Naoko Moriya

Omics Studies of the Murine Intestinal Ecosystem Exposed to Subchronic and Mild Social Defeat Stress

The microbiota-gut-brain axis plays an important role in the development of stress-induced mental disorders. We previously established the subchronic and mild social defeat stress (sCSDS) model, a murine experimental model of depression, and investigated the metabolomic profiles of plasma and liver. Here we used omics approaches to identify stress-induced changes in the gastrointestinal tract. Mice exposed to sCSDS for 10 days showed the following changes: (1) elevation of cholic acid and reduction of 5-aminovaleric acid among cecal metabolites; (2) downregulation of genes involved in the immune response in the terminal ileum; (3) a shift in the diversity of the microbiota in cecal contents and feces; and (4) fluctuations in the concentrations of cecal metabolites produced by gut microbiota reflected in plasma and hepatic metabolites. Operational taxonomic units within the family Lachnospiraceae showed an inverse correlation with certain metabolites. The social interaction score correlated with cecal metabolites, IgA, and cecal and fecal microbiota, suggesting that sCSDS suppressed the ileal immune response, altering the balance of microbiota, which together with host cells and host enzymes resulted in a pattern of accumulated metabolites in the intestinal ecosystem distinct from that of control mice.

Ingestion of medium chain fatty acids by lactating dairy cows increases concentrations of plasma ghrelin.

The purpose of this study was to elucidate the effects of medium-chain fatty acids (MCFAs) on plasma ghrelin concentration in lactating dairy cows. Five early-lactating Holstein cows were randomly assigned to 2 dietary treatments in a crossover design with 2-wk periods. Treatments consisted of diets supplemented or not (control) with calcium salts of MCFAs (MCFA-Ca; 1.5% dry matter). Plasma hormone and metabolite concentrations in blood samples taken from the jugular vein were measured on the morning of feeding on day 14 of each period. Dry matter intake, milk protein, and lactose content of cows fed the MCFA-Ca diet were decreased compared with controls, but with no change in milk yield. Plasma ghrelin concentrations were higher in cows fed the MCFA-Ca diet; however, no significant effect was found on glucagon-like peptide-1 concentrations in plasma. Plasma insulin concentrations decreased, but plasma glucagon concentrations remained unchanged in cows fed the MCFA-Ca diet. The concentrations of nonesterified FAs, total cholesterol, and β-hydroxybutyrate in plasma increased in these cows. In conclusion, dietary MCFAs increase the plasma ghrelin concentrations in lactating dairy cows.

Nitric oxide suppresses preadipocyte differentiation in 3T3-L1 culture

Nitric oxide (NO) is an important chemical messenger controlling many physiological functions, involving cell proliferation, and differentiation. The purpose of this study was to investigate the effect of NO on adipocyte differentiation using a murine preadipocyte cell line, 3T3-L1. The treatment with a NO donor, 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), reduced some markers of adipocyte differentiation such as glycerol-3-phosphate dehydrogenase activity, and intracellular lipid accumulation. To examine whether these effects of NOC18 on adipocyte differentiation markers are due to its cytotoxity, lactate dehydrogenase (LDH) release from the cells were measured. NOC18 did not affect LDH release into the culture medium. Thus, the suppressive actions of NO donor were unlikely to result from its cytotoxicity. Peroxisome proliferator-activated receptor (PPAR) γ is a critical transcription factor for adipocyte differentiation and adipocyte fatty acid binding protein (aP2) gene is one of its targets. Protein expression of PPARγ was not diminished by NOC18 treatment, although mRNA expression of aP2 was reduced. Electrophoretic mobility shift assay showed that NOC18 interfered with the DNA binding activity of PPARγ. Therefore, the present experiment suggest that NO suppresses adipocyte differentiation through suppressing the transcriptional activity of PPARγ, without suppressing its expression level.

Effects of calcium salts of long-chain fatty acids and rumen-protected methionine on plasma concentrations of ghrelin, glucagon-like peptide-1 (7 to 36) amide and pancreatic hormones in lactating cows

Our objective was to determine the effects of calcium salts of long-chain fatty acids (CLFAs) and rumen-protected methionine (RPM) on plasma concentrations of ghrelin, glucagon-like peptide-1 (7 to 36) amide, and pancreatic hormones in lactating cows. Four Holstein cows in midlactation were used in a 4 by 4 Latin square experiment in each 2-wk period. Cows were fed corn silage-based diets with supplements of CLFAs (1.5% added on dry matter basis), RPM (20 g/d), CLFAs plus RPM, and without supplement. Jugular blood samples were taken from 1 h before to 2 h after morning feeding at 10-min intervals on day 12 of each period. CLFAs decreased dry matter intake, but RPM did not affect dry matter intake. Both supplements of CLFAs and RPM did not affect metabolizable energy intake and milk yield and composition. Plasma concentrations of NEFAs, triglyceride (TG), and total cholesterol (T-Cho) were increased with CLFAs alone, but increases of plasma concentrations of TG and T-Cho were moderated by CLFAs plus RPM. Calcium salts of long-chain fatty acids increased plasma ghrelin concentration, and the ghrelin concentration with CLFAs plus RPM was the highest among the treatments. Plasma concentrations of glucagon-like peptide-1, glucagon, and insulin were decreased with CLFAs, whereas adding RPM moderated the decrease of plasma glucagon concentration by CLFAs. These results indicate that the addition of methionine to cows given CLFAs increases plasma concentrations of ghrelin and glucagon associated with the decrease in plasma concentrations of TG and T-Cho.

Effects of amino acids infused into the vein on ghrelin-induced GH, insulin and glucagon secretion in lactating cows

To investigate the effects of amino acids on ghrelin-induced growth hormone (GH), insulin and glucagon secretion in lactating dairy cattle, six Holstein cows were randomly assigned to two infusion treatments in a cross-over design. Mixture solution of amino acids (AMI) or saline (CON) was continuously infused into the left side jugular vein via catheter for 4 h. At 2 h after the start of infusion, synthetic bovine ghrelin was single injected into the right side jugular vein through the catheter. Ghrelin injection immediately increased plasma GH, glucose and non-esterified fatty acids (P<0.05) with no difference between both treatments. Additionally, plasma insulin and glucagon concentrations were increased by ghrelin injection in both treatments. The peak value of plasma insulin concentration was greater in AMI compared with CON (P<0.05). Plasma glucagon concentration showed no difference in the peak value reached at 5 min between both treatments, and then the plasma levels in AMI compared with CON showed sustained higher values (P<0.05). After plasma glucose concentration reached the peak, the decline was greater in AMI compared with CON (P<0.05). These results showed that the increased plasma amino acids may enhance ghrelin action which in turn enhances insulin and glucagon secretions in lactating cows.

New lactic acid bacterial strains from traditional Mongolian fermented milk products have altered adhesion to porcine gastric mucin depending on the carbon source

Attachment of lactic acid bacteria to the mucosal surface of the gastrointestinal tract is a major property of probiotics. Here, we examined the ability of 21 lactic acid bacterial strains isolated from traditional fermented milk products in Mongolia to adhere to porcine gastric mucin in vitro. Higher attachment was observed with Lactobacillus delbrueckii subsp. bulgaricus strains 6-8 and 8-1 than with Lactobacillus rhamnosus GG (positive control). Lactococcus lactis subsp. cremoris strain 7-1 adhered to mucin as effectively as did strain GG. Heat inactivation decreased the adhesive ability of strains 6-8 and 8-1 but did not affect strain 7-1. The adhesion of strains 6-8, 7-1 and 8-1 was significantly inhibited when the cells were pretreated with periodate and trypsin, indicating that proteinaceous and carbohydrate-like cell surface compounds are involved in the adhesion of these strains. The adhesion of strain 7-1 was affected by the type of carbohydrate present in the growth medium, being higher with fructose than with lactose, galactose or xylose as the carbon source. The sugar content of 7-1 cells grown on various carbohydrates was negatively correlated with its adhesive ability. We provide new probiotic candidate strains and new information regarding carbohydrate preference that influences lactic acid bacterial adhesion to mucin.

Differential localization of lingual antimicrobial peptide in the digestive tract mucosal epithelium of calves.

Immunolocalization of lingual antimicrobial peptide (LAP), a member of the β-defensin family, in the digestive tract of calves was investigated to determine its distribution in the digestive tract of Holstein-Friesian calves. Various regions of the digestive tract were collected from slaughtered calves, and fixed in 10% formalin in PBS. Paraffin sections were stained with anti-LAP antibody, followed by visualization of immunoreactions products utilizing the avidin-biotin complex method. Expression of LAP mRNA was analyzed with reverse transcription-PCR. Immunoreactive LAP was localized in the stratum corneum of the stratified squamous epithelium of the tongue, esophagus, rumen, reticulum and omasum but not in their basal layer and lamina propria. In the gastric glands of the abomasum, only chief cells showed LAP positive reaction at the apical side of their cytoplasm. Lamina propria and Peyers patch of the ileum had some leukocyte-like cells immunopositive for LAP. Weak immunoreaction of LAP was also detected in the mucosal epithelium of the intestinal gland of the cecum, colon and rectum. All regions of digestive tract showed LAP mRNA expression with PCR. These results indicate differential localization of LAP in the mucosal epithelium of digestive tracts in calves. The LAP expressed in stratum corneum of stratified squamous epithelium and chief cells of abomasum specifically may play role in the innate immune function in these tissues.

Influence of orally administered bovine lactoferrin on lipid metabolism in lipopolysaccharide-injected preruminant calves.

ABSTRACT The aim of this study was to investigate the influence of oral lactoferrin (LF) administration on lipid metabolism changes in calves given lipopolysaccharide (LPS). Twenty-one 4-day-old Holstein calves were divided into three groups, with each group receiving one of three oral doses of LF (0, 1, 3 g/day) for 10 consecutive days (day -10 to day -1). All calves were intravenously injected with LPS (50 ng/kg BW) on day 0, the day after LF treatment ended. Plasma triglyceride concentrations were lower (P < 0.05) in the LF-treated calves than in the control calves given 0 g/day of LF at 12 and 24 h after LPS injection. Plasma NEFA concentrations were elevated between 6 and 24 h after LPS treatment. At 12 h, the concentration of plasma NEFA was lower (P < 0.05) in the calves given LF 3 g/day than in the control calves. On day 0, plasma total cholesterol and phospholipid concentrations tended to be lower in the LF groups administered 1 and 3 g of LF/day than in the control group, but did not differ significantly among the groups. The plasma very-low-density and low-density lipoprotein concentrations were lower (P < 0.05) at 12, 24, and 72 h in the LF groups than in the control calves. The concentrations of plasma high-density lipoprotein tended to be lower in the LF groups than in the control group between day 0 and 96 h, though there were no significant group differences. The concentration of plasma interleukin-1beta was lower (P < 0.05) in the calves fed LF 3 g/day than in the control calves at 2 and 12-48 h after LPS injection. These data suggest that LF inhibits LPS-induced alterations in lipid metabolism in preruminant calves.

Fermentation of Shiikuwasha (Citrus depressa Hayata) pomace by lactic acid bacteria to generate new functional materials

Citrus depressa Hayata, known as Shiikuwasha, has a high content of flavonoids (e.g. nobiletin) and beneficial effects on human health. Because Shiikuwasha is mainly consumed as squeezed juice, the pomace is usually discarded, despite its high content of nobiletin. Here, we developed Shiikuwasha pomace as a functional food through fermentation with probiotic lactic acid bacteria. Lactobacillus plantarum E58 was able to ferment Shiikuwasha pomace. This strain had good survival at low pH and was able to adhere to porcine gastric mucin, a beneficial property of probiotics. Analysis with a taste‐sensing system indicated that Shiikuwasha pomace fermented by this strain, compared with unfermented pomace, was more acidic but less umami, and had less of an anionic bitter aftertaste. The fermentation did not decrease the nobiletin content. Thus, fermented Shiikuwasha pomace is a new functional by‐product that contains both flavonoids and probiotics and may facilitate waste reduction.

Complete Genome Sequence of Lactococcus lactis subsp. lactis G50 with Immunostimulating Activity, Isolated from Napier Grass

ABSTRACT Lactococcus lactis subsp. lactis G50 is a strain with immunostimulating activity, isolated from Napier grass (Pennisetum purpureum). We determined the complete genome sequence of this strain using the PacBio RS II platform. The single circular chromosome consists of 2,346,663 bp, with 35.03% G+C content and no plasmids.