Free Linoleic Acid and Oleic Acid Reduce Fat Digestion and Absorption In Vivo as Potent Pancreatic Lipase Inhibitors Derived from Sesame Meal.

Pancreatic lipase catalyzes the cleavage of triacylglycerols at the oil-water interface, and is known as the dominant determiner of dietary fat digestion. Reducing dietary fat digestion and absorption by modulating the activity of pancreatic lipase has become a favorable strategy to tackle obesity. Orlistat is, at present, the only pancreatic lipase inhibitor approved for the treatment of obesity; however, an array of gastrointestinal adverse effects associated with orlistat limits its tolerability. As a safe alternative to orlistat, a number of natural product-derived compounds with varying degrees of pancreatic lipase inhibitory activity have been reported. We herein reported that bioactivity-guided fractionation of sesame meal led to the identification of free linoleic acid and oleic acid as potent inhibitors of porcine pancreatic lipase in vitro with an IC50 of 23.1 µg/mL (82.4 µM) and 11.7 µg/mL (41.4 µM), respectively. In rats, a single oral dose of the mixture of these fatty acids significantly suppressed the elevation of blood triacylglycerol level following fat intake. These results substantiate the role of free linoleic acid and oleic acid as a novel class of natural product-derived functional molecules that act as pancreatic lipase inhibitors, and their potential for healthy, routine-based weight management.

Dietary Gamma-Aminobutyric Acid (GABA) Induces Satiation by Enhancing the Postprandial Activation of Vagal Afferent Nerves.

Gamma-aminobutyric acid (GABA) is present in the mammalian brain as the main inhibitory neurotransmitter and in foods. It is widely used as a supplement that regulates brain function through stress-reducing and sleep-enhancing effects. However, its underlying mechanisms remain poorly understood, as it is reportedly unable to cross the blood-brain barrier. Here, we explored whether a single peroral administration of GABA affects feeding behavior as an evaluation of brain function and the involvement of vagal afferent nerves. Peroral GABA at 20 and 200 mg/kg immediately before refeeding suppressed short-term food intake without aversive behaviors in mice. However, GABA administration 30 min before refeeding demonstrated no effects. A rise in circulating GABA concentrations by the peroral administration of 200 mg/kg GABA was similar to that by the intraperitoneal injection of 20 mg/kg GABA, which did not alter feeding. The feeding suppression by peroral GABA was blunted by the denervation of vagal afferents. Unexpectedly, peroral GABA alone did not alter vagal afferent activities histologically. The coadministration of a liquid diet and GABA potentiated the postprandial activation of vagal afferents, thereby enhancing postprandial satiation. In conclusion, dietary GABA activates vagal afferents in collaboration with meals or meal-evoked factors and regulates brain function including feeding behavior.

Exosome-Mediated Activation of Neuronal Cells Triggered by γ-Aminobutyric Acid (GABA).

γ-Aminobutyric acid (GABA) is a potent bioactive amino acid, and several studies have shown that oral administration of GABA induces relaxation, improves sleep, and reduces psychological stress and fatigue. In a recent study, we reported that exosomes derived from GABA-treated intestinal cells serve as signal transducers that mediate brain-gut interactions. Therefore, the purpose of this study was to verify the functionality of GABA-derived exosomes and to examine the possibility of improving memory function following GABA administration. The results showed that exosomes derived from GABA-treated intestinal cells (Caco-2) activated neuronal cells (SH-SY5Y) by regulating genes related to neuronal cell functions. Furthermore, we found that exosomes derived from the serum of GABA-treated mice also activated SH-SY5Y cells, indicating that exosomes, which are capable of activating neuronal cells, circulate in the blood of mice orally administered GABA. Finally, we performed a microarray analysis of mRNA isolated from the hippocampus of mice that were orally administered GABA. The results revealed changes in the expression of genes related to brain function. Gene Set Enrichment Analysis (GSEA) showed that oral administration of GABA affected the expression of genes related to memory function in the hippocampus.

Effects of Banafine® , a fermented green banana-derived acidic glycoconjugate, on influenza vaccine antibody titer in elderly patients receiving gastrostomy tube feeding.

Immunosenescence can negatively affect cytokine production in elderly and may impair poor antibody responses to influenza vaccination and infection. Herein, the effects of Banafine® administration on influenza vaccine antibody titer in elderly patients (average age ∼80 years) receiving gastrostomy tube feeding were examined. In the double-blind, single-center, randomized clinical studies, 30 elderly bedridden patients were administered Banafine® or placebo for 8 weeks. At week 4, all patients received influenza vaccination against H1N1, H3N2, B/Yamagata, or B/Victoria. Blood biochemical indices and serum antibody titers were assessed. Banafine® administration significantly increased hemagglutination inhibition titers in response to vaccination against H1N1, H3N2, and B/Yamagata in the elderly patients (P < 0.05). Moreover, the seroconversion rate against H1N1 (47.1%) and H3N2 (29.4%) and seroprotection rate against H1N1 (71.4%) and both B strains (31.3% and 12.5%, respectively) were increased for the Banafine® group. These results suggest that Banafine® administration can increase antibody responses to influenza vaccination in bedridden hospitalized patients, and potentially modulate immune function in the elderly. PRACTICAL APPLICATION: Literature review suggested that most of the synbiotics are based on innate immunity, strain specific (probiotics), and are not consistently observed. Herein, in clinical studies we demonstrate that administration of Banafine® , a plant-based glycoconjugate, can increase antibody levels in bedridden hospitalized elderly patients following influenza vaccination.

γ-Aminobutyric acid (GABA) activates neuronal cells by inducing the secretion of exosomes from intestinal cells.

The oral administration of γ-aminobutyric acid (GABA) has been shown to affect brain functions. However, the molecular mechanisms underlying GABA-induced gut-brain interactions have not yet been fully elucidated. As the blood-brain barrier is impermeable to GABA, we hypothesized that the gut-brain interaction might be stimulated by some secretory factors derived from the gut. Then we focused on exosomes as a secretory mediator. In the present study, we investigated whether exosomes derived from GABA-treated intestinal cells activate neuronal cells. Our results revealed that secretory factors derived from GABA-treated intestinal cells (Caco-2) activated neuronal cells (SH-SY5Y). Further investigation revealed that exosomes derived from GABA-treated Caco-2 cells were responsible for activating the SH-SY5Y cells. These results suggested that GABA-activated intestinal cells induce the secretion of exosomes that activate neuronal cells. MicroRNAs in the exosomes derived from GABA-treated intestinal cells may play a key role in the activation of neuronal cells.

Study of the cartilage matrix production-promoting effect of chicken leg extract and identification of the active ingredient.

BACKGROUND/OBJECTIVES: Osteoarthritis (OA) is a major public health issue in Japan and other countries, and foods that prevent or treat OA are in strong demand. Proteins and peptides in chicken meat and bones are known for being rich in functional and nutritional ingredients for the improvement of osteoporosis. We speculated that chicken legs, a food consumed in many regions of the world, may also contain such ingredients. In this study, we aim to (i) evaluate the effect of chicken leg extract (CLE) on the promotion of cartilage matrix production and (ii) identify the active ingredient in CLE that contributes to this function. MATERIALS/METHODS: Artificial CLE digest was prepared, and the acid mucopolysaccharide production-promoting activity of the CLE digest was evaluated by alcian blue staining of ATDC5 cells. CLE was orally administered to rabbits with burr holes in the knee joint of the femur, and the degree of regeneration of cartilage matrix was evaluated. Furthermore, we investigated orally administered CLE-derived peptides in human plasma using LC-MS. From measuring the acid mucopolysaccharide production-promotion activity of these peptides, a molecule considered to be an active ingredient in the CLE digest was identified. RESULTS: CLE digest promoted acid mucopolysaccharide production and facilitated regeneration of cartilage matrix in in vitro and in vivo experiments. Four peptides including phenylalanyl-hydroxyproline (Phe-Hyp) were detected as CLE-derived peptides in human plasma. The effect of CLE was inferred to be due to Phe-Hyp, which was confirmed to be present in the CLE digest. CONCLUSIONS: It was shown that CLE stimulated the production of articular cartilage matrix both in vitro and in vivo, and that CLE could be an effective food for preventing or treating OA. Furthermore, only Phe-Hyp was confirmed as the active compound in the CLE digest, suggesting that the activity of CLE was due to Phe-Hyp.

Effect of oral γ-aminobutyric acid (GABA) administration on sleep and its absorption in humans.

The effects of γ-aminobutyric acid (GABA) on sleep and its levels in blood after oral administration were investigated in humans. A randomized, single-blind, placebo-controlled crossover-designed study was conducted to evaluate the effect of GABA on sleep. Sleep was evaluated by electroencephalography (EEG) after oral GABA administration. GABA significantly shortened sleep latency and increased the total non-rapid eye movement (non-REM) sleep time. Questionnaires showed that subjects receiving GABA realized its effects on sleep. In addition, the blood level of GABA after administration was investigated, and the absorption and metabolism rates of GABA were determined. GABA was quickly absorbed, and the blood level of GABA was the highest 30 min after oral administration, with a subsequent decrease in concentration. As GABA strongly affected the early stage of sleep, the effect of GABA on sleep may be connected to its levels in blood.

The Improvement of Sleep by Oral Intake of GABA and Apocynum venetum Leaf Extract.

The effects of two food materials, γ-aminobutyric acid (GABA) produced by natural fermentation and Apocynum venetum leaf extract (AVLE), on the improvement of sleep were investigated in humans. The electroencephalogram (EEG) test revealed that oral administration of GABA (100 mg) and AVLE (50 mg) had beneficial effects on sleep. GABA shortened sleep latency by 5.3 min and AVLE increased non-rapid eye movement (REM) sleep time by 7.6%. Simultaneous intake of GABA and AVLE shortened sleep latency by 4.3 min and increased non-REM sleep time by 5.1%. The result of questionnaires showed that GABA and AVLE enabled subjects to realize the effects on sleep. These results mean that GABA can help people to fall asleep quickly, AVLE induces deep sleep, and they function complementarily with simultaneous intake. Since both GABA and AVLE are materials of foods and have been ingested for a long time, they can be regarded as safe and appropriate for daily intake in order to improve the quality of sleep.

Subchronic toxicity evaluation of γ-aminobutyric acid (GABA) in rats.

γ-Aminobutyric acid (GABA) is an amino acid compound contained in vegetables such as tomatoes and also widely distributed in mammals. GABA acts as an inhibitory neurotransmitter and promotes parasympathetic activity to provide several beneficial effects, for instance, relaxation, anti-stress, and insomnia. GABA, produced via a fermentation process, has been available as a functional food ingredient. As part of a program to assess its safety, GABA was administered by oral gavage at doses of 500, 1250, and 2500mg/kg body weight to groups of 10 male and 10 female Sprague-Dawley rats for 13weeks. Treatment was not associated with the test substance-related mortality and appeared to be well tolerated. There were no toxicologically and statistically significant changes in urinalysis, hematology, clinical chemistry parameters, and in necropsy findings. A few statistically significant changes in food consumption and body weights were noted in the male groups while any significant changes were not noted in female groups. There was no effect of treatment on organ weights or on the results of the histopathological examinations. The results of toxicity evaluation support the safety use of GABA and the potential use as a functional food ingredient.

Thermococcus kodakarensis as a host for gene expression and protein secretion.

Taking advantage of the gene manipulation system developed in Thermococcus kodakarensis, here, we developed a system for gene expression and efficient protein secretion using this hyperthermophilic archaeon as a host cell. DNA fragments encoding the C-terminal domain of chitinase (ChiAΔ4), which exhibits endochitinase activity, and the putative signal sequence of a subtilisin-like protease (TK1675) were fused and positioned under the control of the strong constitutive promoter of the cell surface glycoprotein gene. This gene cassette was introduced into T. kodakarensis, and secretion of the ChiAΔ4 protein was examined. ChiAΔ4 was found exclusively in the culture supernatant and was not detected in the soluble and membrane fractions of the cell extract. The signal peptide was specifically cleaved at the C-terminal peptide bond following the Ala-Ser-Ala sequence. Efficient secretion of the orotidine-5'-monophosphate decarboxylase protein was also achieved with the same strategy. We next individually overexpressed two genes (TK1675 and TK1689) encoding proteases with putative signal sequences. By comparing protein degradation activities in the host cells and transformants in both solid and liquid media, as well as measuring peptidase activity using synthetic peptide substrates, we observed dramatic increases in protein degradation activity in the two transformants. This study displays an initial demonstration of cell engineering in hyperthermophiles.

Phosphoenolpyruvate synthase plays an essential role for glycolysis in the modified Embden-Meyerhof pathway in Thermococcus kodakarensis.

We have carried out a genetic analysis on pyruvate kinase (PykTk) and phosphoenolpyruvate synthase (PpsTk) in the hyperthermophilic archaeon, Thermococcus kodakarensis. In principle, both enzymes can catalyse the final step of the modified Embden-Meyerhof (EM) pathway found in Thermococcales, the conversion of phosphoenolpyruvate (PEP) to pyruvate, with the former utilizing ADP, while the latter is dependent on AMP and phosphate. Enzyme activities and transcript levels of both PykTk and PpsTk increased in T. kodakarensis under glycolytic conditions when compared with cells grown on pyruvate or amino acids. Using KW128, a tryptophan auxotrophic mutant with a trpE gene disruption, as a host strain, we obtained mutant strains with single gene disruptions in either the pykTk (Deltapyk strain) or ppsTk (Deltapps strain) gene. Specific growth rates and cell yields were examined in various media and compared with the host KW128 strain. The results indicated that both enzymes participated in pyruvate metabolism, but were not essential. In the presence of maltooligosaccharides, the Deltapyk strain displayed a 15% decrease in growth rate compared with the host strain, indicating that PykTk does participate in glycolysis. However an even more dramatic effect was observed in the Deltapps strain in that the strain could not grow at all on maltooligosaccharides. The results clearly indicate that, in contrast to the conventional EM pathway dependent on pyruvate kinase, PEP synthase is the essential enzyme for the glycolytic conversion of PEP to pyruvate in T. kodakarensis. The physiological roles of the two enzymes under various growth conditions are discussed.

Isolation and characterization of a novel poly(vinyl alcohol)-degrading bacterium, Sphingopyxis sp. PVA3.

We have isolated a poly(vinyl alcohol) (PVA)-degrading bacterium from an activated sludge sample obtained from the drainage of a dyeing factory. Enrichment cultures were performed in media containing PVA as the sole or major carbon source. After several rounds of cultivation on liquid and solid media, we were able to isolate a single colony with PVA-degrading ability (strain PVA3). The bacterium could degrade PVA in the absence of symbionts or cofactors such as pyrroloquinoline quinone (PQQ). Over 90% of PVA, at an initial concentration of 0.1%, was degraded within a 6-day cultivation. Degradation was confirmed by both iodometric methods and gel permeation chromatography. Examination of the PVA attached to the cells revealed a large increase in carbonyl groups, suggesting the oxidation of hydroxyl groups of the polymer on the surfaces of cells. Addition of PQQ to the culture medium did not enhance the growth and the PVA-degrading rates of strain PVA3. Furthermore, we found that cells grown on PVA generated hydrogen peroxide upon the addition of PVA. The results strongly suggest that the initial oxidation of PVA is mediated via a PVA oxidase, and not a PQQ-dependent dehydrogenase. A biochemical and phylogenetic characterization of the bacterium was performed. The sequence of the 16S ribosomal RNA gene of the bacterium indicated a phylogenetic position of the strain within the genus Sphingopyxis, and the strain was therefore designated Sphingopyxis sp. PVA3.