fumonisin-b1 and thermozymocidin

Topics: Acyltransferases; Animals; Carboxylic Acids; Depsipeptides; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Fumonisins; Glycolipids; Molecular Probes; Oxidoreductases; Peptides, Cyclic; Serine C-Palmitoyltransferase; Sphingolipids; Tetrahydronaphthalenes

Curcumin, a hydrophobic polyphenol found in the rhizome of Curcuma longa, has been shown to reduce intracellular lipid accumulation in mouse models of lysosomal storage diseases such as Niemann-Pick type C. Exosomes are small extracellular vesicles secreted by cells in response to changes in intracellular ceramide composition. Curcumin can induce exosome/microvesicle release in cellular models of lipid deposition; however, the mechanism by which curcumin stimulates this release is unknown. In a model of lipid trafficking impairment in C6 glia cells, we show that curcumin stimulated ceramide synthesis by increasing the intracellular concentration of ceramide-dihydroceramide. Ceramide overload increased exosome/microvesicle secretion 10-fold, thereby reducing the concentration of lipids in the endolysosomal compartment. These effects were blocked by inhibitors of serine palmitoyltransferase (myriocin) and ceramide synthase (fumonisin B1). It is concluded that the decrease in intracellular lipid deposition induced by curcumin is mediated by increased ceramide synthesis and exosome/microvesicle release. This action may represent an additional health benefit of curcumin.

Topics: Animals; Cell Line, Tumor; Cell-Derived Microparticles; Ceramides; Curcumin; Exosomes; Fatty Acids, Monounsaturated; Fumonisins; Humans; Lipid Metabolism; Lipoproteins, LDL; Lysosomes; Neuroglia; Niemann-Pick Disease, Type C; Oxidoreductases; Rats; Serine C-Palmitoyltransferase

Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

Topics: Aging; Aniline Compounds; Animals; Benzylidene Compounds; Ceramides; Fatty Acids, Monounsaturated; Fumonisins; Glucose; Glycogen; Hepatocytes; Imipramine; Insulin; Insulin Resistance; Male; Oxidoreductases; Rats; Serine C-Palmitoyltransferase; Sphingolipids; Sphingomyelin Phosphodiesterase

It is well established that +ssRNA viruses manipulate cellular lipid homoeostasis and distribution to facilitate efficient replication. Here, we show that the cellular lipid ceramide is redistributed to the West Nile virus strain Kunjin virus (WNVKUN) replication complex (RC) but not to the dengue virus serotype 2 strain New Guinea C (DENVNGC) RC. We show that prolonged chemical inhibition of serine palmitoyltransferase with myriocin had a significant deleterious effect on WNVKUN replication but enhanced DENVNGC replication. Additionally, inhibition of ceramide synthase with Fumonisin B1 had a detrimental effect on WNVKUN replication and release of infectious virus particles but contrastingly an enhancing effect on DENVNGC replication and virus production. These observations suggest that ceramide production via the de novo and salvage pathway is a requirement for WNVKUN replication but inhibitory for DENVNGC replication. Thus, although these two viruses are from the same genus, they have a differential ceramide requirement for replication.

Topics: Animals; Antiviral Agents; Base Composition; Ceramides; Chlorocebus aethiops; Dengue Virus; Fatty Acids, Monounsaturated; Fumonisins; Oxidoreductases; Serine C-Palmitoyltransferase; Vero Cells; Virus Replication; West Nile virus

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis. Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1. In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.

Topics: 3T3-L1 Cells; Adipocytes; Adipokines; Animals; Cell Line, Tumor; Ceramides; Chemokine CCL2; Coculture Techniques; Fatty Acids, Monounsaturated; Fumonisins; Interleukin-6; Macrophages; Mice; Palmitates; RNA, Messenger; Serine C-Palmitoyltransferase; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

The terpene alcohols geranyllinalool, phytol (diterpene alcohol), and farnesol (sesquiterpene alcohol) were newly found to inhibit sphingolipid de novo biosynthesis in LLC-PK₁ cells (pig kidney epithelial cells). A simple chromatographic bioassay was established for the screening of inhibitory compounds able to reduce the amount of sphinganine, an intermediate metabolite of sphingolipid biosynthesis. The screening strategy was based on the degree of suppression of fumonisin B₁ (FB₁-induced sphinganine accumulation following co-treatment with selected terpene alcohols. L-cycloserine and ISP-1, specific serine palmitoyltransferase (SPT) inhibitors, were used as positive controls. Our results show that measuring reduced sphinganine levels after treatment with 2 µM FB₁ in combination with the putative inhibitory compounds provides a useful screening bioassay for evaluating compounds causing sphingolipid depletion. Intracellular sphinganine concentrations were analyzed using the fluorescent peak areas of the O-phthalaldehyde (OPA) derivatives of sphinganine eluted with 87 % acetonitrile on a reversed-phase column. Geranyllinalool, phytol, and farnesol were identified as novel SPT inhibitors that reduce FB₁-induced sphinganine accumulation and thus inhibit the first step of sphingolipid de novo synthesis.

Topics: Acyclic Monoterpenes; Animals; Biological Assay; Cycloserine; Diterpenes; Drug Synergism; Farnesol; Fatty Acids, Monounsaturated; Fumonisins; Humans; LLC-PK1 Cells; Molecular Structure; Phytol; Sphingolipids; Sphingosine; Swine; Terpenes

Chlamydia trachomatis L2 invasively attacks lymphatic and subepithelial tissues of the genital tract during the formation of primary lesions. This subsequently results in lymphadenopathy, and suggests a greater propensity for systemic dissemination. However, whether lymphocytes are a potential vehicle cell for the dissemination of this infection remains unknown. We therefore assessed the growth properties of C. trachomatis L2 in lymphoid Jurkat cells compared with those observed in epithelial HeLa cells. Both cells supported the growth of C. trachomatis with a similar increase in infective progenies. Enriched human-blood lymphocytes also supported the C. trachomatis growth as well as Jurkat cells. Bacteria infecting the Jurkat cells were more susceptible to antibiotics (doxycycline, azithromycin, ofloxacin) than those in HeLa cells. Of the sphingomyelin biosynthesis inhibitors tested, both myriocin and fumonisin B1 significantly inhibited bacterial growth in both cells types. A Jurkat cell mutant that impaired bacterial growth was established using ethylmethanesulfonate treatment. DNA microarray analysis with real-time reverse transcription-polymerase chain reaction revealed that the mutant cells over-expressed granzyme K gene. Immunofluorescence staining also indicated that granzyme K irregularly over-expressed among the mutant cells as compared with that of the wild cells, suggesting a possible mechanism refractory to C. trachomatis infection. Thus, we concluded that C. trachomatis L2 could infect Jurkat cells with lymphoid properties, providing a new tool for studying C. trachomatis dissemination to tissues via lymphocyte movement.

Topics: Anti-Bacterial Agents; Chlamydia trachomatis; Fatty Acids, Monounsaturated; Fumonisins; Gene Expression Profiling; HeLa Cells; Humans; Jurkat Cells; Microarray Analysis; Models, Biological; Real-Time Polymerase Chain Reaction; T-Lymphocytes

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) uniquely displays broad cancer-specific apoptosis-inducing activity through induction of endoplasmic reticulum (ER) stress. We hypothesize that ceramide, a promoter of apoptosis, might contribute to mda-7/IL-24 induction of apoptosis. Ad.mda-7-infected tumor cells, but not normal cells, showed increased ceramide accumulation. Infection with Ad.mda-7 induced a marked increase in various ceramides (C16, C24, C24:1) selectively in prostate cancer cells. Inhibiting the enzyme serine palmitoyltransferase (SPT) using the potent SPT inhibitor myriocin (ISP1), impaired mda-7/IL-24-induced apoptosis and ceramide production, suggesting that ceramide formation caused by Ad.mda-7 occurs through de novo synthesis of ceramide and that ceramide is required for mda-7/IL-24-induced cell death. Fumonisin B1 (FB1) elevated ceramide formation as well as apoptosis induced by Ad.mda-7, suggesting that ceramide formation may also occur through the salvage pathway. Additionally, Ad.mda-7 infection enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin in cancer cells. ASMase silencing by RNA interference inhibited the decreased cell viability and ceramide formation after Ad.mda-7 infection. Ad.mda-7 activated protein phosphatase 2A (PP2A) and promoted dephosphorylation of the anti-apoptotic molecule BCL-2, a downstream ceramide-mediated pathway of mda-7/IL-24 action. Pretreatment of cells with FB1 or ISP-1 abolished the induction of ER stress markers (BiP/GRP78, GADD153 and pospho-eIF2alpha) triggered by Ad.mda-7 infection indicating that ceramide mediates ER stress induction by Ad.mda-7. Additionally, recombinant MDA-7/IL-24 protein induced cancer-specific production of ceramide. These studies define ceramide as a key mediator of an ER stress pathway that may underlie mda-7/IL-24 induction of cancer-specific killing.

Topics: Apoptosis; Carcinoma; Cell Line, Tumor; Cell Survival; Ceramides; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Humans; Interleukins; Male; Phosphorylation; Prostatic Neoplasms; Protein Phosphatase 2; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; RNA Interference; Serine C-Palmitoyltransferase; Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingomyelins; Stress, Physiological; Time Factors; Transduction, Genetic; Up-Regulation

Ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMP-activated protein kinase (AMPK). This study shows that the inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of the phosphorylation of upstream kinases and the activation of protein phosphatase 2A (PP2A).Inhibition of AMPK phosphorylation by palmitate was attributed to ceramide-dependent PP2A activation. We hypothesized that the inhibitory effect of ethanol on AMPK phosphorylation was mediated partly through the generation of ceramide. The effect of ethanol and inhibitors of ceramide synthesis on AMPK phosphorylation, ceramide levels, and PP2A activity were assessed in rat hepatoma cells (H4IIEC3). The effect of ethanol on hepatic ceramide levels was also studied in C57BL/6J mice fed the Lieber-DeCarli diet. In H4IIEC3 cells, ceramide reduced AMPK phosphorylation when they were treated for between 4 and 12 h. The basal level of AMPK phosphorylation in hepatoma cells was increased with the treatment of ceramide synthase inhibitor, fumonisin B1. Ethanol treatment significantly increased cellular ceramide content and PP2A activity by approximately 18-23%, when the cells were treated with ethanol for between 4 and 12 h. These changes in intracellular ceramide concentrations and PP2A activity correlated with the time course over which ethanol inhibited AMPK phosphorylation. The activation of PP2A and inhibition of AMPK phosphorylation caused by ethanol was attenuated by fumonisin B1 and imipramine, an acid sphingomyelinase (SMase) inhibitor. There was a significant increase in the levels of ceramide and acid SMase mRNA in the livers of ethanol-fed mice compared with controls. We concluded that the effect of ethanol on AMPK appears to be mediated in part through increased cellular levels of ceramide and activation of PP2A.

Topics: Adenylate Kinase; Aniline Compounds; Animals; Benzylidene Compounds; Cell Line; Ceramides; Diet; Ethanol; Fatty Acids, Monounsaturated; Fumonisins; Gene Expression Regulation; Hydrogen Peroxide; Imipramine; Male; Mice; Phosphorylation; Protein Phosphatase 2; Rats

In metabolic syndrome, down-regulation of the insulin signaling leads to insulin-regulated metabolism and cardiovascular dyfunctions. Free fatty acids (FFAs) in the circulation are increased in this disorder and inhibit insulin signaling. Lipid oversupply contributes to the development of insulin resistance, likely by promoting the accumulation of lipid metabolites capable of inhibiting signal transduction.. This study was designed to examine the effects of FFAs and their metabolites on the insulin signaling pathway that leads to the activation of endothelial nitric oxide synthase (eNOS) and increase in nitric oxide (NO) production in endothelial cells.. Here we demonstrate that exposing human umbilical vein endothelial cells (HUVECs) to palmitate inhibits activation of Akt/eNOS signal pathway by insulin, and subsequently insulin-stimulated NO generation. Palmitate concomitantly induced the accumulation of ceramide, a product of acyl-CoA that has been shown to accumulate in insulin-resistant tissues and to inhibit insulin signaling. Preventing de novo ceramide synthesis abolished the antagonistic effect of palmitate toward the Akt/ eNOS pathway. Moreover, inducing ceramide buildup augmented the inhibitory effect of palmitate.. Taken together, we have demonstrated that palmitic acid induces accumulation of ceramide, which appears to mediate palmitic acid's inhibitory effects on the Akt/eNOS pathway, leading to a significant decrease in NO generation. Therefore, ceramide is a necessary and sufficient intermediate mediating the inhibition of the AKT/eNOS signaling pathway by palmitate in endothelial cells.

Topics: Animals; Cells, Cultured; Ceramides; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fatty Acids, Nonesterified; Fumonisins; Humans; Immunosuppressive Agents; Insulin; Insulin Resistance; Metabolic Syndrome; Nitric Oxide; Nitric Oxide Synthase Type III; Palmitates; Proto-Oncogene Proteins c-akt; Signal Transduction

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.

Topics: Antifungal Agents; Cell Line; Ceramides; Endoplasmic Reticulum; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Gene Expression Regulation, Enzymologic; Humans; Membrane Proteins; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Serine C-Palmitoyltransferase; Sphingosine

Fumonisin B(1) (FB(1)) is a fungal toxin produced by Fusarium verticillioides that inhibits ceramide synthase (CS), a key enzyme in the de novo sphingolipid biosynthesis pathway. In LLC-PK(1) cells, FB(1) inhibits cell proliferation and induces apoptosis, which can be prevented by inhibitors of serine palmitoyltransferase (SPT). Inhibition of SPT prevents the FB(1)-induced accumulation of free sphinganine, a precursor of ceramide biosynthesis. However, not all of the effects of FB(1) in LLC-PK(1) cells can be explained solely by the increase in free sphingoid bases. The downstream signaling pathways that are affected by FB(1)-induced disruption of sphingolipid biosynthesis are not well understood. This study determined, in LLC-PK(1) cells, changes in p42 MAP kinase (phosphorylated ERK2 [pERK2]) phosphorylation in response to various inhibitors of key enzymes of the de novo sphingolipid biosynthesis pathway (CS, SPT, and glucosylceramide synthase [GlcCer synthase]). The results show that inhibition of any of the three enzymes caused a similar decrease in the extent of phosphorylation of ERK2 with no reduction in total ERK2. The co-treatment of FB(1) (CS inhibitor) with SPT inhibitors or the GlcCer synthase inhibitor had no effect on the FB(1)-induced reduction in pERK2 phosphorylation, indicating that FB(1)-mediated changes in phosphorylation of pERK2 was independent of increases in free sphinganine or its metabolites or a reduction in ceramide. Nonetheless, the decrease in pERK2 phosphorylation was dependent on inhibition of de novo sphingolipid biosynthesis. Decreased pERK2 activity could contribute to the physiological effects of FB(1) in LLC-PK(1) cells that are not due to alteration in pathways modulated by free sphingoid bases and their metabolites but are sensitive to inhibition of glycosphingolipid biosynthesis.

Topics: Acyltransferases; Animals; Apoptosis; Cell Division; Cycloserine; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Glucosyltransferases; LLC-PK1 Cells; Mitogen-Activated Protein Kinase 1; Morpholines; Oxidoreductases; Phosphorylation; Serine C-Palmitoyltransferase; Sphingolipids; Swine

Fumonisin B(1) (FB(1)), a mycotoxin produced by Fusarium verticillioides present on corn and corn-based products, causes species- and organ-specific diseases. The hepatotoxic effects of FB(1) in mice have been closely correlated with the accumulation of free sphinganine, a marker for ceramide synthase inhibition, and reduced biosynthesis of more complex sphingolipids. It has been shown that FB(1) modulates expression of many cell signaling factors. In the current study we used myriocin, a specific inhibitor of serine palmitoyltransferase, to investigate the role of free sphinganine accumulation in FB(1)-induced hepatotoxicity and increased expression of selected signaling genes in BALB/c mice. The mice were pretreated daily with intraperitoneal injection of 1.0 mg/kg myriocin 30 min before subcutaneous injections of 2.25 mg/kg of FB(1) for 3 days. Results showed that myriocin alone was not hepatotoxic and the combination of myriocin plus FB(1) completely prevented the FB(1)-induced elevation of hepatic free sphinganine and prevented the FB(1)-induced induction of selected cell signaling genes, suggesting that accumulation of free sphinganine and/or its metabolites contribute to the FB(1)-modulation of the cell signaling factors. However, the combination of myriocin and FB(1) did not prevent FB(1)-increased concentration of plasma alanine aminotransferase and only slightly attenuated aspartate aminotransferase; it did not affect the FB(1)-induced hepatocyte apoptosis or increased cell proliferation. A longer combined treatment of myriocin and FB(1) was highly toxic. The hepatotoxic effects in mice seen in this study are most likely due to a combination of factors including accumulation of free sphinganine, depletion of more complex sphingolipids and sphingomyelin, or other unknown mechanisms.

Topics: Acyltransferases; Animals; Antifungal Agents; Chemical and Drug Induced Liver Injury; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Female; Fumonisins; Mice; Mice, Inbred BALB C; Serine C-Palmitoyltransferase; Sphingolipids; Sphingosine

Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase-deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, sphingosine-1-phosphate, prevented lung apoptosis, implying that a balance between ceramide and sphingosine-1-phosphate is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.

Topics: Acyltransferases; Animals; Apoptosis; Cells, Cultured; Ceramides; Dose-Response Relationship, Drug; Emphysema; Fatty Acids, Monounsaturated; Fumonisins; Humans; Lung; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Oxidoreductases; Rats; Rats, Sprague-Dawley; Serine C-Palmitoyltransferase; Smoking; Sphingosine; Up-Regulation; Vascular Endothelial Growth Factor A

Fumonisin B(1) (FB(1)), a potent and naturally occurring mycotoxin produced by the fungus Fusarium verticillioides, has been implicated in fatal and debilitating diseases in animals and humans. FB(1) affects a variety of cell signaling proteins including protein kinase C (PKC); a serine/threonine kinase, involved in a number of signal transduction pathways that include cytokine induction, carcinogenesis and apoptosis. The aim of this study was to investigate the short-term temporal and concentration-dependent effects of FB(1) on PKC isoforms present in LLC-PK(1) cells in relation to the FB(1)-induced accumulation of sphinganine and sphingosine utilizing various inhibitors and activators. Our studies demonstrated that FB(1) (0.1-1 microM) selectively and transiently activated PKCalpha at 5 min, without affecting PKC-delta, -epsilon and -zeta isoforms. At higher FB(1) concentrations and later time points (15-120 min), PKCalpha membrane concentrations declined to untreated levels. The observed increase in cytosol PKCalpha protein expression at 15 min was not associated with an increase in its activity or protein biosynthesis. Calphostin C, a PKC inhibitor, abrogated the FB(1)-induced translocation of PKCalpha. Pre-incubation with the PKC activator, phorbol 12-myristate 13-acetate, resulted in an additive effect on membrane translocation of PKCalpha. Intracellular sphinganine and sphingosine concentrations were unaltered at the time points tested. Myriocin, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, did not prevent the FB(1)-induced PKCalpha cytosol to membrane redistribution. Altering PKCalpha and its signal transduction pathways may be of importance in the ability of FB(1) to exert its toxicity via apoptosis and/or carcinogenesis.

Topics: Animals; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Fatty Acids, Monounsaturated; Female; Fumonisins; Humans; Isoenzymes; Kidney; Male; Molecular Structure; Mycotoxins; Oxidoreductases; Protein Kinase C; Protein Kinase C-alpha; Protein Transport; Sphingolipids; Subcellular Fractions; Swine

Sterol-regulatory element-binding proteins (SREBPs) regulate transcription of genes of lipid metabolism. Ceramide decreases transcriptionally active SREBP levels independently of intracellular cholesterol levels. Mechanisms of the ceramide-mediated decrease of SREBP levels were investigated.. Experiments were performed in Chinese hamster ovary cells. Inhibition of ceramide synthesis with myriocin, cycloserine, or fumonisin decreases levels of transcriptionally active SREBP and reduces SRE-mediated gene transcription. When ceramide synthesis is increased through exogenous sphingosine or inhibition of sphingosine kinase, SRE-mediated gene transcription is increased. The important role of ceramide synthesis in SRE-mediated gene transcription is confirmed in LY-B cells that do not synthesize ceramide de novo. LY-B cells fail to increase SRE-mediated gene transcription in sterol depletion.. Ceramide synthesis correlates with the generation of transcriptionally active SREBP and SRE-mediated gene transcription. Inhibition of ceramide synthesis decreases levels of transcriptionally active SREBP and SRE-mediated gene transcription. It is hypothesized that the process of ongoing ceramide synthesis contributes to the physiological processing of SREBP, perhaps affecting ER-to-Golgi trafficking. Taken together, modification of ceramide synthesis could be a novel target for drug development in the pharmacologic modification of SRE-dependent pathways.

Topics: 1-Deoxynojirimycin; Acyltransferases; Amidohydrolases; Animals; CCAAT-Enhancer-Binding Proteins; Ceramides; CHO Cells; Cholesterol; Cricetinae; Cricetulus; Cycloserine; DNA-Binding Proteins; Enzyme Induction; Fatty Acids, Monounsaturated; Fumonisins; Genes, Reporter; Hydroxymethylglutaryl-CoA Synthase; Morpholines; Myristates; Phosphotransferases (Alcohol Group Acceptor); Propanolamines; RNA Processing, Post-Transcriptional; Serine C-Palmitoyltransferase; Sphingolipids; Sphingosine; Sterol Regulatory Element Binding Protein 1; Transcription Factors; Transcription, Genetic; Transfection

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

Topics: Animals; Biological Transport; Cell Line; Cell Membrane; Cells, Cultured; Cytosol; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Repression; Epithelial Cells; Fatty Acids, Monounsaturated; Fumonisins; Isoenzymes; Kidney; Models, Biological; Molecular Structure; Mycotoxins; Phosphates; Protein Kinase C; Sphingosine; Swine; Time Factors

Recent studies are beginning to implicate sphingolipids in the heat stress response. In the yeast Saccharomyces cerevisiae, heat stress has been shown to activate de novo biosynthesis of sphingolipids, whereas in mammalian cells the sphingolipid ceramide has been implicated in the heat shock responses. In the current study, we found an increase in the ceramide mass of Molt-4 cells in response to heat shock, corroborating findings in HL-60 cells. Increased ceramide was determined to be from de novo biosynthesis by two major lines of evidence. First, the accumulation of ceramide was dependent upon the activities of both ceramide synthase and serine palmitoyltransferase. Second, pulse labeling studies demonstrated increased production of ceramide through the de novo biosynthetic pathway. Significantly, the de novo sphingolipid biosynthetic pathway was acutely induced upon heat shock, which resulted in a 2-fold increased flux in newly made ceramides within 1-2 min of exposure to 42.5 degrees C. Functionally, heat shock induced the dephosphorylation of the SR proteins, and this effect was demonstrated to be dependent upon the accumulation of de novo-produced ceramides. Thus, these studies disclose an evolutionary conserved activation of the de novo pathway in response to heat shock. Moreover, SR dephosphorylation is emerging as a specific downstream target of accumulation of newly made ceramides in mammalian cells.

Topics: Biological Transport; Cell Survival; Ceramides; Fatty Acids, Monounsaturated; Fumonisins; HL-60 Cells; Hot Temperature; Humans; Kinetics; Microsomes; Mycotoxins; Nuclear Proteins; Palmitic Acid; Phosphoproteins; Precursor Cell Lymphoblastic Leukemia-Lymphoma; RNA-Binding Proteins; Saccharomyces cerevisiae; Serine-Arginine Splicing Factors; Sphingolipids; Tumor Cells, Cultured

Sphingolipids have been implicated in apoptosis after various stress inducers. To assess the involvement of the de novo sphingolipid pathway in apoptosis, photodynamic therapy (PDT) with the photosensitizer Pc 4 was used as a novel stress inducer. Here we provide biochemical and genetic evidence of the role of the de novo sphingolipids in apoptosis post-Pc 4-PDT. In Jurkat cells PDT-induced intracellular sphinganine accumulation, DEVDase activation, PARP cleavage, and apoptosis were suppressed by the de novo sphingolipid synthesis inhibitor ISP-1 (Myriocin). Coincubation with sphinganine, sphingosine, or C16-ceramide specifically reversed the antiapoptotic actions of ISP-1 or the singlet oxygen scavenger L-histidine. PDT-induced cytochrome c release from mitochondria into the cytosol was inhibited by L-histidine, but not by ISP-1. Cotreatment with sphinganine did not reverse the inhibitory effect of L-histidine. In addition, PDT-induced sphinganine accumulation and apoptosis were ISP-1-sensitive in A431 human epidermoid and HT29 human carcinoma cells. Furthermore, in LY-B cells, CHO-derived mutants deficient in the de novo sphingolipid synthesis enzyme serine palmitoyltransferase (SPT) activity, DEVDase activation and apoptosis were delayed and suppressed post-PDT. Hence, the data are consistent with the partial involvement of the de novo sphingolipid pathway in apoptosis via DEVDase activation downstream of mitochondrial cytochrome c release post-Pc 4-PDT.

Topics: Acyltransferases; Animals; Apoptosis; Carboxylic Acids; Caspases; CHO Cells; Cricetinae; Cytochrome c Group; Fatty Acids, Monounsaturated; Fumonisins; Histidine; Humans; Indoles; Jurkat Cells; Kinetics; Mitochondria; Models, Chemical; Neoplasms; Photosensitizing Agents; Poly(ADP-ribose) Polymerases; Serine C-Palmitoyltransferase; Sphingolipids; Sphingosine; Tumor Cells, Cultured

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.

Topics: 3T3 Cells; Animals; Apoptosis; Calcium-Binding Proteins; Calnexin; Carboxylic Acids; Cell Line; Cells, Cultured; Ceramides; Endoplasmic Reticulum; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Humans; Membrane Proteins; Mice; Models, Biological; Palmitic Acid; Phosphoric Monoester Hydrolases; Sphingolipids; Sphingosine

These studies determined (1) the time course for sphingoid base elevation in the small intestines, liver, and kidney of mice following a single 25 mg/kg body weight (bw) oral dose (high dose) of fumonisin B(1) (FB(1)), (2) the minimum threshold dose of FB(1) that would prolong the elevated sphingoid base concentration in kidney following the single high dose, and (3) the importance of the balance between the rate of sphingoid base biosynthesis and degradation in the persistence of sphingoid base accumulation. Following the high dose of FB(1), there was an increase in sphinganine in intestinal cells and liver that peaked at 4 to 12 h and declined to near the control level by 48 h. In kidney, sphinganine peaked at 6-12 h but remained elevated until 72 h, approaching control levels at 96-120 h. Oral administration of 0.03 mg FB(1)/kg bw (low dose) for 5 days had no effect on the sphingoid bases in kidney. However, following an initial high dose, daily administration of the low dose prolonged the elevation in kidney sphinganine compared to mice receiving a single high dose. Thus, a single exposure to a high dose of FB(1) followed by daily exposure at low levels will prolong the elevation of sphinganine in kidney. In cultured renal cells FB(1) was rapidly eliminated, but elevated sphinganine was persistent. This persistence in renal cells was rapidly reversed in the presence of the serine palmitoyltransferase inhibitor (ISP-1), indicating that the persistence was due to differences in the rates of sphinganine biosynthesis and degradation. The in vivo persistence in kidney may be due to similar differences.

Topics: Acyltransferases; Administration, Oral; Animals; Carboxylic Acids; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Intestine, Small; Kidney; Liver; LLC-PK1 Cells; Male; Mice; Mycotoxins; Oxidoreductases; Serine C-Palmitoyltransferase; Sphingosine; Swine

Fumonisin B1 is a mycotoxin commonly found on corn. It is hepatotoxic and nephrotoxic in domestic and experimental animals, and causes equine leukoencephalomalacia and porcine pulmonary oedema. It is a potent inhibitor of ceramide synthase. Inhibition leads to accumulation of free sphingoid bases in cells and tissues. In pig kidney epithelial cells (LLC-PK1), fumonisin B1 induces increased tumour necrosis factor alpha (TNFalpha) expression independent of the accumulation of sphingoid bases. The objective of this study was to investigate pharmacological approaches for intervening in fumonisin B1 toxicity using the LLC-PK1 cell model. The toxicity of fumonisin B1 was assayed using cell viability and lactate dehydrogenase (lactate dehydrogenase) release. Pretreatment of cells with myriocin, preventing sphinganine accumulates, prevented the fumonisin B1-induced decrease in cell viability and increased lactate dehydrogenase release. Modulation of adenosine receptor activity did not reduce the fumonisin B1 cytotoxicity. As with myriocin, silymarin pretreatment prevented the fumonisin B1-induced effects on cell viability and lactate dehydrogenase release. When added 6 or 24 hr after treatment of cells with fumonisin B1, both myriocin and silymarin reversed the decreased cell viability and suppressed the increased lactate dehydrogenase release. Myriocin, but not silymarin, blocked the accumulation of sphinganine in fumonisin B1-treated cells. Silymarin, unlike myriocin, induced expression of TNFalpha to an extent similar to fumonisin B1, but pretreatment with silymarin decreased the fumonisin B1-induced TNFalpha expression in LLC-PK1 cells. Results suggest that the mechanisms by which myriocin and silymarin protect renal cells are different, and silymarin potentially prevents fumonisin B1-induced toxicity by modulating TNFalpha expression or signals downstream of the inhibition of ceramide synthase.

Topics: Acyltransferases; Adenosine; Animals; Antioxidants; Carcinogens, Environmental; Cell Culture Techniques; Cell Survival; Fatty Acids, Monounsaturated; Flavins; Fumonisins; L-Lactate Dehydrogenase; LLC-PK1 Cells; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Reverse Transcriptase Polymerase Chain Reaction; Serine C-Palmitoyltransferase; Silymarin; Sphingosine; Swine; Tumor Necrosis Factor-alpha

Previous studies have shown that fumonisin B1 (FB1) inhibits ceramide synthase, resulting in accumulation of free sphinganine and sphingosine. Tumor necrosis factor-alpha (TNFalpha) plays an important role in FB1 toxicity and the expression of TNFalpha mRNA in liver and kidney is increased following FB1 exposure in mice. The objective of the current study was to investigate whether these two events (sphingoid bases accumulation and TNFalpha induction) are dependent on each other. An increase in expression of TNFalpha mRNA was detected in LLC-PK1 cells as early as 4 h after FB1 treatment but decreased to the control levels after 8 h. A positive linear correlation was observed between the expression of TNFalpha mRNA and FB1 concentration. Increases of intracellular sphingoid bases were also detected after 4 h of FB1 treatment and progressively increased until 24 h. Exposure of the cells to sphinganine or sphingosine did not significantly alter the expression of TNFalpha. Inhibition of sphingoid base biosynthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, efficiently blocked the accumulation of free sphingoid bases in response to FB1, but it did not prevent the induction of TNFalpha expression. Results indicate that FB1-induced increase in TNFalpha expression is independent of sphingoid base accumulation-induced by ceramide synthase inhibition in LLC-PK1 cells.

Topics: Acyltransferases; Animals; Carboxylic Acids; Cell Line; Ceramides; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Gene Expression; Kidney; Kinetics; Mitogen-Activated Protein Kinases; Naphthalenes; Oxidoreductases; Protein Kinase C; RNA, Messenger; Serine C-Palmitoyltransferase; Sphingosine; Swine; Tumor Necrosis Factor-alpha

Topics: Acyltransferases; Carboxylic Acids; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Fungi; Serine C-Palmitoyltransferase; Sphingosine

The first discovered naturally occurring inhibitor of de novo sphingolipid biosynthesis was fumonisin B1. There are now 11 identified fungal inhibitors of ceramide synthase or 'fumonisin B1-like' compounds. With the exception of the australifungins, all other fungal ceramide synthase inhibitors are structurally sphingoid-like. There are several recently discovered fungal inhibitors of another enzyme in the de novo sphingolipid biosynthesis pathway: serine palmitoyltransferase (SPT). One of the SPT inhibitors is named ISP-I. While ceramide synthase inhibitors are toxic to animals, plants and fungi, the SPT inhibitors are not known to cause animal or plant disease, but are potent inhibitors of fungal growth. Very little is known about their toxicity in animals. There are at least 24 fungal SPT inhibitors produced by a variety of fungi. Given that the fungal inhibitors of sphingolipid biosynthesis are chemically and biologically diverse, two bioassays have been developed to screen for fumonisin-like or ISP-I-like activity in naturally contaminated products or fungal culture materials. These bioassays are based on the changes in free sphingoid base concentration that occur when the ceramide synthase or SPT are inhibited. The bioassays have the advantage that they are functionally rather than chemically specific and thus will detect ceramide synthase and SPT inhibitors regardless of their chemical structure.

Topics: Acyltransferases; Animals; Biological Assay; Carboxylic Acids; Cells, Cultured; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fumonisins; Kidney; Liver; Oxidoreductases; Rats; Serine C-Palmitoyltransferase; Sphingolipids; Zea mays

Fumonisin B1 (FB1) and aminopentol (AP1) (which is formed by hydrolysis of FB1) are found in corn contaminated with some strains of Fusarium moniliforme. Incubation of HT29 cells (a human colonic cell line) with FB1 or AP1 caused a significant reduction in cell number; AP1 was less potent, with 50 microM AP1 causing the same reduction (ca. 30% after 24 h) as 10 microM FB1. The reduction in cell number reflected increases in DNA fragmentation and the percentage of apoptotic cells. Both FB1 and AP1 caused the accumulation of sphinganine (25- and 35-fold by 10 microM FB1 and 50 microM AP1, respectively); thus, concentrations of FB1 and AP1 that caused comparable reductions in cell number were also similar with respect to elevation of sphinganine, a compound that is growth inhibitory and cytotoxic. Inhibition of the first step of sphingolipid biosynthesis with ISP-1 prevented the elevation in sphinganine, DNA fragmentation, and apoptosis induced by FB1. Therefore, these effects of FB1 on HT29 cells can be attributed to the accumulation of sphinganine. Since consumption of food contaminated with Fusarium moniliforme (Sheldon) exposes colonic cells to these mycotoxins, the possibility that FB1 and AP1 are toxic for intestinal cells in vivo should be evaluated, especially in the light of the recent report (Bhat et al., Clin. Toxicol. 35, 249, 1997) describing intestinal disturbances in humans after consumption of moldy corn and sorghum containing fumonisins.

Topics: Antifungal Agents; Apoptosis; Carboxylic Acids; Carcinogens, Environmental; Cell Count; Cell Survival; DNA Fragmentation; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Food Contamination; Fumonisins; HT29 Cells; Humans; Mycotoxins; Sphingolipids; Sphingosine; Zea mays

GM-95, a mutant cell line derived from mouse melanoma MEB-4 cells, is deficient in glycosphingolipids (GSLs) due to the lack of ceramide glucosyltransferase-1 activity (Ichikawa, S., Nakajo, N., Sakiyama, H., and Hirabayashi, Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2703-2707). In this study, we examined the involvement of the complex sphingolipids in cell to substratum adhesion. Immunofluorescent and chemical analyses revealed that the complex sphingolipids were significantly concentrated in the detergent-insoluble substrate attachment matrix of both GM-95 and MEB-4 cells. In spite of the absence of GSLs, GM-95 cells retained the ability to adhere to extracellular matrix (ECM) proteins such as fibronectin, collagen, and laminin. When both GM-95 and MEB-4 cells were treated with neutral sphingomyelinase, GM-95 cells were rounded up and detached from all ECM proteins examined. In contrast, neither the morphology nor the adherence of MEB-4 cells was altered. Under this treatment, sphingomyelin (SM) became undetectable in both cells. A similar inhibition was observed upon pretreatment of cells with fumonisin B1 or ISP-1, both of which block the synthesis of ceramide, a common precursor of both GSLs and SM. Stable transfectants expressing GSLs, which were established by transfection of glucosyltransferase-1 cDNA into GM-95 cells, became resistant to neutral sphingomyelinase-mediated rounding up and detachment from ECM proteins. In conclusion, the complex sphingolipids play critical roles in cell to substratum adhesion, and the presence of either GSLs or SM is sufficient for the adhesion.

Topics: Animals; Cell Adhesion; Ceramides; Extracellular Matrix Proteins; Fatty Acids, Monounsaturated; Fumonisins; Glucosyltransferases; Glycosphingolipids; Kinetics; Melanoma, Experimental; Membrane Lipids; Mice; Mutagenesis; Mycotoxins; Recombinant Proteins; Sphingolipids; Sphingomyelin Phosphodiesterase; Transfection