipid structures, arising in part from C4 hydroxylation and Δ4 and Δ8 desaturation of the component long-chain bases (LCBs). Typically, 85-90% of sphingolipid LCBs in Arabidopsis leaves contain a cis or transΔ8 double bond produced by sphingoid LCB Δ8 desaturase (SLD). To understand the metabolic and physiological significance of Δ8 unsaturation, studies were performed using mutants of the Arabidopsis SLD genes AtSLD1 and AtSLD2. Our studies revealed that both genes are constitutively expressed, the corresponding polypeptides are ER-localized, and expression of these genes in Saccharomyces cerevisiae yields mixtures of cis/transΔ8 desaturation products, predominantly as trans isomers. Consistent in part with the higher expression of AtSLD1 in Arabidopsis plants, AtSLD1 T-DNA mutants showed large reductions in Δ8 unsaturated LCBs in all organs examined, whereas AtSLD2 mutants showed little change in LCB unsaturation. Double mutants of AtSLD1 and AtSLD2 showed no detectable LCB Δ8 unsaturation. Comprehensive analysis of sphingolipids in rosettes of these mutants revealed a 50% reduction in glucosylceramide levels and a corresponding increase in glycosylinositolphosphoceramides that were restored by complementation with a wild-type copy of AtSLD1. Double sld1 sld2 mutants lacked apparent growth phenotypes under optimal conditions, but displayed altered responses to certain stresses, including prolonged exposure to low temperatures. These results are consistent with a role for LCB Δ8 unsaturation in selective channeling of ceramides for the synthesis of complex sphingolipids and the physiological performance of Arabidopsis.

Modulation of Sphingolipid Metabolism by the Phosphatidylinositol-4-phosphate Phosphatase Sac1p through Regulation of Phosphatidylinositol in Saccharomyces cerevisiae

Sphingolipids and phosphoinositides both play signaling roles in Saccharomyces cerevisiae. Although previous data indicate independent functions for these two classes of lipids, recent genetic studies have suggested interactions between phosphatidylinositol (PtdIns) phosphate effectors and sphingolipid biosynthetic enzymes. The present study was undertaken to further define the effects of phosphatidylinositol 4-phosphate (PtdIns(4)P) metabolism on cell sphingolipid metabolism. The data presented indicate that deletion of SAC1, a gene encoding a PtdIns(4)P phosphatase, increased levels of most sphingolipid species, including sphingoid bases, sphingoid base phosphates, and phytoceramide. In contrast, sac1Delta dramatically reduced inositol phosphosphingolipids, which result from the addition of a PtdIns-derived phosphoinositol head group to ceramides through Aur1p. Deletion of SAC1 decreased PtdIns dramatically in both steady-state and pulse labeling studies, suggesting that the observed effects on sphingolipids may result from modulation of the availability of PtdIns as a substrate for Aur1p. Supporting this hypothesis, acute attenuation of PtdIns(4)P production through Stt4p immediately increased PtdIns and subsequently reduced sphingoid bases. This reduction was overcome by the inhibition of Aur1p. Moreover, modulation of sphingoid bases through perturbation of PtdIns(4)P metabolism initiated sphingolipid-dependent biological effects, supporting the biological relevance for this route of regulating sphingolipids. These findings suggest that, in addition to potential signaling effects of PtdInsP effectors on sphingolipid metabolism, PtdIns kinases may exert substantial effects on cell sphingolipid profiles at a metabolic level through modulation of PtdIns available as a substrate for complex sphingolipid synthesis.

A Sphingolipid Inhibitor Induces a Cytokinesis Arrest and Blocks Stage Differentiation in Giardia lamblia

Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 muM. The inhibition of parasite replication was irreversible at 10 muM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents.

The Cryptococcal Enzyme Inositol Phosphosphingolipid-Phospholipase C Confers Resistance to the Antifungal Effects of Macrophages and Promotes Fungal Dissemination to the Central Nervous System

In recent years, sphingolipids have emerged as critical molecules in the regulation of microbial pathogenesis. In fungi, the synthesis of complex sphingolipids is important for the regulation of pathogenicity, but the role of sphingolipid degradation in fungal virulence is not known. Here, we isolated and characterized the inositol phosphosphingolipid-phospholipase C1 (ISC1) gene from the fungal pathogen Cryptococcus neoformans and showed that it encodes an enzyme that metabolizes fungal inositol sphingolipids. Isc1 protects C. neoformans from acidic, oxidative, and nitrosative stresses, which are encountered by the fungus in the phagolysosomes of activated macrophages, through a Pma1-dependent mechanism(s). In an immunocompetent mouse model, the C. neoformans Deltaisc1 mutant strain is almost exclusively found extracellularly and in a hyperencapsulated form, and its dissemination to the brain is remarkably reduced compared to that of control strains. Interestingly, the dissemination of the C. neoformans Deltaisc1 strain to the brain is promptly restored in these mice when alveolar macrophages are pharmacologically depleted or when infecting an immunodeficient mouse in which macrophages are not efficiently activated. These studies suggest that Isc1 plays a key role in protecting C. neoformans from the intracellular environment of macrophages, whose activation is important for preventing fungal dissemination of the Deltaisc1 strain to the central nervous system and the development of meningoencephalitis.

The Protozoan Inositol Phosphorylceramide Synthase: A NOVEL DRUG TARGET THAT DEFINES A NEW CLASS OF SPHINGOLIPID SYNTHASE

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects.

Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes

Complex glycophosphosphingolipids present in plants are composed of ceramide, inositolphosphate, and diverse polar oligosaccharide substituents. The activity of inositolphosphorylceramide (IPC) synthase (phosphatidylinositol:ceramide inositolphosphate transferase), the enzyme proposed to catalyze the initial committed step in the formation of these complex sphingolipids, was characterized in wax bean hypocotyl microsomes. Enzyme activity was assayed by monitoring the incorporation of fluorescent NBD-C 6 ceramide or [ 3H]inositolphosphate from radiolabeled phosphatidylinositol (PI) into product identified by TLC. IPC synthase was found to utilize nonhydroxy fatty acid-containing ceramide, hydroxy fatty acid-containing ceramide, and NBD-C 6 ceramide as substrate. Maximum product formation was observed at PI concentrations in excess of 600 μM (with half-maximum activity at approximately 200 μM). Both endogenous PI and ceramide appeared to serve as substrates. Aureobasidin A and rustmicin, two potent inhibitors of fungal IPC synthase, inhibited enzyme activity in bean microsomes with values for IC 50 of 0.4–0.8 and 16–20 nM, respectively. IPC synthase activity appeared most closely associated with the Golgi based on results using selected marker enzymes. Enzyme activity was detected in a variety of plant tissues. This report, the first to characterize IPC synthase in plant tissues, demonstrates the similarities between the plant enzyme and its yeast counterpart, and provides insight into plant glycophosphosphingolipid biology.

The Uracil Transporter Fur4p Associates with Lipid Rafts

Sphingolipids are abundant components of eucaryotic membranes, where they perform essential functions. To uncover new roles for sphingolipids, we studied Saccharomyces cerevisiae lcb1-100 cells, which have a temperature-sensitive block in the first step in sphingolipid synthesis. We find that the level of all five species of the sphingoid long chain base intermediates is reduced 2-7-fold in cells grown at a permissive temperature, and the level of complex sphingolipids is reduced 50%. In addition, lcb1-100 cells make no detectable phosphorylated sphingoid bases. After transfer to a restrictive temperature (a heat shock), the level of the major sphingoid bases drops rather than transiently rising, as in wild type cells. These changes affect lcb1-100 cells in multiple ways. Basal uracil transport by Fur4p is reduced 25%, and when cells are heat-shocked, uracil transport activity falls rapidly and is not restored as it is in wild type cells. Restoration requires a functional secretory pathway and synthesis of complex sphingolipids, leading us to hypothesize that Fur4p associates with lipid rafts. The finding that Fur4p is insoluble in TritonX-100 at 4 degrees C and behaves like a raft-associated protein on a density gradient supports this hypothesis. Raft association may be essential for regulating breakdown of Fur4p in response to stresses and other factors that govern uracil transport activity. Our results show that long chain bases do not contribute to the inactivation of Fur4p transport activity after heat stress, but they are essential for some later, but unknown, process that leads to degradation of the protein. Further studies using lcb1-100 cells should reveal new roles of sphingolipids in nutrient uptake and other membrane-dependent processes.

New approaches to synthesis of stereospecific sphingomyelin

Enormous progress in the asymmetric synthesis of stereochemically and chemically pure d- erythro-sphingosine and ceramides led to the development of a practical, efficient, easily scaleable process to provide industrial quantities of chiral sphingosine and ceramides. This established a new platform of chiral starting materials which facilitate the synthesis of complex sphingolipids. Utilizing stereochemically homogeneous, fully synthetic ceramides, two efficient synthetic methods were developed for the preparation of ultra pure stereochemically homogeneous sphingomyelins. The first method adapted highly efficient phosphoramidite technology from oligonucleotide chemistry. This method allows selective insertion of a phosphocholine moiety into 3- O-protected ceramide through phosphitylation, followed by choline attachment, phosphite oxidation and deprotection. This route provides stereochemically homogeneous sphingomyelin in 35–79% yield. The second route is based on the reaction of selectively protected ceramides with cyclic chlorophosphate followed by treatment with trimethylamine to give the desired sphingomyelins in 50% yield. Multigram quantities of 14C-labeled N-palmitoyl- d- erythro-sphingomyelin were produced with specific activity >1000 dpm/nmol.

1-Methylthiodihydroceramide, a Novel Analog of Dihydroceramide, Stimulates Sphinganine Degradation Resulting in Decreased de Novo Sphingolipid Biosynthesis

1-Methylthiodihydroceramide (10 microM) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons. Accordingly, de novo formation of sphingomyelin and of glycosphingolipids, all of which contain ceramide in their backbone, was reduced in a time- and concentration-dependent manner by up to 80%. Complex sphingolipid synthesis was restored upon addition of dihydroceramide or ceramide, in micromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases involved in glycosphingolipid biosynthesis is inhibited by this analog. Assays of the enzymes catalyzing sphinganine biosynthesis, as well as its N-acylation to form dihydroceramide, revealed that they were also not affected. In contrast, there was a 2.5-fold increase in the activity of sphinganine kinase. Reduction of de novo sphingolipid biosynthesis by 1-methylthiodihydroceramide is therefore due to its ability to deplete cells of newly formed free sphinganine. As a consequence of depletion of sphinganine levels, 1-methylthiodihydroceramide disrupted axonal growth in cultured hippocampal neurons in a manner similar to that reported for direct inhibitors of sphingolipid synthesis; thus, there was essentially no axon growth after incubation with 1-methylthiodihydroceramide between days 2 and 3, and co-incubation with short acyl chain analogs of ceramide (5 microM) antagonized the inhibition of growth. Interestingly, the D-erythro and the L-threo isomere were equally effective, but the corresponding free base as well as other structurally related compounds did not affect either sphingolipid biosynthesis or neuronal growth.

Mitochondrial Activity and Morphology are Affected by Sphinganine inNeurospora Crassa.

It has been proposed that sphinganine may function as a natural antifungal barrier in mammals. This sphingoid base is found highly concentrated in epidermis, where it has been documented to inhibit the colonization of pathogenic fungi such as Epidermophyton and Candida. SinceNeurospora crassais sensitive to the effects of sphinganine within the same concentration range as infectious fungi, it is being employed as the model to study the mechanism of sphinganine toxicity in fungal pathogens. With the dramatic increase in fungal mycoses seen in immunocompromised individuals to date, this natural constituent of mammalian epidermis may be employed as a potent, novel antifungal drug.During this project, radioactive labelling with [3H]sphinganine was employed to observe metabolism of exogenous sphinganine into complex sphingolipids. Transmission electron microscopy analysis was utilized to observe changes in cellular ultrastructure, while fluorescence microscopic techniques were used to assay certain cell functions.Within 1 hr, exogenous sources of sphinganine are metabolized into dihydroceramides and cerebrosides by the enzymes of complex sphingolipid synthesis.

Biosynthesis of gangliosides containing C18:1 and C20:1 [3‐14C]sphingosine after administrating [1‐14C]palmitic acid and [1‐14C]stearic acid to rat cerebellar granule cells in culture

The biosynthesis of ganglioside molecular species containing sphingosine of different structure was investigated by administrating rat cerebellar granule cells in culture with [1-14C]palmitic and [1-14C]stearic acids which are the precursors for sphingosine biosynthesis. The incorporation of radioactivity into the sphingosine of the ganglioside species containing C20:1 sphingosine after administrating [1-14C]stearic acid was low in comparison with the incorporation of radioactivity into the sphingosine of ganglioside species containing C18:1 sphingosine after administration of [1-14C]palmitic acid, but the ratio between the radioactivity incorporated in the C20:1 and the C18:1 sphingosine of C20 and C18 ganglioside species progressively increased when the cell culture was prolonged. Ceramide-containing radioactive sphingosine was found after palmitic or stearic acid administration. Ceramide-containing C20:1 sphingosine found after adding stearic acid was about 5% of that synthesized starting from palmitic acid and containing C18:1 sphingosine. Free radioactive C18:1 and C20:1 sphingosine were found after adding radioactive palmitic or stearic acid. This is representative of a catabolic process occurring after biosynthesis of the complex sphingolipid starting from the radioactive precursor. In fact it has been proved that only saturated sphingosines are used for the synthesis of complex sphingolipids, the introduction of the double bond at position four of the sphingoid chain occurring at the level of ceramide [Rother, J., van Echten, G., Schwarzmann, G. & Sandhoff, K. (1992) Biochem. Biophys. Res. Commun. 189, 14-20]. Saturated sphingosines were not present. The lack of free C20:0 sphingosine confirms the hypothesis that the C20:0 sphingosine synthesis and the process (C20:0 sphingosine-->C20:0 ceramide-->C20:1 ceramide) occur in the correct quantity for the synthesis of C20:1 gangliosides. Moreover, we found only traces of free C20:1 sphingosine, at days 8 and 15 of cell culture when the biosynthesis of complex C20:1 gangliosides and the related catabolic processes occur to a higher extent, thus excluding the idea that a large amount of C20:0 sphingosine can be acylated to C20:0 ceramide and dehydrogenated to C20:1 ceramide which, being not used for ganglioside biosynthesis, is immediately catabolised to C20:1 sphingosine.

Uptake and Metabolism of Sphingolipids in Isolated Intestinal Loops of Mice

Sphingolipids are found in all eukaryotic organisms. However, little is known about the digestion, uptake and subsequent metabolism of these constituents of food. In this study, radiolabeled sphingolipids were placed in isolated intestinal segments of female CF1 mice, and the metabolism and distribution of the radiolabel were followed. Most of the sphingomyelin was degraded to ceramide and other products in all regions of the intestine, and increasing amounts of several [3H]-labeled sphingolipids appeared in the tissues. Small amounts of the radiolabel disappeared from the intestinal loops and appeared in liver within the first 30 to 60 min implying that neither intact sphingomyelin nor its metabolites are transported very efficiently from the intestine to other organs. There were different degrees of uptake and metabolism of sphingomyelin, [4,5-3H-sphinganyl]ceramide, and [3H]sphingosine. The [3H]sphingomyelin was also administered by gavage and the appearance along the intestine measured. After 90 min, 12% was found in the cecum and colon. These results establish that some of the sphingomyelin that enters the gastrointestinal tract is hydrolyzed and taken up by the intestine, with the lipid backbone being degraded or reutilized for complex sphingolipid synthesis; however, at least a portion passes into the large intestine. The appearance of bioactive compounds throughout the gastrointestinal tract may alter the behavior of intestinal cells.

Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ.

Fumonisins, mycotoxins produced by Fusarium moniliforme and a number of other fungi, cause neuronal degeneration, liver and renal toxicity, cancer, and other injury to animals. Recent work with rat hepatocytes (Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H., Jr. (1991) J. Biol. Chem. 266, 14486-14490) found that fumonisins block sphingosine biosynthesis by inhibiting the conversion of sphinganine to dihydroceramides, which precedes introduction of the 4,5-trans-double bond of sphingosine. The current study utilized mouse cerebellar neurons in culture to evaluate how this affects the distribution of newly synthesized ceramides among different complex sphingolipids. Fumonisin B1 inhibited ceramide synthase in mouse brain microsomes with a competitive-like kinetic behavior with respect to both sphinganine and stearoyl-CoA. Fumonisin B1 inhibited sphingolipid biosynthesis in cultured cerebellar neurons in situ as reflected by accumulation of free sphinganine, a reduction in the mass of total sphingolipids, reductions in the incorporation of [14C]serine into glucosylceramide, lactosylceramide, sphingomyelin, and gangliosides (GM1, GD3, GD1a, GD1b, GT1b, and GQ1b), and inhibition of the incorporation of [14C]galactose and [3H]sphinganine into complex sphingolipids. Dose-response studies revealed that the labeling of sphingomyelin (IC50 of 0.7 microM) was more sensitive to inhibition by fumonisin B1 than was glycolipid formation (IC50 of approximately 7 microM) in these cells. A similar effect was seen when beta-fluoroalanine was added to inhibit the activity of serine palmitoyltransferase, the first enzyme of the pathway. The inhibition of complex sphingolipid synthesis was reversible, and nearly normal labeling profiles were obtained 48 h after removing the mycotoxin. These studies establish that fumonisin B1 inhibits de novo sphingolipid biosynthesis by neuronal cells and, moreover, that limiting ceramide synthesis differentially affects the formation of sphingomyelin versus glycosphingolipids.