Prosaposin Reduces α-Synuclein in Cells and Saposin C Dislodges it from Glucosylceramide-enriched Lipid Membranes.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder affecting over 1% of the 65 + age population. Saposin C, a lysosomal protein required for the normal activity of glucocerebrosidase (GCase), may serve as a disease modifier in PD. Saposin C is cleaved from its precursor, Prosaposin (PSAP), which is secreted as an uncleaved protein and exerts neuroprotective effects. In this study, we aim to elucidate the neuroprotective roles of PSAP and saposin C in PD by evaluating their effects on α-synuclein accumulation in human neuroblastoma cells. Stable overexpression of PSAP reduced monomeric α-synuclein levels in SH-SY5Y cells, while PSAP knockdown by small interfering RNA led to the opposite effect, and those effects were independent of GCase activity. Autophagy flux was decreased by stable PSAP overexpression. Furthermore, a flow-through assay revealed that recombinant saposin C was able to detach α-synuclein from artificial glucosylceramide-enriched lipid membranes at the lysosomal pH. Taken together, our findings provide further evidence that PSAP and saposin C as key proteins involved in α-synuclein clearance by dislodging it from lipid membranes.

Anti-melanogenic effects of glucosylceramides and elasticamide derived from rice oil by-products in melanoma cells, melanocytes, and human skin.

Glucosylceramides (GlcCer), which are present in many edible plants, suppress melanin production in mouse melanocytes. Rice GlcCer consist of multiple molecules that comprise different types of sphingoid bases as well as diverse lengths and stereotypes of free fatty acids. Adjacent to the GlcCer fraction, there are free ceramides (Cer) as minor constituents. However, the anti-melanogenic activities of individual GlcCer and Cer remain unknown. Therefore, we herein isolated 13 GlcCer and elasticamide, a Cer [AP] from the gummy by-products of rice bran oil, and examined their anti-melanogenic activities. In theophylline-induced melanogenesis in B16 melanoma cells, GlcCer [d18:2(4E,8Z)/18:0], GlcCer [d18:2(4E,8Z)/20:0], and elasticamide significantly suppressed melanin production with IC50 values of 6.6, 5.2, and 3.9 µM, respectively. Elasticamide, but not GlcCer [d18:2 (4E,8Z)/20:0], suppressed melanogenesis in human 3D-cultured melanocytes and the expression of tyrosinase-related protein 1 in normal human melanocytes. Based on these results, we conducted a clinical trial on the effects of rice ceramide extract (Oryza ceramide®), containing 1.2 mg/day of GlcCer and 56 µg/day of elasticamide, on UV-B-induced skin pigmentation. The ingestion of Oryza ceramide® for 8 weeks significantly suppressed the accumulation of melanin 7 days after UV irradiation (1288 and 1546 mJ/cm2 ·S). Rice-derived GlcCer and elasticamide, which exhibited anti-melanogenic activities, were suggested to contribute to the suppressive effects of Oryza ceramide® on UV-induced skin pigmentation. Although the mechanisms underlying the anti-melanogenic activities of GlcCer remain unclear, elasticamide was identified as a promising Cer that exhibits anti-melanogenic activity. PRACTICAL APPLICATIONS: The anti-melanogenic activities of rice-derived GlcCer and elasticamide currently remain unclear. We herein demonstrated the inhibitory effects of individual GlcCer and elasticamide on melanogenesis in melanoma cells, melanocytes, and human skin.

GBA1-dependent membrane glucosylceramide reprogramming promotes liver cancer metastasis via activation of the Wnt/ß-catenin signalling pathway.

The effect of glucosylceramide (GlcCer) reprogramming on liver cancer metastasis remains poorly understood. In this study, we demonstrated that the protein expression of GBA1, which catalyses the conversion of GlcCer to ceramide, was downregulated in liver cancer tissue. A clinical relevance analysis revealed that low expression of GBA1 was associated with the metastatic potential of liver cancer cells. Furthermore, loss- and gain-of-function studies confirmed that low expression of GBA1 promoted metastasis of liver cancer both in vitro and in vivo. Mechanistic studies indicated that low expression of GBA1 enhanced the metastatic ability of liver cancer by promoting the epithelial-mesenchymal transition (EMT), in which Wnt signalling pathway is involved. In the plasma membrane (PM), GBA1-dependent GlcCer reprogramming increased LRP6 location in the PM leading to an interaction between GlcCer and LRP6, subsequently promoting LRP6 phosphorylation at Ser1490, and finally activating the Wnt/ß-catenin signalling pathway. To our knowledge, this is the first time to be found that GlcCer interacted with a protein. In addition, the results of mass spectrometry indicated that GlcCer d18:1/18:0 was the most notably increased studied species in the PM when GBA1 was downregulated, suggesting that GlcCer d18:1/18:0 may be the major functional lipid that promotes GBA1-dependent liver cancer metastasis. Thus, GBA1-mediated GlcCer reprogramming in the PM promotes metastasis of liver cancer via activation of the Wnt/ß-catenin signalling pathway, upregulation of GBA1 may be a potential therapeutic strategy to combat liver cancer metastasis.

Sphingoid base in pineapple glucosylceramide suppresses experimental allergy by binding leukocyte mono-immunoglobulin-like receptor 3.

BACKGROUND: The increase in patients suffering from type I hypersensitivity, including hay fever and food allergy, is a serious public health issue around the world. Recent studies have focused on allergy prevention by food factors with fewer side effects. The purpose of this study was to evaluate the effect of dietary glucosylceramide from pineapples (P-GlcCer) on type I hypersensitivity and elucidate mechanisms. RESULTS: Oral administration of P-GlcCer inhibited ear edema in passive cutaneous anaphylaxis reaction. In a Caco-2/RBL-2H3 co-culture system, P-GlcCer inhibited ß-hexosaminidase release from RBL-2H3 cells. The direct treatment of P-GlcCer on RBL-2H3 did not affect ß-hexosaminidase release, but sphingoid base moiety of P-GlcCer did. These results predicted that sphingoid base, a metabolite of P-GlcCer, through the intestine inhibited type I hypersensitivity by inhibiting mast cell degranulation. In addition, the inhibitory effects of P-GlcCer on ear edema and degranulation of RBL-2H3 cells were canceled by pretreatment of leukocyte mono-immunoglobulin-like receptor 3 (LMIR3)-Fc, which can block LMIR3-mediated inhibitory signals. CONCLUSION: It was demonstrated that a sphingoid base, one of the metabolites of P-GlcCer, may inhibit mast cell degranulation by binding to LMIR3. The oral administration of P-GlcCer is a novel and attractive food factor that acts directly on mast cells to suppress allergy. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Anti-allergic property of 4,8-sphingadienine stereoisomers in vivo and in vitro model.

4,8-Sphingadienines (SD), metabolites of glucosylceramides (GlcCer), are sometimes determined as key mediators of the biological activity of dietary GlcCer, and cis/trans geometries of 4,8-SD have been reported to affect its activity. Since regulating excessive activation of mast cells seems an important way to ameliorate allergic diseases, this study was focused on cis/trans stereoisomeric-dependent inhibitory effects of 4,8-SD on mast cell activation. Degranulation of RBL-2H3 was inhibited by treatment of 4-cis-8-trans- and 4-cis-8-cis-SD, and their intradermal administrations ameliorated ear edema in passive cutaneous anaphylaxis reaction, but 4-trans-8-trans- and 4-trans-8-cis-SD did not. Although the activation of mast cells depends on the bound IgE contents, those stereoisomers did not affect IgE contents on RBL-2H3 cells after the sensitization of anti-TNP IgE. These results indicated that 4-cis-8-trans- and 4-cis-8-cis-SD directly inhibit the activation of mast cells. In conclusion, it was assumed that 4,8-SD stereoisomers with cis double bond at C4-position shows anti-allergic activity by inhibiting downstream pathway after activation by the binding of IgE to mast cells.

Dietary Ceramide Prepared from Soy Sauce Lees Improves Skin Barrier Function in Hairless Mice.

Dietary sphingolipids such as glucosylceramide and sphingomyelin are known to improve the skin barrier function of damaged skin. In this study, we focused on free-ceramide prepared from soy sauce lees, which is a byproduct of soy sauce production. The effects of dietary soy sauce lees ceramide on the skin of normal mice were evaluated and compared with those of dietary maize glucosylceramide. We found that transepidermal water loss value was significantly suppressed by dietary supplementation with soy sauce lees ceramide as effectively as or more effectively than maize glucosylceramide. Although the content of total and each subclass of ceramide in the epidermis was not significantly altered by dietary sphingolipids, that of 12 types of ceramide molecules, which were not present in dietary sources, was significantly increased upon ingestion of maize glucosylceramide and showed a tendency to increase with soy sauce lees ceramide intake. In addition, the mRNA expression of ceramide synthase 4 and involucrin in the skin was downregulated by sphingolipids. This study, for the first time, demonstrated that dietary soy sauce lees ceramide enhances skin barrier function in normal hairless mice, although further studies are needed to clarify the molecular mechanism.

Tyrosine-phosphorylation and activation of glucosylceramide synthase by v-Src: Its role in survival of HeLa cells against ceramide.

Sphingolipids represent a family of cellular lipid-molecules that regulate physiological and pathophysiological processes. Glucosylceramide (GlcCer), the simplest glycosphingolipid (GSL), is synthesized from ceramide and UDP-glucose by GlcCer synthase (GCS). Both GlcCer (and resulting GSLs) and ceramide regulate various cellular functions including cell death and multiple drug resistance. Src family tyrosine kinases are up-regulated in various human cancer cells. We examined the effect of v-Src expression on GCS activity, the formation of 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled GlcCer from NBD-ceramide, and the effect of tyrosine132 mutation in GCS on ceramide-induced cytotoxicity in HeLa cells. Expression of v-Src increased the formation of NBD-GlcCer in both intact cells without marked changes in other sphingolipid metabolites and cell homogenates without changing affinities of NBD-ceramide and UDP-glucose. Expression of v-Src also increased tyrosine-phosphorylated levels in GCS proteins in HeLa and HEK293T cells. In HEK293T cells transiently expressing the GCS mutant, GCS-Y132F-HA, showing replacement of the tyrosine132 residue with phenylalanine, tyrosine-phosphorylated levels in GCS proteins were significantly lower than those in control cells expressing the GCS-wild-type-HA. The formation of NBD-GlcCer in HeLa cells stably expressing GCS-Y132F-HA was significantly lower than that in the control. Ceramide-induced cytotoxicity in HeLa-GCS-Y132F-HA cells was significantly greater than in the control. In this study, we showed for the first time that expression of v-Src up-regulated GCS activity via tyrosine phosphorylation of the enzyme in a post-translational manner. Mechanisms of Src-induced resistance to ceramide-induced cytotoxicity are discussed in relation to the Src-induced up-regulation of GCS activity.

A synergistic effect of Ambroxol and Beta-Glucosylceramide in alleviating immune-mediated hepatitis: A novel immunomodulatory non-immunosuppressive formulation for treatment of immune-mediated disorders.

BACKGROUND: Ambroxol hydrochloride is being used in respiratory diseases as a broncholytic therapy. Beta-Glucosylceramide (GC) is a naturally occurring glycosphingolipid that exerts an immune protective effect. The aim of the present study was to determine the synergistic immunomodulatory effect between these two compounds. METHODS: Immune-mediated hepatitis was induced in the mice by administration of Con A. Mice were treated with either Ambroxol or GC alone or with the combination of both. Mice were followed for their effect on the liver injury, cytokine profile, and the immune system. RESULTS: Coadministration of Ambroxol and GC significantly alleviated the liver injury induced by ConA, as demonstrated by the decreased liver enzymes. The combined treatment had a statistically significant synergistic effect on the suppression of intrahepatic CD8+CD25+, an increase in the CD4/CD8 lymphocyte ratio and in the CD8+ intrahepatic lymphocyte trapping, as well as on change of serum in the IL4 levels. The beneficial effect was associated with the promotion of regulatory T lymphocytes subsets, and with a trend for a pro-inflammatory to an anti-inflammatory cytokine shift. CONCLUSIONS: Coadministration of Ambroxol with GC exerted a synergistic immunoprotective effect in a model of immune-mediated acute liver damage. Considering the high safety profile of both agents, the combination may become a novel immunomodulatory non-immunosuppressive therapeutic agent. SIGNIFICANCE STATEMENT: Coadministration of Ambroxol with glucocerebroside exerted a synergistic immunoprotective effect in a model of immune-mediated acute liver damage.

Effects of Dietary Ethanol Extracts from Sake Rice and Sake Lees on Intestinal Impairment in Mice.

Glucosylceramide (GlcCer), a major sphingolipid in plants and fungi, is known to have food functions, such as preventing intestinal impairment and enhancing the moisture content of skin. This study investigated the influence of fermentation on the composition and function of lipophilic components containing GlcCer in plant-based foods; we compared the effects of ethanol extracts from sake rice (SR) and sake lees (SL) on colon impairment in mice. GlcCer and ceramide (Cer) levels in SL were much higher than those in SR, and GlcCer in SL contained 9-methyl-trans-4,trans-8-sphingadienine as a fungi-specific sphingoid base. 1,2-dimethylhydrazine (DMH) treatment markedly increased the formation of aberrant crypt foci (ACF) and the levels of TNF-α and lipid oxidation in mice colons. However, dietary SR or SL significantly suppressed these DMH-induced changes, and SR demonstrated stronger effects than SL. In addition, dietary SR or SL suppressed the expression of apoptotic and anti-apoptotic proteins induced by DMH treatment. This study suggests that SR or SL intake could reduce colon ACF formation via the suppression of inflammation and oxidation-induced cell cycle disturbances. When compared to SR, the weaked effects of SL rich in GlcCer may be the result of the changes in sphingolipid composition (sphingoid base and Cer) and differences in the concentration of other bioactive compounds produced or digested during fermentation.

Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain.

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and a-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of mM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand-receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

Plant sphingolipids promote extracellular vesicle release and alleviate amyloid-ß pathologies in a mouse model of Alzheimer's disease.

The accumulation of amyloid-ß protein (Aß) in brain is linked to the early pathogenesis of Alzheimer's disease (AD). We previously reported that neuron-derived exosomes promote Aß clearance in the brains of amyloid precursor protein transgenic mice and that exosome production is modulated by ceramide metabolism. Here, we demonstrate that plant ceramides derived from Amorphophallus konjac, as well as animal-derived ceramides, enhanced production of extracellular vesicles (EVs) in neuronal cultures. Oral administration of plant glucosylceramide (GlcCer) to APP overexpressing mice markedly reduced Aß levels and plaque burdens and improved cognition in a Y-maze learning task. Moreover, there were substantial increases in the neuronal marker NCAM-1, L1CAM, and Aß in EVs isolated from serum and brain tissues of the GlcCer-treated AD model mice. Our data showing that plant ceramides prevent Aß accumulation by promoting EVs-dependent Aß clearance in vitro and in vivo provide evidence for a protective role of plant ceramides in AD. Plant ceramides might thus be used as functional food materials to ameliorate AD pathology.

Torula yeast (Candida utilis)-derived glucosylceramide contributes to dermal elasticity in vitro.

Glucosylceramide (GlcCer) is derived from several plants, such as rice, maize, and wheat, and has been reported to retain moisture by functioning as a barrier between the epidermis and the environment. However, there is insufficient research on the effect of GlcCer on dermal elasticity and wrinkles. In this study, we investigated the effects of torula yeast extract and torula yeast-derived GlcCer on dermal elasticity. We measured cell proliferation, collagen production, and collagen gel contraction using human dermal fibroblasts. Torula yeast extract and torula yeast-derived GlcCer increased dermal fibroblast proliferation and collagen production. Collagen gel contraction was promoted by torula yeast extract and torula yeast-derived GlcCer. These results indicate that GlcCer may affect dermal elasticity. Torula yeast extract and torula yeast-derived GlcCer may contribute to the maintenance of dermal elasticity. PRACTICAL APPLICATIONS: In this study, we found that torula yeast-derived glucosylceramide (GlcCer) has an additional function of improving dermal elasticity. With improved elasticity, skin becomes more resilient, thus preventing wrinkles. GlcCer has already been used in cosmetic products to retain skin moisture. Therefore, torula yeast-derived GlcCer can be expected to have several cosmetic applications.

Extraction of Lipophilic Fraction from Polished Rice Improves Its Ameliorative Effect on Intestinal Impairment.

Glucosylceramide (GlcCer), a major sphingolipid in plants and fungi, is known to have food functions such as preventing intestinal impairment and enhancing the moisture content of skin. However, there is little information about functions of GlcCer in food sources as most of the studies on GlcCer functions are done using purified GlcCer. This study was performed to investigate the effects of GlcCer contained in food on intestinal impairment; polished rice flour (RF) and this ethanol extract (RE) were used as sources of GlcCer, and these were evaluated by studying the formation of aberrant crypt foci (ACF) in 1,2-dimethylhydrazine (DMH)-treated mice, which is a model of colon cancer. Mice were fed with either a control diet, a RF diet where RF replaces cornstarch (150 g/kg), or a plus RE diet (0.5 g/kg; RE was extracted from the same amount of RF present in the RF diet). The amount of GlcCer was similar in both the RF and RE diets (3.0 and 2.7 mg/kg, respectively). DMH treatment induced the formation of ACF and the production of inflammation-related cytokines. Both dietary RF and RE suppressed ACF formation and RE, in particular, showed a significant suppressive effect. Dietary RE inhibited the production of almost all of the inflammation-related cytokines studied, while RF suppressed only a few of these cytokines. The present study suggests that the lipophilic fraction including GlcCer, present in polished rice has protective effects against intestinal impairment, but it requires extraction since digestion alone is not enough to elicit its complete protective action.

Long-chain glucosylceramides crosstalk with LYN mediates endometrial cell migration.

Endometriosis is a disease characterized by regurgitated lesions which are invasive and migratory, embedding at ectopic, extra-uterine locations. Extracellular glucosylceramides (GlcCers), bioactive sphingolipids potentiating signals for cell migration, are found in elevated levels in endometriosis; however underlying mechanisms that result in cellular migration are poorly defined. Here, we demonstrated that internalized GlcCer induced migratory activity in immortalized human endometrial stromal cells (HESCs), with highest potency observed in long-chain GlcCer. Long-chain ceramide (Cer) similarly induced cellular migration and mass spectrometry results revealed that the migratory behavior was contributed through glycosylation of ceramides. Cells treated with GlcCer synthase inhibitor, or RNAi-mediated knockdown of glucosylceramide synthase (GCS), the enzyme catalyzing GlcCer production attenuated cell motility. Mechanistic studies showed that GlcCer acts through stromal cell-derived factor-1 alpha and its receptor, CXC chemokine receptor 4 (SDF-1α-CXCR4) signaling axis and is dependent on phosphorylation of LYN kinase at Tyr396, and dephosphorylation of Tyr507. Migration was prominently attenuated in cells exposed to CXCR4 antagonist, AMD3100, yet can be rescued with diprotin A, which prevents the degradation of SDF-1α. Furthermore, blocking of LYN kinase activity in the presence of SDF-1α and GlcCer reduced HESC migration, suggesting that LYN acts downstream of GlcCer-SDF-1α-CXCR4 axis as part of its intracellular signal transduction. Our results reveal a novel role of long-chain GlcCer and the dialog between GlcCer, LYNpTyr396 and SDF-1α-CXCR4 in inducing HESC migration. This finding may improve our understanding how endometriotic lesions invade to their ectopic sites, and the possibility of using GlcCer to modulate the SDF-1α-CXCR4-LYNpTyr396 axis in endometriosis.

Konjac Ceramide (kCer) Regulates NGF-Induced Neurite Outgrowth via the Sema3A Signaling Pathway.

The tuber of the konjac plant is a source enriched with GlcCer (kGlcCer), and has been used as a dietary supplement to improve the dry skin and itching that are caused by a deficiency of epidermal ceramide. Previously, we showed chemoenzymatically prepared konjac ceramide has a neurite-outgrowth inhibitory effect that is very similar to that of Sema3A and is not seen with animal-type ceramides. While, it has been unclear whether kCer may act on Sema3A or TrkA signaling pathway. In the present study, we showed kCer induces phosphorylation of CRMP2 and microtubules depolymerization via Sema3A signaling pathway not TrkA. It is concluded that kCer may be a potential Sema3A-like agonist that activates Sema3A signaling pathway directly.

Characterization of Konjac Ceramide (kCer) Binding to Sema3A Receptor Nrp1.

Konjac ceramide (kCer) can be prepared by a chemoenzymatic method as previously published (Usuki, S.; Tamura, N.; Sakai, S.; Tamura, T.; Mukai, K.; Igarashi, Y. Biochem. Biophys. Rep. 5, 160-167 (2016)). Thus prepared kCer showed an activation effect on Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerizaion, resulting in opposing NGF-induced neurite outgrowth. In the present study, we have shown that kCer is a potential Sema3A-like ligand that has a competitive effect on Sema3A binding to a cell surface receptor Nrp1, but animal-type ceramides have no effect on Sema3A binding to Nrp1. In addition, kCer showed a direct molecular interaction with Nrp1, but animal-type ceramides, C16Cer, C18Cer, and C24Cer show no specific bindings to Nrp1. Further, kCer showed an additive effect to activate the Sema3A signaling pathway together with low-dose Sema3A but a reversed effect to inhibit this pathway when combined with high-dose Sema3A.

Effects of Plant Sphingolipids on Inflammatory Stress in Differentiated Caco-2 Cells.

To determine the mechanism underlying the anti-inflammatory effects of plant sphingolipids, especially plant glucosylceramide (GlcCer), the effects of plant sphingolipids on inflammatory stress in differentiated Caco-2 cells were compared to those of a sphingolipid of animal origin, galactosylceramide (GalCer). Addition of GlcCer or GalCer suppressed cell injury caused lipopolysaccharide (LPS)- and TNF-α-induced inflammatory stress and induction of apoptosis in differentiated Caco-2 cells. There was no difference in the suppressive effect between GlcCer and GalCer. The inflammatory cytokines and chemokines induced by LPS were suppressed by GlcCer. GlcCer remained on the cell surface. The results of this study can be summarized as follows: 1) sphingolipids such as GlcCer have potent anti-inflammatory effects; 2) GlcCer suppresses LPS-induced production of cytokines and apoptosis; 3) sphingolipids may remain on the surface of cells, and 4) the chemical properties of sphingolipids may prevent the interaction between LPS and its receptor.

Recovery Effects of Oral Administration of Glucosylceramide and Beet Extract on Skin Barrier Destruction by UVB in Hairless Mice.

Purified glucosylceramide from beet extract (beet GlcCer) and beet extract containing an equal amount of GlcCer were administered orally to ultra violet B (UVB)-irradiated mice, and differences in the protective effects against skin barrier dysfunction caused by UVB irradiation were compared. In the beet GlcCer group, epidermal thickening and the decrease in stratum corneum (SC) ceramide content caused by UVB irradiation were reduced. In the group that was orally administered beet extract containing glucosylceramide, effects similar to those in the beet GlcCer group were observed. Oral administration of beet GlcCer had no obvious effects against an increase in TEWL or decrease in SC water content after UVB irradiation, but there was improvement in the beet extract group. Oral administration of beet GlcCer is effective in improving skin barrier function in UVB-irradiated mice. Beet extract contains constituents other than GlcCer that are also effective in improving skin barrier function.

Permeability Barrier and Microstructure of Skin Lipid Membrane Models of Impaired Glucosylceramide Processing.

Ceramide (Cer) release from glucosylceramides (GlcCer) is critical for the formation of the skin permeability barrier. Changes in ß-glucocerebrosidase (GlcCer'ase) activity lead to diminished Cer, GlcCer accumulation and structural defects in SC lipid lamellae; however, the molecular basis for this impairment is not clear. We investigated impaired GlcCer-to-Cer processing in human Cer membranes to determine the physicochemical properties responsible for the barrier defects. Minor impairment (5-25%) of the Cer generation from GlcCer decreased the permeability of the model membrane to four markers and altered the membrane microstructure (studied by X-ray powder diffraction and infrared spectroscopy), in agreement with the effects of topical GlcCer in human skin. At these concentrations, the accumulation of GlcCer was a stronger contributor to this disturbance than the lack of human Cer. However, replacement of 50-100% human Cer by GlcCer led to the formation of a new lamellar phase and the maintenance of a rather good barrier to the four studied permeability markers. These findings suggest that the major cause of the impaired water permeability barrier in complete GlcCer'ase deficiency is not the accumulation of free GlcCer but other factors, possibly the retention of GlcCer bound in the corneocyte lipid envelope.

Glucosylceramide Critically Contributes to the Host Defense of Cystic Fibrosis Lungs.

BACKGROUND: Cystic fibrosis (CF) is the most common autosomal-recessive disorder in western countries. Previous studies have demonstrated an important role of sphingolipids in the pathophysiology of cystic fibrosis. It has been shown that ceramide has a central role in various pulmonary infections, including those with Pseudomonas aeruginosa (P. aeruginosa). Ceramide is accumulated in the airways of CF mice and patients. However, little is known about a potential role of glucosylceramide in cystic fibrosis. METHODS: We investigated the expression of glucosylceramide and lactosylceramide in the respiratory tract of murine and human CF samples by immunohistochemistry and analyzed effects of glucosylceramide on P. aeruginosa in vitro. We performed pulmonary infections with P. aeruginosa and tested inhalation with glucosylceramide. RESULTS: We demonstrate that glucosylceramide is down-regulated on the apical surface of bronchial and tracheal epithelial cells in cystic fibrosis mice. Although glucosylceramide did not have a direct bactericidal effect on Pseudomonas aeruginosa in vitro, inhalation of CF mice with glucosylceramide protected these mice from infection with P. aeruginosa, while non-inhaled CF mice developed severe pneumonia. CONCLUSION: Our data suggest that glucosylceramide acts in vivo in concert with ceramide and sphingosine to determine the pulmonary defense against P. aeruginosa.