Isolation of an isoflavone-metabolizing, Clostridium-like bacterium, strain TM-40, from human faeces.

Recently, the biological effects of isoflavones have attracted much attention. Intestinal microbiota plays an important role in the metabolism and bioavailability of isoflavones. However, few reports have discussed intestinal bacteria that metabolize daidzein into dihydrodaidzein. In this study, we isolated the dihydrodaidzein-producing intestinal bacterium TM-40 from a healthy boy's faeces. The bacteria from faecal samples were incubated with daidzein. Among all tested bacteria, one strain (strain TM-40) produced dihydrodaidzein both from daidzein and daidzin. However, in our experimental conditions, strain TM-40 did not produce equol from daidzein. The 16S rRNA partial sequence of strain TM-40 (AB249652) exhibited a 93% similarity to that of Coprobacillus catenaformis (AB030218). This strain seems to be a new species.

Effect of dietary fat on skin reactivity against histamine, Th1 and Th2 cytokine levels and some serum parameters in mice.

Changes in diet may be associated with the increase in allergic disease; change to high-calorie and high-fat diets may be a factor. In this study our objective was to determine skin reactivity of histamine and serum cytokine concentrations in mice fed diets containing different amounts of fat. Histamine reactivity was performed on mice back skin and serum cytokine concentrations were measured by ELISA in mice injected with anti-CD3 antibody. We measured serum interferon-gamma as a Th1-type cytokine and interleukin-4 as a Th2-type cytokine. Mice fed a high fat diet displayed enhanced skin reactivity of histamine and higher IL-4 levels in serum. These data suggest that a high fat diet may play a role in enhancing allergic reactions.

Comparative effects of non-gelatinized corn and rice starches on the life-span of ICR mice.

We compared the effects of non-gelatinized rice and corn starches on the life-span of ICR mice. Six groups of male ICR mice consisting of 30 animals each were maintained on purified experimental diets containing either corn or rice starch and different amounts of soybean oil (6, 12 or 24%) throughout their life-time. Plots of the survival rates of the mice indicate that rice compared to corn starch conferred a longer life-span to ICR mice, although a significant difference due to the starch type was only observed in the mice fed on the 24% fat diet (p=0.012). A divergent effect of rice and corn starches on the survival rate was apparent when observations were combined with respect to the starch type regardless of the dietary fat level (p=0.005). In addition, two-way ANOVA data indicate that the mean survival time was longer for the mice given rice starch (593-645 days) than for those fed corn starch (538-580 days) (p=0.011). However, no significant difference in these parameters due to dietary fat levels was observed. The results of our study indicate that starch type is one of the determinants of longevity in mice.

Lactobacillus gasseri: effects on mouse intestinal flora enzyme activity and isoflavonoids in the caecum and plasma.

The effects of Lactobacillus gasseri JCM 1131(T) on isoflavonoid levels within the caecum and plasma were assessed in adult mice. Male 5-week-old mice were fed an AIN 93M diet for 30 d. Two groups of mice were administered either L. gasseri JCM 1131(T) (the LGI group) or physiological saline solution (the control (CI) group) daily for 5 d before dissection. The plasma daidzein concentration was significantly higher in the LGI group, however, their plasma equol concentration was significantly less than in the CI group. The total amount of equol present as aglycone in the caecum was significantly greater in the CI group, but there was no significant difference in the total daidzein present as caecal aglycone. In an in vitro incubation of daidzein with the faecal flora of mice, the equol concentration was significantly higher in the CI group. The numbers of lactobacilli present were significantly higher in the LGI group. The present data suggest that the administration of L. gasseri is likely to influence the effect of isoflavonoids on the host via changes in the gastrointestinal environment.

Activity and mRNA levels of enzymes involved in hepatic fatty acid synthesis and oxidation in mice fed conjugated linoleic acid.

The effects of dietary conjugated linoleic acid (CLA) on the activity and mRNA levels of hepatic enzymes involved in fatty acid synthesis and oxidation were examined in mice. In the first experiment, male ICR and C57BL/6J mice were fed diets containing either a 1.5% fatty acid preparation rich in CLA or a preparation rich in linoleic acid. In the second experiment, male ICR mice were fed diets containing either 1.5% linoleic acid, palmitic acid or the CLA preparation. After 21 days, CLA relative to linoleic acid greatly decreased white adipose tissue mass but caused hepatomegaly accompanying an approximate 10-fold increase in the tissue triacylglycerol content irrespective of mouse strain. CLA compared to linoleic acid greatly increased the activity and mRNA levels of various lipogenic enzymes in both experiments. Moreover, CLA increased the mRNA expression of Delta6- and Delta5-desaturases, and sterol regulatory element binding protein-1 (SREBP-1). The mitochondrial and peroxisomal palmitoyl-CoA oxidation rate was about 2.5-fold higher in mice fed CLA than in those fed linoleic acid in both experiments. The increase was associated with the up-regulation of the activity and mRNA expression of various fatty acid oxidation enzymes. The palmitic acid diet compared to the linoleic acid diet was rather ineffective in modulating the hepatic lipid levels or activity and mRNA levels of enzymes in fatty acid metabolism. It is apparent that dietary CLA concomitantly increases the activity and mRNA levels of enzymes involved in fatty acid synthesis and oxidation, and desaturation of polyunsaturated fatty acid in the mouse liver. Both the activation of peroxisomal proliferator alpha and up-regulation of SREBP-1 may be responsible for this.

Docosahexaenoic acid suppresses nitric oxide production and inducible nitric oxide synthase expression in interferon-gamma plus lipopolysaccharide-stimulated murine macrophages by inhibiting the oxidative stress.

N-3 polyunsaturated fatty acids (PUFAs) are known to have anti-inflammatory effects. Excess production of nitric oxide (NO) is associated with inflammation. Therefore, we examined the effects of PUFAs on NO production and inducible NO synthase (iNOS) expression by stimulated murine macrophages. One typical n-3 PUFA docosahexaenoic acid (DHA) strongly inhibited NO production and iNOS expression in RAW264 macrophages and mouse peritoneal macrophages in a dose-dependent manner. This inhibition was accompanied by inhibiting the oxidative stress-sensitive transcription factor nuclear factor (NF)-kappaB activation. In stimulated macrophages, intracellular peroxides level was enhanced, but pretreatment of DHA dose-dependently inhibited this enhancement. These results suggest that DHA has an antioxidative effect based on the inhibition of the accumulation of intracellular peroxides, and this inhibition caused the suppression of the activation of NF-kappaB, resulting in the inhibition of NO production and iNOS expression. On the other hand, DHA treatment enhanced the level of intracellular glutathione (GSH), and this enhancement is thought to mediate the activity of DHA because lowering the GSH level by inhibiting GSH biosynthesis reversed the DHA-induced suppression of NO production, NF-kappaB activation, and the accumulation of intracellular peroxides. Our results demonstrate that DHA inhibits NO production in macrophages and this inhibition is, in part, mediated by upregulation of GSH.

Dietary conjugated linoleic acid reduces body fat mass and affects gene expression of proteins regulating energy metabolism in mice.

ICR and C57BL/6J mice were fed experimental diets containing either a 2% fatty acid preparation rich in conjugated linoleic acid (CLA) or a preparation rich in linoleic acid and free of CLA for 21 days. CLA greatly decreased weights of white adipose tissue and interscapular brown adipose tissue in the two strains. CLA reduced mRNA levels of glucose transporter 4 (Glut 4) in white and brown adipose tissue of both strains. A CLA-dependent decrease in mRNA levels of peroxisome proliferator activated receptor (PPAR) gamma was seen in interscapular brown adipose tissue of both strains and in white adipose tissue of C57BL/6J but not ICR mice. Dietary CLA was found to cause a decrease in the mRNA levels of uncoupling protein (UCP) 1 in brown adipose tissue when the value was corrected for the expression of a house-keeping gene (beta-actin) in the two strains. Uncorrected values were, however, indistinguishable between the animals fed the CLA diet and CLA-free diet. UCP 3 expression in brown adipose tissue was much lower in mice fed the CLA diet than in those fed the control diet in both strains. In contrast, CLA greatly up-regulated the gene expression of UCP 2 in brown adipose tissue. Dietary CLA also increased UCP 2 mRNA level in skeletal muscle. It is apparent that dietary CLA decreases white and brown adipose tissue mass, accompanying changes in the gene expression of proteins regulating energy metabolism in white and brown adipose tissues, and skeletal muscle of mice.

Effects of rice starch-isoflavone diet or potato starch-isoflavone diet on plasma isoflavone, plasma lipids, cecal enzyme activity, and composition of fecal microflora in adult mice.

The effects of rice starch-isoflavone diet or potato starch-isoflavone diet on plasma concentration of isoflavones, plasma lipids, cecal enzyme activity, and intestinal microflora were studied. Male 15-wk-old mice were fed a rice-starch-based or potato-starch-based diet supplemented with isoflavones for 4 wk, and plasma samples, cecal contents, and feces were collected individually. Plasma equol concentration was significantly higher in the potato-isoflavone diet group than in the rice-isoflavone diet group, but no significant difference was observed in plasma daidzein or genistein concentrations. Plasma total cholesterol concentration was higher in the potato-isoflavone diet group, but no significant difference was observed in plasma triglyceride concentration. Both cecal beta-glucuronidase and beta-glucosidase activities were significantly higher in the rice-isoflavone diet group. The number of bifidobacteria was significantly higher in the potato-isoflavone diet group. These results indicate that different types of starches have different influences on plasma isoflavone and suggest that the influences might be through the change of host physiology and/or the metabolism and composition of intestinal microflora.

mRNA expression of enzymes involved in taurine biosynthesis in rat adipose tissues.

mRNA levels of the enzymes involved in taurine synthesis were compared among 12 rat tissues. Using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern-blotting, high levels of mRNA for cysteine dioxygenase were confirmed in the liver and kidney. Unexpectedly, the mRNA levels in epididymal and perirenal white adipose tissues and interscapular brown adipose tissue were remarkably high, at least compared with those observed in the liver and kidney. Cysteine dioxygenase mRNA levels in other tissues were very low. Using Northern blotting, significant amounts of cysteine sulfinic acid decarboxylase mRNA were detected in the liver, kidney, epididymal and perirenal white adipose tissues, and brown adipose tissue, but not in other tissues. Again, the mRNA levels of cysteine sulfinic acid decarboxylase in adipose tissues were comparable to or even higher than those in the liver and kidney. The activity of cysteine sulfinic acid decarboxylase in white and brown adipose tissues was 50% to 80% of that in the liver and much higher than the values observed in kidney, lung, and brain. However, the occurrence of cysteine dioxygenase activity in adipose tissues was not confirmed.

Comparison of the effects of dietary alpha-linolenic, stearidonic, and eicosapentaenoic acids on production of inflammatory mediators in mice.

The effects of dietary stearidonic acid (18:4n-3) on inflammatory mediator release in whole blood and splenocytes was investigated in Balb/c mice, and the effects were compared with those of two other n-3 PUFA: alpha-linolenic acid (18:3n-3) and EPA (20:5n-3). TAG mixtures containing 10% of 18:4n-3, 18:3n-3, or 20:5n-3 as the respective sole n-3 PUFA were enzymatically synthesized. Diets containing synthesized TAG mixtures were fed to Balb/c mice for 3 wk. The release of prostaglandin F2 (PGE2) and tumor necrosis factor (TNF) were measured in whole blood and splenocytes stimulated with lipopolysaccharide. In whole blood, the production of TNF was suppressed by all dietary n-3 PUFA (18:3n-3, 18:4n-3, and 20:5n-3) as compared with the control diet, which contained TAG prepared from safflower oil. PGF2 production was not significantly changed. Differences among the n-3 PUFA (18:3n-3, 18:4n-3, and 20:5n-3) were not observed. In splenocytes, PGE2 production was suppressed by dietary n-3 PUFA, but TNF production was not. GC analysis of plasma and splenocyte FA profiles showed an increase in the levels of 20:4n-3, 20:5n-3, and 22:6n-3 in mice fed the diet containing 18:4n-3.