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PubMed1982

Metastable phase behavior of a sphingolipid analogue.

The phase behavior of the sphingolipid analogue 1-palmitoyl-2-tridecanylcarbamyloxy-sn-glycero-3-phosphocholine (CM-PC) has been studied by differential scanning calorimetry. When CM-PC is cooled at rates greater than 5 K/min, subsequent heating runs exhibit metastable behavior: a low enthalpy exotherm is observed at about 9 degrees C (delta H = -(1-2)kcal/mol), followed by a high enthalpy endotherm at 38 degrees C (delta H = 13 kcal/mol). Systematic variation of cooling/heating protocols indicates that CM-PC exhibits two low temperature states, one metastable and the other stable. Cooling from the liquid crystalline state results in formation of the metastable low-temperature polymorph I, which must transform into the stable low-temperature polymorph II before the liquid crystalline state can be reached again. This metastable thermal behavior is virtually identical to that recently reported for synthetic palmitoyl cerebroside (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966) and for bovine brain n-acylcerebrosides (Curatolo, W. (1982) Biochemistry, 21, 1761-1764). The observation of the metastable phase behavior of CM-PC indicates that the sphingosine backbone is not a prerequisite for such metastable behavior. Furthermore, the carbamyl group in CM-PC is reversed in orientation compared with the amide of sphingolipids (-NH-CO- vs. -CO-NH-), suggesting that the intermolecular hydrogen bonding potential, rather than some highly specific steric or conformational constraint, is responsible for the observed metastability of sphingolipids.

Calorimetry↗
PubMed1995

Intracellular transport of inositol-containing sphingolipids in the yeast, Saccharomyces cerevisiae.

Organelles of the early protein secretion pathway (ER, Golgi) are involved in biosynthesis and intracellular migration of the yeast sphingolipids, inositolphosphorylceramide (IPC), mannosylinositolphosphorylceramide (MIPC), and mannosyldiinositolphosphorylceramide (M(IP)2C). Cycloheximide and nocodazole neither block biosynthesis of sphingolipids, nor ER to Golgi transport of IPC. In contrast, treatment of yeast cells with brefeldin A, which affects integrity of the Golgi, decreases formation of IPC and MIPC. Interruption of late steps of protein secretion (Golgi to plasma membrane transport) in temperature-sensitive secretory mutants prevents sphingolipids from being transported to the cell periphery.

Biological Transport↗
PubMed1996

Disruption of sphingolipid metabolism in non-human primates consuming diets of fumonisin-containing Fusarium moniliforme culture material.

The fumonisin mycotoxins are produced by Fusarium moniliforme Sheldon, a contaminant of corn worldwide. The two most abundant analogues (fumonisins B1 and B2) are known to be potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) and hence to disrupt de novo sphingolipid biosynthesis. The sphingoid bases, sphingosine and sphinganine (and hence their ratio), were measured at varying intervals over a period of 60 weeks in the serum of non-human primates (vervet monkeys; Cercopithecus aethiops) which were consuming diets containing 'low' and 'high' amounts of F. moniliforme culture material, such that their total daily fumonisin intake was approximately 0.3 and 0.8 mg/kg body weight/day, respectively. Although no significant differences were found in the serum levels of sphingosine compared to controls, serum sphinganine levels in the experimental groups (mean of 219 nM and 325 nM, respectively) were significantly (P = 0.02) elevated above the levels in controls (mean 46 nM). As a consequence, the ratio sphinganine:sphingosine was significantly (P = 0.003) elevated from a mean of 0.43 in the control group to 1.72 and 2.57 in the experimental groups, respectively. Similar changes in sphingolipid profiles were also measured in urine with an increase of the ratio from 0.87 in controls to 1.58 and 2.17 in the experimental groups, although the differences were not statistically significant. Hence, the disruption of sphingolipid biosynthesis in vervet monkeys by fumonisins in culture material added to their diet can effectively be monitored in the serum as an elevation of the sphinganine:sphingosine ratio.

Animals↗
PubMed2006

Dietary sphingolipids suppress a subset of preneoplastic rat liver lesions exhibiting high PTEN, low phospho-Akt and high levels of ceramide species.

Rat liver glutathione-S-transferase Pi-(GST-P)-positive enzyme-altered foci (EAF) are preneoplastic lesions that develop in response to carcinogenic stress. They are often used as endpoints in e.g. chemopreventive studies. In this study we characterize a pAkt-negative/ceramide-positive (pAkt-/cer+) EAF phenotype, as defined by immunohistochemistry for pAkt and ceramide species, in diethylnitrosamine(DEN)-, phenobarbital- or aflatoxinB1-treated rats. There was a close to 100% overlap for the pAkt and the ceramide marker. Furthermore, serial sections stained for PTEN indicated a close correlation between PTEN-positive and pAkt-negative lesions in DEN-treated rats. Experiments with DEN-treated rats given sphingomyelin in the diet suggested that sphingomyelin selectively targeted these lesions. In in vitro experiments sphingosine rapidly decreased pAkt levels in hepatocytes, and in experiments with hepatocytes from DEN-treated rats sphingosine selectively killed EAF cells. Furthermore, pretreatment with antisense Akt oligonucleotides in vitro sensitized non-EAF hepatocytes, so that EAF and non-EAF cells became equally sensitive to sphingosine. It is concluded that rat liver, in response to carcinogenic stress, develops a distinct EAF phenotype exhibiting low pAkt levels and concomitant alterations in sphingolipid metabolism. Our data also suggest that pAkt-/cer+ EAF are selectively targeted by sphingolipids in the diet and that lesions with this phenotype should be of particular interest for future studies on chemopreventive effects that may affect sphingolipid metabolism.

Animals↗
PubMed2004

Myriocin, a serine palmitoyltransferase inhibitor, alters regional brain neurotransmitter levels without concurrent inhibition of the brain sphingolipid biosynthesis in mice.

Myriocin is a specific serine palmitoyltransferase (SPT) inhibitor whose effect on the brain is unknown. Brain amine metabolism and sphingolipid biosynthesis were studied in mice treated intraperitoneally with 0, 0.1, 0.3 or 1 mg/kg per day of myriocin for 5 days. Regional concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT, serotonin), 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine (NE), were determined. Sphinganine (Sa) and sphingosine (So) concentrations and SPT activity in brain and liver were used to evaluate the impact of myriocin on sphingolipid biosynthesis. Myriocin treatment increased DA in striatum and hippocampus and reduced it in cortex. NE concentration decreased in cerebellum and 5-HT levels were reduced in cortex and in medulla oblongata. Changes in ratios for DOPAC/DA and HVA/DA were observed in hippocampus, cortex and midbrain. Brain Sa, So and SPT activity remained unchanged, whereas Sa and SPT activity decreased in liver. Results showed that myriocin may alter the levels and metabolism of brain amines and this effect is not related with inhibition of sphingolipid biosynthesis in the nervous system.

Acyltransferases↗
PubMed2002

sigma(2) Receptors regulate changes in sphingolipid levels in breast tumor cells.

sigma(2) Receptors induce apoptosis in various cell types. The sphingolipid, ceramide as well as the sphingoid bases are involved in cell proliferation. Sphingolipids of MCF-7/Adr- and T47D breast tumor cells were metabolically radiolabeled. The sigma(2) receptor agonists (+)-1R,5R-E-8-(3,4-dichlorobenzylidene)-5-(3-hydroxyphenyl)-2-methylmorphan-7-one (CB-184) and 1S,2R-(--)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)-cyclohexylamine (BD737) caused dose-dependent increases in [(3)H]ceramide, with concomitant decreases in [(3)H]sphingomyelin. Both effects were attenuated by the novel sigma(2) receptor antagonist, N-phenethylpiperidine oxalate (AC927). sigma(2) Receptors may produce effects on cell growth and apoptosis by regulating the sphingolipid pathway.

Benzylidene Compounds↗
PubMed2003

The roles of ceramide and complex sphingolipids in neuronal cell function.

The roles of sphingolipids, and particularly of the complex glycosphingolipids (GSLs), the gangliosides, have been studied for many years in neurons, glia, and cell lines derived from these tissues, due to their abundance in tissues of neuronal origin. More recently, significant attention has been paid to the simple sphingolipids, particularly ceramide, glucosylceramide (GlcCer), and sphingosine-1-phosphate (S1P), each of which appears to be involved in the regulation of specific aspects of neuronal proliferation, differentiation, survival and apoptosis. In this review, we will summarize studies performed in our laboratory over the past few years using cultured hippocampal neurons in an attempt to define the precise roles of these lipids, and to define their mechanisms of action by identifying down-stream targets with which they interact. We will also discuss work suggesting that complex GSLs, such as gangliosides GM2 and GD3, can also regulate neuronal development, although the down-stream targets with which they interact are less well defined. Our work will be reviewed in light of studies from other laboratories, with particular emphasis on the use of models of sphingolipid storage diseases to determine how these lipids affect neuronal function.

Animals↗
PubMed2001

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol.

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.

Brain Chemistry↗
PubMed2000

Sphingolipids signal heat stress-induced ubiquitin-dependent proteolysis.

Sphingolipids are essential eukaryotic membrane lipids that are structurally and metabolically conserved through evolution. Sphingolipids have also been proposed to regulate eukaryotic stress responses as novel second messengers. Here we show that, in Saccharomyces cerevisiae, phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-dependent proteolysis via the endocytosis vacuolar degradation and 26 S proteasome pathways. Inactivation of serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress. Addition of phytosphingosine bypasses the requirement for serine palmitoyltransferase and restores proteolysis. Phytosphingosine-induced proteolysis requires multiubiquitin chain formation through the stress-responsive lysine 63 residue of ubiquitin. We propose that heat stress increases phytosphingosine and activates ubiquitin-dependent proteolysis.

Acyltransferases↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2003

Effects of inhaled CdO particles on the sphingolipid synthesis of rat lungs.

Surfactant lipids of the alveolar space protect the lung from various environmental stimuli. We investigated the influence of ultrafine (UF) CdO particles inhalation on two key enzymes involved in lung sphingolipid metabolism, serine palmitoyltransferase (SPT), and sphingomyelinase (SMase). Rats inhaled either 0.63 mg UF-CdO/m(3) for 6 h (group 1), or 1.08 mg UF-CdO/m(3) 12 h/day for 10 days (group 2). Two corresponding control groups inhaled filtered clean air. Additional rats intratracheally instilled with lipopolysaccharide (LPS) were used as positive controls. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) of lung tissue showed a significant increase in the level of SPT mRNA (LCB2 subunit) expression in group 2 compared to the corresponding controls (p <.01). Group 1 and LPS were not statistically different from control. No alteration in the mRNA level of SMase was detected in any exposure group. The immunohistochemical analysis showed that SPT (LCB2 subunit) localization was stronger in the alveolar type II cells of group 2 lungs compared to the corresponding controls. These results were correlated with alterations in BALF cellular and biochemical parameters and lung morphology. Since SPT is the key enzyme for de novo sphingolipid synthesis in lung surfactant and SMase is responsible for sphingomyelin catabolism, we can postulate that high-dose UF-CdO exposure for 10 days induces an increase in sphingolipid synthesis in the type II cells of rat lungs that would not be promptly followed by its degradation.

Acyltransferases↗
PubMed2001

A cross-sectional study of human serum sphingolipids, diet and physiologic parameters.

Sphinganine and sphingosine, the two sphingoid base backbones of sphingolipids, are highly bioactive compounds that are of increasing interest to nutritionists because they occur in food and their metabolism can be altered by fungal toxins that contaminate some foods. Nonetheless, no studies of diet and sphinganine or sphingosine concentrations in serum have yet been reported. Here we describe a cross-sectional study of 265 residents of Linxian, People's Republic of China, which examines potential demographic, physiologic and dietary correlates of serum sphinganine and sphingosine in this population. Median concentrations of serum sphinganine and sphingosine were compared among strata for 29 different variables. For sphinganine, no significant differences were found. For sphingosine, significant differences were seen among strata of age, menstruation status, serum cholesterol, carotenoids, retinol, tocopherols, fresh and dried vegetable and fresh fruit consumption. Using multivariate linear regression with stepwise selection, we found that the significant predictors for serum sphingosine included total tocopherols, age, serum selenium and retinol, with a final R(2) = 0.22; for sphinganine, tooth loss was the sole correlate, with R(2) = 0.015. Analyses using ranked sphingolipid data or principal components analysis, to simplify the food variables, did not materially alter these results. This study represents the largest report of human serum sphingolipid concentrations to date and provides insight into potential explanatory variables that can be incorporated into future studies.

Adult↗
PubMed2003

Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration.

Mutations in proteins of the Drosophila phototransduction cascade, a prototypic guanine nucleotide-binding protein-coupled receptor signaling system, lead to retinal degeneration and have been used as models to understand human degenerative disorders. Here, modulating the sphingolipid biosynthetic pathway rescued retinal degeneration in Drosophila mutants. Targeted expression of Drosophila neutral ceramidase rescued retinal degeneration in arrestin and phospholipase C mutants. Decreasing flux through the de novo sphingolipid biosynthetic pathway also suppressed degeneration in these mutants. Both genetic backgrounds modulated the endocytic machinery because they suppressed defects in a dynamin mutant. Suppression of degeneration in arrestin mutant flies expressing ceramidase correlated with a decrease in ceramide levels. Thus, enzymes of sphingolipid metabolism may be suitable targets in the therapeutic management of retinal degeneration.

Acyltransferases↗
PubMed2005

Mannosyl-diinositolphospho-ceramide, the major yeast plasma membrane sphingolipid, governs toxicity of Kluyveromyces lactis zymocin.

Kluyveromyces lactis zymocin, a trimeric (alphabetagamma) protein toxin complex, inhibits proliferation of Saccharomyces cerevisiae cells. Here we present an analysis of kti6 mutants, which resist exogenous zymocin but are sensitive to intracellular expression of its inhibitory gamma-toxin subunit, suggesting that KTI6 encodes a factor needed for toxin entry into the cell. Consistent with altered cell surface properties, kti6 cells resist hygromycin B, syringomycin E, and nystatin, antibiotics that require intact membrane potentials or provoke membrane disruption. KTI6 is allelic to IPT1, coding for mannosyl-diinositolphospho-ceramide [M(IP)(2)C] synthase, which produces M(IP)(2)C, the major plasma membrane sphingolipid. kti6 membranes lack M(IP)(2)C and sphingolipid mutants that have reduced levels of M(IP)(2)C precursors, including the sphingolipid building block ceramide survive zymocin. In addition, kti6/ipt1 cells allow zymocin docking but prevent import of its toxic gamma-subunit. Genetic analysis indicates that Kti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1, a previously identified zymocin sensitivity factor. In sum, M(IP)(2)C operates in a plasma membrane step that follows recognition of cell wall chitin by zymocin but precedes the involvement of elongator, the potential toxin target.

Alleles↗
PubMed1997

Accumulation of sphingolipids in SAP-precursor (prosaposin)-deficient fibroblasts occurs as intralysosomal membrane structures and can be completely reversed by treatment with human SAP-precursor.

The degradation of glycosphingolipids takes place in lysosomes by action of specific exohydrolases, with the assistance of sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins A to D), are synthesized from a single protein, the SAP-precursor (prosaposin). Deficiency in this precursor protein, a rare inherited disease in humans, results in the storage of sphingolipids with short oligosaccharide head groups within the patients' tissues, and electron microscopy revealed the accumulation of large multivesicular storage organelles. In this study we analyze the multivesicular storage organelles in cultivated fibroblasts from these patients. The results support our hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures that are then digested within the lysosomes (Sandhoff, K., T. Kolter, Trends in Cell Biol. 6, 98-103 (1996). First, we show that the storage compartment consists of late endosomes and lysosomes by immunogold labeling for marker proteins of these organelles. The transport of endocytosed bovine serum albumin-colloidal gold or cationized ferritin into the compartment occurs with the timing expected for transport to late endocytic organelles. Second, complementation of the medium of the SAP-precursor-deficient fibroblasts with only nanomolar concentrations of purified SAP-precursor nearly completely reversed the aberrant accumulation of multivesicular structures, thereby abolishing most of the intralysosomal membrane structures. Analysis of the sphingolipid pattern of the cells after metabolic labeling with [14C]serine reveals that the cells' ability to degrade glycosphingolipids is completely restored by feeding of SAP-precursor at the same concentrations. This is the first demonstration in vivo that endocytosed SAP-precursor is processed into functional active SAPs A,- B,- C, and D and that the degradation of the vesicular structures within the lysosomes depends on the presence of the SAPs. Moreover, these studies suggest that a therapy program based on feeding purified SAP-precursor may be valuable in treating the entire family of diseases which result from the loss of one or more of the SAPs.

Endosomes↗
PubMed1995

Immunological studies on sphingolipid activator proteins in the neuronal ceroid-lipofuscinoses.

The neuronal ceroid-lipofuscinoses constitute an important group of progressive encephalopathies leading to severe psychomotor retardation, blindness, and early death. They are characterized by accumulation of autofluorescent, electron-dense storage bodies within the cytoplasm of neurons and many other cell types. We have recently identified sphingolipid activator proteins A and D as major components of the storage cytosomes in the infantile form of NCL. Using an immunological approach we have shown that sphingolipid activator proteins also constitute an integral component of the storage bodies in the other major forms of the disease.

Blotting, Western↗
PubMed2001

High-performance liquid chromatography analysis of molecular species of sphingolipid-related long chain bases and long chain base phosphates in Saccharomyces cerevisiae after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

The molecular species of dihydrosphingosines and phytosphingosines and their 1-phosphates with carbon chain lengths from 16 to 20 have been tagged with the fluorescent amino group reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. All these derivatives could be resolved by reversed phase HPLC on a C18 column. A convenient one-pot method is described whereby lipid extracts from strains of Saccharomyces cerevisiae containing carbon chain length homologs of sphingolipid long chain bases and their phosphorylated derivatives were directly reacted with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, ester lipids were deacylated, and the reaction mixtures were subjected to liquid chromatography. Five molecular species of both sphingolipid long chain bases and their phosphorylated derivatives are for the first time separated and analyzed. The procedure is quite sensitive, requiring only approximately 10(8) wild-type cells.

Aminoquinolines↗
PubMed1994

Sphingolipid activator protein D (sap-D) stimulates the lysosomal degradation of ceramide in vivo.

Glycosphingolipids of cultured fibroblasts from patients with total sphingolipid activator proteins (SAPs) deficiency (Schnabel et al. J. Biol. Chem. 267:3312-3315, 1992) were labeled biosynthetically with [14C]serine. After a chase period of 120h the patients' fibroblasts showed increased labeling of ceramide, glucosylceramide, lactosylceramide and ganglioside GM3 in comparison to normal control cells. Addition of sap-D to the chase-media of the patients' fibroblasts led to the degradation of the accumulated ceramide down to nearly normal levels, whereas the levels of other labeled sphingolipids remained unaffected. In contrast, addition of sap-B to the chase-media of the patients' fibroblasts did not reduce the increased ceramide levels but resulted, as expected from in vitro experiments, in a specific decrease of levels of lactosylceramide and ganglioside GM3. Therefore, we conclude that sap-D stimulates in vivo the lysosomal degradation of ceramide.

Cells, Cultured↗