Kei Sonoyama

Lactobacillus plantarum strain No. 14 reduces adipocyte size in mice fed high-fat diet.

Because gut microbiota has recently attracted much attention as an environmental factor involved in the development of obesity, probiotics may be useful in preventing and/or improving obesity and its related disorders. The present study aimed to investigate the effects of Lactobacillus plantarum strain No. 14 (LP14), a bacterial strain reported to decrease body fat percentage in healthy volunteers, on adipocyte size in mice. Female C57BL/6 mice were fed either normal- or high-fat diet and administered intragastrically with LP14 (1 × 108 colony-forming units/mouse) or vehicle daily for 11 weeks. High dietary fat intake increased body weight gain, white adipose tissue weight, mean adipocyte size and serum total cholesterol and leptin concentrations, and decreased serum adiponectin concentration. In mice fed the high-fat diet, LP14 administration significantly reduced the mean adipocyte size and tended to reduce the white adipose tissue weight and serum total cholesterol and leptin concentrations as compared with the vehicle-administered mice. All mice had undetectable serum levels of conjugated linoleic acids that reportedly exert antiobesity action. In a separate experiment, LP14 ingestion had no influence on serum triacylglycerol accumulation following olive oil administration in Triton WR1339-treated mice, suggesting that dietary fat absorption is unaffected by LP14. In conclusion, we propose that LP14 may exert a beneficial effect on the onset of diet-induced obesity by reducing the cell size of white adipose tissues, and it seems unlikely that previously reported mechanisms for other bacterial strains are involved in the action of LP14.

Cholesterol-Lowering Effects of Maitake (Grifola frondosa) Fiber, Shiitake (Lentinus edodes) Fiber, and Enokitake (Flammulina velutipes) Fiber in Rats:

The effects of mushroom fibers on serum cholesterol and hepatic low-density lipoprotein (LDL) receptor mRNA in rats were investigated. Rats were fed a cholesterol-free diet with 50 g/kg cellulose powder (CP), 50 g/kg maitake (Grifola frondosa) fiber (MAF), 50 g/kg shiitake (Lentinus edodes) fiber (SF), or 50 g/kg enakitake (Flammulina velutipes) fiber (EF) for 4 weeks. There were no significant differences in the body weight, food intake, liver weight, cecum weight, and cecum pH among the groups. Cecal acetic acid, butyric acid, and total short-chain fatty acid (SCFA) concentrations in the SF and EF groups were significantly higher than those in the other groups. The serum total cholesterol concentration in the CP group was significantly higher than that in the MAF and EF groups. The very LDL (VLDL) + intermediate-density lipoprotein (IDL) + LDL-cholesterol concentration in the CP group was significantly higher than that in the MAF, SF, and EF groups, whereas the high-density lipoprotein (HDL)-cholesterol concentration in the EF group was significantly lower than that in the other groups at the end of the 4-week feeding period. The hepatic LDL receptor mRNA level in the EF group was significantly higher than that in the CP group. The fecal cholesterol excretion in the MAF, SF, and EF groups was significantly higher than that in the CP group. The results of this study demonstrate that MAF and EF lowered the serum total cholesterol level by enhancement of fecal cholesterol excretion, and in particular, by enhancement of hepatic LDL receptor mRNA in EF group.

Response of gut microbiota to fasting and hibernation in Syrian hamsters.

ABSTRACT Although hibernating mammals wake occasionally to eat during torpor, this period represents a state of fasting. Fasting is known to alter the gut microbiota in nonhibernating mammals; therefore, hibernation may also affect the gut microbiota. However, there are few reports of gut microbiota in hibernating mammals. The present study aimed to compare the gut microbiota in hibernating torpid Syrian hamsters with that in active counterparts by using culture-independent analyses. Hamsters were allocated to either torpid, fed active, or fasted active groups. Hibernation was successfully induced by maintaining darkness at 4°C. Flow cytometry analysis of cecal bacteria showed that 96-h fasting reduced the total gut bacteria. This period of fasting also reduced the concentrations of short chain fatty acids in the cecal contents. In contrast, total bacterial numbers and concentrations of short chain fatty acids were unaffected by hibernation. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments indicated that fasting and hibernation modulated the cecal microbiota. Analysis of 16S rRNA clone library and species-specific real-time quantitative PCR showed that the class Clostridia predominated in both active and torpid hamsters and that populations of Akkermansia muciniphila, a mucin degrader, were increased by fasting but not by hibernation. From these results, we conclude that the gut microbiota responds differently to fasting and hibernation in Syrian hamsters.

Maternal consumption of fructo-oligosaccharide diminishes the severity of skin inflammation in offspring of NC/Nga mice

Strategies to manipulate the gut microbiota in infancy have been considered to prevent the development of allergic diseases later in life. We aimed to elucidate the effects of maternal dietary supplementation with a prebiotic oligosaccharide on gut microbiota and spontaneously developing atopic dermatitis-like skin lesions in the offspring of NC/Nga mice. Female NC/Nga mice were fed diets either with or without fructo-oligosaccharide supplementation during pregnancy and lactation. After weaning, offspring were fed the diets supplemented with or without fructo-oligosaccharide for 11 weeks in an air-uncontrolled conventional room. Changes in gut microbiota were assessed by denaturing gradient gel electrophoresis of the PCR-amplified 16S rRNA gene. Skin lesions were evaluated by a clinical score and scratching behaviour. Serum antibody levels were measured by ELISA, and expression levels of cytokines and chemokines in lesional tissue were evaluated by quantitative RT-PCR. Maternal supplementation with fructo-oligosaccharide modulated the gut microbiota in sucklings. Although maternal supplementation with fructo-oligosaccharide suppressed the increase in clinical skin severity score and scratching behaviour in offspring, dietary fructo-oligosaccharide after weaning was less effective. The diminution of skin lesions was accompanied by lower serum concentrations of total IgG1 and lower expression levels of TNF-alpha in the lesional tissue. These data suggest that maternal consumption of fructo-oligosaccharide diminishes the severity of atopic dermatitis-like skin lesions in the offspring of NC/Nga mice.

Gastrointestinal Candida colonisation promotes sensitisation against food antigens by affecting the mucosal barrier in mice

Backgrounds and aims: Controversy still exists as to whether gastrointestinal colonisation by Candida albicans contributes to aggravation of atopic dermatitis. We hypothesised that Candida colonisation promotes food allergy, which is known to contribute to a pathogenic response in atopic dermatitis. We tested this using a recently established murine Candida colonisation model. Methods:Candida colonisation in the gastrointestinal tract was established by intragastric inoculation with C albicans in mice fed a synthetic diet. To investigate sensitisation against food antigen, mice were intragastrically administered with ovalbumin every other day for nine weeks, and antiovalbumin antibody titres were measured weekly. To examine gastrointestinal permeation of food antigen, plasma concentrations of ovalbumin were measured following intragastric administration of ovalbumin. Results: Ovalbumin specific IgG and IgE titres were higher in BALB/c mice with Candida colonisation than in normal mice. Gastrointestinal permeation of ovalbumin was enhanced by colonisation in BALB/c mice. Histological examination showed that colonisation promoted infiltration and degranulation of mast cells. Candida colonisation did not enhance ovalbumin permeation in mast cell deficient W/Wv mice but did in congenic littermate control +/+ mice. Reconstitution of mast cells in W/Wv mice by transplantation of bone marrow derived mast cells restored the ability to increase ovalbumin permeation in response to Candida colonisation. Conclusions: These results suggest that gastrointestinal Candida colonisation promotes sensitisation against food antigens, at least partly due to mast cell mediated hyperpermeability in the gastrointestinal mucosa of mice.

Degree of Polymerization of Inulin-Type Fructans Differentially Affects Number of Lactic Acid Bacteria, Intestinal Immune Functions, and Immunoglobulin A Secretion in the Rat Cecum

This study examined the role of degree of polymerization (DP) of inulin-fructans in modulating the interaction between lactic acid bacteria and IgA cecal secretion. Rats were fed a control diet or a diet containing one of the fructans with different DP. Consuming fructans increased the cecal IgA concentrations in the order DP4 > DP8 > DP16. Cecal lactobacilli counts were higher in DP4, DP8, and DP16, whereas bifidobacteria were higher in DP8, DP16, and DP23. Cecal IgA concentrations were correlated with cecal lactobacilli counts (P < 0.01). DP4, DP8, and DP16, but not DP23, significantly increased IgA-producing plasma cells in the cecal mucosa. IFN-γ and IL-10 production in the cecal CD4(+) T cells was enhanced solely in DP4. The results show that fructans with lower DP enhance cecal IgA secretion and increase the plasma cells and suggest that the increased lactobacilli may contribute to the stimulation of cecal IgA secretion.

Consumption of fructo-oligosaccharide reduces 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice

Strategies to manipulate the intestinal microbiota have been considered to promote immune health. The aim of the present study was to examine whether fructo-oligosaccharide, a typical prebiotic, could suppress antigen-specific skin inflammation by favourably changing the population of intestinal microbiota. Female BALB/c mice were fed a synthetic diet with or without fructo-oligosaccharide supplementation for 3 weeks and were then epicutaneously immunised with 2,4-dinitrofluorobenzene. Afterwards, mice continued to receive their respective diets. At 5 d after immunisation, the mice were ear challenged with the hapten. Ear swelling after the challenge was significantly reduced in the mice fed the diet supplemented with fructo-oligosaccharide than in mice fed the control diet. To characterise the change in the intestinal microbiota, DNA samples isolated from fresh faeces were subjected to PCR-denaturing gradient gel electrophoresis and real-time PCR based on 16S rDNA gene sequences. Dietary fructo-oligosaccharide altered the composition of intestinal microbiota. The numbers of bifidobacteria, but not lactobacilli, were significantly higher in mice fed the fructo-oligosaccharide-supplemented diet than in mice fed the control diet. Ear swelling was negatively correlated with the numbers of bifidobacteria in the faeces. Sequence analysis revealed that Bifidobacterium pseudolongum was the most predominant bifidobacteria in the intestine of mice fed the fructo-oligosaccharide-supplemented diet. These results suggest that consumption of fructo-oligosaccharide reduces contact hypersensitivity, which is associated with proliferation of B. pseudolongum in the intestinal tract of mice.

Assessing changes in composition of intestinal microbiota in neonatal BALB/c mice through cluster analysis of molecular markers

The present study introduced a molecular biological approach to demonstrate changes in the composition of intestinal microbiota in neonatal mice. Female BALB/c mice were fed either a control diet or a diet supplemented with fructo-oligosaccharide (FOS) at 50 g/kg diet, and then mated to male mice. A cultivation-independent approach, denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene, was performed to characterise changes in intestinal microbial populations in pups at 0, 7, 14 and 21 d old and their dams. Comparisons of DGGE profiles were performed using the Dice similarity coefficient and the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis based on numbers, positions and intensities of bands. DGGE profiles differed between dams fed control and FOS-supplemented diets. Although profiles in pups on the day of birth showed a high similarity with dams, profiles in 7-d-old pups differed from dams and showed high similarity to littermates. In 14- and 21-d-old pups, profiles again showed high similarity with dams. DGGE profiles in pups were divided into two large clusters of control and FOS-supplemented diet groups in the range of 0- to 21-d-old, suggesting modulation of intestinal microbiota in infants by manipulation of microbiota in dams. The present study shows a useful technique for demonstrating changes in intestinal microbiota and provides a mouse model for modulation of intestinal microbiota in neonatal life.

Reduction of allergic airway eosinophilia by dietary raffinose in Brown Norway rats.

Oral administration of raffinose, a naturally occurring indigestible oligosaccharide, has reportedly ameliorated atopic dermatitis in human subjects although the mechanism is unknown. The present study investigated the effect of dietary raffinose on allergen-induced airway eosinophilia in ovalbumin-sensitised Brown Norway rats as an atopic disease model. Brown Norway rats were immunised by subcutaneous injection with ovalbumin on day 0 and fed either a control diet or the diet supplemented with raffinose (50 g/kg diet). The rats were exposed to aerosolised ovalbumin on day 20, and broncho-alveolar lavage fluid was obtained on the next day. The number of eosinophils in the fluid was significantly lower in the rats fed the raffinose diet than in those fed the control diet. Dietary raffinose significantly reduced IL-4 and IL-5 mRNA levels in lung tissue and tended to lower ovalbumin-specific Ig E levels. Suppression of eosinophilia by dietary raffinose was still observed in caecectomised and neomycin-administered rats, suggesting little contribution by the colonic bacteria to the effect of raffinose. Intraperitoneal administration of raffinose also suppressed eosinophilia. Significant concentrations of raffinose were detected in portal venous and abdominal arterial plasma after the intragastric administration of raffinose. Overall, the findings suggest that dietary raffinose ameliorates allergic airway eosinophilia at least partly via post-absorptive mechanisms in Brown Norway rats.

Adiponectin is partially associated with exosomes in mouse serum.

Exosomes are membrane vesicles 30-120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo.