Characterization of Ceramides and Glucosylceramides of the Satsuma Mandarin(Citrus unshiu) Fruit.

Ceramide (Cer) and glucosylceramide (GlcCer) were isolated from Satsuma mandarin (Citrus unshiu) fruits and characterized. 2,3-Dihydroxy fatty acids with C20 or longer acyl chains were found in Cer. GlcCers from the flesh of the fruit contained sphingosine (4-trans-sphingenine) as a major component. Notably, the Cer content was 1.5-fold higher than GlcCer content. The ratio of Cer plus GlcCer to the total lipid content in Satsuma mandarin was higher than that in the other citrus fruits analyzed in this study. Collectively, the pomace of the Satsuma mandarin fruit can be a good source of sphingolipids as functional components in foods.

(4Z,15Z)-Octadecadienoic Acid Inhibits Glycogen Synthase Kinase-3ß and Glucose Production in H4IIE Cells.

Many uncommon non-methylene-interrupted fatty acids (NMI FA) are present in limpet gonads, but their biological properties remain unknown. To investigate new biological effects of naturally occurring NMI FA in eukaryotic cells, the biological activities of structurally analogous (4Z,15Z)-octadecadienoic acid (1), (9Z,20Z)-tricosadienoic acid (2), and (12Z,23Z)-hexacosadienoic acid (3) were examined by using a yeast-based drug-screening system using the Ca2+-sensitive mutant strain, Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ). Among 1-3, 1 showed restored growth activity at a dose of 80 µg/disc in the mutant yeast strain. This phenotype suggests that 1 suppresses Ca2+-signaling of the mutant yeast through inhibition of glycogen synthase kinase-3ß (GSK-3ß) or calcineurin pathways or both. From this result, the inhibitory activity of 1-3 against GSK-3ß was further determined. 1-3 showed potent inhibitory activity against GSK-3ß with IC50 values ranging from 8.7 to 21.9 µM. Inhibition of GSK-3ß reduces gene expression of the gluconeogenic key enzymes in liver, so we analyzed glucose production in rat hepatoma H4IIE cells to assess GSK-3ß inhibitory activity of 1-3. Acid 1 inhibited glucose production at 25 µM in H4IIE cells. Our results would open up new possibilities for an anti-diabetic effect of 1 and might provide important insights into understanding the biological properties of naturally occurring NMI FA.

Novel Odd-Chain Fatty Acids with a Terminal Double Bond in Ovaries of the Limpet Cellana toreuma.

Our previous study characterized highly diverse dienoic fatty acids (FA), in particular an uncommon non-methylene-interrupted (NMI) FA, in the ovaries of the Japanese limpet Cellana toreuma belonging to the archaeogastropods, but many minor chemically unidentified FA remain. In this study, among previously unidentified FA (less than 0.1% of total FA), four novel NMI FA with a terminal double bond [7,18-nonadecadienoic (19:2Δ7,18), 11,18-nonadecadienoic (19:2Δ11,18), 7,20-heneicosadienoic (21:2Δ7,20), and 11,20-heneicosadienoic (21:2Δ11,20) acids] were found, along with known 14-pentadecenoic (15:1Δ14), 16-heptadecenoic (17:1Δ16), and 9,18-nonadecadienoic (19:2Δ9,18) acids, based on capillary GC-MS of their methyl esters, 3-pyridylcarbinol derivatives, and argentation thin-layer chromatography. From our findings, possible biosynthetic pathways for the novel FA are discussed.

An Unprecedented Occurrence of Δ5,9- and Δ9,15-Dienoic Fatty Acids in Ovaries of the Archaeogastropod Limpet Cellana toreuma.

A detailed structural diversity of dienoic fatty acids (FA), including non-methylene-interrupted dienoic FA, of triacylglycerols and polar lipids in ovaries of Cellana toreuma was clarified for the first time by using capillary gas chromatography-mass spectrometry of their 3-pyridylcarbinol esters and argentation thin-layer chromatography. Interestingly, in addition to 5,9-octadecadienoic (18:2Δ5,9), 5,9-eicosadienoic (20:2Δ5,9), 5,9-heneicosadienoic (21:2Δ5,9), 5,9-docosadienoic (22:2Δ5,9), 5,9-tricosadienoic (23:2Δ5,9), and 5,9-tetracosadienoic (24:2Δ5,9) acids, previously identified in ovaries of C. grata, rare FA 5,9-hexadecadienoic (16:2Δ5,9), 5,9-nonadecadienoic (19:2Δ5,9), and 21-methyl-5,9-docosadienoic (iso 23:2Δ5,9) were newly recognized in ovaries of C. toreuma. Detectable amounts of four Δ9,15-dienoic FA were present in the ovary lipids. The FA identified were one novel 9,15-heneicosadienoic (21:2Δ9,15) acid and known 9,15-docosadienoic (22:2Δ9,15), 9,15-tricosadienoic (23:2Δ9,15), and 9,15-tetracosadienoic (24:2Δ9,15) acids. The findings help to explain the broad evidence of the structural diversity in marine gastropods and suggest biomarkers to evaluate marine food web relations.

Cytoprotective Effects of Lysophospholipids from Sea Cucumber Holothuria atra.

Lysophospholipids are important signaling molecules in animals and metazoan cells. They are widely distributed among marine invertebrates, where their physiological roles are unknown. Sea cucumbers produce unique lysophospholipids. In this study, two lysophospholipids were detected in Holothuria atra for the first time, lyso-platelet activating factor and lysophosphatidylcholine, with nuclear magnetic resonance and liquid chromatography-time-of-flight mass spectrometric analyses. The lipid fraction of H. atra contained lyso-platelet activating factor and lysophosphatidylcholine, and inhibited H2O2-induced apoptosis in the macrophage cell line J774A.1. The antioxidant activity of the lysophospholipid-containing lipid fraction of H. atra was confirmed with the oxygen radical absorbance capacity method. Our results suggest that the lysophospholipids from H. atra are potential therapeutic agents for the inflammation induced by oxidative stress.

Effects of Dietary Plant-Origin Glucosylceramide on Bowel Inflammation in DSS-Treated Mice.

The effects of dietary plant-origin glucosylceramide (GlcCer) on symptoms similar to those of inflammatory bowel diseasewere investigated in dextran sulfate sodium salt (DSS)-treated mice. Dietary GlcCer suppressed decreases in body weight due to DSS administration. To determine its effects on the colon, we examined its surface under a microscope following toluidine blue staining. Dietary GlcCer decreased DSS-induced chorionic crypt injury and elevated myeloperoxidase levels. Moreover, dietary GlcCer significantly suppressed the production of cytokines by the intestinal mucosa. These results provide evidence for the suppression of DSS-induced inflammation by dietary GlcCer.

Determination of carbon isotopic measurement conditions for ceramide in skin using gas chromatography-combustion-isotope ratio mass spectrometry.

The ceramide (Cer) content of skin and glucosylceramide (GlcCer) intake affect skin moisture conditions, but their mutual relation in skin remains unclear. For clarification of that mutual relation, carbon stable isotopes ((12)C and (13)C) are useful as a tracer. However, carbon isotopic measurement has not been applied to the study of clarifying their skin moisturizing effects. Therefore, we used gas chromatography / combustion / isotope ratio mass spectrometry (GC-C-IRMS) to ascertain the appropriate conditions for carbon isotopic measurements using synthesized Cer (SCer) in substitution for very low concentrations of Cer in skin. SCer was derivatized to trimethylsilylated SCer (TMS-SCer) quantitatively using N-trimethylsilylimidazole (TMSI) depending on the amount of SCer. The derivatization rates were 75-85%. Excess TMSI was removed using three cycles of hexane-water distribution. Under these conditions, carbon isotopic measurements of TMS-SCer conducted using GC-C-IRMS showed high repeatability and good inter-day variation (S.D. < 0.3‰). The carbon stable isotope ratio value (δ(13)C) of SCer calculated using a mass balance equation was compared with δ(13)C of underivatized SCer, which was regarded as the actual δ(13)C of SCer obtained using sealed tube combustion method. The difference between the calculated δ(13)C of SCer and δ(13)C of the underivatized SCer depended on the TMSI reagent supplier and on the number of hydroxyl groups to be derivatized in SCer. For accurate δ(13)C of Cer in skin using GC-C-IRMS, the measured δ(13)C of a target TMS-Cer must be calculated using a correction factor representing the difference in δ(13)C of underivatized standard SCer from that of TMS-standard SCer having a structure resembling that of the target Cer in skin. In addition, we show that the same lot of TMSI reagent from a specific supplier must be used throughout the experiments.

The Effect of Oral Intake of Low-Temperature-Processed Whey Protein Concentrate on Colitis and Gene Expression Profiles in Mice.

Inflammatory bowel disease (IBD) is an autoimmune disease of unknown etiology and can lead to inflammation and cancer. Whey proteins contain many bioactive peptides with potential health benefits against IBD. We investigated the effect of low-temperature-processed whey protein concentrate (LWPC) on the suppression of IBD by using a dextran sodium sulfate (DSS)-induced colitis model in BALB/c mice. Oral intake of LWPC resulted in improved recovery of body weight in mice. Histological analysis showed that the epithelium cells of LWPC-treated mice were healthier and that lymphocyte infiltration was reduced. The increase in mucin due to the LWPC also reflected reduced inflammation in the colon. Transcriptome analysis of the colon by DNA microarrays revealed marked downregulation of genes related to immune responses in LWPC-fed mice. In particular, the expression of interferon gamma receptor 2 (Ifngr2) and guanylate-binding proteins (GBPs) was increased by DSS treatment and decreased in LWPC-fed mice. These findings suggest that LWPCs suppress DSS-induced inflammation in the colon by suppressing the signaling of these cytokines. Our findings suggest that LWPCs would be an effective food resource for suppressing IBD symptoms.

Effect of dietary Maitake (Grifola frondosa) mushrooms on plasma cholesterol and hepatic gene expression in cholesterol-fed mice.

To investigate the effects of dietary Grifola frondosa on cholesterol, normal mice were fed a diet containing 1% cholesterol (HC group) or 1% cholesterol and 10% freeze-dried G. frondosa powder (HC+G group) for 4 weeks and hepatic and plasma lipid levels were compared with those of a cholesterol-free diet-fed mice (N group). Hepatic total cholesterol (TC), triacylglycerol contents were considerably increased and plasma TC / phospholipid (PL) was also increased significantly in the HC group compared with the N group. However, plasma TC content decreased in the HC+G group compared with the HC group. To characterize the mechanisms responsible for lowered plasma cholesterol in G. frondosa-supplemented mice, hepatic gene expression was profiled using DNA microarray and gene ontology. Genome analyses revealed that de novo cholesterol synthesis genes were suppressed following cholesterol intake. However, expression of bile acid biosynthesis and low-density lipoprotein receptor genes showed little change. Scarb1, Abcg5, and Abcg8, involved in cholesterol transport and excretion, were slightly upregulated in the HC+G group compared with the HC group. These data indicate the plasma cholesterol-lowering effect of G. frondosa. Moreover, fatty acid (FA) ß-oxidation was promoted via adipocytokine signaling pathways, and Saa, encodes serum amyloid A related to arteriosclerosis, was suppressed in the HC+G group.

Efficient conversion of crude glycerol into triacylglycerol by the yeast Pseudozyma sp. TYC-2187 for biodiesel production.

Crude glycerol is a byproduct of biodiesel production. In this study, we isolated a yeast strain that grows vigorously using crude glycerol as a carbon source, and tested the effects of culture conditions on triacylglycerol (TG) production. TYC-2187, isolated from wild grapes and identified as Pseudozyma sp., converted high-concentration crude glycerol into TG more efficiently than other strains of the genus Pseudozyma. Sequence analysis of the 26S rRNA gene D1/D2 domain indicated that TYC-2187 diverged from other strains of the genus Pseudozyma. Optimum culture temperature and optimum initial pH of culture medium, that maximized TG yield of TYC-2187, were 25°C and pH 6.0, respectively. Optimum nitrogen source was yeast extract and optimum concentration was 10 g/L. Optimum concentration of glycerol, when the yeast was cultured for 48 h under optimum conditions, was 80 g/L. TG yield peaked at 15.7 g/L at 48 h and then remained at this level until 66 h. In addition, much of the glycerol in the medium had been consumed by 48 h. Fatty acid composition of TG produced by TYC-2187, including linolenic acid methyl ester content, iodine value, and cetane number, complied with biodiesel standards. These results suggest that the TYC-2187 yeast strain is superbly able to produce TG that is suitable for biodiesel production using crude glycerol.

Distribution of unusual cholesterol precursors, 4-methyl- and 4, 4-dimethylsterols with δ8 unsaturation, in gonads of marine archaeogastropods.

In the present study, we show that four unusual cholesterol precursors, 5α-cholesta-8,14-dien-3ß-ol, 4-methyl-5α-cholesta-8,24-dien-3ß-ol (4-methylzymosterol), 4,4,14-trimethyl-5α-cholesta-8,24-dien-3ß-ol (lanosterol), and 4,4-dimethyl-5α-cholesta-8,24-dien-3ß-ol (4,4-dimethylzymosterol) are present in testes of the limpet Cellana grata, which is part of one of the most primitive gastropod families (Nacelllidae Family). The distribution of these sterols in testes and ovaries of four dominant species of limpets, Cellana grata, Cellana toreuma, Nipponacmea concinna, and Nipponacmea fuscoviridis, was examined by capillary gas chromatography-mass spectrometry. Based on the data, we discuss about the biological roles and possible application as bio-resources of these sterols. This is the first identification of 5α-cholesta-8, 14-dien-3ß-ol, 4-methylzymosterol, and 4,4-dimethylzymosterol in marine invertebrate gonads.

Novel heneicosadienoic and tricosadienoic acid isomers in ovaries of marine archaeogastropods.

To investigate unusual odd-chain fatty acids (FA) from gonads of archaeogastropods, limpets Cellana grata and Cellana toreuma, a subfraction enriched in FA with two double bonds extracted from the gonads was obtained by using argentation thin-layer chromatography. The resulting fraction was analyzed by using capillary gas chromatography-mass spectrometry of its methyl esters, 3-pyridylcarbinol esters and pyrrolidide derivatives. Six novel all-cis diene isomers were identified as 7,18-heneicosadienoic (21:2Δ7,18), 8,14-tricosadienoic (23:2Δ8,14), 9,15-tricosadienoic (23:2Δ9,15), 7,18-tricosadienoic (23:2Δ7,18), 9,18-tricosadienoic (23:2Δ9,18), and 9,20-tricosadienoic (23:2Δ9,20) acids. In the present study, the differences in the proportion of tricosadienoic acid isomers between the ovary lipids of C. grata and C. toreuma were recognized.

Evaluation of the effects of dietary organic germanium, ge-132, and raffinose supplementation on caecal flora in rats.

Poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) is the most common organic germanium compound. The ingestion of Ge-132 promotes bile secretion. We assessed the rat caecal characteristics after the administration of Ge-132 and raffinose, a prebiotic oligosaccharide, because both Ge-132 and some prebiotics can change the fecal color to yellow. We also compared the changes in the caecal flora caused by the two compounds. In addition, we evaluated the simultaneous administration of Ge-132 and raffinose and their effects on ß-glucuronidase activity, which is known to be a factor related to colon cancer. Male Wistar rats (three weeks old) were given one of the following diets: 1) a control diet (control group), 2) a diet containing 0.05% Ge-132 (Ge-132 group), 3) a diet containing 5% raffinose (RAF group) or 4) a diet containing 0.05% Ge-132 + 5% raffinose (GeRAF group). The Bifidobacterium, Lactobacillus and total bacteria counts were significantly increased by the dietary raffinose, and Ge-132 did not suppress this increase. The raffinose intake increased caecal acetic acid production significantly. The activity of ß-glucuronidase in the caecal contents was increased by dietary Ge-132, whereas dietary raffinose decreased the ß-glucuronidase activity significantly. These results indicate that the simultaneous intake of dietary raffinose and Ge-132 does not inhibit the effects of either compound on intestinal fermentation and bile secretion. Additionally, the simultaneous intake of both raffinose and Ge-132 could abrogate the increase in ß-glucuronidase activity induced by Ge-132 alone.

Mechanism of visceral fat reduction in Tsumura Suzuki obese, diabetes (TSOD) mice orally administered ß-cryptoxanthin from Satsuma mandarin oranges (Citrus unshiu Marc).

The carotenoid ß-cryptoxanthin (ß-CRX) is abundant in Satsuma mandarins (Citrus unshiu Marc). Several studies have shown a relationship between Satsuma mandarin consumption and a low risk of several diseases, for example, diabetes, gout, and hypertension, suggesting ß-CRX involvement in disease prevention. We investigated the effect of ß-CRX on mildly obese males. ß-CRX administration reduced visceral adipose tissue, body weight, and abdominal circumference. However, the detailed mechanism by which ß-CRX mediates these changes remains unknown. To identify this mechanism, we used an obese model mouse (TSOD). Oral ß-CRX administration repressed body weight, abdominal adipose tissue weight, and serum lipid concentrations in TSOD; these results are identical to previous human trial results. ß-CRX administration significantly repressed adipocyte hypertrophy. Gene expression analysis strongly indicated that ß-CRX can alter cytokine secretion and cell proliferation. These results suggest that ß-CRX derived from Satsuma mandarins can help prevent obesity by repressing hypertrophy of abdominal adipocytes.

Differences in sterol composition of gonads of the lottiid limpets Nipponacmea concinna and Nipponacmea fuscoviridis from northeastern Japan.

This is the first report on the sterol composition in Nipponacmea concinna and Nipponacmea fuscoviridis, 2 dominant species of lottiid limpets. There were significant differences in sterol composition between male and female gonads of the limpets. Previous studies have shown that zymostenol and zymosterol are major lipid components of male gonads of the nacellid limpets Cellana grata and Cellana toreuma. In contrast, in this study, only trace amounts of zymosterol were detected in male gonads of N. fuscoviridis.

Profiling of hepatic gene expression of mice fed with edible japanese mushrooms by DNA microarray analysis: comparison among Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus.

To compare and estimate the effects of dietary intake of three kinds of mushrooms (Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus), mice were fed a diet containing 10-14% of each mushroom for 4 weeks. Triacylglycerol in the liver and plasma decreased and plasma cholesterol increased in the P. ostreatus-fed group compared with those in the control group. Cholesterol in the liver was lower in the G. frondosa-fed group than in the control group, but no changes were found in the H. marmoreus-fed group. DNA microarray analysis of the liver revealed differences of gene expression patterns among mushrooms. Ctp1a and Fabp families were upregulated in the P. ostreatus-fed group, which were considered to promote lipid transport and ß-oxidation. In the G. frondosa-fed group, not only the gene involved in signal transduction of innate immunity via TLR3 and interferon but also virus resistance genes, such as Mx1, Rsad2, and Oas1, were upregulated.

Content and fatty acid composition of sulfoquinovosyldiacylglycerol in conifer leaves grown in Hokkaido, Japan.

Sulfoquinovosyldiacylglycerol (SQDG) contents in conifer leaves and their fatty acid (FA) compositions were determined. The SQDG content was 16-36 mg/100 g, and was high in Picea glehnii. Palmitic and alpha-linolenic acid were the usually predominant FAs. In Picea, the proportion of alpha-linolenic acid was low, and those of oleic and linoleic acid were high. The essential oil residues of Abies sachalinensis leaves were found to be a potential source of SQDG material.

Dietary sphingolipids ameliorate disorders of lipid metabolism in Zucker fatty rats.

Dietary sphingolipids (SL) inhibit colon carcinogenesis, reduce serum cholesterol, and improve skin barrier function and are considered to be "functional lipids". For comparative determination of the effects of SL with different chemical compositions on lipid metabolism and its related hepatic gene expression, Zucker fatty rats were fed pure sphingomyelin (SM) of animal origin and glucosylceramide (GC) of plant origin. After 45 days, the SM and GC diets led to significant reductions in hepatic lipid and plasma non-HDL cholesterol. Both SM and GC diets decreased plasma insulin levels, whereas only the GC diet increased the plasma adiponectin level. Hepatic gene expression analysis revealed increased expression of adiponectin receptor 2 (Adipor2), peroxisome proliferator-activated receptor alpha (PPARalpha), and pyruvate dehydrogenase kinase 4 (Pdk4). However, expression of stearoyl CoA desaturase (Scd1) was significantly decreased. These results suggest that dietary SL, even of different origins and chemical compositions, may prevent fatty liver and hypercholesterolemia through improvement of adiponectin signaling and consequent increases in insulin sensitivity.

Invariant Valpha14 natural killer T cell activation by edible mushroom acidic glycosphingolipids.

Invariant natural killer T (iNKT) cells regulate multi-immune response through Th1/Th2 cytokine release triggered by the recognition of CD1d-restricted glycosphingolipid antigens. Here we report that acidic glycosphingolipids (AGLs) of mushroom (Hypsizigus marmoreus and Pleurotus eryngii) presented by murine CD1d-transfected rat basophilic leukocytes induced interleukin-2 (IL-2) release from iNKT hybridoma cells. AGL-1, one of the AGLs, containing mannose at the non-reducing ends, induced CD1d-dependent IL-2 release. Al-though alpha-galactosylceramide (alpha-GalCer) presented by CD11c-positive cells induced both interferon-gamma (IFN-gamma) and IL-4 release, all of AGLs presented by CD11c-positive cells and AGL-1 presented by B cells induced IL-4 release from iNKT hybridoma cells. A single intravenous injection of AGLs into B6 mice induced only a little elevation of IL-4 in serum but repeated intravenous injection of AGLs induced prolonged retention of IL-4 in serum; therefore, these results suggested that edible mushroom AGLs might contribute to the retention of immunohomeostasis through the minimum induction of iNKT cell activation in vivo.

Screening for beneficial effects of oral intake of sweet corn by DNA microarray analysis.

To identify novel functions of the oral intake of sweet corn, we performed DNA microarray analysis of the livers of sweet corn-fed mice. Functional annotation clustering 1600 genes with expression levels that were affected (more than 1.5-fold change) by dietary sweet corn indicated that both cell proliferation and programmed cell death were modulated by sweet corn intake. In the Wnt signaling pathway, which is involved in cell proliferation, the levels of Jun and beta-catenin expression were downregulated by dietary sweet corn. The mRNA levels of Rb and p53, negative regulators of the cell cycle, were increased in mice fed with sweet corn. Dietary corn upregulated expression levels of genes that regulate apoptosis positively (for example, BOK, BID, CASP4). These results suggested that sweet corn is a valuable food for suppressing cancer. Oral administration of sweet corn inhibited tumor growth (36.6% reduce in tumor weight, P < 0.05) in mice inoculated with Ehrlich tumor cells.