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Ceramides belong to the sphingolipid family and represent the central hub of the sphingolipid network. In obesity, oversupply of saturated fatty acids including palmitate raises ceramide levels which can be detrimental to cells. Elevated ceramides can cause insulin resistance, endoplasmic reticulum stress, and mitochondrial dysfunction. Studies over the last few decades have highlighted the role played by ceramides in pancreatic islet β-cell apoptosis, especially under glucolipotoxic and inflammatory conditions. This review focuses on ceramides and adiponectin receptor signaling, summarizing recent advancements in our understanding of their roles in islet β-cells and the discovery of zinc-dependent lipid hydrolase (ceramidase) activity of adiponectin receptors. The therapeutic potential of targeting these events to prevent islet β-cell loss for treating diabetes is discussed.

Ceramidases catalyze hydrolysis of ceramides to generate sphingosine (SPH), which is phosphorylated to form sphingosine-1-phosphate (S1P). Ceramide, SPH, and S1P are bioactive lipids that mediate cell proliferation, differentiation, apoptosis, adhesion, and migration. Presently, 5 human ceramidases encoded by 5 distinct genes have been cloned: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). Each human ceramidase has a mouse counterpart. AC, NC, and ACER1-3 have maximal activities in acidic, neutral, and alkaline environments, respectively. ACER1-3 have similar protein sequences but no homology to AC and NC. AC and NC also have distinct protein sequences. The human AC (hAC) was implicated in Farber disease, and hAC may be important for cell survival. The mouse AC (mAC) is needed for early embryo survival. NC is protective against inflammatory cytokines, and the mouse NC (mNC) is required for the catabolism of ceramides in the digestive tract. ACER1 is critical in mediating cell differentiation by controlling the generation of SPH and S1P and that ACER2's role in cell proliferation and survival depends on its expression or the cell type in which it is found. Here, we discuss the role of each ceramidase in regulating cellular responses mediated by ceramides, SPH, and S1P.

Aim: We aimed at gaining more insight into the mechanisms underlying exercise-induced alterations in myocardial ceramide (CER) content by employing physical activity of various durations and examining all key pathways of CER metabolism., Methods: The experiments were carried out on male Wistar rats divided into four groups (n = 6 in each case): control, exercised for 30 and 90 min and until exhaustion on the electrically driven treadmill moving with a speed of 1200 m h(-1) and set at +10 degrees incline. The animals were anaesthetized and samples of the heart's left ventricle were excised., Results: Thirty-minute exercise decreased the level of CER in the heart by 15%. However, after 90 min of running it returned to the baseline and at the point of exhaustion it exceeded that of the control animals by 26%. The initial reduction in the content of CER was probably a result of its augmented degradation, as a concomitant elevation in the activity of acid ceramidase and the level of sphingosine was observed. The transition from reduction in CER content after 30 min of exercise to its accumulation at the point of exhaustion was a consequence of gradual reduction in the activity of acid ceramidase and simultaneous increase in the rate of de novo CER synthesis, as evidenced by progressive activation of serine palmitoyltransferase and accumulation of sphinganine., Conclusion: We conclude that the effect of physical effort on myocardial CER content and metabolism depends to a large extent on exercise duration.

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias.

Several investigations have shown that acid ceramidase inhibitors are potential antiproliferative and cytostatic drugs for cancer chemotherapy. The combinatorial chemistry approach for the discovery of acid ceramidase inhibitors requires the availability of a high-throughput enzyme assay. The synthesis of a novel fluorogenic ceramidase substrate, and its processing both in vitro and in cultured cells in a microtiter plate layout, are reported in this article. This coumarinic substrate was hydrolyzed in vitro (rat liver lysosomes) with Km and Vmax values of 113 microM and 3.6 pmol min-1 mg-1, respectively. Similarly, hydrolysis occurred in intact cultured cells that overexpressed acidic ceramidase. The assay was validated for the identification and characterization of acidic ceramidase inhibitors by using several alpha-ketoamide ceramide analogues, whose inhibitory activity had been previously described.

It was shown that high-fat feeding of mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor (PPAR) alpha but not wild type animals leads to the accumulation of ceramide (an important mediator of lipotoxicity) in the heart [Finck et al. 2003 Proc Natl Acad Sci USA]. To investigate the mechanism of this phenomenon we examined the effects of PPARalpha activation on ceramide metabolism in the myocardium. Male Wistar rats were fed either a standard chow or a high-fat diet. Each group was divided into two subgroups: control and treated with selective PPARalpha activator - WY-14643. In the rats fed on the standard diet WY-14643 did not affect the myocardial content of sphingomyelin and ceramide but reduced the content of sphinganine and sphingosine. It also inhibited the activity of neutral sphingomyelinase and increased the activity of acid sphingomyelinase, whereas the activity of ceramidases and serine palmitoyltransferase (SPT) remained stable. High-fat diet itself did not affect the content of the examined sphingolipids. However, it reduced the activity of sphingomyelinases and ceramidases having no effect on the activity of SPT. Administration of WY-14643 to this group significantly increased the content of myocardial free palmitate, ceramide, sphingomyelin and the activity of SPT. Our results demonstrated that PPARalpha activation modulates myocardial ceramide metabolism and leads to the accumulation of ceramide in the heart of the high-fat fed rats due to its increased synthesis de novo.

Ceramide (CER) is an important mediator of lipotoxicity in the heart. It was found that Zucker diabetic fatty rats develop an age-dependent accumulation of myocardial CER leading to cardiomyocyte apoptosis. However, administration of peroxisome proliferator-activated receptor (PPAR) gamma agonist decreased the content of CER and prevented cardiomyocyte apoptosis [Zhou et al. Proc Natl Acad Sci USA 2000;97:1784-9]. These data suggest that PPARgamma activators affect myocardial CER metabolism. Therefore, the aim of our study was to examine the effects of pioglitazone, a selective PPARgamma agonist, on the content of CER and its metabolites and on the activity of key enzymes of CER metabolism in the heart. The experiments were conducted on rats fed either a standard chow (STD) or a high-fat diet (HFD) for 21 days. Each group was divided into two subgroups: control and treated with pioglitazone for 14 days. Surprisingly, administration of PPARgamma agonist significantly increased myocardial CER content in both STD and HFD rats. In the latter group an elevation in the amount of sphingomyelin was also observed. In STD rats pioglitazone treatment increased the activity of neutral sphingomyelinase and acid ceramidase. However, in HFD group the compound did not affect the activity of the aforementioned enzymes. Interestingly, the activity of serine palmitoyltransferase in both STD and HFD rats increased two-fold after pioglitazone treatment. We conclude that pioglitazone induced accumulation of CER in rat myocardium as a result of augmented CER synthesis de novo. However, in the STD group increased activity of neutral sphingomyelinase could also contributed to this effect.

Sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate, have emerged as a new class of lipid biomodulators of various cell functions. These metabolites are known to function not only as intracellular second messengers, but also in the extracellular space. Sphingosine 1-phosphate especially has numerous functions as an important extracellular mediator that binds to cell surface S1P receptors. Recent studies have also shown that sphingolipid-metabolizing enzymes function not only in intracellular organelles but also in the extracellular spaces, including the outer leaflet of the plasma membrane. This review focuses on the metabolic enzymes (acid and alkaline sphingomyelinases, neutral ceramidase, and sphingosine kinase) that are involved in the production of the sphingolipid metabolites in these extracellular spaces, and on the metabolic pathway itself.

Ceramide is involved in the pathogenesis of insulin resistance in skeletal muscles of humans and rodents. However, there are conflicting reports in the literature on the effect of thiazolidinediones (a new class of insulin sensitizing drugs) on skeletal muscle ceramide content. Therefore, the aim of our study was to examine the effect of pioglitazone on the level of ceramide and its metabolites and on the activity of the key enzymes of ceramide metabolism in different skeletal muscle types of the rat. The experiments were carried out on rats fed either a standard chow or a high-fat diet for 21 days. Each group was divided into two subgroups: control and treated with pioglitazone for 14 days. High-fat diet increased the content of ceramide in the soleus and in the red section of the gastrocnemius, but not in the white section of the latter. The activity of neutral Mg(2+)-dependent sphingomyelinase and acid sphingomyelinase was simultaneously reduced in all examined muscles. Administration of pioglitazone decreased ceramide level in the soleus and in the red section of the gastrocnemius in rats fed either diet. This effect could not be attributed to decreased rate of ceramide formation from sphingomyelin or to its augmented deacylation to sphingosine. Pioglitazone treatment reduced the concentration of plasma free fatty acids in rats fed on either diet. Therefore, we conclude that the drug decreased the muscle content of ceramide by reducing its de novo synthesis. The results of our study indicate that reduction in ceramide level may be one of the mechanisms by which pioglitazone improves skeletal muscle insulin sensitivity.

In recent years sphingolipids have emerged as important signaling molecules regulating fundamental cell responses such as cell death and differentiation, proliferation and aspects of inflammation. Especially ceramide has been a main focus of research since it possesses pro-apoptotic capacity in many cell types. A counterplayer of ceramide was found in sphingosine-1-phosphate (S1P), which is generated from ceramide by the consecutive actions of ceramidase and sphingosine kinase. S1P can potently induce cell proliferation via binding to and activation of the Edg family of receptors which have now been renamed as S1P receptors. Obviously, a delicate balance between ceramide and sphingosine-1-phosphate determines whether cells undergo apoptosis or proliferate, two cell responses that are critically involved in tumor development. Directing the balance in favor of ceramide, i.e. by inhibiting ceramidase or sphingosine kinase activities may support the pro-apoptotic action of ceramide and thus may have beneficial effects in cancer therapy. This review will summarize novel insights into the regulation of sphingolipid formation and their potential involvement in tumor development. Finally, we will pinpoint potential new targets for tumor therapy.

Neutral ceramidase is a type II integral membrane protein, which is occasionally secreted into the extracellular milieu after the processing of its N-terminal anchor. We found that when overexpressed in CHOP cells, neutral ceramidase hydrolyzed cell surface ceramide, which increased in amount after the treatment of cells with bacterial sphingomyelinase, leading to an increase in the cellular level of sphingosine and sphingosine 1-phosphate. On the other hand, knockdown of the endogenous enzyme by siRNA decreased the cellular level of both sphingolipid metabolites. The treatment of cells with bovine serum albumin significantly reduced the cellular level of sphingosine, but not sphingosine 1-phosphate, generated by overexpression of the enzyme. The cellular level of sphingosine 1-phosphate increased with overexpression of the cytosolic sphingosine kinase. These results suggest that sphingosine 1-phosphate is mainly produced inside of the cell after the incorporation of sphingosine generated on the plasma membranes. The enzyme also seems to participate in the hydrolysis of serum-derived ceramide in the vascular system. Significant amounts of sphingosine as well as sphingosine 1-phosphate were generated in the cell-free conditioned medium of ceramidase transfectants, compared with mock transfectants. No increase in these metabolites was observed if serum or bacterial sphingomyelinase was omitted from the conditioned medium, suggesting that the major source of ceramide is the serum-derived sphingomyelin. A sphingosine 1-phosphate receptor, S1P(1), was internalized much faster by the treatment of S1P(1)-overexpressing cells with conditioned medium of ceramidase transfectants than that of mock transfectants. Collectively, these results clearly indicate that the enzyme is involved in the metabolism of ceramide at the plasma membrane and in the extracellular milieu, which could regulate sphingosine 1-phosphate-mediated signaling through the generation of sphingosine.

Ceramide and sphingosine are sphingolipids with important functional and structural roles in cells. In this paper we report a new enzyme-based method to simultaneously quantify the levels of ceramide and sphingosine in biological samples. This method utilizes purified human recombinant acid ceramidase to completely hydrolyze ceramide to sphingosine, followed by derivatization of the latter with naphthalene-2,3-dialdehyde (NDA) and quantification by reverse-phase high-performance liquid chromatography. The limits of detection for sphingosine-NDA and ceramidase-derived sphingosine-NDA were 9.6 and 12.3 fmol, respectively, and the limits of quantification were 34.2 and 45.7 fmol, respectively. The recovery of sphingosine and ceramide standards quantified by this assay were between 95.6 and 104.6%. The relative standard deviations for the intra- and interday sphingosine assay were 2.1 and 4.5%, respectively, and those for the ceramide assay were 3.3 and 4.1%, respectively. To validate this procedure, we quantified ceramide and sphingosine in mouse plasma, white blood cells, and hemoglobin, the first reported time that the amounts of these lipids have been documented in individual blood components. We also used this technique to evaluate the ability of a novel ceramide analog, AD2646, to inhibit the hydrolytic activity of acid ceramidase. The results demonstrate that this new procedure can provide sensitive, reproducible, and simultaneous ceramide and sphingosine quantification. The technique also may be used for determining the activity and inhibition of ceramidases and may be adapted for quantifying sphingomyelin and sphingosine-1-phosphate levels. In the future it could be an important tool for investigators studying the role of ceramide/sphingosine metabolism in signal transduction, cell growth and differentiation, and cancer pathogenesis and treatment.

Ceramide is a bioactive lipid involved in the induction of apoptosis and is the precursor to several sphingolipids, including sphingomyelin, the gangliosides, and sphingosine. Ceramide production is increased in response to stress and toxic agents. Because modulation of ceramide levels has been shown to affect sensitivity and/or resistance to therapeutic agents, it will be important to assess the activity of sphingolipid metabolic pathways when investigating the mode of action of antitumor drugs. This chapter summarizes protocols for quantitating the level of apoptosis, the activities of acidic sphingomyelinase, neutral sphingomyelinase, glycosylceramide synthase, sphingomyelin synthase, and ceramidase, and the amount of ceramide in tumor xenografts in nude mice.

Mammalian spermatozoa must undergo acrosomal exocytosis prior to penetration of the oocyte at fertilization. The mechanisms underlying acrosomal exocytosis have not yet been fully elucidated. This study explored the possible involvement of ceramide in exocytosis of the boar sperm acrosome. Ejaculated boar spermatozoa, stored with the Beltsville TS extender at 17 degrees C for up to 3 days, were washed and preincubated for 10 min with C2-ceramide, an analogue of endogenous ceramide, C2-dihydroceramide (C2-DH-ceramide), a negative control to C2-ceramide, or with (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP), an inhibitor of alkaline ceramidase, followed by incubation and stimulation with 3 mM Ca2+ and 0.3 microM A23187 (Ca2+/A23187) at 37 degrees C in air in a water bath. Spermatozoa fixed at specific intervals were examined, and the % of acrosomal exocytosis was monitored. Stimulation of spermatozoa with Ca2+/A23187 resulted in a time-dependent increase. There were no obvious changes at 5 min, but this was followed by a rapid increase at 10 min, reaching nearly a maximum level after 15 min or more of incubation. Preincubation with C2-ceramide or D-erythro-MAPP enhanced acrosomal exocytosis triggered by Ca2+/A23187 in a dose-dependent manner, whereas C2-DH-ceramide was without effect. These results suggest the possibility that ceramide may be involved in the mechanisms underlying acrosomal exocytosis.

Neutral ceramidase activity has previously been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine but has never been fully characterized. We have purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9% NaCl and 3 mM sodium taurodeoxycholate. Using a combination of acetone precipitation and ion-exchange, hydrophobic-interaction, and gel chromatographies, we obtained a homogenous enzyme protein with a molecular mass of approximately 116 kDa. The enzyme acts on both [14)]octanoyl- and [14C]palmitoyl-sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate but is inhibited by 2 mM or more of other bile salts. It is a glycosylated protein stable to trypsin and chymotrypsin exposure, is not influenced by Ca2+, Mg2+, or Mn2+, and is inhibited by Zn2+ and Cu2+. Mass fragmentographic analysis identified 12 fragments covering 17.5% of the sequence for neutral/alkaline ceramidase 2 purified (Mitsutake S, Tani M, Okino N, Mori K, Ichinose S, Omori A, Iida H, Nakamura T, and Ito M. J Biol Chem 276: 26249-262459, 2001) from rat kidney and located in apical membrane of renal tubular cells. Intestinal and kidney ceramidases also have similar molecular mass and ion dependence. Intestinal ceramidase thus is a neutral ceramidase 2 released by bile salts and resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.

The sphingolipid, ceramide, is a natural dietary constituent and a potent mediator of apoptosis. If left undegraded, it may induce apoptosis and cause disruption of cellular integrity. A potential mechanism to prevent ceramide-induced apoptosis in various organs may involve ceramidases that facilitate the degradation of ceramide. In this study, we first isolated and characterized the murine neutral ceramidase (N-CDase) gene, mapped its chromosomal location and determined its developmental and organ-specific expression. Then we used cultured mesangial cells as our in vitro model and mouse gastrointestinal (GI) tract as the in vivo model to determine the effects of an inhibitor of N-CDase, D-erythro-MAPP, to delineate whether N-CDase plays a role in preventing ceramide-induced apoptosis. Our results show that: (i) the structure of the murine neutral ceramidase gene is virtually identical to that of the human gene; (ii) it is localized on chromosome 19 at bands C2-C3 that is syntenic to human chromosome 10q24-26; (iii) N-CDase expression is developmentally regulated and it is expressed at high levels in cultured mesangial cells and in specific regions of the mouse small intestine; (iv) inhibition of N-CDase by D-erythro-MAPP leads to increased ceramide levels and consequent apoptosis in cultured mesangial cells; (v) mice treated with D-erythro-MAPP alone also caused apoptosis in the small intestine; and (vi) mice treated with D-erythro-MAPP prior to feeding C2 ceramide manifest markedly elevated levels of apoptosis in the GI tract raising the possibility that neutral ceramidase plays a detoxifying role against inadvertent stimulation of ceramide-induced apoptosis in organs that come in contact with this sphingolipid. We propose that N-CDase is an essential component of an innate detoxifying mechanism to prevent ceramide-induced apoptosis.

Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids. In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity. maCER1 consists of 287 amino acids, and it has a 28 and 32% identity to the Saccharomyces alkaline ceramidases (YPC1p and YDC1p) and the human alkaline phytoceramidase, respectively. Reverse transcriptase-PCR analysis demonstrated that maCER1 was predominantly expressed in skin. maCER1 was localized to the endoplasmic reticulum as revealed by immunocytochemistry. In vitro biochemical characterization determined that maCER1 hydrolyzed D-erythro-ceramide exclusively but not D-erythro-dihydroceramide or D-ribo-phytoceramide. Similar to other alkaline ceramidases, maCER1 had an alkaline pH optimum of 8.0, and it was activated by Ca2+ but inhibited by Zn2+,Cu2+, and Mn2+. maCER1 was also inhibited by sphingosine, one of its products. Metabolic labeling studies showed that overexpression of maCER1 caused a decrease in the incorporation of radiolabeled dihydrosphingosine into ceramide and complex sphingolipids but led to a concomitant increase in sphingosine-1-P (S1P) in HeLa cells. Mass measurement showed that overexpression of maCER1 selectively lowered the cellular levels of D-erythro-C24:1-ceramide, but not other ceramide species and caused an increase in the levels of S1P. Taken together, these data suggest that maCER1 is a novel alkaline ceramidase with a stringent substrate specificity and that maCER1 is selectively expressed in skin and may have a role in regulating the levels of bioactive lipids ceramide and S1P, as well as complex sphingolipids.

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.

Recent years have witnessed significant advances in the understanding of the role of ceramide in apoptosis. This review summarizes these recent findings and discusses insights from studies of ceramide metabolism, topology, and effector actions. The recent identification of several genes for enzymes of ceramide metabolism, the development of mass spectrometric methods for ceramide analysis, and the increasing molecular and pharmacological tools to probe ceramide metabolism and function promise an accelerated phase in defining the molecular and biochemical details of the role of ceramide in apoptosis.

We previously reported that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblast-like MC3T3-E1 cells and that mitogen-activated protein (MAP) kinases are involved in bone morphogenetic protein (BMP)-4-stimulated osteocalcin synthesis in these cells. In the present study, we investigated whether sphingomyelinase affects BMP-4-stimulated synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. Sphingomyelinase significantly enhanced the BMP-4-stimulated osteocalcin synthesis. Among sphingomyelin metabolites, C(2)-ceramide enhanced the BMP-4-stimulated osteocalcin synthesis while sphingosine and sphingosine 1-phosphate had little effect on the synthesis. D-erythro-MAPP, an inhibitor of ceramidase, amplified the sphingomyelinase-effect on the osteocalcin synthesis. C(2)-ceramide suppressed the BMP-4-induced phosphorylation of p44/p42 MAP kinase, while having little effect on the phosphorylation of Smad1 and p38 MAP kinase. Taken together, our results strongly suggest that extracellular sphingomyelinase enhances the BMP-stimulated osteocalcin synthesis via ceramide in osteoblasts and that the effect of ceramide is exerted at a point upstream from p44/p42 MAP kinase., (Copyright 2002 Elsevier Science Inc.)

We have reported previously that the ceramidase from Pseudomonas aeruginosa AN17 isolated from a patient with atopic dermatitis requires detergents for hydrolysis of ceramide (Cer) [Okino, Tani, Imayama and Ito (1998) J. Biol. Chem. 273, 14368--14373]. In the present study, we report that some glycerophospholipids strongly activated the hydrolysis of Cer by Pseudomonas ceramidase in the absence of detergents. Among the glycerophospholipids tested, cardiolipin was most effective in stimulating hydrolysis of Cer followed by phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol, whereas phosphatidylcholine, lysophosphatidic acid and diacylglycerol were less effective. Interestingly, Staphylococcus aureus-derived lipids, which contain cardiolipin and phosphatidylglycerol as major lipid components, also strongly enhanced the hydrolysis of normal Cer, as well as the human skin-specific omega-hydroxyacyl Cer, by the enzyme in the absence of detergents. It was confirmed that several strains of P. aeruginosa, including AN17, secrete a significant amount of staphylolytic proteases to lyse S. aureus cells, resulting in the release of cardiolipin and phosphatidylglycerol. Since both P. aeruginosa and S. aureus are suspected of being present in microflora of atopic skin, we speculate that S. aureus-derived glycerophospholipids stimulate the hydrolysis of Cer in atopic skin by bacterial ceramidase.

Ceramidase (CDase) hydrolyses the N-acyl linkage of the sphingolipid ceramide. We synthesized the non-fluorescent ceramide analogue (4E,2S,3R)-2-N-(10-pyrenedecanoyl)-1,3,17-trihydroxy-17-(3,5-dinitrobenzoyl)-4-heptadecene (10) that becomes fluorescent upon hydrolysis of its N-acyl bond. This novel substrate was used to study several kinetic aspects of the recombinant CDase from the pathogenic bacterium Pseudomonas aeruginosa PA01. Maximum CDase activity was observed above 1.5 microM substrate, with an apparent K(m) of 0.5+/-0.1 microM and a turnover of 5.5 min(-1). CDase activity depends on divalent cations without a strong specificity. CDase is inhibited by sphingosine and by several sphingosine analogues. The lack of inhibition by several mammalian CDase inhibitors such as D-erythro-MAPP, L-erythro-MAPP or N-oleoylethanolamine points to a novel active site and/or substrate binding region. The CDase assay described here offers the opportunity to develop and screen for specific bacterial CDase inhibitors of pharmaceutical interest.

In this study, the substrate specificity of a newly identified rat brain ceramidase (CDase) was investigated. To this end, the major functional groups and stereochemistry of ceramide (Cer) were evaluated for their influence on the hydrolysis of substrate by this CDase. The results showed that, of the four possible stereoisomers of Cer, only the natural d-e-C(18)-Cer isomer was used as substrate (K(m) of 1.1 mol% and V(max) of 5 micromol/min/mg). Removal of the 4-5 trans double bond to generate dihydroceramide decreased the affinity of the enzyme toward its substrate by around 90%, whereas changing the configuration of the double bond from the natural trans configuration into cis or introduction of a hydroxyl group (phytoceramide) resulted in loss of hydrolysis. Shortening the chain length of the sphingosine backbone resulted in decreased affinity. Methylation of either the primary or the secondary hydroxyl groups resulted in loss of activity. Results also indicated that Cer species that harbor long saturated or monounsaturated fatty acyl chains are preferred substrates of the enzyme. alpha-Hydroxylated Cer demonstrated considerably higher affinity, indicating a preference of the enzyme to those Cer molecular species. These results disclose a very high specificity of nonlysosomal CDase for its substrate, Cer.

Ceramide and sphingosine-1-phosphate (S1P) are important dietary lipids involved in cell growth, differentiation, apoptosis and cell survival. Treatment of C3H10T1/2 murine fibroblast cells (10T1/2) with ceramide did not induce apoptosis, a commonly observed effect of ceramide treatment. To determine whether the metabolism of ceramide played a role in this resistance to apoptosis, inhibitors of ceramidase and sphingosine kinase, two important enzymes in sphingolipid metabolism, were used. Treatment of 10T1/2 cells both without or with ceramide plus N-oleoyl-ethanolamine (NOE) and (1S,2R)-D-erythro-s-(N-myristoylamino)-1-phenol-1-propanol (MAPP), two ceramidase inhibitors, resulted in fourfold and eightfold increases, respectively, in apoptosis. Cells treated without or with ceramide plus N,N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, resulted in fourfold and sixfold increases, respectively, in apoptosis. In all treatments the induction of apoptosis was prevented by the addition of S1P. With the addition of S1P with NOE and MAPP as well as with ceramide, treatments reduced the apoptotic response by 30 and 35%, respectively; whereas the addition of S1P with the DMS only and ceramide with DMS treatments reduced the apoptotic response by 60 and 70%, respectively. Studies using labeled ceramide demonstrated ceramide was metabolized to S1P. In addition, a 14-fold increase in apoptosis occurred in cells treated with a nonhydrolyzable analog of ceramide, ceramine, compared with vehicle control. Because inhibiting the conversion of ceramide to S1P resulted in apoptosis, the lack of an apoptotic response to ceramide alone for C3H10T1/2 cells is attributable to the conversion of this pro-apoptotic sphingolipid to the anti-apoptotic metabolite S1P, which is essential for cell survival.

The effects of structural analogues of ceramide on rat brain mitochondrial ceramidase (mt-CDase) were investigated. Design of target compounds was mainly based on modifications of the key elements in ceramide and sphingosine, including stereochemistry, the primary and secondary hydroxyl groups, the trans double bond in the sphingosine backbone, and the amide bond. Mt-CDase was inhibited by (1) all stereoisomers of D-erythro-ceramide (D-e-Cer) with an IC50 of 0.11, 0.21, and 0.26 mol % for the L-threo, D-threo, and L-erythro isomers, respectively; (2) all stereoisomers of sphingosine with IC50 ranging from 0.04 to 0.14 mol %, N-methyl-D-erythro-sphingosine (N-Me-Sph, IC50 0.13 mol %); and (3) D-erythro-urea-C16-ceramide (C16-urea-Cer IC50 0.33 mol %). The enzyme was not inhibited by N-methyl ceramide (N-Me-C16-Cer), 1-O-methyl ceramide (1-O-Me-C16-Cer), 3-O-methyl ceramide (3-O-Me-C16-Cer), cis-D-erythro ceramide (cis-D-e-C16-Cer) and 3-O-methyl-D-erythro-sphingosine (3-O-Me-Sph). It was less potently inhibited by D-erythro-sphinganine (D-e-dh-Sph, IC50 0.20 mol %), D-erythro-dehydro sphingosine (D-e-deh-Sph, IC50 0.25 mol %), (2S)-3-keto-sphinganine (3-keto-dh-Sph, IC50 0.34 mol %), (2S) 3-keto-ceramide (3-keto-C16-Cer, IC50 0.60 mol %), and ceramine (C18-ceramine, IC50 0.62 mol %), 1-O-methyl-D-erythro-sphingosine (1-O-Me-Sph), cis-D-erythro-sphingosine (cis-D-e-Sph), (2S)-3-ketosphingosine (3-keto-Sph), (2S)-3-keto-dehyrosphingosine (3-keto-deh-Sph), and N,N-dimethyl-D-erythrosphingosine (N,N-diMe-Sph) were weak inhibitors whereas ceramide-1-phosphate (Cer-1-P) and sphingosine-1-phosphate (Sph-1-P) stimulated the enzyme. Thus, for inhibition, the enzyme requires the primary and secondary hydroxyl groups, the C4-C5 double bond, the trans configuration of this double bond, and the NH-protons from either the amide of ceramide or the amine of sphingosine. Therefore, these results provide important information on the requirements for ceramide-enzyme interaction, and they suggest that ligand interaction with the enzyme occurs in a high affinity low specificity manner, in contrast to catalysis which is highly specific for D-erythro-ceramide (D-e-Cer) but occurs with a lower affinity. In addition, this study identifies two competitive inhibitors of mt-CDase; urea-ceramide (C16-urea-Cer) and ceramine (C18-ceramine) that may be further developed and used to understand the mechanism of mt-CDase in vitro and in biologic responses.

Dysfunction in the physiological pathways of programmed cell death may promote proliferation of malignant cells, and correction of such defects may selectively induce apoptosis in cancer cells. We measured the levels of ceramide, a candidate lipid mediator of apoptosis, in human metastatic colorectal cancer and tested in vitro and in vivo effects of various ceramide analogues in inducing apoptosis in metastatic colon cancer. Human colon cancer showed a > 50% decrease in the cellular content of ceramide when compared with normal colon mucosa. Application of ceramide analogues and ceramidase inhibitors induced rapid cell death through activation of various proapoptotic molecules, such as caspases and release of cytochrome c. Ceramidase inhibition increases the ceramide content of tumor cells, resulting in maximum activation of the apoptotic cascade. Normal liver cells were completely resistant to inhibitors of ceramidases. Treatment of nude mice with B13, the most potent ceramidase inhibitor, completely prevented tumor growth using two different aggressive human colon cancer cell lines metastatic to the liver. Therefore, B13 and related analogues of ceramide and inhibitors of ceramidases offer a promising therapeutic strategy with selective toxicity toward malignant but not normal cells. These studies also suggest that the ceramide content in cancer cells might be involved in the pathogenesis of tumor growth in vitro and in vivo.

In the present study, a possible role of a ceramide-dependent pathway in the regulation of Leydig cell function was investigated. Intracellular ceramide levels were increased by: (a) adding ceramide analogs; (b) inhibiting ceramidase activity; and (c) adding sphingomyelinase (SMase). The cell-permeable ceramide analogs N-acetyl-, N-hexanoyl- and N-octanoylsphingosine (C2, C6 and C8) were used. As inhibitor of ceramidase activity 1S,2R-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP) was used. Sphingomyelinase from S. aureus origin was utilized. Leydig cells were cultured for 3 or 24 h with or without the different drugs (10 microM) and SMase (0.3 U/ml) in the presence or absence of hCG (10 ng/ml). Basal testosterone production was not modified under any of the experimental conditions. A decrease in hCG-stimulated testosterone production was observed at 3 and 24 h in all cases. The inactive analog (N-hexanoyl dihydrosphingosine) did not produce inhibition in hCG-stimulated testosterone production. TNFalpha and IL1beta, two possible inducers of sphingomyelin hydrolysis, produced similar effects on hCG-stimulated testosterone production. In experiments performed in the presence of C6, inhibition in hCG-stimulated cAMP production was observed. The inhibitory effect of ceramide was also observed in dbcAMP-stimulated cultures indicating that this pathway inhibits post-cAMP formation events. To study possible loci for the action of ceramide on the steroidogenic pathway, cells were incubated with C6 and MAPP in the presence of different testosterone precursors. The drugs inhibited testosterone produced from 22(R)-hydroxycholesterol (22R-OHChol), pregnenolone and 17alpha-hydroxyprogesterone (17OHP4) but not from androstenedione (Delta4). These results suggest that a ceramide-dependent pathway regulates hCG-stimulated Leydig cell steroidogenesis at the level of cAMP production and at post-cAMP events.

Aging leads to a decreased ability of liver to metabolize drugs and increased expression and secretion of acute phase proteins, such as serum amyloid A (SAA), C-reactive protein (CRP), and alpha-1-acid glycoprotein (AGP). This phenomenon resembles some aspects of the acute phase response of host to inflammation; however, the molecular basis for the similarity is unclear. Ceramide and sphingosine are second messenger mediators of cellular responses to stress and inflammation. In liver, they play important role in mediating acute phase responses to IL1-beta. In this study, we use HPLC and thin layer chromatography to evaluate the effects of aging on steady-state levels of ceramide and sphingosine. We report that both lipids are elevated in liver of old (24 months) as compared to young (5 months) male Fisher 344 rats. To elucidate the mechanism(s) for ceramide elevation, we test the acidic (ASMase) and neutral sphingomyelinase (NSMase) in vitro using NBD-sphingomyelin as an exogenous substrate. SM synthase is also analyzed in vitro using NBD-ceramide and [3H]-dipalmitoylphosphatidylcholine (DPPC) as exogenous substrates. In accordance with the increases in the mass of ceramide, the activity of acid and neutral SMase is elevated in old animals. Michaelis-Menten analysis of NSMase implies that the apparent activation of this enzyme is caused by an increase in the Vmax of the enzyme. In contrast, SM synthase activity is lower in old animals as compared to young ones. These results show that aging is accompanied by an elevation in SM turnover and a decrease in its synthesis, resulting in accumulation of pro-inflammatory and growth inhibitory second messenger ceramide. Ceramidase, the only enzyme leading to sphingosine generation, is also measured in vitro using NBD-ceramide as a substrate and liver homogenate as an enzyme source. Its activity is higher in the old rats, as compared to young ones. The acid and neutral forms of the enzyme are affected the most, while the changes in the alkaline enzyme are not significant. The increases in the basal levels of ceramide and sphingosine in old animals may contribute to the onset of an inflammatory like state in liver during aging, exemplified by decreased P4502C11 mRNA expression and chronic induction of acute phase protein expression.

In a previous study, we reported that the Saccharomyces cerevisiae gene YPC1 encodes an alkaline ceramidase with a dual activity, catalyzing both hydrolysis and synthesis of yeast ceramide (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876-6884). In this study, we have identified a YPC1 homologue in S. cerevisiae that also encodes an alkaline ceramidase. We show that these two ceramidases have different substrate specificity, such that YPC1p preferentially hydrolyzes phytoceramide, whereas the new ceramidase YDC1p hydrolyzes dihydroceramide preferentially and phytoceramide only slightly. Neither enzyme hydrolyzes unsaturated mammalian-type ceramide. In contrast to YPC1p, YDC1p had only minor in vitro reverse activity of catalyzing dihydroceramide formation from a free fatty acid and dihydrosphingosine and no activity with phytosphingosine. Overexpression of YDC1p had no reverse activity in non-stressed yeast cells, but like YPC1p suppressed the inhibition of growth by fumonisin B1 albeit more modestly. Deletion of YDC1 and YPC1 or both did not apparently affect growth, suggesting neither gene is essential. However, the Deltaydc1 deletion mutant but not the Deltaypc1 deletion mutant was sensitive to heat stress, indicating a role for dihydroceramide but not phytoceramide in heat stress responses, and suggesting that the two enzymes have distinct physiological functions.

To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated the endogenous ceramide levels and concomitantly induced apoptosis. Addition of RA caused an increase in ceramide levels within 3-5 h, which reached a maximum (up to 3.5-fold of control) between days 1 and 3 of differentiation. Differentiated PCC7-Mz1 cells did not respond with ceramide formation and apoptosis to RA treatment. The acidic sphingomyelinase contributed only weakly and the neutral Mg(2+)-dependent and Mg(2+)-independent sphingomyelinases not at all to the RA-mediated production of ceramides. However, ceramide increase was sensitive to the ceramide synthase inhibitor fumonisin B(1), suggesting a crucial role for the de novo synthesis pathway. Enzymatic assays revealed that ceramide synthase activity remained unaltered, whereas serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, was activated approximately 2.5-fold by RA treatment. Activation of SPT seemed to be mediated via a post-translational mechanism because levels of the mRNAs coding for the two SPT subunits were unaffected. Expression of marker proteins shows that ceramide regulates apoptosis, rather than differentiation, during early neural differentiation.

We report here a novel ceramidase that was purified more than 150, 000-fold from the membrane fraction of mouse liver. The enzyme was a monomeric polypeptide having a molecular mass of 94 kDa and was highly glycosylated with N-glycans. The amino acid sequence of a fragment obtained from the purified enzyme was homologous to those deduced from the genes encoding an alkaline ceramidase of Pseudomonas aeruginosa and a hypotheical protein of the slime mold Dictyostelium discoideum. However, no significant sequence similarities were found in other known functional proteins including acid ceramidases of humans and mice. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, sulfatide, GM1a, or sphingomyelin. The enzyme exhibited the highest activity around pH 7.5 and was thus identified as a type of neutral ceramidase. The apparent K(m) and V(max) values for C12-4-nitrobenzo-2-oxa-1, 3-diazole-ceramide and C16-(14)C-ceramide were 22.3 microM and 29.1 micromol/min/mg and 72.4 microM and 3.6 micromol/min/mg, respectively. This study also clearly demonstrated that the purified 94-kDa ceramidase catalyzed the condensation of fatty acid to sphingosine to generate ceramide, but did not catalyze acyl-CoA-dependent acyl-transfer reaction.

We have identified the aspartic protease cathepsin D as a novel intracellular target protein for the lipid second messenger ceramide. Ceramide specifically binds to and induces CTSD proteolytic activity. A-SMase deficient cells derived from Niemann-Pick patients show decreased CTSD activity that was reconstituted by transfection with A-SMase cDNA. Ceramide accumulation in cells derived from A-ceramidase defective Farber patients correlates with enhanced CTSD activity. These findings suggest that A-SMase-derived ceramide targets endolysosomal CTSD.

Experiments were designed to examine the role of sphingosine, PP2A phosphatases, and protein kinase C (PKC) inhibition in mediating the vasodilatory effects of ceramide in rat thoracic aorta. Sphingosine did not cause vasorelaxation, and oleoylethanol-amine, a ceramidase inhibitor, did not affect sphingomyelinase-induced relaxation. Okadaic acid potentiated the relaxation response to ceramide. These observations rule out involvement of sphingosine and PP2A phosphatases in mediating ceramide-induced relaxation. Sphingomyelinase attenuated contractile and single-cell intracellular calcium responses to phorbol ester. Chelerythrine incubation potentiated the relaxation response to ceramide. These observations support a role for PKC inhibition in mediating the vasodilatory effects of ceramide.

Background/aims: Portal vein branch ligation induces apoptosis of hepatocytes in the ligated lobes in rat liver. Sphingomyelin degradation was studied during the process to evaluate its possible involvement in apoptosis in vivo., Methods: DNA scissions were detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and an agarose gel electrophoresis of DNA. Using both ligated and non-ligated lobes, we measured activities of sphingomyelin degradation enzymes and contents of their products in purified nuclei and plasma membrane., Results: DNA fragmentation was detectable in the ligated lobes at 90 min after the portal vein branch ligation by gel electrophoresis. At 15 h after the ligation, 27% of hepatocytes became TUNEL-positive. Prior to the onset of apoptosis, the activity of neutral sphingomyelinase increased in the nuclei of hepatocytes in ligated lobes (30 min after the ligation). The increase in sphingomyelinase paralleled its reaction product, ceramide. This was followed by the elevation of ceramidase activity in nuclei (60 min after the ligation) in association with an increase of its reaction product, sphingosine. Activities of these two enzymes and their products increased for at least 90 min. These changes were not observed in nuclei of the non-ligated lobes, or in the plasma membranes from either ligated or non-ligated lobes., Conclusions: These results, specific to the liver where apoptosis is being generated, suggest that nuclear sphingomyelin breakdown with an accumulation of ceramide and/or sphingosine in nuclei may induce the apoptosis of hepatocytes in vivo.

Ceramide is recognized as an intracellular mediator of cell growth, differentiation and apoptosis. Tumour necrosis factor, anti-fas antibody, radiation and anticancer drugs such as actinomycin D are known to induce apoptosis in several cell types through generation of ceramide by activation of the sphingomyelinase pathway or ceramide synthetase. In this study, we examined the occurrence of apoptosis in fibroblasts from patients with Farber disease and from sphingolipid activator protein-deficient (sap -/-) mouse. These cells accumulate ceramide as the result of genetic deficiency of acid ceramidase and the ceramidase activator (sap-D), respectively. Amounts of ceramide in fibroblasts from Farber patients and in fibroblasts from sap -/- mouse were increased 2.9-fold and 2.8-fold, respectively, over the level of controls. Despite the similar degree of ceramide accumulation, cells exhibiting apoptotic features were increased only in fibroblasts from the sap -/- mouse but not those from the Farber patients. Thymidine uptake of Farber fibroblasts was normal while that of sap -/- mouse fibroblasts was twice normal, consistent with the apparently normal growth and the different rates of apoptotic cell death in these two cell lines. These data suggest that intralysosomal accumulation of ceramide due to defective acid ceramidase or its activator may not play an important role as a mediator of apoptosis. The increased apoptosis in the cultured fibroblasts from the sap -/- mouse may be caused by mechanisms other than the ceramide accumulation. Although more frequent than normal, significant apoptotic cell death was not observed in sap -/- mouse brain in vivo.

A fluorescent analogue of ceramide, C12-NBD-ceramide, was found to be hydrolyzed much faster than 14C-labeled ceramide by alkaline ceramidase from Pseudomonas aeruginosa and neutral ceramidase from mouse liver, while this substrate was relatively resistant to acid ceramidase from plasma of the horseshoe crab. The radioactive substrate was used more preferentially by the acid ceramidase. It should be noted that C6-NBD-ceramide, which is usually used for ceramidase assays, was hardly hydrolyzed by any of the enzymes examined, compared to C12-NBD-ceramide. For the alkaline and neutral enzymes, the Vmax and k (Vmax/Km) with C12-NBD-ceramide were much higher than those with 14C-ceramide. In contrast, for the acid enzyme these parameters with C12-NBD-ceramide were less than half those with the radioisotope-labeled substrate. It is noteworthy that the labeling of ceramide with NBD did not itself reduce the Km of the alkaline enzyme, but did that of the neutral enzyme. It was also found that C12-NBD-ceramide was preferentially hydrolyzed by the alkaline and neutral enzymes, but not the acid one, in several mammalian cell lines. This study clearly shows that the attachment of NBD, but not dansyl, increases the susceptibility of ceramide to alkaline and neutral enzyme, and decreases that to acid enzymes. Thus the use of this substrate provides a specific and sensitive assay for alkaline and neutral ceramidases.

Sphingolipids, ceramide in particular, have come to be regarded as having roles in cellular signaling, most recently being associated with stress and the cellular responses to stress. In the present study we first examined the mechanisms involved in the changes in cellular ceramide levels in normal human mesangial cells (NHMC) in the growth, quiescent, and senescent phases as well as those resulting from environmental stimuli. We found that in NHMC total ceramide levels increase in response to cellular stresses as a result of a combination of enzyme activities. Furthermore, different stresses cause different alterations in various enzyme activities, with age and growth influencing acidic enzymes, but cell density affecting neutral, resulting in final ceramide level increases which most likely are associated with distinct pools of ceramide. Secondly, we examined the influence of changes in ceramide levels on apoptosis induced by sphingosine and its methylated derivative N, N-dimethylsphingosine. We found that increases in cellular ceramide levels prohibited the apoptosis and caused a quiescent state in the cells. The data presented here provide additional insight into the roles of ceramide and related enzymes in cellular responses to stress and suggest a possible relevance to in vivo disease states.

We report that N-oleoylethanolamine (NOE), widely employed as a ceramidase inhibitor, also inhibits glucosylation of naturally occurring ceramides. When CHP-100 neuroepithelioma cells were exposed for 18h to non-toxic NOE concentrations (i.e. up to 70 microM), basal incorporation of labelled hexose into glucosylceramide (GlcCer) and higher order neutral glycosphingolipids was significantly inhibited. In cells treated with 30 microM N-hexanoylsphingosine (C6-Cer), NOE affected only marginally short-chain glucocerebroside accumulation, but markedly decreased accumulation of glucocerebrosides originating from glucosylation of a long-chain ceramide (Lc-Cer) pool produced upon C6-Cer treatment. Evidence is provided that NOE effects neither are mediated by their effects on ceramidase nor are due to enhanced long-chain GlcCer (Lc-GlcCer) conversion to higher order glycosylated derivatives. NOE inhibition of Lc-GlcCer generation was accompanied by enhanced accumulation of Lc-Cer and by potentiation of apoptosis induced by C6-Cer; the possible causal relationships between these two phenomena are discussed., (Copyright 1999 Academic Press.)

A marked decrease in the content of ceramide has been reported in the horny layer of the epidermis in atopic dermatitis (AD). This decrease impairs the permeability barrier of the epidermis, resulting in the characteristic dry and easily antigen-permeable skin of AD, since ceramide serves as the major water-holding molecule in the extracellular space of the horny layer. On the other hand, the skin of such patients is frequently colonized by bacteria, most typically by Staphylococcus aureus, possessing genes such as those for sphingomyelinase, which are related to sphingolipid metabolism. We therefore tried to identify a possible correlation between the ceramide content and the bacterial flora obtained from the skin of 25 patients with AD versus that of 24 healthy subjects, using a thin-layer chromatographic assay of the sphingomyelin-associated enzyme activities secreted from the bacteria. The findings of the assay demonstrated that ceramidase, which breaks ceramide down into sphingosine and fatty acid, was secreted significantly more from the bacterial flora obtained from both the lesional and the nonlesional skin of patients with AD than from the skin of healthy subjects; sphingomyelinase, which breaks sphingomyelin down into ceramide and phosphorylcholine, was secreted from the bacterial flora obtained from all types of skin at similar levels for the patients with AD and the healthy controls. The finding that the skin of patients with AD is colonized by ceramidase-secreting bacteria thus suggests that microorganisms are related to the deficiency of ceramide in the horny layer of the epidermis, which increases the hypersensitivity of skin in AD patients by impairing the permeability barrier.

Background: There is a renewed interest in metabolism of sphingolipids because of their role in signal transduction. Sphingomyelin is the dominating phospholipid in human milk but its metabolism and possible function in the gastrointestinal tract of breast fed infants is unknown. We explored whether bile salt-stimulated milk lipase has a role in sphingolipid metabolism., Methods: In vitro assays of sphingomyelinase and ceramidase activities, using radiolabeled substrates, human milk samples and purified native and recombinant variants of bile salt-stimulated milk lipase with or without known activators or inhibitors., Results: Human whey and purified lipase catalysed hydrolysis of palmitoyl-labeled ceramide with the highest rate around pH 8.5-9.0. 1 mg of lipase hydrolysed 0.7 micromol ceramide in one hour at pH 8.5 in presence of 4 mM bile salt. The activity of whey was inhibited by antibodies towards human bile salt-stimulated milk lipase, indicating that this lipase accounted for virtually all ceramidase activity in the milk. In contrast, bile salt-stimulated milk lipase showed no activity against sphingomyelin. However we give evidence of a separate, hitherto unknown, acid sphingomyelinase in human milk. Under the used in vitro conditions this sphingomyelinase could account for hydrolysis of half of milk sphingomyelin in one hour., Conclusions: Human milk bile salt-stimulated milk lipase hydrolyses ceramide and may thus have a role in sphingomyelin digestion, but only after initial hydrolysis to ceramide and phosphorylcholine. Part of the latter could be carried out in the stomach by the acid milk sphingomyelinase now described. We speculate that these two milk enzymes may be of importance for optimal use of human milk sphingolipids.

Sphingolipid ceramide N-deacylase catalyzes the reversible reactions in which the N-acyl linkage of ceramides of various sphingolipids is hydrolyzed or synthesized under different conditions. We report here a new method for preparation of ceramide containing omega-amino-fatty acid by using the condensation reaction of the enzyme. omega-Aminododecanoic acids were efficiently condensed by the enzyme to sphingosine in 25 mM glycine-NaOH buffer, pH 10, containing 0.3% Triton X-100 when the amino residue at the omega position of the fatty acid was blocked with trifluoroacetate. The reaction product was purified sequentially from the reaction mixture on a C18 reversed-phase column and Sep-Pak Plus Silica and Sep-Pak QMA cartridges with an overall yield of 80% and determined to be omega-aminododecanoylsphingosine by thin-layer chromatography and fast atom bombardment-mass spectrometry analyses after removing the block of trifluoroacetate by alkaline treatment. The enzyme can also be applied successfully to the synthesis of various glycosphingolipids and sphingomyelin containing omega-aminododecanoic acids. The 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled N-dodecanoylsphingosine was easily prepared from the omega-amino-ceramide by coupling with NBD-fluoride. This fluorescent ceramide was found to be hydrolyzed by ceramidase of B16 melanoma cells much faster than NBD-labeled N-hexanoylsphingosine in vitro as well as in vivo, indicating that the former is an excellent substrate for the assay of ceramidase., (Copyright 1998 Academic Press.)

Protein kinase C (PKC) activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Recent investigations showed that activation of PKC alpha by 12-O-tetradecanoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate cancer cells. The present studies examine the mechanism of this effect and show that de novo ceramide generation through the enzyme ceramide synthase is required. TPA induced rapid ceramide generation, which was detectable by 1 h and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations into the mechanism of TPA-induced ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, TPA induced an increase in ceramide synthase activity that persisted for at least 16 h. Treatment with fumonisin B1, a specific natural inhibitor of ceramide synthase, abrogated both ceramide production and TPA-induced apoptosis. Ceramide analogues bypassed fumonisin B1 inhibition to initiate apoptosis directly. Thus, ceramide appears to be a necessary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.

Since the generation upon cell stimulation of the second messenger ceramide has been reported to occur in an endosomal/lysosomal compartment, we investigated whether ceramide formed in the lysosomes can escape this compartment. The metabolic fate of radiolabelled ceramide produced by intralysosomal hydrolysis of LDL-associated [ceramide-3H]sphingomyelin or [stearoyl-1-(14)C]sulfatide was examined in fibroblasts from control individuals and a patient with inborn lysosomal ceramidase deficiency (Farber disease). The behavior of this radioactive ceramide was compared to that of a fluorescent (lissamine-rhodaminyl) ceramide analogue deriving from sulfatide degradation. While in Farber cells the natural, radiolabelled ceramide remained completely undegraded and accumulated in the lysosomes, the fluorescent derivative was rapidly converted to sphingomyelin. These findings strongly suggest that, in contrast to fluorescent derivatives, endogenous long-chain ceramide is unable to exit from lysosomes, therefore making the lysosomal ceramide unlikely to be a biomodulatory molecule.

De novo synthesis and turnover of endogenous ceramide in cultured skin fibroblasts from patients affected with Farber lipogranulomatosis were studied by biosynthetical labeling of cellular sphingolipids with [14C]serine. The cellular uptake of [14C]serine and incorporation into de novo synthesized ceramide was similar in normal and Farber fibroblasts, with a half life of newly synthesized ceramide of 2.7 h in normal and diseased cells. Newly synthesized ceramide was found to be channeled directly into biosynthesis of complex sphingolipids rather than contributing to the pool of accumulated ceramide in Farber fibroblasts. The degradation of ceramide generated by the catabolism of complex sphingolipids in Farber cells was greatly delayed compared with control fibroblasts, with differences in the amount of radiolabeled cellular ceramide becoming evident after 6 h chase time. Individual Farber cell lines differed from each other in the amount of accumulated ceramide; however, no correlation was found between ceramide accumulation and residual acid ceramidase activity as determined in vitro. In addition, the amount of radiolabeled sphingomyelin was significantly increased in Farber fibroblasts suggesting a delayed degradation of this compound in this ceramide storage disorder. We propose biosynthetical labeling of endogenous ceramide with [14C]serine, in addition to other established methods, as a highly sensitive and reliable method for the diagnosis of Farber disease, allowing semiquantitative measurement of ceramide accumulation in cultured skin fibroblasts of patients affected with Farber lipogranulomatosis.