Megumi Nishimukai

Asaccharobacter celatus gen. nov., sp. nov., isolated from rat caecum

An obligately anaerobic and equol-producing bacterium, designated strain do03T, was isolated from the caecal content of a rat. Cells were Gram-positive, non-spore-forming rods. The results from a phylogenetic analysis based on 16S rRNA gene sequences showed that strain do03T formed a separate line of descent in the phylogenetic cluster of the family Coriobacteriaceae. The strain was unable to metabolize glucose or other carbohydrates as sole carbon sources; growth was enhanced in the presence of arginine. The cell wall contained meso-diaminopimelic acid. The major fatty acid was C18 : 1cis9 (54.0 %). The strain had one unidentified predominant (91.9 %) quinone that was not menaquinone, methylmenaquinone, demethylmenaquinone, ubiquinone or rhodoquinone. The DNA G+C content was 63 mol%. The data presented in this work show that strain do03T differs from members of the related recognized genera Eggerthella and Denitrobacterium at both the phylogenetic and phenotypic level. Therefore, the strain constitutes a novel genus and species, for which the name Asaccharobacter celatus gen. nov., sp. nov. is proposed. The type strain of the type species is do03T (=JCM 14811T=DSM 18785T=AHU 1763T).

Prebiotic Properties of Epilactose

We recently reported that cellobiose 2-epimerase from Ruminococcus albus effectively converted lactose to epilactose. In this study, we examined the biological effects of epilactose on intestinal microbiota, bile acid metabolism, and postadministrative plasma glucose by animal tests. Dietary supplementation with epilactose or fructooligosaccharide (4.5% each) increased cecal wall weight and cecal contents and decreased the pH of the cecal contents in Wistar-ST rats. The number of total anaerobes tended to be greater in rats fed epilactose and fructooligosaccharide than in those fed the control diet. Lactobacilli and bifidobacteria were more numerous in rats fed epilactose and fructooligosaccharide diets than in those fed the control diet. Analysis of clone libraries of 16S rRNA suggests that supplementation with epilactose did not induce the proliferation of harmful bacteria belonging to classes Clostridia or Bacteroidetes. Epilactose, as well as fructooligosaccharide, inhibited the conversion of primary bile acids to secondary bile acids, which are suggested to be promoters of colon cancer. In addition, oral administration of epilactose did not elevate the plasma glucose concentration in ddY mice. These results clearly indicate that epilactose is a promising prebiotic. We also showed that cellobiose 2-epimerase converted lactose in cow milk and a spray-dried ultrafiltrate of cheese whey to epilactose. Cellobiose 2-epimerase may increase the value of dairy products by changing lactose to epilactose possessing prebiotic properties.

Effects of Epilactose on Calcium Absorption and Serum Lipid Metabolism in Rats

Epilactose (4-O-beta-galactopyranosyl-D-mannnose) is a rare disaccharide in cow milk that can be synthesized from lactose by the cellobiose 2-epimerase of Ruminococcus albus. In this study, we examined the biological activities of epilactose using male Wistar-ST rats. The apparent rates of calcium and magnesium absorption of rats fed epilactose and fructooligosaccharide diets were greater than those fed control and lactose diets, accompanied by greater weight gain of the cecal wall and higher levels of short-chain fatty acids and other organic acids. Epilactose also increased the calcium absorption in everted small intestinal sacs. In addition, the levels of plasma total cholesterol and nonhigh-density lipoprotein cholesterol were lower in epilactose-fed rats. These results indicate that epilactose promotes calcium absorption in the small intestine and possibly lowers the risk of arteriosclerosis. Cecal microbes may efficiently utilize epilactose and contribute to these biological activities.

Ingestion of plasmalogen markedly increased plasmalogen levels of blood plasma in rats

Plasmalogens, a subclass of phospholipids, are widely distributed in human and animals, and are taken into the body as food. However, no data exist on the intestinal absorption or fate of ingested plasmalogen. Here, we determined whether dietary plasmalogen is absorbed and whether blood and tissue concentrations increased in normal male Wistar rats by using four separate experiments. Phospholipids containing more than 20 wt% of plasmalogen extracted from the bovine brain were incorporated into test diets (10–15 wt%). In experiment 1, we estimated the absorption rate by measuring the plasmalogen vinyl ether bonds remaining in the alimentary tract of rats after the ingestion of 2 g of test diet containing 91 μmol plasmalogen. The absorption rate of plasmalogen was nearly 80 mol% after 4 h, comparable to the total phospholipid content in the test diet. In experiment 2, we observed no degradation of the plasmalogen vinyl ether bonds under in vitro conditions simulating those of the stomach and small intestinal lumen. In experiment 3 we confirmed a comparable absorption (36 mol%) by using a closed loop of the upper small intestine in anesthetized rats 90 min after injecting a 10 wt% brain phospholipid emulsion. Feeding a test diet containing 10 wt% brain phospholipids for 7 d increased plasmalogen concentration threefold in blood plasma and by 25% in the liver; however, no increases were seen in blood cells, skeletal muscle, brain, lungs, kidneys, or adipose tissue (experiment 4). We concluded that dietary plasmalogen is absorbed from the intestine and contributes to a large increase in plasmalogen levels in blood plasma.

Enteral administration of soyabean lecithin enhanced lymphatic absorption of triacylglycerol in rats

As the physiological roles of dietary lecithin have not yet been clearly defined, we examined the effects of lecithin on lipid absorption in male Wistar rats with a mesenteric lymph cannula. Lymphatic absorption was observed after the infusion of 1 ml emulsion containing 100 mg test oil emulsified with sodium taurocholate (10 g/l) in three separate experiments. Test oils (100 mg) were: soyabean oil (triacylglycerol (TG) source, SO) and soyabean oil+lecithin (75 mg soyabean oil+25 mg lecithin, LE) in Expt 1; SO, LE or soyabean oil+lysolecithin (75 mg soyabean oil plus 25 mg lysolecithin, LY) in Expt 2; hydrolysed soyabean oil (HSO) or HSO+lysolecithin (75 mg HSO+25 mg lysolecithin, HLY) in Expt 3. After LE and LY infusions, lymph flow and the lymphatic output of TG was higher than after SO infusion at 0-30 min and 0-90 min respectively (Expts 1 and 2). Lecithin-induced increases in lymph TG output remained constant when HSO was infused (Expt 3). There were no differences in the TG:phospholipid ratio in the lymph after infusion among the groups; nevertheless, the lymphatic output of TG was much higher after infusion with LE than with SO. Fatty acid was released more efficiently from SO than from LE and LY by in vitro digestion with rat bile-pancreatic juice. These present results demonstrate that a TG emulsion containing soyabean lecithin or its hydrolysates promote lymphatic TG output and suggest that the increases in TG absorption do not depend on TG digestion.

Serum choline plasmalogens, particularly those with oleic acid in sn-2, are associated with proatherogenic state

Serum plasmalogens (Pls) (1-O-alk-1’-enyl-2-acyl glycerophospholipids) are of particular interest for studies on metabolic disorders associated with oxidative stress and chronic inflammation. Serum levels of Pls are known to correlate positively with HDL-cholesterol (HDL-C); however, few studies have examined serum Pls molecular species in association with pathophysiological conditions and their clinical significance. To clarify these, we determined serum levels of individual ether glycerophospholipids in Japanese asymptomatic cohorts (n = 428; 362 male and 66 female subjects) by LC/MS/MS, and examined their correlations with clinical parameters. We found that the proportion of choline Pls (PlsCho) among total serum phospholipids was significantly lower in the male group over 40 years old and was associated with multiple risk parameters more strongly than HDL-C. The abundance of serum PlsCho with oleic acid (18:1) in sn-2 exhibited the strongest positive correlation with serum concentrations of adiponectin and HDL-C, while being inversely associated with waist circumference and the serum levels of TG and small dense LDL-cholesterol. The characterization of serum ether glycerophospholipids verified the specificity of PlsCho, particularly the ones with 18:1 in sn-2, as a sensitive biomarker for the atherogenic state.

The Nondigestible Disaccharide Epilactose Increases Paracellular Ca Absorption via Rho-Associated Kinase- and Myosin Light Chain Kinase-Dependent Mechanisms in Rat Small Intestines

We previously showed that epilactose, a nondigestible disaccharide, increased calcium (Ca) absorption in the small intestines of rats. Here, we explored the mechanism(s) underlying the epilactose-mediated promotion of Ca absorption in a ligated intestinal segment of anesthetized rats. The addition of epilactose to the luminal solution increased Ca absorption and chromium (Cr)-EDTA permeability, a paracellular indicator, with a strong correlation (R = 0.93) between these changes. Epilactose induced the phosphorylation of myosin regulatory light chains (MLCs), which is known to activate the paracellular route, without any change in the association of tight junction proteins with the actin cytoskeleton. The epilactose-mediated promotion of the Ca absorption was suppressed by specific inhibitors of myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). These results indicate that epilactose increases paracellular Ca absorption in the small intestine of rats through the induction of MLC phosphorylation via MLCK- and ROCK-dependent mechanisms.

Massive large bowel resection decreases bone strength and magnesium content but not calcium content of the femur in rats

We examined the effects of massive large bowel resection (cecocolonectomy) on calcium and magnesium absorption and bone characteristics in rats. Male Sprague-Dawley rats were divided into two groups: sham-operated and cecocolonectomized rats. The rats were fed a sucrose-based diet containing casein at 250 g/kg diet for 10 d after a 9- to 10-d postoperative recovery period. Apparent magnesium absorption but not calcium absorption was lower in the resection group than in the sham group. There was a tendency of lower serum magnesium concentration (P = 0.070) but not calcium concentration (P = 0.418) in the resection group compared with the sham group. The maximum breaking force and magnesium content but not the calcium content of the femur were lower in the resection group than in the sham group. These results suggest that massive large bowel resection influences magnesium kinetics and decreases bone strength through reduction of the magnesium content of the femur in rats. Femoral breaking force was positively correlated (r = 0.617, P = 0.011) with only the magnesium content. We conclude that the changes in magnesium kinetics caused by cecocolonectomy could contribute to the fragility of bone.

Tysnd1 Deficiency in Mice Interferes with the Peroxisomal Localization of PTS2 Enzymes, Causing Lipid Metabolic Abnormalities and Male Infertility

Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the in vivo functions and physiological role of Tysnd1, we analyzed the phenotype of Tysnd1−/− mice. Male Tysnd1−/− mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. Tysnd1−/− mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of Tysnd1 interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.

Ingestion of epilactose, a non-digestible disaccharide, improves postgastrectomy osteopenia and anemia in rats through the promotion of intestinal calcium and iron absorption.

Gastrectomy often results in osteopenia and anemia because of calcium (Ca) and iron (Fe) malabsorption. Here, we investigated the effects of feeding epilactose, a non-digestible disaccharide, on gastrectomy-induced osteopenia, anemia, and Ca and Fe malabsorption in male Sprague-Dawley rats. Totally gastrectomized or sham-operated rats were fed the control or epilactose (50 g/kg) diets for 30 days. Gastrectomy severely decreased intestinal Ca and Fe absorption, femoral bone strength, Ca content, hemoglobin concentration, and hematocrit. These decreases were partly or totally restored by feeding epilactose. Feeding epilactose increased the cecal tissue weight and the soluble Ca concentration and short-chain fatty acid pools of the cecal contents. Collectively, the increases in cecal mucosal area and/or soluble Ca concentration of the cecal contents, resulting from short-chain fatty acid production by intestinal microbes, are thought to be responsible for the epilactose-mediated promotion of Ca and Fe absorption in the gastrectomized rats.