Time-fixed feeding prevents obesity induced by chronic advances of light/dark cycles in mouse models of jet-lag/shift work.

Recent findings have uncovered intimate relationships between circadian clocks and energy metabolism. Epidemiological studies have shown that the frequency of obesity and metabolic disorders increases among shift-workers. Here we found that a chronic shift in light/dark (LD) cycles comprising an advance of six hours twice weekly, induced obesity in mice. Under such conditions that imitate jet lag/shift work, body weight and glucose intolerance increased, more fat accumulated in white adipose tissues and the expression profiles of metabolic genes changed in the liver compared with normal LD conditions. Mice fed at a fixed 12 h under the LD shift notably did not develop symptoms of obesity despite isocaloric intake. These results suggest that jet lag/shift work induces obesity as a result of fluctuating feeding times and it can be prevented by fixing meal times. This rodent model of obesity might serve as a useful tool for understanding why shift work induces metabolic disorders.

Effects of subchronic and mild social defeat stress on the intestinal microbiota and fecal bile acid composition in mice.

The gut microbiota plays a crucial role in both the pathogenesis and alleviation of host depression by modulating the brain-gut axis. We have developed a murine model of human depression called the subchronic and mild social defeat stress (sCSDS) model, which impacts not only behavior but also the host gut microbiota and gut metabolites, including bile acids. In this study, we utilized liquid chromatography/mass spectrometry (LC/MS) to explore the effects of sCSDS on the mouse fecal bile acid profile. sCSDS mice exhibited significantly elevated levels of deoxycholic acid (DCA) and lithocholic acid (LCA) in fecal extracts, leading to a notable increase in total bile acids and 7α-dehydroxylated secondary bile acids. Consequently, a noteworthy negative correlation was identified between the abundances of DCA and LCA and the social interaction score, an indicator of susceptibility in stressed mice. Furthermore, analysis of the colonic microbiome unveiled a negative correlation between the abundance of CDCA and Turicibacter. Additionally, DCA and LCA exhibited positive correlations with Oscillospiraceae and Lachnospiraceae but negative correlations with the Eubacterium coprostanoligenes group. These findings suggest that sCSDS impacts the bidirectional interaction between the gut microbiota and bile acids and is associated with reduced social interaction, a behavioral indicator of susceptibility in stressed mice.

Bile acids induced hepatic lipid accumulation in mice by inhibiting mRNA expression of patatin-like phospholipase domain containing 3 and microsomal triglyceride transfer protein.

Preliminary studies have shown that a lithogenic diet (LG), which contains cholesterol and cholic acid, induces gallstones and hepatic lipid accumulation (HLA), and reduction of blood triglyceride in mice. We hypothesized that an LG induces HLA by diminishing hepatic triglyceride excretion; however, there is no clear understanding of the mechanism of LG-induced HLA. This study aimed to investigate transcript expression related to the synthesis, expenditure, and efflux of hepatic triglyceride, in mice fed an LG for 4 weeks. Results showed lower plasma concentrations of triglyceride in the LG group than in the control group, but no symptoms of hepatic injury were observed. Hepatic mRNA expressions of patatin-like phospholipase domain containing 3 (Pnpla3), microsomal triglyceride transfer protein (Mttp), and acyl-CoA oxidase 1 (Acox1) were also reduced in the LG group. Deoxycholic acid and lithocholic acid promoted intracellular lipid accumulation, reduced triglyceride concentration in media, and suppressed expression of PNPLA3 and MTTP in HepG2 human hepatoma cells. These findings suggest that deoxycholic acid and lithocholic acid promote HLA by inhibiting the expression of PNPLA3, ACOX1, and MTTP that are involved in lipid metabolism.

Repeated oral administration of a squeezed ginger (Zingiber officinale) extract augmented the serum corticosterone level and had anti-inflammatory properties.

We investigated the ability of a ginger extract to induce an immune response in RAW 264 cells and after a repeated oral administration to mice. The squeezed ginger extract augmented the production of tumor necrosis factor-α, interleukin-6, and monocyte chemotactic protein-1 when added to RAW 264 cells. This extract was collected as its ethanol-insoluble fraction. The oral administration of the squeezed ginger extract or its ethanol-insoluble fraction once or twice to mice also augmented the tumor necrosis factor-α production in peritoneal cells; however, its long-term administration had the opposite effect. The serum corticosterone level had increased after orally administering the squeezed ginger extract and was maintained during the administration period. Oral administration of the squeezed ginger extract also inhibited arachidonic acid-induced ear edema, but its repeated administration was needed to achieve an anti-inflammatory effect. These results suggest that the repeated administration of the aqueous constituents of ginger augmented the serum corticosterone level and that this may have gradually induced anti-inflammatory activity.

Involvement of the hepatic branch of the vagus nerve in the regulation of plasma adipokine levels in rats fed a high-fructose diet.

High-fructose diets are associated with not only fat accumulation in liver but also blood adipokine levels. Some studies have shown the involvement of humoral factors in the regulation of adipokines. However, the role of the vagus nerve in expression of adipokines is not fully understood. We attempted to investigate the involvement of the hepatic branch of the vagus nerve (HBVN) in the regulation of plasma adipokine levels in rats fed a high-fructose (HFr) diet. Rats underwent hepatic vagotomy (Vx) or sham operation; thereafter, they were fed a control diet (CT) or HFr diet for 6 weeks. At the sixth week, the oral glucose tolerance test (OGTT) was performed. In the sham-operated group, plasma leptin and adiponectin levels were significantly lower in the HFr group than those in the control group. In contrast, in the Vx group, there was no difference in the respective adipokine levels of the two dietary groups. In OGTT, plasma leptin levels were significantly correlated to the area under the curve (AUC) of plasma insulin levels and insulin levels at some points. Further, the ratio of plasma leptin levels to plasma adiponectin levels was correlated with the AUC of plasma insulin levels. However, the plasma adiponectin level itself did not correlate with plasma insulin levels and insulin AUC. Thus, we showed that HBVN played a key role in down-regulating plasma leptin and adiponectin levels in HFr-fed rats.

Absolute quantification of the α, α', and ß subunits of ß-conglycinin from soybeans by liquid chromatography/tandem mass spectrometry using stable isotope-labelled peptides.

ß-Conglycinin, a major protein in soybeans, shows improvement effect of lipid metabolism. Moreover, this protein influences the processing properties of soybeans. ß-Conglycinin is a hetero-trimer constituted by α, α', and ß subunits. In this work, a method for the selective quantification of these subunits was developed by means of protein absolute quantification (AQUA) technology using liquid chromatography/tandem mass spectrometry with the stable isotope-labelled internal standard peptides LQSGDALR[13C6,15N4], NILEASYDTK[13C6,15N2], and NPIYSNNFGK[13C6,15N2]. This method exhibited linear relationships (r2 > 0.99) in the concentration range of 1.2-300 fmol/µL for LQSGDALR[13C6,15N4] and NILEASYDTK[13C6,15N2], and of 4.7-300 fmol/µL for NPIYSNNFGK[13C6,15N2]. As a result, the content of these subunits in ß-conglycinin-rich and both α and α' subunit-deficient soybean cultivars was successfully determined. This quantitative assay is promising for the evaluation of the food functionality and processing properties of soybeans.

Lowering effects of onion intake on oxidative stress biomarkers in streptozotocin-induced diabetic rats.

The protective effect of onion against oxidative stress in streptozotosin-induced diabetic rats was investigated in comparison with that of quercetin aglycone. We measured oxidative stress biomarkers involving the susceptibility of the plasma against copper ion-induced lipid peroxidation, which was estimated by the amounts of thiobarbituric acid-reactive substances (TBARS) and cholesteryl ester hydroperoxides, and urine TBARS and 8-hydroxydeoxyguanosine contents. After the 12-week feeding period, plasma glucose levels and these biomarkers increased in diabetic rats compared to normal rats. In diabetic rats fed a 6.0% onion diet (quercetin equivalent: 0.023%), quercetin metabolites accumulated in the plasma at concentrations of approximately 35 microM. Onion intake decreased plasma glucose levels and lowered the oxidative stress biomarkers. On the other hand, quercetin metabolites in the plasma of rats fed a diet with 0.023% quercetin aglycone were found at lower concentrations (14.2 microM) than the rats fed the onion diet. Furthermore, oxidative stress biomarkers were higher in the quercetin diet group compared to the onion diet group. These results strongly suggest that onion intake suppresses diabetes-induced oxidative stress more effectively than the intake of the same amount of quercetin aglycone alone.

Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/µL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods.

Quercetin suppresses immune cell accumulation and improves mitochondrial gene expression in adipose tissue of diet-induced obese mice.

SCOPE: To examine the effect of dietary quercetin on the function of epididymal adipose tissue (EAT) in Western diet-induced obese mice. METHODS AND RESULTS: C57BL/6J mice were fed a control diet; a Western diet high in fat, cholesterol, and sucrose; or the same Western diet containing 0.05% quercetin for 18 weeks. Supplementation with quercetin suppressed the increase in the number of macrophages, the decrease in the ratio of CD4(+) to CD8(+) T cells in EAT, and the elevation of plasma leptin and tumor necrosis factor α levels in mice fed the Western diet. Comprehensive gene expression analysis revealed that quercetin suppressed gene expression associated with the accumulation and activation of immune cells, including macrophages and lymphocytes in EAT. It also improved the expression of the oxidative stress-sensitive transcription factor NFκB, NADPH oxidases, and antioxidant enzymes. Quercetin markedly increased gene expression associated with mitochondrial oxidative phosphorylation and mitochondrial DNA content. CONCLUSION: Quercetin most likely universally suppresses the accumulation and activation of immune cells, including antiinflammatory cells, whereas it specifically increased gene expression associated with mitochondrial oxidative phosphorylation. Suppression of oxidative stress and NFκB activity likely contributed to the prevention of the accumulation and activation of immune cells and resulting chronic inflammation.

Dietary intake of heat-killed Lactococcus lactis H61 delays age-related hearing loss in C57BL/6J mice.

Age-related hearing loss (AHL) is a common disorder associated with aging. In this study, we investigated the effect of the intake of heat-killed Lactococcus lactis subsp. cremoris H61 (strain H61) on AHL in C57BL/6J mice. Measurement of the auditory brainstem response (ABR) demonstrated that female mice at 9 months of age fed a diet containing 0.05% strain H61 for 6 months maintained a significantly lower ABR threshold than control mice. The age-related loss of neurons and hair cells in the cochlea was suppressed by the intake of strain H61. Faecal analysis of bacterial flora revealed that the intake of strain H61 increased the prevalence of Lactobacillales, which is positively correlated with hearing ability in mice. Furthermore, plasma fatty acid levels were negatively correlated with hearing ability. Overall, the results supported that the intake of heat-killed strain H61 for 6 months altered the intestinal flora, affected plasma metabolite levels, including fatty acid levels, and retarded AHL in mice.

Absolute quantification of protein NP24 in tomato fruit by liquid chromatography/tandem mass spectrometry using stable isotope-labelled tryptic peptide standard.

Protein NP24 is a thaumatin-like protein contained in tomato (Lycopersicon esculentum Mill.). This protein is reported to be a putative tomato allergen and is listed as a food allergen in Structural Database of Allergenic Proteins (SDAP). In this research, we developed the quantitative analysis of NP24 by employing the protein absolute quantification (AQUA) technology composed of stable isotope-labelled internal standard (SIIS) peptide (GQTWVINAPR[(13)C6,(15)N4]) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). A linear relationship (r(2)>0.99) was found throughout the concentration range (2.0-500 fmol/µL). The coefficients of variation (CVs) measured on each of the five days when NP24 contained in the tomato skin was analysed did not exceed 13%. Our developed assay of NP24 will contribute to the allergological examination of tomato and its derived products.

Estimated daily intake and seasonal food sources of quercetin in Japan.

Quercetin is a promising food component, which can prevent lifestyle related diseases. To understand the dietary intake of quercetin in the subjects of a population-based cohort study and in the Japanese population, we first determined the quercetin content in foods available in the market during June and July in or near a town in Hokkaido, Japan. Red leaf lettuce, asparagus, and onions contained high amounts of quercetin derivatives. We then estimated the daily quercetin intake by 570 residents aged 20-92 years old in the town using a food frequency questionnaire (FFQ). The average and median quercetin intakes were 16.2 and 15.5 mg day(-1), respectively. The quercetin intakes by men were lower than those by women; the quercetin intakes showed a low correlation with age in both men and women. The estimated quercetin intake was similar during summer and winter. Quercetin was mainly ingested from onions and green tea, both in summer and in winter. Vegetables, such as asparagus, green pepper, tomatoes, and red leaf lettuce, were good sources of quercetin in summer. Our results will help to elucidate the association between quercetin intake and risks of lifestyle-related diseases by further prospective cohort study and establish healthy dietary requirements with the consumption of more physiologically useful components from foods.

Effect of naturally occurring organosulfur compounds on nitric oxide production in lipopolysaccharide-activated macrophages.

Excessive nitric oxide (NO) production is involved in cellular injury and possibly in the multistage process of carcinogenesis. In this study, we investigated the effect of organosulfur compounds (S-allyl cysteine, allyl sulfide, diallyl disulfide, allyl isothiocyanate, phenyl isothiocyanate, and benzyl isothiocyanate) that are found in allium or cruciferous vegetables on NO production in J774.1 macrophages activated with lipopolysaccharide (LPS). Diallyl disulfide, allyl, phenyl, and benzyl isothiocyanates inhibited NO production, as evaluated by nitrite formation at 25 microM. Allyl and benzyl isothiocyanates, the most active of the six organosulfur compounds, exhibited dose-dependent inhibition and had IC(50) values of 1.6 and 2.7 microM, respectively. Western blot analysis suggested that suppression of the induction of inducible NO synthase (iNOS) expression is responsible for the inhibition of NO production by allyl and benzyl isothiocyanates. In contrast, these isothiocyanates increased LPS-stimulated tumor necrosis factor alpha (TNF-alpha) release, suggesting their selective action on genes activated by LPS. Our results demonstrate that certain organosulfur compounds inhibit NO synthesis in LPS-activated macrophages, and the inhibitory effect may be a significant component of their anticarcinogenic activity.

[6]-Gingerol inhibits nitric oxide synthesis in activated J774.1 mouse macrophages and prevents peroxynitrite-induced oxidation and nitration reactions.

Reactive nitrogen species (RNS), such as nitric oxide (NO) and its derivatives, e.g. peroxynitrite (ONOO-), have been proposed as being able to influence signal transduction and cause DNA damage, contributing to carcinogenic processes. In this study, the effect of [6]-gingerol, a pungent phenolic compound present in ginger (Zingiber officinale Roscoe), on NO synthesis in lipopolysaccharide (LPS)-activated J774.1 macrophages was tested, and the protective ability of this compound against peroxynitrite-mediated oxidation and nitration reactions were evaluated. [6]-Gingerol exhibited dose-dependent inhibition of NO production and significant reduction of inducible NO synthase (iNOS) in LPS-stimulated J774.1 cells. Moreover, [6]-gingerol effectively suppressed peroxynitrite-induced oxidation of dichlorodihydrofluorescein, oxidative single strand breaks in supercoiled pTZ 18U plasmid DNA, and formation of 3-nitrotyrosine in bovine serum albumin (BSA) and J774.1 cells. Our results indicate that [6]-gingerol is a potent inhibitor of NO synthesis and also an effective protector against peroxynitrite-mediated damage.

Combination of lipids and emulsifiers enhances the absorption of orally administered quercetin in rats.

The effects of lipids, emulsifiers, and ethanol on the absorption of orally administered quercetin in rats were investigated for its efficient intestinal absorption. Rats were administered 150 micromol/kg quercetin in water supplemented with lipids and/or emulsifiers, or ethanol, and blood was collected from the tail for 6 h after administration. Co-administration of lipids such as lecithin and soybean oil or emulsifiers including sucrose fatty acid ester, polyglycerol fatty acid ester, and sodium taurocholate had no statistically significant effects on quercetin absorption, although these constituents rather increased the accumulation of conjugated forms of quercetin and those of isorhamnetin in rat plasma. However, the combination of lipids and emulsifiers enhanced the absorption of quercetin significantly. Thirty and fifty percent (v/v) of the ethanol in the vehicle raised the efficiency of quercetin absorption in a concentration-dependent manner. Quercetin absorption-enhancing effects of these constituents seemed to be affected by quercetin's solubility in respective vehicles used for the administration. Ethanol is not helpful for the effective absorption of quercetin, as a high concentration is required. In conclusion, a combination of lipids and emulsifiers is necessary for enhancing quercetin absorption.

Milk prevents the degradation of daikon (Raphanus sativus L.) isothiocyanate and enhances its absorption in rats.

Epidemiological and experimental researches show that isothiocyanate (ITC), a class of phytochemical compounds that imparts a characteristic biting taste and pungent odour to cruciferous vegetables, such as daikon (Japanese white radish, Raphanus sativus L. Daikon Group), broccoli, cabbage, and Chinese cabbage, possesses anticancer and anti-inflammatory properties. The concentration of daikon ITC, which degrades in aqueous solution, was measured in mixtures of daikon juice and water, corn oil, or milk. Daikon juice mixed with corn oil or milk showed a higher concentration (1.4-fold) of daikon ITC than that in mixture with water; thus, corn oil and milk prevent the degradation of daikon ITC. Moreover, orally administered daikon juice with milk increased daikon ITC absorption in rats. Therefore, dishes or drinks that include raw daikon with corn oil or milk may promote the possible health benefits of daikon ITC by preventing ITC degradation and enhancing its absorption in vivo.

Corn oil and milk enhance the absorption of orally administered allyl isothiocyanate in rats.

Allyl isothiocyanate, a chief component of mustard oil, exhibits anticancer effects in both cultured cancer cells and animal models. The accumulation of the N-acetylcysteine conjugate of allyl isothiocyanate, the final metabolite of allyl isothiocyanate, in urine was evaluated in rats that were orally coadministered allyl isothiocyanate with fluids (e.g., water, green tea, milk, and 10% ethanol) or corn oil. The N-acetylcysteine conjugate of allyl isothiocyanate content in urine when allyl isothiocyanate (2 or 4µmol) was coadministered with corn oil or milk showed a greater increase (1.4±0.22 or 2.7±0.34µmol or 1.2±0.32 or 2.5±0.36µmol, 1.6- to 1.8-fold or 1.5-fold, respectively) than when allyl isothiocyanate (2 or 4µmol) was coadministered with water (0.78±0.10 or 1.7±0.17µmol). This result demonstrates that corn oil and milk enhance the absorption of allyl isothiocyanate in rats.

Evaluation of tolerable levels of dietary quercetin for exerting its antioxidative effect in high cholesterol-fed rats.

The tolerable level of dietary quercetin for exerting its antioxidative effect was evaluated in high cholesterol-fed rats, using quercetin-containing diets (31-1260 mg quercetin/kg body weight/day) and onion diets (19-94 mg quercetin aglycone equivalent/kg body weight/day), from the viewpoint of a safety assessment. After feeding for 4 weeks, the urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels of the quercetin-containing diet groups fed more than 157 mg quercetin/kg body weight/day were higher than the group fed a quercetin-free diet, although the plasma quercetin metabolite levels and plasma antioxidative activity were elevated depending on the amounts of quercetin or onion diet intake. No significant effect on body weight gain by quercetin-containing diets or onion diets was observed. However, ratios of the liver and kidney weights to the body weight were significantly increased in the quercetin-containing diet groups fed more than 314 mg and 157 mg quercetin/kg body weight/day, respectively, and in the onion diet groups fed more than 47 mg quercetin aglycone equivalent/kg body weight/day. These results indicated that the tolerable level for dietary quercetin for exerting its antioxidative effect was between 126 and 157 mg/kg/day for the quercetin diet and between 19 and 34 mg/kg/day for the onion diet.

Structures and antioxidant activity of anthocyanins in many accessions of eggplant and its related species.

Anthocyanins were detected in extracts from the peels of 123 accessions of eggplant (Solanum melongena) and its related species. Their anthocyanin profiles were classified into four types, including known Japanese eggplant type (type 1) and non-Japanese eggplant type (type 2). Although most of the eggplant accessions had one of the two known profiles, one accession had a novel profile (type 3). Two accessions of related species showed another novel profile (type 4). The major anthocyanins were identified as delphinidin 3-(p-coumaroylrutinoside)-5-glucoside (nasunin) (type 1), delphinidin 3-rutinoside (type 2), delphinidin 3-glucoside (type 3), and petunidin 3-(p-coumaroylrutinoside)-5-glucoside (petunidin 3RGc5G) (type 4). Delphinidin 3-caffeoylrutinoside-5-glucoside (delphinidin 3RGcaf5G) was isolated from the hybrid (F1) plants of a type 1 cultivar and a type 3 germplasm. Among the five purified anthocyanins, delphinidin 3RGcaf5G showed the highest radical-scavenging activities toward both 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and linoleic acid radical, followed in order by nasunin and petunidin 3RGc5G.

Mechanism of inhibition of peroxynitrite-induced oxidation and nitration by [6]-gingerol.

[6]-Gingerol potently inhibits peroxynitrite-induced oxidation and nitration reactions, but its mechanism of action is unclear. In order to discover the mechanism of inhibition, [6]-gingerol was reacted with peroxynitrite and the reaction mixture was analyzed using HPLC. The HPLC chromatogram showed one novel peak, indicative of the formation of a reaction product between [6]-gingerol and peroxynitrite. This compound was purified and identified as a symmetrical dimer of [6]-gingerol covalently linked at the aromatic ring. It has been assumed that this dimer is generated from a phenoxyl radical intermediate produced from [6]-gingerol via one-electron oxidation by peroxynitrite-derived radicals. We propose a mechanism in which [6]-gingerol scavenges peroxynitrite-derived radicals and consequently inhibits peroxynitrite-induced oxidation and nitration reactions.