Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis.
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We report that apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells associates with accumulation of monohexosylsphingolipids produced not only by short-chain ceramide glycosylation but also through glycosylation of a ceramide pool endogenously produced. By high-performance thin layer chromatography on borate silica gel plates, newly formed monohexosylsphingolipids were identified as glucosylceramides (GluCer); however, accumulation of lactosylceramide or higher-order glycosphingolipids was not observed. GluCer accumulation was fully suppressed by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; moreover, while this inhibitor had no effect on cell viability when administered alone, it markedly potentiated the apoptotic effect of C6-Cer. These results provide evidence that activation of GluCer synthesis is an important mechanism through which CHP-100 cells attempt to escape ceramide-induced apoptosis. (+info)
Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice.
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The epidermal permeability barrier for water is essentially maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the main components of these membranes, derive in large part from hydrolysis of glucosylceramides mediated by the lysosomal enzyme beta-glucocerebrosidase. As analyzed in this work, the beta-glucocerebrosidase deficiency in type 2 Gaucher mice (RecNci I) resulted in an accumulation of all epidermal glucosylceramide species accompanied with a decrease of the related ceramides. However, the levels of one ceramide subtype, which possesses an alpha-hydroxypalmitic acid, was not altered in RecNci I mice suggesting that the beta-glucocerebrosidase pathway is not required for targeting of this lipid to interstices of the stratum corneum. Most importantly, omega-hydroxylated glucosylceramides which are protein-bound to the epidermal cornified cell envelope of the transgenic mice accumulated up to 35-fold whereas levels of related protein-bound ceramides and fatty acids were decreased to 10% of normal control. These data support the hypothesis that in wild-type epidermis omega-hydroxylated glucosylceramides are first transferred enzymatically from their linoleic esters to proteins of the epidermal cornified cell envelope and then catabolized to protein-bound ceramides and fatty acids, thus contributing at least in part to the formation of the lipid-bound envelope. (+info)
Structure and properties of totally synthetic galacto- and gluco-cerebrosides.
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The structural and thermal properties of aqueous dispersions of the totally synthetic cerebrosides, D-erythro-N-palmitoyl galactosyl- and glucosyl-C18-sphingosine (C16:0-GalCer and C16:0-GluCer, respectively) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction. Over the temperature range 0-100 degrees C, both C16:0-GalCer and C16:0-GluCer show complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating, hydrated C16:0-GalCer undergoes an exothermic bilayer-bilayer transition at 59 degrees C to produce a stable bilayer crystal form. X-ray diffraction at 70 degrees C reveals a bilayer structure with an ordered hydrocarbon chain-packing arrangement. This ordered bilayer phase undergoes an endothermic chain-melting transition at 85 degrees C to the bilayer liquid crystalline state. Similar behavior is exhibited by hydrated C16:0-GluCer which undergoes the exothermic transition at 49 degrees C and a chain-melting transition at 87 degrees C. The exothermic transitions observed on heating hydrated C16:0-GalCer and C16:0-GluCer are irreversible and dependent upon previous chain melting, prior cooling rate, and time of incubation at low temperatures. Thus, the structure and properties of totally synthetic C16:0-GalCer and C16:0-GluCer with identical sphingosine (C18:1) and fatty acid (C16:0) chains are quite similar, suggesting that the precise isomeric structure of the linked sugar plays only a minor role in regulating the properties of hydrated cerebrosides. Further, these studies indicate that the complex thermal behavior and bilayer phase formation exhibited by these single-sugar cerebrosides are intrinsic properties and not due to the heterogeneity of the sphingosine base found in natural and partially synthetic cerebrosides. (+info)
Regulation of glycosphingolipid metabolism in liver during the acute phase response.
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The host response to infection is associated with multiple alterations in lipid and lipoprotein metabolism. We have shown recently that endotoxin (lipopolysaccharide (LPS)) and cytokines enhance hepatic sphingolipid synthesis, increase the activity and mRNA levels of serine palmitoyltransferase, the first committed step in sphingolipid synthesis, and increase the content of sphingomyelin, ceramide, and glucosylceramide (GlcCer) in circulating lipoproteins in Syrian hamsters. Since the LPS-induced increase in GlcCer content of lipoproteins was far greater than that of ceramide or sphingomyelin, we have now examined the effect of LPS and cytokines on glycosphingolipid metabolism. LPS markedly increased the mRNA level of hepatic GlcCer synthase, the enzyme that catalyzes the first glycosylation step of glycosphingolipid synthesis. The LPS-induced increase in GlcCer synthase mRNA levels was seen within 2 h, sustained for 8 h, and declined to base line by 24 h. LPS-induced increase in GlcCer synthase mRNA was partly accounted for by an increase in its transcription rate. LPS produced a 3-4-fold increase in hepatic GlcCer synthase activity and significantly increased the content of GlcCer (the immediate product of GlcCer synthase reaction) as well as ceramide trihexoside and ganglioside GM3 (products distal to the GlcCer synthase step) in the liver. Moreover, both tumor necrosis factor-alpha and interleukin-1beta, cytokines that mediate many of the metabolic effects of LPS, increased hepatic GlcCer synthase mRNA levels in vivo as well as in HepG2 cells in vitro, suggesting that these cytokines can directly stimulate glycosphingolipid metabolism. These results indicate that LPS and cytokines up-regulate glycosphingolipid metabolism in vivo and in vitro. An increase in GlcCer synthase mRNA levels and activity leads to the increase in hepatic GlcCer content and may account for the increased GlcCer content in circulating lipoproteins during the acute phase response. (+info)
Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons.
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Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca(2+)](i) response to glutamate, and a large increase in [Ca(2+)](i) release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co-incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B(1), completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca(2+)](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C(8)-glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre-incubation with ryanodine, suggesting that [Ca(2+)](i) release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease. (+info)
Pitfalls in the use of artificial substrates for the diagnosis of Gaucher's disease.
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A patient with Gaucher's disease is described, in whom the disease could not be diagnosed enzymically in liver and leucocytes using artificial substrate 4-methylumbelliferyl beta-glucoside. Normal activity was found in the liver, and about 60% of control activity was determined in the patient's leucocytes. In contrast, when [14C]-N-stearoyl glucocerebroside was employed as a substrate, activity as low as 5% of control has been found in all the proband's tissues, and carrier levels were determined in the proband's parents and maternal uncle. (+info)
Preferential killing of multidrug-resistant KB cells by inhibitors of glucosylceramide synthase.
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This study has compared the preferential killing of three multidrug-resistant (MDR) KB cell lines, KB-C1, KB-A1 and KB-V1 by two inhibitors of glucosylceramide synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP), to the killing produced by these compounds in the drug-sensitive cell line, KB-3-1. Both of the inhibitors caused much greater induction of apoptosis in each of the three MDR cell lines than in the drug-sensitive cell line, as judged by morphological assay and confirmed by poly-(ADP-ribose)-polymerase cleavage. The highest level of apoptosis was produced following 24-h exposure to 5 microM PPPP. This treatment produced 75.8 (+/- 7.1)%, 73.6 (+/- 9.8)% and 75.3 (+/- 6.4)% apoptotic cells in the three MDR cell lines respectively, compared to 19.0 (+/- 9.8)% in the drug-sensitive cell line. A reduction in glucosylceramide level following inhibitor treatment occurred in KB-3-1 cells as well as in the MDR cell lines, suggesting that the increased apoptotic response in the MDR cells reflected a different downstream response to changes in the levels of this lipid in these cells compared to that in the drug-sensitive cells. These results suggest that the manipulation of glucosylceramide levels may be a fruitful way of causing the preferential killing of MDR cells in vitro and possibly in vivo. (+info)
Polarized sphingolipid transport from the subapical compartment: evidence for distinct sphingolipid domains.
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In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains at the luminal side of the SAC membrane. Furthermore, as revealed by the use of various modulators of membrane trafficking, such as calmodulin antagonists and dibutyryl-cAMP, it is shown that the fate of these separate sphingolipid domains is regulated by different signals, including those that govern cell polarity development. Thus under conditions that stimulate apical plasma membrane biogenesis, SM is rerouted from a SAC-to-basolateral to a SAC-to-apical pathway. The latter pathway represents the final leg in the transcytotic pathway, followed by the transcytotic pIgR-dIgA protein complex. Interestingly, this pathway is clearly different from the apical recycling pathway followed by glucosylceramide, further indicating that randomization of these pathways, which are both bound for the apical membrane, does not occur. The consequence of the potential coexistence of separate sphingolipid domains within the same compartment in terms of "raft" formation and apical targeting is discussed. (+info)