Takaji Yajima

Comparative expression of hexose transporters (SGLT1, GLUT1, GLUT2 and GLUT5) throughout the mouse gastrointestinal tract

Hexose transporters play a pivotal role in the absorption of food-derived monosaccharides in the gastrointestinal tract. Although a basic knowledge of the hexose transporters has already been gained, their detailed distribution and comparative intensities of expression throughout the gastrointestinal tract have not been fully elucidated. In this study, we quantitatively evaluated the expression of SGLT1, GLUT1, GLUT2, and GLUT5 by in situ hybridization and real-time PCR techniques using a total of 28 segments from the gastrointestinal tract of 9-week-old mice. GLUT2 and GLUT5 mRNA expressions were detected predominantly from the proximal to middle parts of the small intestine, showing identical expression profiles, while SGLT1 mRNA was expressed not only in the small intestine but also in the large intestine. Notably, GLUT1 mRNA was expressed at a considerable level in both the stomach and large intestine but was negligible in the small intestine. Immunohistochemistry demonstrated the polarized localization of hexose transporters in the large intestine: SGLT1 on the luminal surface and GLUT1 on the basal side of epithelial cells. The present study provided more elaborate information concerning the localization of hexose transporters in the small intestine. Furthermore, this study revealed the significant expression of glucose transporters in the large intestine, suggesting the existence of the physiological uptake of glucose in that location in mice.

Non‐neuronal release of ACh plays a key role in secretory response to luminal propionate in rat colon

Non‐technical summary ACh is the best characterized neurotransmitter that is synthesized in cholinergic neurons in the brain and gut wall. In the gut, acetylcholine is released from the nerve endings in response to luminal stimuli and regulates the movement of gut contents via stimulating muscle contraction and epithelial ion secretion. We show that acetylcholine is synthesized in colonic epithelial cells and released on the serosal side by luminal chemical stimulation of the short chain fatty acid propionate and causes chloride secretion. These results suggest that non‐neuronal release of acetylcholine in response to luminal stimuli plays a role in colonic chloride secretion.

Simplified preparation of a refined milk formula comparable to rat's milk : Influence of the formula on development of the gut and brain in artificially reared rat pups

BACKGROUND Milk formulas for artificially reared (AR) rat pups are mostly based on complex cows milk products, prepared by laborious and time-consuming processes. The aim of this study was to develop a simplified procedure for preparing a refined formula and to examine its influences on gut and brain development. METHODS The formula comprised a combination of purified cows casein and whey proteins, five kinds of edible oil, minerals, and vitamins. Detailed analyses showed that the composition of macro- and micro-nutrients, osmolarity, and pH of the new formula closely resembled those of rats milk. Rat pups, each with an intragastric cannula implanted at age 5 days, were artificially reared for the following 10-15 days. RESULTS The body weight gain of AR pups matched that of mother-reared (MR) pups. Histoplanimetrical analyses showed that the small intestine in AR pups was more developed in relation to area of a transverse section, number and length of villi, and thickness of tunica muscularis than that of MR pups. Fat components in the formula influenced the fatty acid composition and the cholesterol-to-phospholipid ratio in the small intestinal microvillus membrane (MVM) of AR pups, but not the MVM fluidity. Brain weight was not significantly different between the two groups at age 15-20 days. CONCLUSION This formula is useful for artificial rearing of rats and for identifying dietary components contributing to metabolic adaptation during the suckling period.

Bacterial Translocation in Neonatal Rats: The Relation Between Intestinal Flora, Translocated Bacteria, and Influence of Milk

Background A high incidence of bacterial translocation in neonates results not only from immaturity of host-defense functions, but also from the dominant colonization of aerobic bacteria in the intestine. Bacterial colonization develops differently among breast-fed, formula-fed, premature, and full-term infants. The purpose of this study was to examine the incidence of bacterial translocation and to identify the translocated bacterial species, relating these findings to the intestinal microflora and to the type of feeding in neonatal rats. Methods Animals were divided into three groups: breast-fed normal pups (MR group), formula-fed pups fed via an intragastric cannula implanted esophageally (AR group), and breast-fed pups after the removal of the cannula (Sham group). Artificial rearing was achieved using a machine feeding system. Culture and identification of the bacteria in the intestine, mesenteric lymph nodes, liver, portal blood, and lungs were made using a simplified version of Mitsuokas method. Results At 14 days of age, the dominant bacteria in the feces of the MR and Sham Groups were Enterobacteriaceae, Lactobacillus, and Enterococcus, but Enterobacteriaceae and Clostridium were significantly more common in the AR group than in the MR group. The dominant bacteria in the mesenteric lymph nodes were Enterobacteriaceae, Lactobacillus, and Staphylococcus. The extent of systemic bacterial translocation decreased earlier in the Sham group than in the AR group. Conclusions The frequency with which species of bacteria were cultured from mesenteric lymph nodes and other peripheral sites did not mirror the composition of the intestinal flora. Among the translocated bacteria, Staphylococcus may be especially hard to recognize and difficult for the host-defense systems to destroy. Breast-feeding inhibited systemic bacterial translocation in the suckling period of the rat.

Lactoferrin Protects against Development of Hepatitis Caused by Sensitization of Kupffer Cells by Lipopolysaccharide

ABSTRACT BALB/c mice were intravenously injected with lipopolysaccharide (LPS) (0.05 μg/g of body weight) 7 days after being primed with zymosan. Recombinant human lactoferrin (250 μg/g of body weight), intravenously administered 1 day before the injection of LPS, significantly lessened the severity of hepatitis, as assessed by levels of serum alanine transaminase compared to those seen when casein was administered. The transient rise of serum tumor necrosis factor alpha (TNF-α) after LPS treatment was also significantly lowered by the intravenous administration of lactoferrin, suggesting that the effect of lactoferrin was due to the suppression of TNF-α production. The following results indicate that the sites of action of lactoferrin for the suppression of the development of this type of hepatitis are Kupffer cells. Gadolinium chloride, a substance known to eliminate Kupffer cells, administered 1 day before LPS, inhibited the transient rise of TNF-α and protected against the development of hepatitis. Kupffer cells isolated from mice intraperitoneally injected with recombinant human lactoferrin became refractory to LPS. The specific interaction of recombinant human lactoferrin with the Kupffer cells was shown by a binding assay, which revealed two types of binding sites on mouse Kupffer cells. Of the two dissociation constants determined in this way, the lower dissociation constant, 0.47 × 10−6 M, was within the range of the 50% effective doses for the suppression of TNF-α production. These results suggest that recombinant human lactoferrin administered to mice suppresses the production of TNF-α by Kupffer cells by directly associating with the binding sites on these cells.

Dietary Nucleotides Can Up-Regulate Antigen-Specific Th1 Immune Responses and Suppress Antigen-Specific IgE Responses in Mice

Background: It has been reported that dietary nucleotides enhance T helper cell activities. In this study, we have determined the effects of dietary nucleotides on antigen-specific Th1 and Th2 responses and IgE responses. Methods: Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OVA-TCR Tg) mice, 3 weeks old, were fed a nucleotide-free diet (NT(–) diet) or the NT(–) diet supplemented with dietary nucleotides (NT(+) diet) for 4 weeks. Cytokine production by spleen cells and macrophages obtained from these mice was measured in vitro. BALB/c mice, 3 weeks old, immunized intraperitoneally with OVA adsorbed onto alum, were fed the NT(–) diet or the NT(+) diet for 4 weeks. Serum levels of antigen-specific antibodies in the BALB/c mice were determined by ELISA. Results: The level of production of antigen-specific interferon-γ by spleen cells was significantly higher in the OVA-TCR Tg mice fed the NT(+) diet than in the control mice. The levels of secretion of bioactive IL-12 by spleen cells and peritoneal macrophages were also significantly increased in the NT(+) diet group. The serum OVA-specific IgE level was significantly decreased in BALB/c mice fed the NT(+) diet compared with those fed the NT(–) diet. Conclusion: These results show that dietary nucleotides up-regulate the antigen-specific Th1 immune response through the enhancement of IL-12 production and suppress the antigen-specific IgE response.

Characteristic Transfer of Colostral Components into Cerebrospinal Fluid via Serum in Neonatal Pigs

In order to evaluate the possibility of modification of brain function by colostral suckling, the characteristic transfer of colostral components into serum and cerebrospinal fluid (CSF) has been studied by SDS electrophoresis, immunoblot and ELISA methods in nonsuckling pigs. Total protein concentrations in the serum increased immediately after oral administration of bovine colostrum, reaching a peak value (7.0 ± 0.7 g/dl) at 24 h after administration, corresponding to a 3-fold increase compared to preinfusion levels. IgG and other macromolecular components (MW 19,000–58,000) were recognized in serum by electrophoretic and ELISA analysis. Total protein concentrations in the CSF collected from the cisterna magna also increased steeply after colostral administration, reaching a maximal value (54.1 ± 5.0 mg/dl) at 4 h, corresponding to a 4-fold increase compared to preinfusion levels. Two colostral components (MW 19,000 and 31,000) in serum were confirmed to be present in the CSF by electrophoresis. The component of MW 19,000 was identified by immunoblot as β-lactoglobulin. IgG in serum transferred from colostrum could not be detected in the CSF by ELISA. Lactoferrin administered into the intestine was also detected in the CSF via serum. These results indicate that some components of colostrum can be transported into the CSF via the serum, suggesting the possibility of modification of immature brain functions by colostral suckling in neonatal pigs.

Effects of dietary nucleotides on serum antibody and splenic cytokine production in mice

Abstract We examined the effects of dietary nucleotides on the immune response balance in two subtypes of T helper cells, type 1 (Th1) and type 2 (Th2) cells. In experiment 1, BALB/c mice were maintained on a nucleotide-free diet (NT(−) diet) or the NT(−) diet supplemented with dietary nucleotides (NT(+) diet) from 4 weeks prior to mating and throughout the experiments. The second generations of these mice (F1) were maintained on the same diet as the respective dams. Male F1-generation mice were used to determine the serum immunoglobulin levels. The serum IgE levels were significantly decreased in mice fed the NT(+) diet ( p p

Intestinal Adherent Bacteria and Bacterial Translocation in Breast-Fed and Formula-Fed Rats in Relation to Susceptibility to Infection

The barrier function of the intestinal mucosa is immature in the newborn mammal, and is strengthened by breast milk. We investigated this effect of breast milk by comparing the susceptibility to infection assessed in terms of adherent bacterial colonization of the intestinal tissue (AdC) and bacterial translocation (BT) between breast-fed and formula-fed newborn rats. Three-day-old rat pups were assigned to one of three groups: mother-reared (MR), pseudo-cannulated (sham), and artificially reared (AR). AR rats were infused with formula through an intragastric cannula, under the control of a computer-regulated pumping machine. MR and sham rat pups were reared with their respective dams and received breast milk until weaning in a specially designed cage. In 10-d-old rats, there was no significant difference in the fecal or cecal flora between the AR and MR groups, whereas the AdC and the BT to the liver were greater in the AR than MR group. Enterobacteriaceae, Streptococcus and/or Enterococcus, and Staphylococcus were dominantly detected as microorganisms in AdC flora and BT. The AdC flora did not directly reflect the bacterial colonization flora. These findings suggest that AR rat pups mature normally, although there is a greater colonization of Enterobacteriaceae and BT in AR than MR pups. Consequently, the intestinal barrier function of the pups reared by artificial feeding may become susceptible to BT, and AdC may be more indicative than bacterial colonization of the susceptibility to BT.

Postnatal changes in the expression of genes for cryptdins 1–6 and the role of luminal bacteria in cryptdin gene expression in mouse small intestine

Although there have been many fascinating studies on cryptdins, the information for each cryptdin isoform was not completely provided. In this study, the postnatal changes in the gene expression of cryptdin 1-6 were evaluated, and the patterns of change were compared between conventional and germ-free mice. Two patterns of postnatal change were observed: gene expression of cryptdins 1, 3 and 6 increased gradually, and that of cryptdins 2 and 5 increased rapidly. Gene expression of cryptdin 4 increased gradually in the ileum but rapidly in the jejunum. Conventional mice showed significantly higher gene expression for all isoforms than germ-free mice. Interestingly, the difference in the gene expression for cryptdin 2, 4 and 5 between the jejunum and ileum seemed to be increased by the presence of the luminal bacteria. The results indicate that cryptdin isoforms develop differently depending on the isoform type, and that the gene expression of all cryptdin isoforms was affected by the presence of the luminal bacteria.