A new procedure for synthesis of 3-keto derivatives of sphingolipids and its application for study of fatty acid composition of brain ceramides and cerebrosides containing dihydrosphingosine of sphingosine

3-Keto derivatives were prepared in good yield by the oxidative procedure with 2,3-dichloro-5,6-dicyanobenzoquinone from N-acetyl sphingosine, N-palmitoyl sphingosine, N-lignoceroyl sphingosine, and N-lignoceroyl psychosine. None of these 3-keto derivatives, except the one from N-acetyl sphingosine, have been previously reported. Ceramides were isolated from a calf brain and reacted with 2,3-dichloro-5, 6-dicyanobenzoquinone. Ceramides containing sphingosine (4-sphingenine) were converted to 3-keto derivative, while those containing dihydrosphingosine (sphinganine) remained intact under these conditions. The 3-keto ceramides were then separated from the ceramides containing dihydrosphingosine by preparative thin layer chromatography. Similarly cerebrosides from the same calf brain were oxidized and fractionated to 3-ketocerebrosides (from cerebrosides containing sphingosine) and unreacted cerebrosides (cerebrosides containing dihydrosphingosine). The fatty acid composition of these four sphingolipids were determined. Both the ceramides and the cerebrosides containing sphingosine had more unsaturated fatty acids than the corresponding dihydrosphingosine-containing compounds. The ratio of C₁₆-C₂₀ fatty acids to C₂₂-C₂₆ acids was higher in the ceramides containing sphingosine than in ceramides containing dihydrosphingosine, while the ratio was reversed in cerebrosides. The possible precursor-product relationship among these lipids is discussed.     

Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system

Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C_18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C_18:1-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C_18:1-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C_18:1-ceramide, but not C_18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C_18:1-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C_18:1-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C_18:1-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.Ceramides are the central lipid in the metabolic network of sphingolipids, and are generated through the de novo, catabolic and salvage pathways.¹ In the de novo pathway, ceramides are synthesized through multiple steps catalyzed sequentially by serine palmitoyltransferase (SPT), keto-dihydrosphingosine reductase, (dihydro)ceramide synthases (CerSs) and dihydroceramide desaturases. In the catabolic pathways, ceramides are derived from the hydrolysis of sphingomyelins by sphingomyelinases (SMases) or the hydrolysis of glycosphingolipids. In the salvage pathway, ceramides are synthesized from sphingosine (SPH) and fatty acyl-CoA by CerSs. As CerSs (CerS1-6) have distinct specificity toward acyl-CoA chain length and degree of unsaturation, ceramides with various acyl-chains are found in mammalian cells. Upon generation, ceramides can be hydrolyzed by five ceramidases encoded by five distinct genes (ASAH1, ASAH2, ACER1, ACER2 and ACER3). These ceramidases vary in pH optimum for catalytic activity, tissue distribution, cellular localization and substrate specificity,² allowing for regulation of specific ceramides in a cell- or tissue-specific manner.Recent studies have implicated ceramides in regulating the innate immune response. Sakata et al.³ demonstrated that lipopolysaccharides (LPS), a potent inducer of the innate immune response, increases C₁₆-ceramide by activating acid SMase and that inhibition of SMase attenuates LPS-induced production of pro-inflammatory cytokines in THP-1 macrophages. Andreyev et al.⁴ found that ceramides are increased by Toll-like receptor 4 (TLR4)-specific LPS in RAW 264.7 macrophages. Schilling et al.⁵ revealed that LPS and palmitic acid synergistically increase C₁₆-ceramide in primary mouse peritoneal macrophages (PMs) by activating de novo biosynthesis of ceramides and that inhibiting the C₁₆-ceramide increase attenuates LPS-induced production of TNF-α and IL-1β in PMs. A recent study found that LPS increases ceramides in Raw 264.7 macrophages through nuclear factor kappa B (NF-κB)-dependent upregulation of SPT long chain base subunit 2 Sptlc2, a regulator of SPT.⁶ These results suggest that ceramides mediate the immune response in part by enhancing the production of pro-inflammatory cytokines in innate immune cells.Emerging evidence suggests that dysregulation in the innate immune response in inflammatory bowel disease (IBD) contributes to the pathogenesis of the disease.⁷ Consistent with the role of ceramides in potentiating the innate immune response, several studies found that ceramides may have a role in the pathogenesis of IBD. Sakata et al.³ demonstrated that blocking the generation of ceramides with the SMase inhibitor hinders mouse colitis. Fischbeck et al.⁸ showed that increasing ceramides in the gut by supplying mice with dietary sphingomyelins, a precursor of ceramides, aggravates mouse colitis. These results suggest that increased levels of ceramides may contribute to the pathogenesis of IBD.Although the role of ceramides and their generating enzymes in the innate immune response have been well studied, much remains unclear about the role of ceramidases involved in the catabolism of ceramides in this biological response. In this study, we investigated the role of alkaline ceramidase 3 (ACER3)/Acer3 and its substrates in immune response. We demonstrated that Acer3 is downregulated, whereas its substrate, C_18:1-ceramide, is upregulated in murine immune cells and colonic epithelial cells (CECs) during the innate immune response to LPS. Using Acer3 null mice (Acer3^−/−) and their wild-type (Acer3^+/+) littermates, we further discovered that the inverse regulation of Acer3 and C_18:1-ceramide potentiates LPS-induced production of pro-inflammatory cytokines in innate immune cells. More importantly, we found that Acer3 deficiency aggravates dextran sulfate sodium (DSS)-induced colitis and colitis-associated colorectal cancer (CAC) in a murine model. These findings indicate that Acer3/ACER3 and C_18:1-ceramide are novel modulators in the innate immune response and that their dysregulation may contribute to the pathogenesis of inflammatory diseases.     

Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C₆, C₁₂, C₁₆, C_18:1, and C_24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe²⁺. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.     

Sphingolipids and cell signaling: Involvement in apoptosis and atherogenesis

This review considers various functional aspects of cell sphingolipids (sphingomyelin, ceramides) and lysosphingolipids (sphingosine-1-phosphate (S1P) and sphingosine phosphorylcholine). Good evidence now exists that they are actively involved in numerous cell-signaling processes. The enzymes responsible for formation and interconversion of cell sphingolipids (sphingomyelinases, ceramidase, sphingosine kinase, S1P-lyase) exhibit high sensitivity to various stimulating factors. This determines the content of individual cell sphingolipids and therefore the mode of cell response. Special attention is paid to preferential localization of sphingolipids in the rigid plasma membrane domains (rafts) coupled to many signal proteins. The suggestion is discussed that ceramide signaling may be based on the modification of fine molecular interactions in lipid rafts, resulting in its clusterization inducing the signal transduction. The review also highlights involvement of sphingolipids in cell proliferation, apoptosis, and in processes implicated to atherosclerosis.     

Sphingolipid Biosynthesis Is Necessary for Dendrite Growth and Survival of Cerebellar Purkinje Cells in Culture

Abstract: The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 µM fumonisin B₁, a fungal inhibitor of mammalian ceramide synthase, inhibited incorporation of [³H]galactose/glucosamine and [¹⁴C]serine into complex sphingolipids of cultured cerebellar neurons. Under this condition, the expression of Purkinje cell-enriched sphingolipids, including GD1α, 9-O-acetylated LD1 and GD3, and sphingomyelin, was significantly decreased. After 2 weeks' exposure to fumonisin B₁, dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag, but their branches began to degenerate. In some cells, formation of elongated dendrite trees was severely impaired. However, treatment with fumonisin B₁ also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells, morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B₁. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed, these effects of fumonisin B₁ were reversed, but not completely, by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]caproyl]sphingosine (C₆-NBD-ceramide), a synthetic derivative of ceramide. Thus, we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells.     

Blood sphingolipidomics in healthy humans: impact of sample collection methodology

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C₁₄-C₂₆ N-acyl part) of “normal” sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (C_n-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C₁₆-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C₁₆-LacCer (10.0 µM) and the abundant HexCer was C₂₄-HexCer (2.5 µM). The abundant Cer, C₂₄-Cer (4.0 µM), was not influenced by fasting; however, levels of C₁₆-C₂₀ Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL₃ was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.     

Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations

Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (∼10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (∼70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C₂₀-, C₂₂-, and C₂₄-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.     

Ceramide and Its Interconvertible Metabolite Sphingosine Function as Indispensable Lipid Factors Involved in Survival and Dendritic Differentiation of Cerebellar Purkinje Cells

Abstract: 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.     

Recycling of sphingosine is regulated by the concerted actions of sphingosine-1-phosphate phosphohydrolase 1 and sphingosine kinase 2

In yeast, the long-chain sphingoid base phosphate phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous sphingosine into ceramide in mammalian cells was regulated by the homologue of Lcb3p, sphingosine-1-phosphate phosphohydrolase 1 (SPP-1), an endoplasmic reticulum resident protein. Sphingosine incorporation into endogenous long-chain ceramides was increased by SPP-1 overexpression, whereas recycling of C(6)-ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B(1), an inhibitor of ceramide synthase, but not by ISP-1, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-1 expression increased the incorporation of sphingosine into all ceramide acyl chain species, particularly enhancing C16:0, C18:0, and C20:0 long-chain ceramides. The increased recycling of sphingosine into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. Sphingosine kinase 2, but not sphingosine kinase 1, acted in concert with SPP-1 to regulate recycling of sphingosine into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of sphingosine to ceramide and more complex sphingolipids.     

Sphingolipids of developing human central nervous tissue: changes in composition of sphingosine bases

—Developmental changes in composition of sphingosine bases of cerebrosides, sulphatides and sphingomyelins were investigated in samples from brain stem or corpus callosum from premature infants and patients aged 14 months, 39 and 59 years who died of non-neurological causes. Since insufficient material was obtainable from the premature brains, sulphatides were studied only in older cases. Individual sphingolipids were isolated by combinations of column chromatography and TLC, and were examined for purity by analytical TLC. Sphingosine bases were released by acid-catalysed methanolysis, and analysed as aldehydes by GLC. Effectiveness and limitations of methods used for analyses of sphingosine bases were evaluated. In contrast to adult sphingolipids in which approximately 95 per cent of the sphingosine bases was 18: sphingosine, as much as 10 per cent of the total was 18: dihydrosphingosine in immature sphingolipids. A compound tentatively identified as 20: sphingosine was present in foetal, infant and adult sphingomyelin at 5, 3 and 1 per cent of the total, respectively. Composition of sphingosine bases of non-ganglioside sphingolipids in human brain varies with age, presumably in a complex manner.     

FATTY ACID COMPOSITION OF CEREBROSIDES, SULPHATIDES AND CERAMIDES IN MURINE SUDANOPHILIC LEUCODYSTROPHY: THE JIMPY MUTANT

The fatty acid composition of cerebrosides, sulphatides and ceramides was determined at 15-16 days post partum in the brain of the Jimpy mutant and in littermate controls. There was a marked deficit in the long chain fatty acids (C₂₂-C₂₄) of cerebrosides and sulphatides of Jimpy brain, with the unsubstituted fatty acids affected more than the alpha-hydroxy fatty acids. A decrease of long chain normal fatty acids was also found in the ceramides of Jimpy brain. The deficit of long chain fatty acids in these sphingolipids of the Jimpy brain was more severe than that found in the Quaking mutant which has a less extensive disorder of myelin formation.     

Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C_12:0), N-myristoyl- (C_14:0) and N-palmitoyl- (C_16:0) sphingosine (C_18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C_18:0) substrate. The activity towards N-linolenoylsphingosine (C_18:3/C_18:1), N-linoleoylsphingosine (C_18:2/C_18:1) and N-oleoylsphingosine (C_18:1/C_18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C_18:0/C_18:1). The activity towards N-stearoyldihydrosphingosine (C_18:0/C_18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C_6:0/C_18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher K_m values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.     

Sphingolipids and glycerophospholipids – The “ying and yang” of lipotoxicity in metabolic diseases

Sphingolipids in general and ceramides in particular, contribute to pathophysiological mechanisms by modifying signalling and metabolic pathways. Here, we present the available evidence for a bidirectional homeostatic crosstalk between sphingolipids and glycerophospholipids, whose dysregulation contributes to lipotoxicity induced metabolic stress. The initial evidence for this crosstalk originates from simulated models designed to investigate the biophysical properties of sphingolipids in plasma membrane representations. In this review, we reinterpret some of the original findings and conceptualise them as a sort of “ying/yang” interaction model of opposed/complementary forces, which is consistent with the current knowledge of lipid homeostasis and pathophysiology. We also propose that the dysregulation of the balance between sphingolipids and glycerophospholipids results in a lipotoxic insult relevant in the pathophysiology of common metabolic diseases, typically characterised by their increased ceramide/sphingosine pools.     

Metabolomic profiling of sphingolipids in human glioma cell lines by liquid chromatography tandem mass spectrometry.

Sphingolipids participate in membrane structure and signaling in neuronal cells, and an emerging strategy for control of gliomas is to inhibit growth and/or induce apoptosis using ceramide and ceramide analogs. Nonetheless, some sphingolipids (ceramides and sphingosine) induce and others (sphingosine 1-phosphate) inhibit apoptosis; therefore, when testing putative anti-cancer agents, it is critical to obtain precise knowledge of the types and quantities of not only the test compounds, but also their effects on endogenous species. Combination of liquid chromatography and tandem mass spectrometry affords a "metabolomic" profile of all of the intermediates of ceramide biosynthesis (3-ketosphinganine, sphinganine and dihydroceramides) and the direct products of ceramide metabolism (sphingomyelins and monohexosylceramides as well as sphingosine and sphingosine 1-phosphate). This method has been applied to four human glioma cell lines (LN18, LN229, LN319 and T98G), and differences in the amounts and types of sphingolipids were found. For example, LN229 and LN319 have approximately twice the sphingosine 1-phosphate of LN18 and T98G; LN229 and LN319 have more monohexosylceramides than lactosylceramides, whereas the opposite is the case for LN18 and T98G; and the fatty acyl chain distributions of the sphingolipids differ among the cell lines. The ability to obtain this type of "metabolomic" profile allows studies of how anti-cancer agents (especially sphingolipids and sphingolipid analogs) affect the amounts of these bioactive species, and may lead to a better understanding of the abnormal phenotypes of gliomas.     

Early fumonisin B1 toxicity in relation to disrupted sphingolipid metabolism in male BALB/c mice

Fumonisin B₁ is a mycotoxin produced by Fusarium moniliforme, a common fungus in corn. It is known to cause a variety of diseases, including hepatic and renal degeneration in many species of laboratory and domestic animals. The known biochemical events in fumonisin B₁ toxicity involve inhibition of ceramide synthase leading to disruption of sphingolipid metabolism. The effect of fumonisin B₁ on ceramide and more complex sphingolipids in mice is not known. Groups of five male BALB/c mice each were injected with fumonisin B₁ subcutaneously at doses of 0, 0.25, 0.75, 2.25, and 6.75 mg/kg body weight daily for 5 days. This protocol has been shown to produce a dose-dependent increase in apoptosis in liver and kidney of these animals. In the present study, liver, kidney, and brain were sampled and analyzed for free sphingoid bases and complex sphingolipids one day after the last treatment. A dose-related accumulation of free sphinganine and sphingosine was observed in liver and kidney, but not brain. The maximal increase in free sphinganine in kidney was 10-fold greater than in liver. Total phospholipids increased only in liver, whereas ceramide levels were not consistently altered in liver, kidney, or brain. In liver and kidney, fumonisin B₁ treatment increased the sphinganine-containing complex sphingolipids, but no effect was observed on sphingosine-containing complex sphingolipids. No changes in complex sphingolipids were observed in brain. In liver, there was a close correlation between the extent of free sphinganine accumulation, and apoptosis and hepatopathy. This correlation was also evident in kidney but to a lessor extent. Nonetheless, the apoptosis and nephropathy occurred with little or no change in the levels of ceramide or more complex sphingolipids. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 281–289, 1998     

Comparative quantification of sphingolipids and analogs in biological samples by high-performance liquid chromatography after chloroform extraction

Sphingosine 1-phosphate (S1P) is an extra- and intracellular messenger that specifically activates five G-protein-coupled cell surface receptors designated S1P_1-5. The S1P₁ receptor is particularly important for the maintenance of immune surveillance by regulating egress of lymphocytes from thymus and secondary lymphoid organs. S1P is generated through phosphorylation of sphingosine which is catalyzed by sphingosine kinase types 1 and 2. The immunosuppressant and sphingosine analog Fingolimod (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol, FTY720) can also be phosphorylated and induces lymphopenia by downregulating cell surface expression of the S1P₁ receptor on lymphocytes. To analyze the role of S1P in lymphocyte circulation and distribution we established a high-performance-liquid-chromatography-based method for parallel detection and quantification of Fingolimod, sphingosine, and dihydrosphingosine together with their phosphorylated derivatives Fingolimod-phosphate, S1P, and dihydrosphingosine 1-phosphate. Phosphorylated and nonphosphorylated lipids were efficiently isolated from biological samples such as cells, tissues, serum, plasma, and media by simple chloroform extraction. Fluorescence labeling with 9-fluorenylmethyl chloroformiate ensured high selectivity and enhanced sensitivity for sphingolipid detection. The described method provides an accurate approach to investigate phosphorylation, dephosphorylation, hydrolyzation, and dehydrolyzation of sphingolipids and analogs. In addition it works independently from enzymatic conversions, measuring actual concentrations rather than enzymatic activities.     

The ceramide structure of GM1 ganglioside differently affects its recovery in low-density membrane fractions prepared from HL-60 cells with or without triton-X100

Gangliosides are characteristically enriched in various membrane domains that can be isolated as low density membrane fraction insoluble in detergents (detergent-resistant membranes, DRMs) or obtained after homogenization and sonication in 0.5 M sodium carbonate (low-density membranes, LDMs). We assessed the effect of the ceramide structure of four [³H]-labeled GM1 ganglioside molecular species (GM1s) taken up by HL-60 cells on their occurrence in LDMs, and compared it with our previous observations for DRMs. All GM1s contained C18 sphingosine, which was acetylated in GM1(18:1/2) or acylated with C₁₄, C₁₈ or C_18:1 fatty acids (Fas)     

Control of inflammatory responses by ceramide,sphingosine 1-phosphate and ceramide 1-phosphate

Inflammation is a network of complex processes involving a variety of metabolic and signaling pathways aiming at healing and repairing damage tissue, or fighting infection. However, inflammation can be detrimental when it becomes out of control. Inflammatory mediators involve cytokines, bioactive lipids and lipid-derived metabolites. In particular, the simple sphingolipids ceramides, sphingosine 1-phosphate, and ceramide 1-phosphate have been widely implicated in inflammation. However, although ceramide 1-phosphate was first described as pro-inflammatory, recent studies show that it has anti-inflammatory properties when produced in specific cell types or tissues. The biological functions of ceramides and sphingosine 1-phosphate have been extensively studied. These sphingolipids have opposing effects with ceramides being potent inducers of cell cycle arrest and apoptosis, and sphingosine 1-phosphate promoting cell growth and survival. However, the biological actions of ceramide 1-phosphate have only been partially described. Ceramide 1-phosphate is mitogenic and anti-apoptotic, and more recently, it has been demonstrated to be key regulator of cell migration. Both sphingosine 1-phosphate and ceramide 1-phosphate are also implicated in tumor growth and dissemination. The present review highlights new aspects on the control of inflammation and cell migration by simple sphingolipids, with special emphasis to the role played by ceramide 1-phosphate in controlling these actions.     

FTY720 Inhibits Ceramide Synthases and Up-regulates Dihydrosphingosine 1-Phosphate Formation in Human Lung Endothelial Cells

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P_1,3–5 receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-¹³C, ¹⁵N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent K_i of 2.15 μm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells.FTY720² is a synthetic analog of sphingosine and is currently being studied as a potent immunosuppressive and immunomodulatory agent (1–3). FTY720-induced immunosuppression is ascribed, in part, to its protective effect on endothelial cell barrier function that results in inhibition of lymphocyte egress from lymph nodes and down-regulation of innate and adaptive immune responses (4). As endothelial cells predominantly express the sphingosine 1-phosphate 1 (S1P₁) receptor and its activation initiates signaling that results in the assembly of VE-cadherin-based adherens junctions (5), it is thought that the phosphorylation of FTY720 and the binding of FTY720-P to the S1P₁ receptor determine its effect on vasculature (1). Recently it became evident that the action of FTY720 is more complex as several other direct protein targets were identified. Thus, FTY720 was found to bind to and inhibit the cannabinoid CB1 receptor (6), to inhibit cytosolic phospholipase A₂ (cPLA₂), and to counteract ceramide 1-phosphate-induced cPLA₂ activation (7). Additionally FTY720 but not FTY720-P was shown to inhibit S1P lyase (8), which degrades S1P to ethanolamine phosphate and (E)-2-hexadecenal and regulates the removal of sphingoid bases from the cumulative pool of sphingolipids. These findings characterize FTY720 as a molecule with a multitargeted mode of action whose cellular effects are complicated by its metabolic transformation to FTY720-P, a structural and functional analog of S1P.Phosphorylation of FTY720 to FTY720-P by sphingosine kinases (SphKs) is the only reported metabolic transformation of FTY720 and has been actively explored because of its link to S1P-mediated signaling (1, 2, 9, 10). Recent studies suggest that the endogenous balance between S1P and ceramide molecules regulates prosurvival and proapoptotic signaling cascades, which determine the outcome of cellular response to different stress conditions (11, 12) or the efficiency of anticancer therapy (12–14). However, despite the fact that FTY720 resembles sphingosine (Sph) and is a substrate of SphK2 (15–17), there are no reported studies on the effect of FTY720 on the intrinsic balance of signaling sphingolipids. Metabolic interconnections between proapoptotic (ceramides) and prosurvival (dihydrosphingosine 1-phosphate (DHS1P)) molecules are expected because it is known that fumonisin B1 (FB1), an inhibitor of (dihydro)ceramide synthases, not only blocks the formation of ceramides and up-regulates the intracellular content of dihydrosphingosine (DHSph) but also increases the cellular level of DHS1P (19, 20).In view of these considerations, it is important to know how compounds with a potential ability to interfere with the sphingolipidome turnover affect the DHS1P-S1P/ceramide balance in cells. To address this question we have investigated the effect of FTY720 on metabolic pathways leading to ceramide and sphingoid base 1-phosphate generation in human pulmonary artery endothelial cells (HPAECs) by using a stable isotope pulse labeling approach and quantitative liquid chromatography-tandem mass spectrometry of signaling sphingolipids. We demonstrate that treatment of HPAECs with FTY720 results in the inhibition of de novo ceramide formation with a concomitant increase in DHSph and DHS1P content in cells. Moreover FTY720 showed a direct inhibition of ceramide synthases in an in vitro assay, albeit it was less efficient compared with the classical inhibitor of ceramide synthases, FB1. Our present findings have identified ceramide synthase isozymes as a novel molecular target for FTY720 action, opening a new direction for its potential therapeutic application through the inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells.     

Associations among circulating sphingolipids, β-cell function,and risk of developing type 2 diabetes: A population-based cohort study in China

BackgroundAnimal studies suggest vital roles of sphingolipids, especially ceramides, in the pathogenesis of type 2 diabetes (T2D) via pathways involved in insulin resistance, β-cell dysfunction, and inflammation, but human studies are limited. We aimed to evaluate the associations of circulating sphingolipids with incident T2D and to explore underlying mechanisms.Methods and findingsThe current study included 826 men and 1,148 women who were aged 50–70 years, from Beijing and Shanghai, and without T2D in 2005 and who were resurveyed in 2011. Cardiometabolic traits were measured at baseline and follow-up surveys. A total of 76 sphingolipids were quantified using high-coverage targeted lipidomics. Summary data for 2-sample Mendelian randomization were obtained from genome-wide association studies of circulating sphingolipids and the China Health and Nutrition Survey (n = 5,731). During the 6-year period, 529 participants developed T2D. Eleven novel and 3 reported sphingolipids, namely ceramides (d18:1/18:1, d18:1/20:0, d18:1/20:1, d18:1/22:1), saturated sphingomyelins (C34:0, C36:0, C38:0, C40:0), unsaturated sphingomyelins (C34:1, C36:1, C42:3), hydroxyl-sphingomyelins (C34:1, C38:3), and a hexosylceramide (d18:1/20:1), were positively associated with incident T2D (relative risks [RRs]: 1.14–1.21; all P < 0.001), after multivariate adjustment including lifestyle characteristics and BMI. Network analysis further identified 5 modules, and 2 modules containing saturated sphingomyelins showed the strongest associations with increased T2D risk (RR_{Q4 versus Q1} = 1.59 and 1.43; both P_trend < 0.001). Mediation analysis suggested that the detrimental associations of 13 sphingolipids with T2D were largely mediated through β-cell dysfunction, as indicated by HOMA-B (mediation proportion: 11.19%–42.42%; all P < 0.001). Moreover, Mendelian randomization evidenced a positive association between a genetically instrumented ceramide (d18:1/20:1) and T2D (odds ratio: 1.15 [95% CI 1.05–1.26]; P = 0.002). Main limitations in the current study included potential undiagnosed cases and lack of an independent population for replication.ConclusionsIn this study, we observed that a panel of novel sphingolipids with unique structures were positively associated with incident T2D, largely mediated through β-cell dysfunction, in Chinese individuals.