Dietary Glucosylceramide Enhances Cornified Envelope Formation via Transglutaminase Expression and Involucrin Production

In this study, we investigated whether dietary glucosylceramide (GlcCer) and its metabolite sphingoid bases, sphingosine (SS), phytosphingosine (PS), sphingadienine (SD) and 4-hydroxysphingenine (4HS), influence cornified envelope (CE) formation. CE is formed during terminal differentiation of the epidermis through crosslinking of specific precursor proteins by transglutaminases (TGases), and is essential for the skin's barrier function. Oral administration of GlcCer (0.25 mg/day) for 14 consecutive days dramatically reduced transepidermal water loss, an indicator of the skin barrier condition, in hairless mice with barrier perturbation induced by single-dose ultraviolet B (UVB) irradiation. The GlcCer treatment also increased the level of TGase-1 mRNA in UVB-irradiated murine epidermis approximately 1.6-fold compared with the control. Further, all four sphingoid bases at 1 μM concentration enhanced CE formation of cultured normal human keratinocyte cells. Among them, SS, PS and SD, but not 4HS, stimulated production of involucrin, one of the CE major precursor proteins. SD increased the expression of TGase-1 mRNA, while SS increased the expression of TGase-3 mRNA. These results indicate that the skin barrier improvement induced by oral GlcCer treatment might be at least partly due to a reinforcement of CE formation in the epidermis mediated by sphingoid bases metabolically derived from GlcCer.     

Oral Glucosylceramide Reduces 2,4-Dinitrofluorobenzene Induced Inflammatory Response in Mice by Reducing TNF-Alpha Levels and Leukocyte Infiltration

Sphingolipids are constituents of cellular membranes and play important roles as second messengers mediating cell functions. As significant components in foods, sphingolipids have been proven to be critical for human health. Moreover, diverse metabolic intermediates of sphingolipids are known to play key roles both in proinflammatory and in anti-inflammatory effects. However, the effect of dietary sphingolipids on inflammation is a complicated field that needs to be further assessed. Our study evaluated the effects of orally administered maize glucosylceramide (GluCer), one of the most conventional dietary sphingolipids, on inflammation using the 2,4-dinitro-1-fluorobenzene-treated BALB/c murine model. Oral administration of GluCer inhibited ear swelling and leukocyte infiltration to the inflammatory site, suggesting that dietary GluCer has anti-inflammatory properties. ELISA analyses revealed that oral administration of GluCer for 6 days had not modified the Th1/Th2 balance, but significantly down-regulated the activation of TNF-α at the inflammatory site. Based on these results, the down-regulation of TNF-α by dietary GluCer may suppress vascular permeability and reduce the migration of inflammatory cells. Our findings increase understanding of the actions of dietary sphingolipids on the balance of the immune response.     

An Improved Method for Analysis of Glucosylceramide Species Having cis-8 and trans-8 Isomers of Sphingoid Bases by LC–MS/MS

High-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) approaches have enabled high selectivity and sensitivity for the identification and quantification of glucosylceramide molecular species. Here we demonstrate that HPLC–ESI–MS/MS is an efficient method for characterizing plant glucosylceramide species having the cis-8 and trans-8 isomers of sphingoid bases. Complete baseline separation was achieved using a high-carbon-content octadecylsilyl column and a simple binary gradient comprising methanol and water. The result of 2-hydroxy fatty acid composition achieved by HPLC–ESI–MS/MS was compared with that achieved by gas chromatography with flame ionization detection (GC–FID), indicating that the two methods yield similar molar compositions. The current method should be applicable to seeking the active components of glucosylceramide species from plant materials in response to biological challenges.     

Sphingolipids in the gut? Which are the important issues?

Glycosphingolipids and sphingomyelin (SM) are important components of the apical brush border and the Golgi and endocytic vesicles of the gut epithelium. In particular, glucosylceramide is abundant in the microvilli. Synthesis and degradation of mucosal sphingolipids and targeting of sphingolipids to distinct cell compartments during cell differentiation are thus important features of intestinal lipid metabolism. Sphingolipids are also present in the ordinary Western diet, and sphingolipid‐rich formulations of dairy, plant or yeast origin are now available for studies of their biological effects in animals and humans. Since sphingolipids account for a large part of the polar lipids in milk, their digestion and effects in the suckling infant is of particular interest. Dietary sphingolipids are slowly digested and the exposure of the whole gut to sphingolipids and their metabolites can be increased by dietary supply. Metabolites from dietary sphingolipids may have anti‐inflammatory and anticarcinogenic effects and undigested sphingolipids may be protective. Dietary sphingolipids inhibit cholesterol absorption and may have beneficial metabolic effects. Some mucosal sphingolipids have blood group A, B and H reactivity and some act as receptors for bacterial toxins and virus. Sphingolipid signaling triggered by mucosal‐bacterial interaction may be important in both the gut and the bronchi.     

Digestion of Ceramide 2-Aminoethylphosphonate,a Sphingolipid from the Jumbo Flying Squid <Emphasis Type="Italic">Dosidicus gigas</Emphasis>, in Mice

Ceramide 2-aminoethylphosphonate (CAEP), a sphingophosphonolipid containing a carbon–phosphorus bond, is frequently found in marine organisms and has a unique triene type of sphingoid base in its structure. CAEP has not been evaluated as a food ingredient, although it is generally contained in Mollusca organisms such as squids and shellfish, which are consumed worldwide. In this study, we aimed to elucidate the effects of CAEP as a food component by evaluating the digestion of CAEP extracted from the skin of the jumbo flying squid Dosidicus gigas. Our results revealed that dietary CAEP was digested to free sphingoid bases via ceramides by the mouse small intestinal mucosa. At pH 7.2, CAEP was hydrolyzed more rapidly than the major mammalian sphingolipid sphingomyelin; however, the hydrolysis of CAEP was similar to that of sphingomyelin at pH 9.0. Thus, the digestion of CAEP may be catalyzed by alkaline spingomyelinase and other enzymes. Our findings provide important insights into the digestion of the dietary sphingophosphonolipid CAEP in marine foods.     

Azide‐Tagged Sphingolipids: New Tools for Metabolic Flux Analysis

Several diseases involve alterations in sphingolipid metabolism, so the development of tools for the analysis of sphingolipid metabolic fluxes is of interest. In this work, ω‐azidosphingolipids 1 – 3 have been synthesized and tested as tracers in live cells. The synthesis starts from (S)‐Garner's aldehyde and uses bromide or tosyloxy precursors for the introduction of the azido group into the sphingoid base. Studies in HGC‐27 cells showed that probes 1 – 3 compete with the natural metabolites and are incorporated into sphingolipid pathways without affecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited by means of click reactions with different azadibenzocyclooctyne tags. This allows the mass spectrometric characterization of azidosphingolipidomes in pooled samples from different cell populations after independent treatments, providing proof of concept of the applicability of this technology in sphingolipid metabolic flux analysis.     

Evaluation of Plant Ceramide Species-Induced Exosome Release from Neuronal Cells and Exosome Loading Using Deuterium Chemistry

The extracellular accumulation of aggregated amyloid-β (Aβ) in the brain leads to the early pathology of Alzheimer’s disease (AD). The administration of exogenous plant-type ceramides into AD model mice can promote the release of neuronal exosomes, a subtype of extracellular vesicles, that can mediate Aβ clearance. In vitro studies showed that the length of fatty acids in mammalian-type ceramides is crucial for promoting neuronal exosome release. Therefore, investigating the structures of plant ceramides is important for evaluating the potential in releasing exosomes to remove Aβ. In this study, we assessed plant ceramide species with D-erythro-(4E,8Z)-sphingadienine and D-erythro-(8Z)-phytosphingenine as sphingoid bases that differ from mammalian-type species. Some plant ceramides were more effective than mammalian ceramides at stimulating exosome release. In addition, using deuterium chemistry-based lipidomics, most exogenous plant ceramides were confirmed to be derived from exosomes. These results suggest that the ceramide-dependent upregulation of exosome release may promote the release of exogenous ceramides from cells, and plant ceramides with long-chain fatty acids can effectively release neuronal exosomes and prevent AD pathology.     

A glucosylceramide with a novel ceramide and three novel ceramides from the basidiomycete <Emphasis Type="Italic">Cortinarius umidicola</Emphasis>

A glucosylceramide with novel ceramide and three novel ceramide homologs were isolated from the basidiomycete Cortinarius umidicola and structurally characterized. The ceramide portion of the glucocerebroside consists of a rare (4E,8E)-9-methyl-4,8-sphingadienine sphingoid base. In contrast, the three ceramide homologs, while having the same sphingoid base, contain as FA residues 2-hydroxydocosanoic acid, 2-hydroxytricosanoic acid, and 2-hydroxytetracosanoic acid.     

Development of an LC–MS/MS Method for the Analysis of Free Sphingoid Bases Using 4-Fluoro-7-nitrobenzofurazan (NBD-F)

The molecular species of sphingoid bases were tagged with the fluorescent amino group reagent, 4-fluoro-7-nitrobenzofurazan (NBD-F). The NBD-sphingoid bases were analyzed by a highly selective and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) technique capable of reliable detection of several fmol of the derivatives. Lipid extracts from plant samples were derivatized with NBD-F, and all nine species of free sphingoid bases present in plant sphingolipids were separated and quantified for the first time; a complete baseline resolution was achieved for cis-8 and trans-8 isomers of sphingoid bases by reversed phase HPLC on a C₁₈ column. The extraction and derivatization procedures and LC–MS/MS method can facilitate the progress of the studies for seeking the active components of sphingoid bases species in response to biological challenges.     

Identification of Glucosylceramides Containing Sphingatrienine in Maize and Rice Using Ion Trap Mass Spectrometry

We characterized the glucosylceramide moieties from maize and rice using liquid chromatography-ion trap mass spectrometry. Glucosylceramides containing 4,8-sphingadienine (d18:2) acylated to hydroxy-fatty acids were detected as the predominant molecules both in maize and in rice. In addition, 4-hydroxy-8-sphingenine (t18:1) and sphingatrienine (d18:3) were found in maize and rice glucosylceramides, and in the case of rice, sphingenine (d18:1) was also detected. Glucosylceramides containing d18:3 were acylated to hydroxyl fatty acids (16–24 carbon atoms). Our results indicate the presence of the triene type of sphingoid base in higher plants.     

Dietary Sphingolipids Contribute to Health via Intestinal Maintenance

As sphingolipids are constituents of the cell and vacuole membranes of eukaryotic cells, they are a critical component acquired from our daily diets. In the present review, we highlight the knowledge regarding how dietary sphingolipids affect our health, particularly our intestinal health. Animal- and plant-derived foods contain, respectively, sphingomyelin (SM) and glucosylceramide (GlcCer) as their representative sphingolipids, and the sphingoid base as a specific structure of sphingolipids also differs depending upon the source and class. For example, sphingosine is predominant among animal sphingolipids, and tri-hydroxy bases are present in free ceramide (Cer) from plants and fungi. Dietary sphingolipids exhibit low absorption ratios; however, they possess various functions. GlcCer facilitates improvements in intestinal impairments, lipid metabolisms, and skin disorders, and SM can exert both similar and different effects compared to those elicited by GlcCer. We discuss the digestion, absorption, metabolism, and function of sphingolipids while focused on the structure. Additionally, we also review old and new classes in the context of current advancements in analytical instruments.     

The glycosylceramides of the nematodeCaenorhabditis elegans contain an unusual,branched-chain sphingoid base

Caenorhabditis elegans was cultured in semi-defined medium containing yeast extract, soy peptone, glucose, hemoglobin, Tween 80, and sitosterol. Monoglycosylceramides were chromatographically purified from nematode extracts. Their structures were elucidated with mass spectrometry, nuclear magnetic resonance spectroscopy, and analysis of methanolysis products of the parent cerebrosides. The glycosylceramides were unusual in that the only long-chain sphingoid base detected was aniso-branched compound with a C-4 double bond (i.e., 15-methyl-2-aminohexadec-4-en-1,3-diol). Glucose was the only sugar moiety detected. The fatty acids consisted of a series of primarily straint-chain, saturated, 2-hydroxylated C₂₀–C₂₆ acids; someiso-branched analogs also occurred. The sphingomyelins ofC. elegans were also hydrolyzed, and the sameiso-branched C₁₇ compound was the only sphingoid base detected. This is the first structural analysis of a nematode glycosphingolipid and the first report of an organism in which the long-chain sphingoid bases are entirelyiso-branched. Fatty acids are represented by a binumeric system in which the first number refers to the chain length, and the second number refers to the number of double bonds.     

Identification of ceramide-phosphorylethanolamine in Oomycete plant pathogens: Pythium ultimum, phytophthora infestans, and Phytophthora capsici

Cellular lipids were extracted from three species of Oomycete plant pathogens (Pythium ultimum, Phytophthora infestans, and Ph. capsici) and analyzed via normal-phase high-performance liquid chromatography with flame-ionization detection. The most abundant polar lipids in each of the three species were the polar membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine, and a phosphosphingolipid that eluted soon after PE. Structural analysis via mass spectrometry and nuclear magnetic resonance spectrometry revealed that the phosphosphingolipid was ceramide phosphorylethanolamine (Cer-PE). The most abundant molecular species of Cer-PE in P. ultimum had a molecular weight of 670.5, contained an unusual 19-carbon branched triunsaturated sphingoid (C19-Δ4, 8, 10, 9-methyl long-chain base) and palmitic acid as the amidelinked fatty acid. The most abundant molecular species of Cer-PE in Ph. infestans has a molecular weight of 714.5, contained a common 16-carbon 1,3 di-OH sphingoid, and erucic (cis 13-docosenoic, C22-Δ13) acid as the amide-linked fatty acid. The Cer-PE in Ph. capsici comprised a mixture of each of the two molecular species found in P. ultimum and Ph. infestans.     

IL-37 Targets TSLP-Primed Basophils to Alleviate Atopic Dermatitis

Atopic dermatitis (AD) represents a severe global burden on physical, physiological and mental health. Innate immune cell basophils are essential for provoking allergic inflammation in AD. However, the roles of novel immunoregulatory cytokine IL-37 in basophils remain elusive. We employed in vitro co-culture of human basophils and human keratinocyte HaCaT cells and an in vivo MC903-induced AD murine model to investigate the anti-inflammatory mechanism of IL-37. In the in vitro model, IL-37b significantly decreased Der p1-induced thymic stromal lymphopoietin (TSLP) overexpression in HaCaT cells and decreased the expression of TSLP receptor as well as basophil activation marker CD203c on basophils. IL-37 could also reduce Th2 cytokine IL-4 release from TSLP-primed basophils ex vivo. In the in vivo model, alternative depletion of basophils ameliorated AD symptoms and significantly lowered the Th2 cell and eosinophil populations in the ear and spleen of the mice. Blocking TSLP alleviated the AD-like symptoms and reduced the infiltration of basophils in the spleen. In CRISPR/Cas9 human IL-37b knock-in mice or mice with direct treatment by human IL-37b antibody, AD symptoms including ear swelling and itching were significantly alleviated upon MC903 challenge. Notably, IL-37b presence significantly reduced the basophil infiltration in ear lesions. In summary, IL-37b could regulate the TSLP-mediated activation of basophils and reduce the release of IL-4. The results, therefore, suggest that IL-37 may target TSLP-primed basophils to alleviate AD.     

Deacetylasperulosidic Acid Ameliorates Pruritus,Immune Imbalance,and Skin Barrier Dysfunction in 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis NC/Nga Mice

The prevalence of atopic dermatitis (AD), a disease characterized by severe pruritus, immune imbalance, and skin barrier dysfunction, is rapidly increasing worldwide. Deacetylasperulosidic acid (DAA) has anti-atopic activity in the three main cell types associated with AD: keratinocytes, mast cells, and eosinophils. Our study investigated the anti-atopic activity of DAA in 2,4-dinitrochlorobenzene-induced NC/Nga mice. DAA alleviated the symptoms of AD, including infiltration of inflammatory cells (mast cells and eosinophils), epidermal thickness, ear thickness, and scratching behavior. Furthermore, DAA reduced serum IgE, histamine, and IgG1/IgG2a ratio and modulated the levels of AD-related cytokines and chemokines, namely interleukin (IL)-1β, IL-4, IL-6, IL-9, IL-10, IL-12, tumor necrosis factor-α, interferon-γ, thymic stromal lymphopoietin, thymus and activation-regulated chemokine, macrophage-derived chemokine, and regulated on activation the normal T cell expressed and secreted in the serum. DAA restored immune balance by regulating gene expression and secretion of Th1-, Th2-, Th9-, Th17-, and Th22-mediated inflammatory factors in the dorsal skin and splenocytes and restored skin barrier function by increasing the expression of the pro-filaggrin gene and barrier-related proteins filaggrin, involucrin, and loricrin. These results suggest DAA as a potential therapeutic agent that can alleviate the symptoms of AD by reducing pruritus, modulating immune imbalance, and restoring skin barrier function.     

Inhibitory effect of topical maize glucosylceramide on skin photoaging in UVA-irradiated hairless mice

We investigated the effect of topical application of the glucosylceramide prepared from maize on photoaged mice. Six-week-old hairless female mice were swabbed on the back with glucosylceramide solution or vehicle, following UVA irradiation for 9 weeks. Wrinkle formation was evaluated at 3, 6, and 9 weeks by skin replica analysis by using a three-dimensional (3-D) imaging system. Moreover, epidermal thickness was analyzed at the end of the experiment. Topical application of glucosylceramide significantly reduced UVA-induced wrinkle formation in the skin as well as epidermal hypertrophy. These results suggest that topical glucosylceramide has an inhibitory effect on UVA-induced photoaging.     

Cyanide as a Powerful Catalyst for Facile Preparation of 2‐Substituted Benzoxazoles via Aerobic Oxidation

A cyanide‐catalyzed synthesis of 2‐substituted benzoxazoles from Schiff bases via aerobic oxidation has been developed. The products from various Schiff bases were obtained in high yields in an open flask under ambient conditions without other external oxidants. We have also developed a simple one‐step protocol for the synthesis of benzoxazoles from aminophenol and the corresponding aldehydes in the presence of cyanide without isolation of imine intermediates.     

Reactivity of Electrogenerated N‐Heterocyclic Carbenes in Room‐Temperature Ionic Liquids. Cyclization to 2‐Azetidinone Ring via C‐3/C‐4 Bond Formation

The intrinsic chemistry of imidazolium‐based room‐temperature ionic liquids, related to the acidity of the C‐2 imidazolium cation, can be modified via cathodic cleavage of the C‐2/hydrogen bond. N‐Heterocyclic carbenes, electrogenerated by electrolysis of imidazolium‐based room‐temperature ionic liquids, are stable bases that are strong enough to deprotonate bromoamides 1a–k yielding the azetidin‐2‐one ring via C‐3/C 4 bond formation. The electrosynthesis of β‐lactams 2a–k has been achieved under mild conditions, elevated yields and avoiding the use of toxic, volatile, molecular solvents.     

Cerebrosides from a Far‐Eastern Glass Sponge Aulosaccus sp.

Nine new cerebrosides 1a–d , 2a , 2b , 3a–c were found in the extract of a Far‐Eastern glass sponge Aulosaccus sp. (class Hexactinellida). These β‐d ‐glucopyranosyl‐(1 → 1)‐ceramides contain sphingoid bases (2S,3S,4R,11Z)‐2‐aminoeicos‐11‐ene‐1,3,4‐triol (in 1a – d ), (2S,3S,4R,13Z)‐2‐aminoeicos‐13‐ene‐1,3,4‐triol (in 2a , b ) and (2S,3S,4R,13S*,14R*)‐2‐amino‐13,14‐methylene‐eicosane‐1,3,4‐triol (in 3a – c ), which are N‐acylated by (2R,15Z)‐2‐hydroxydocos‐15‐enoic (in 1a , 2a , 3a ), (2R,16Z)‐2‐hydroxytricos‐16‐enoic (in 1b , 2b , 3b ), (2R,17Z)‐2‐hydroxytetracos‐17‐enoic (in 1d ) and (2R)‐2‐hydroxydocosanoic (in 1c , 3c ) acids. The monoenoic and cyclopropane‐containing sphingoid bases of compounds 1a–d , 2a , 2b , 3a–c have not been found previously in any sphingolipids. The structures of the cerebrosides were elucidated on the basis of ¹H‐, ¹³C‐NMR spectroscopy, mass spectrometry, optical rotation data and chemical transformations. A simplified method for the assignment of the absolute configuration of 2‐hydroxy fatty acids by GC analysis of their (2R)‐ and (2S)‐oct‐2‐yl esters was proposed.     

Protective effect of dietary perilla oil on allergic inflammation in asthmatic mice

Perilla oil (PER) is rich in α‐linolenic acid (n‐3 fatty acid). To unravel the effects of dietary PER on allergic asthmatic inflammation, three kinds of dietary oil, including PER, corn oil (COR), and perilla compound oil (50% PER and 50% COR), were used for replacing the oil in an AIN76 feed consumed by ovalbumin (OVA)‐sensitized and challenged mice continuously for 5 wk. T‐helper type 1 lymphocyte (Th1)/T‐helper type 2 lymphocyte (Th2) and pro‐/anti‐inflammatory cytokines secreted by the cells from the airway, the lungs, and the spleen of experimental mice were determined by ELISA. The results showed that dietary PER inhibited interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α secretions by lipopolysaccharide (LPS)‐stimulated lung cells, as well as interferon (IFN)‐γ and IL‐6 secretions by LPS‐stimulated splenocytes. Perilla compound oil increased the secretion ratio of IFN‐γ/IL‐5 (Th1/Th2 cytokines) in LPS‐stimulated bronchoalveolar lavage fluid cells, but decreased the ratio of IL‐6/IL‐10 (pro‐/anti‐inflammatory cytokines) in LPS‐stimulated splenocytes. The present study demonstrated that dietary PER and its compound oil protected the airways, the lungs, and the spleen from allergic inflammation in OVA‐challenged asthmatic mice, suggesting that an appropriate n‐6/n‐3 fatty acid ratio at a ratio of 1:1 or less in dietary oil may be beneficial to improve the Th2‐skewed allergic asthmatic inflammation. Practical applications: The present study demonstrated that dietary PER and its compound oil protected the airways, the lungs, and the spleen from allergic inflammation in OVA‐challenged asthmatic mice, suggesting that an appropriate n‐6/n‐3 fatty acid ratio at a ratio of 1:1 or less in dietary oil may be beneficial to improve the Th2‐skewed allergic asthmatic inflammation.