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PubMed1986

Uptake by neuroblastoma cells of glucosylceramide, glucosylceramide glucosidase, its stimulator protein, and phosphatidylserine.

Serum-free cultured neuroblastoma cells (clone NlE-115) have been shown to absorb emulsified glucosylceramide, glucosylceramide glucosidase, an activator protein for the enzyme, and phosphatidylserine from a synthetic medium. Uptake of the enzyme was augmented by phosphatidylserine, and vice versa. Uptake of the enzyme-lipid complex was further augmented by the activator protein. It appears likely that the activator forms a complex only with the enzyme-lipid complex, not with the individual components. Two uptake mechanisms for the enzyme seem to be involved, one of which (the complex with activator proteins and acidic lipid) is sensitive to mannosyl phosphate groups. Hydrolysis of absorbed glucosylceramide was slow unless the medium was supplemented with the acidic phospholipid or glucosidase. The most rapid disappearance of stored glycolipid took place when the ternary mixture was added to the cell medium, enzyme + activator protein + phosphatidylserine. These findings may be relevant to enzyme replacement therapy for Gaucher disease.

Animals↗
PubMed1999

Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells.

Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682-1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells.

Antibiotics, Antineoplastic↗
PubMed2000

The skin of atopic dermatitis patients contains a novel enzyme, glucosylceramide sphingomyelin deacylase, which cleaves the N-acyl linkage of sphingomyelin and glucosylceramide.

We have demonstrated previously that there is an abnormal expression of sphingomyelin (SM) deacylase in the epidermis of patients with atopic dermatitis (ADe). In the present study, we have prepared N-[palmitic acid-1-(14)C]SM and N-[palmitic acid-1-(14)C]glucosylceramide (GCer) to use as substrates and have quantified SM deacylase activity by detecting the release of [(14)C]palmitic acid in extracts of the stratum corneum or the epidermis of ADe patients. In studies using [palmitic acid-1-(14)C]SM as a substrate, a pH dependency of catalytic activity with a peak at pH 5.0 was found. Preparative SDS/PAGE using an extract of ADe epidermis revealed that the molecular mass of SM deacylase is 40000 Da, which is consistent with its apparent molecular mass of 42000 Da estimated by gel-filtration analysis of stratum corneum extracts. Analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, beta-glucocerebrosidase (GlcCDase), sphingomyelinase (SMase) and acid ceramidase were 4.2, 7.4, 7.0 and 5.7, respectively. In enzymic analysis using pI-4.2 SM deacylase partially purified by IEF, which had no detectable contamination with acid ceramidase, GlcCDase or SMase, radio-TLC analysis revealed that radiolabelled sphingosylphosphocholine or [1-(14)C]palmitic acid was enzymically liberated from [choline-methyl-(14)C]SM or N-[palmitoyl-1-(14)C]GCer, respectively, used as substrates. Further the pI-4.2 protein purified from extracts of the stratum corneum of ADe patients was able to hydrolyse N-[palmitoyl-1-(14)C]SM and GCer, but not N-[palmitoyl-1-(14)C]ceramide. These results indicate that a hitherto undiscovered epidermal enzyme, termed here glucosylceramide sphingomyelin deacylase, is expressed in the skin of ADe patients, which plays an important role in ceramide deficiency (including acylceramides) in the stratum corneum.

Carboxylic Ester Hydrolases↗
PubMed2003

Formation of glucosylceramide and sterol glucoside by a UDP-glucose-dependent glucosylceramide synthase from cotton expressed in Pichia pastoris.

In plants, glucosylceramide (GlcCer) biosynthesis is poorly understood. Previous investigations suggested that sterol glucoside (SG) acts as the actual glucose donor for the plant GlcCer synthase (GCS). We addressed this question by generating a Pichia pastoris double mutant devoid of GlcCer and SG. This mutant was used for heterologous expression of the plant GCS. The activity of the GCS resulted in the accumulation of GlcCer and, surprisingly, a small proportion of SG. The synthesis of GlcCer in the transformed double mutant shows that the GCS is SG-independent, while the detection of SG suggests that in addition to the sterol glucosyltransferase, also the GCS may contribute in planta to SG biosynthesis.

Candida albicans↗
PubMed2000

Up-regulation of glucosylceramide synthesis upon stimulation of axonal growth by basic fibroblast growth factor. Evidence for post-translational modification of glucosylceramide synthase.

We have previously shown that ongoing glucosylceramide (GlcCer) synthesis is required for basic fibroblast growth factor (bFGF) and laminin to stimulate axonal growth in cultured hippocampal neurons (Boldin, S., and Futerman, A. H. (1997) J. Neurochem. 68, 882-885). We now demonstrate that stimulation of axonal growth by bFGF leads to an increase in the rate of GlcCer synthesis. Within minutes of incubation with bFGF, a significant increase in the rate of metabolism of [(14)C]hexanoyl ceramide to [(14)C]hexanoyl GlcCer is detected, but there are no changes in the rate of [(14)C]hexanoyl sphingomyelin synthesis. In vitro analysis of GlcCer synthase activity revealed an approximately 2-fold increase in the rate of [(14)C]hexanoyl GlcCer synthesis upon incubation with either bFGF or laminin; other growth factors, which did not effect the rate of axon growth, had no effect on the rate of [(14)C]hexanoyl GlcCer synthesis. The increased rate of [(14)C]hexanoyl GlcCer synthesis was not affected by preincubation with either cycloheximide or actinomycin, and no elevation of GlcCer synthase mRNA levels was detected, suggesting that GlcCer synthase is up-regulated by a post-translational mechanism. The relevance of these results for understanding the regulation of axonal growth is discussed.

Animals↗
PubMed2002

Human epidermal glucosylceramides are major precursors of stratum corneum ceramides.

Ceramides are the major component of the stratum corneum, accounting for 30%-40% of stratum corneum lipids by weight, and are composed of at least seven molecular groups (designated ceramides 1-7). Stratum corneum ceramides, together with cholesterol and fatty acids, form extracellular lamellae that are responsible for the epidermal permeability barrier. Previous studies indicated that beta-glucocerebrosidase- and sphingomyelinase-dependent ceramide production from glucosylceramides and sphingomyelins, respectively, is important for epidermal permeability barrier homeostasis. A recent study indicated that sphingomyelins are precursors of two stratum corneum ceramide molecular groups (ceramides 2 and 5). In this study, we have examined the role of glucosylceramides in the generation of each of the seven stratum corneum ceramide molecular groups. First, the structures of various glucosylceramide species in human epidermis were determined by gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. The results indicate that total epidermal glucosylceramides are composed of six distinct molecular groups, glucosylceramides 1-6. Glucosylceramide 1 contains sphingenine and nonhydroxy fatty acids, glucosylceramide 2, phytosphingosine and nonhydroxy fatty acids, glucosylceramide 3, phytosphingosine with one double bond and nonhydroxy fatty acids, glucosylceramide 4, sphingenine and alpha-hydroxy fatty acids, glucosylceramide 5, phytosphingosine and alpha-hydroxy fatty acids, and glucosylceramide 6, phytosphingosine with one double bond and alpha-hydroxy fatty acids. The nonhydroxy fatty acids typically have 16-24-carbon-length chains, whereas alpha-hydroxy fatty acids are limited to 24-, 25-, and 26-carbon chains. The sphingosine bases are C18 or C20 chains. Next, acylglucosylceramides and glucosylceramides were treated with beta-glucocerebrosidase and the ceramides released were compared with stratum corneum ceramides. Ceramide moieties of acylglucosylceramides and glucosylceramides 1, 2, 4-6 correspond to stratum corneum ceramides 1-7. These results, together with those of our previous reports characterizing epidermal sphingomyelins, indicate that all ceramide species, including omega-hydroxy fatty-acid-containing ceramides, are derived from glucosylceramides, and fractions of ceramides 2 and 5 are from sphingomyelins. Furthermore, structural analysis of glucosylceramides revealed that human epidermal glycosphingolipids display a unique lipid profile that is rich in very long chain hydroxylated (alpha- and omega-hydroxy) fatty acids and phytosphingosine.

Adolescent↗
PubMed2004

Overexpression of glucosylceramide synthase and P-glycoprotein in cancer cells selected for resistance to natural product chemotherapy.

Resistance to natural product chemotherapy drugs is a major obstacle to successful cancer treatment. This type of resistance is often acquired in response to drug exposure; however, the mechanisms of this adverse reaction are complex and elusive. Here, we have studied acquired resistance to Adriamycin, Vinca alkaloids, and etoposide in MCF-7 breast cancer cells, KB-3-1 epidermoid carcinoma cells, and other cancer cell lines to determine if there is an association between expression of glucosylceramide synthase, the enzyme catalyzing ceramide glycosylation to glucosylceramide, and the multidrug-resistant (MDR) phenotype. This work shows that glucosylceramide levels increase concomitantly with increased drug resistance in the KB-3-1 vinblastine-resistant sublines KB-V.01, KB-V.1, and KB-V1 (listed in order of increasing MDR). The levels of glucosylceramide synthase mRNA, glucosylceramide synthase protein, and P-glycoprotein (P-gp) also increased in parallel. Increased glucosylceramide levels were also present in Adriamycin-resistant KB-3-1 sublines KB-A.05 and KB-A1. In breast cancer, detailed analysis of MCF-7 wild-type and MCF-7-AdrR cells (Adriamycin-resistant) demonstrated enhanced glucosylceramide synthase message and protein, P-gp message and protein, and high levels of glucosylceramide in resistant cells. Similar results were seen in vincristine-resistant leukemia, etoposide-resistant melanoma, and Adriamycin-resistant colon cancer cell lines. Cell-free glucosylceramide synthase activity was higher in lysates obtained from drug-resistant cells. Lastly, glucosylceramide synthase promoter activity was 15-fold higher in MCF-7-AdrR compared with MCF-7 cells. We conclude that selection pressure for resistance to natural product chemotherapy drugs selects for enhanced ceramide metabolism through glucosylceramide synthase in addition to enhanced P-gp expression. A possible connection between glucosylceramide synthase and P-gp in drug resistance biology is suggested.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Presence of glucosylceramide in yeast and its relation to alkali tolerance of yeast.

Glycosylceramide is a membrane lipid that has physiological functions in eukaryotic organisms. The presence of glucosylceramide has been confirmed in some yeast; however, the extent of the role of glucosylceramide in yeast is unknown. Thus, the extent of presence of glucosylceramide in yeast was surveyed using 90 strains of 24 genera. The strains were divided into two groups according to whether they had glucosylceramide (45 strains) or not (45 strains). The distribution of the ceramide glucosyltransferase gene (EC 2.4.1.80), which catalyzes glucosylation to a sphingoid lipid in glucosylceramide synthesis, and the phylogenetic classification of the strains were in agreement with those of glucosylceramide. Thus, the presence of glucosylceramide in yeast was caused by the presence of the gene involved in glucosylceramide synthesis and was closely associated with yeast evolution. Furthermore, the relationship between glucosylceramide presence and alkali tolerance of yeast was evaluated. The yeast with glucosylceramide tended to grow at higher pH, and a ceramide-glucosyltransferase-defective mutant from Kluyveromyces lactis did not grow at pH 8.5 even though the parent strain could grow under the same conditions. These results indicate that glucosylceramide in yeast might be a component that enables yeast to grow under alkali conditions.

Chromatography, Thin Layer↗
PubMed1985

The fate of glucosylceramide (glucocerebroside) in genetically impaired (lysosomal beta-glucosidase deficient) Gaucher disease diploid human fibroblasts.

Diploid human infant skin fibroblasts cultured from normal infants and Gaucher disease infants, with genetically defective lysosomal glucosylceramide:beta-glucohydrolase activity, had a full range of homologous glycosphingolipids from the simplest (glucosylceramide) to higher neutral derivatives (lactosyl-, trihexosyl- and tetrahexosylceramide) and anionic sialo derivatives (gangliosides) (sialosyllactosyl-, disialosyllactosyl-, sialosylgangliotriaosyl-, and mono- and disialosylgangliotetraosylceramide). Although excessive storage of glucosylceramide in histiocytes is pathognomonic for Gaucher disease, we found that Gaucher disease fibroblasts contained 1.23 +/- 0.08 nmol of glucosylceramide/mg cell protein; normal infant cells, 1.11 +/- 0.48. When we aged infantile Gaucher disease fibroblasts for 20 days beyond their confluency state, we found no increased accumulation of glucosylceramide, but a 1.5-2-fold increase in trihexosylceramide, sialosylgangliotetraosylceramide, and disialosyllactosylceramide. Gaucher disease fibroblasts took up and could not degrade but, instead, effectively converted pulse-chase 3-O-[3H]glucosylceramide supplied in the growth medium in liposomes into higher glycosphingolipids, especially the plasma membrane ganglioside, sialosyllactosylceramide. When grown with extracellular particulate [3H]glucosylceramide, infantile Gaucher fibroblasts localized it and higher labeled homologues in the plasma membrane; glucosylceramide did not accumulate in the lysosomes. These findings indicate that fibroblasts that are genetically deficient in lysosomal glucosylceramide:beta-glucosidase avoid pathological lysosomal accumulation by relegating undegradable glucosylceramide to an anabolic compartment where glucosylceramide is converted into more highly glycosylated glycosphingolipids.

Cell Fractionation↗
PubMed1999

Improved inhibitors of glucosylceramide synthase.

Previous work has led to the identification of inhibitors of glucosylceramide synthase, the enzyme catalyzing the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. These inhibitors have two identified sites of action: the inhibition of glucosylceramide synthase, resulting in the depletion of cellular glycosphingolipids, and the inhibition of 1-O-acylceramide synthase, resulting in the elevation of cell ceramide levels. A new series of glucosylceramide synthase inhibitors based on substitutions in the phenyl ring of a parent compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was made. For substitutions of single functional groups, the potency of these inhibitors in blocking glucosylceramide synthase was primarily dependent upon the hydrophobic and electronic properties of the substituents. An exponential relationship was found between the IC50 of each inhibitor and the sum of derived hydrophobic (pi) and electronic (sigma) parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced the potency of these compounds in blocking glucosylceramide formation. A novel compound was subsequently designed and observed to be even more active in blocking glucosylceramide formation. This compound, D-threo-4'-hydroxy-P4, inhibited glucosylceramide synthase at an IC50 of 90 nM. In addition, a series of dioxane substitutions was designed and tested. These included 3',4'-methylenedioxyphenyl-, 3',4'-ethylenedioxyphenyl-, and 3'4'-trimethylenedioxyphenyl-substituted homologues. D-threo-3', 4'-Ethylenedioxy-P4-inhibited glucosylceramide synthase was comparably active to the p-hydroxy homologue. 4'-Hydroxy-P4 and ethylenedioxy-P4 blocked glucosylceramide synthase activity at concentrations that had little effect on 1-O-acylceramide synthase activity. These novel inhibitors resulted in the inhibition of glycosphingolipid synthesis in cultured cells at concentrations that did not significantly raise intracellular ceramide levels or inhibit cell growth.

Cells, Cultured↗
PubMed2001

Localization of ceramide and glucosylceramide in human epidermis by immunogold electron microscopy.

Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound omega-hydroxy ceramides and one covalently bound omega-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis.

Cell Membrane↗
PubMed2000

Assay for the transbilayer distribution of glycolipids. Selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals.

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine- egg phosphatidylethanolamine;-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A(2) confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing <1 mol% radiolabeled glucosylceramide or short-chain C(6)-glucosylceramide, carboxy-TEMPO oxidized half the glucosylceramide. However, if surface C(6)-glucosylceramide was first depleted by bovine serum albumin (BSA) (extracting 49 +/- 1%), 94% of the remaining C(6)-glucosylceramide was resistant to oxidation. Carboxy-TEMPO oxidized glucosylceramide on the surface of LUVs without affecting inner leaflet glucosylceramide. At pH 9.5 and at 0 degrees C, the reaction reached completion by 20 min.

Antioxidants↗
PubMed1997

Effect of saposins A and C on the enzymatic hydrolysis of liposomal glucosylceramide.

The degradation of glucosylceramide in lysosomes is accomplished by glucosylceramidase with the assistance of, at least, another protein, saposin C (Sap C), which is generated from a large precursor together with three other similar proteins, saposins A, B, and D. In the present study, we have examined the effects of saposins on the enzymatic hydrolysis of glucosylceramide inserted in large and small phospholipid liposomes. The glucosylceramide contained in large unilamellar vesicles (LUV) was degraded by glucosylceramidase at a rate 7-8-fold lower than glucosylceramide inserted in small unilamellar vesicles (SUV). The separate addition of either Sap A or Sap C to the LUV system partially stimulated the sphingolipid degradation while saposins B and D had no effect. In the presence of both Sap A and Sap C, the rate of sphingolipid degradation was higher than the sum of the rates with the two saposins individually, indicating synergism in their actions. The stimulatory effect of the two saposins depended on the incorporation of an acidic phospholipid such as phosphatidylserine (PS) into LUV. The characteristics of glucosylceramidase activation by Sap C were different from those of Sap A. Sap C increased the rate of hydrolysis of both the artificial water soluble substrate, 4-methylumbelliferyl-beta-D-glucopyranoside, and the lipid substrate, glucosylceramide, while Sap A only stimulated degradation of the sphingolipid. Also the binding properties of Saps A and C were markedly different. At acidic pH values, Sap C bound to PS-containing LUV and promoted the association of glucosylceramidase with the membrane. In contrast, Sap A had poor affinity for the membrane even in the presence of glucosylceramide; moreover, Sap A did not potentiate the capacity of Sap C to mediate glucosylceramidase binding. In conclusion, our results show that both Sap A and Sap C are required for maximal hydrolysis of glucosylceramide inserted in PS-containing LUV, that their effects are synergistic, and that their mode of action is different. Sap C is responsible for the membrane binding of glucosylceramidase, while Sap A stimulation is possibly related to its effect on the conformation of the enzyme. It can be envisaged that Sap A in conjunction with Sap C might have a physiological role in glucosylceramide degradation.

Catalysis↗
PubMed1991

Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.

Animals↗
PubMed2006

Can P-glycoprotein influence the bioavailability of iminosugar-based glucosylceramide synthase inhibitors?

Recently developed glucosylceramide synthase inhibitors with enhanced hydrophobicity display increased bioavailability in the central nervous system (CNS). Have these improvements come at a potential risk given that the improved glucosylceramide synthase inhibitors bear the hallmarks of P-glycoprotein substrates? This question warrants attention given the potential to induce adverse drug interactions or toxicity, if glucosylceramide synthase inhibitors are administered with other P-glycoprotein substrates. The aim of this study was to determine if glucosylceramide synthase inhibitors are substrates for the multidrug transporter P-glycoprotein. Direct measurements of glucosylceramide synthase inhibitors binding to P-glycoprotein were examined, as was their ability to modulate transport by the protein. The more hydrophobic glucosylceramide synthase inhibitors caused a reduction in drug binding to P-glycoprotein. However, the compounds did not achieve this by direct interaction with the protein, but through a general membrane perturbation. Furthermore, the alterations in drug-P-glycoprotein interaction did not manifest as altered cellular accumulation of glucosylceramide synthase inhibitors or altered efficacy to reduce cellular glycolipid levels. Consequently, P-glycoprotein expression will not contribute significantly to the pharmacokinetic profile of the iminosugar glucosylceramide synthase inhibitors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Accumulation of glucosylceramides in multidrug-resistant cancer cells.

Multidrug-resistant (MDR) tumors and cancer cell lines demonstrate a wide variety of biochemical changes. In this study we used drug-sensitive wild-type (wt) cancer cell lines and respective MDR subclones, and we demonstrate the accumulation of distinct lipids in MDR cells. These lipids were either absent or present at very low levels in drug-sensitive cells. The compounds, termed lipid-1 and lipid-2, migrated on thin-layer chromatography as a doublet. They could be radiolabeled by incubating MCF-7-AdrR (Adriamycin-resistant) breast cancer cells with [3H]serine, [3H]palmitic acid, or [3H]galactose. Utilization of these precursors by MCF-7-wt cells for synthesis of lipid-1 and -2 was minimal. Two inhibitors of sphingolipid biosynthesis, L-cycloserine and fumonisin B1, prevented the observed accumulation of the lipid compounds. An inhibitor of glucosylceramide synthesis, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, completely abolished the formation of lipid-1 and -2 in MCF-7-AdrR cells and, to a lesser extent, inhibited the formation of lactosylceramides and gangliosides. Utilizing mass spectrometry, the multidrug resistance-associated lipids were further characterized as monoglycosylceramides of two major species that contained either 16-carbon (palmitic) or 24-carbon (lignoceric and nervonic) fatty acids. The carbohydrate head group of glycosylceramides was identified as glucose, not galactose, thus designating the accumulated lipids as glucosylceramides. Incorporation of [3H]palmitic acid into glucosylceramide was strikingly higher (8-10 times) in MCF-7-AdrR cells compared with MCF-7-wt cells. Since the rate of glucosylceramide degradation in MCF-7-AdrR cells was not attenuated, accelerated glycosphingolipid synthesis in MDR cells is suggested. Glucosylceramide also accumulated in KB-V-1, a vinblastine-resistant epidermoid carcinoma but not in KB-3-1, drug-sensitive wt cells. MDR ovarian adenocarcinoma cells (NIH:OVCAR-3) also contained elevated levels of glucosylceramide. Our results demonstrate a correlation between cellular drug resistance and alterations in glucosylceramide metabolism.

Chromatography, Thin Layer↗
PubMed1992

Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.

In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Specificity of human glucosylceramide beta-glucosidase towards synthetic glucosylsphingolipids inserted into liposomes. Kinetic studies in a detergent-free assay system.

The behaviour of highly purified glucosylceramide beta-glucosidase (glucosylceramidase, EC 3.2.1.45) from human placenta [Furbish, F. S., Blair, H. E., Shiloach, J., Pentchev, P. G. & Brady, R. B. (1977) Proc. Natl Acad. Sci. USA 74, 3560-3563] was investigated in the absence of detergents with structurally modified glucosylceramides inserted into unilamellar liposomes. The reaction between the water-soluble enzyme and the liposomal substrates was significantly dependent on the structure of the lipophilic aglycon moiety of glycolipids: glucosyl-N-acetyl-sphingosines (D-erythro and L-threo) were better substrates than the corresponding glucosylceramides. The L-threo derivatives were poorer substrates with higher apparent Km values than the corresponding D-erythro derivatives. For glucosyl-3-keto-ceramide and glucosyl-dihydro-ceramide (D-erythro), higher Km values were found than for glucosylceramide. Sphingosine, glucosylsphingosine and glucosyl-N-acetyl-sphingosine were the most effective inhibitors of the hydrolysis of glucosylceramide. D-erythro-Ceramide and D-galactosyl-N-acetyl-D-erythro-sphingosine inhibited the hydrolysis of amphiphilic glucosylceramide but not that of water-soluble 4-methyl-umbelliferyl-beta-glucoside, suggesting a hydrophobic binding site of the enzyme for the aglycon moiety of its membrane-bound substrate. Dilution experiments suggested that at least a fraction of the enzyme associates with the liposomes and degrades the lipid substrate even in the absence of activator proteins. Acidic phospholipids incorporated into liposomes caused a powerful stimulation (30-40-fold) of the glucosylceramide beta-glucosidase, whereas acidic sphingolipids (sulphatide, gangliosides GM1 and GD1a) incorporated into liposomes stimulated this enzyme only moderately (3-10-fold).

Cerebrosides↗