Glucose metabolic upregulation via phosphorylation of S6 ribosomal protein affects tumor progression in distal cholangiocarcinoma.

BACKGROUND: The prognosis of distal cholangiocarcinoma (dCCA) remains poor; thus, the identification of new therapeutic targets is warranted. Phosphorylated S6 ribosomal protein indicates a mammalian target of rapamycin complex 1 (mTORC1) activity, and mTORC1 plays a central role in controlling cell growth and regulating glucose metabolism. We aimed to clarify the effect of S6 phosphorylation on tumor progression and the glucose metabolic pathway in dCCA. METHODS: Thirty-nine patients with dCCA who underwent curative resection were enrolled in this study. S6 phosphorylation and the expression of GLUT1 were evaluated by immunohistochemistry, and their relationship with clinical factors was investigated. The effect of S6 phosphorylation on glucose metabolism with PF-04691502 treatment, an inhibitor of S6 phosphorylation, was examined in cancer cell lines by Western blotting and metabolomics analysis. Cell proliferation assays were performed with PF-04691502. RESULTS: S6 phosphorylation and the expression of GLUT1 were significantly higher in patients with an advanced pathological stage. Significant correlations between GLUT1 expression, S6 phosphorylation, and SUV-max of FDG-PET were shown. In addition, cell lines with high S6 phosphorylation levels showed high GLUT1 levels, and the inhibition of S6 phosphorylation reduced the expression of GLUT1 on Western blotting. Metabolic analysis revealed that inhibition of S6 phosphorylation suppressed pathways of glycolysis and the TCA cycle in cell lines, and then, cell proliferation was effectively reduced by PF-04691502. CONCLUSION: Upregulation of glucose metabolism via phosphorylation of S6 ribosomal protein appeared to play a role in tumor progression in dCCA. mTORC1 may be a therapeutic target for dCCA.

GC-MS/MS analysis of metabolites derived from a single human blastocyst.

INTRODUCTION: The field of assisted reproductive technology (ART) has significantly advanced; however, morphological evaluation remains as the chosen method of assessment of embryo quality. OBJECTIVE: We aimed to examine metabolic changes in embryo culture medium to develop a non-invasive method for evaluation of embryo quality. METHODS: We performed metabolic analysis of culture medium obtained from a single blastocyst cultured for freezing. RESULTS: In total, 187 (39.8%) of the 469 detectable organic acid metabolites were identified. A significant change (p < 0.05) was observed in eight metabolites between the good-quality and poor-quality embryo groups. Differences were observed in several metabolic pathways between the good-quality and poor-quality embryo groups. Metabolites that showed significant changes were primarily involved in the metabolism of branched-chain amino acids. CONCLUSION: The quantification of metabolism in human embryos may assist in identification and selection of good-quality embryos with high rates of survival before freezing and implantation in conjunction with morphological classification. This may help to identify embryos with high rates of survival.

Circadian rhythm-dependent induction of hepatic lipogenic gene expression in rats fed a high-sucrose diet.

Metabolic syndrome has become a global health challenge and was recently reported to be positively correlated with increased sucrose consumption. Mechanistic analyses of excess sucrose-induced progression of metabolic syndrome have been focused mainly on abnormal hepatic lipogenesis, and the exact contribution of excess sucrose to metabolic disorders remains controversial. Considering that carbohydrate and lipid metabolisms exhibit clear circadian rhythms, here we investigated the possible contribution of diurnal oscillations to responses of hepatic lipid metabolism to excess sucrose. We found that excess sucrose dose-dependently promotes fatty liver and hyperlipidemia in in rats fed a high-sucrose diet (HSD). We observed that excess sucrose enhances the oscillation amplitudes of the expression of clock genes along with the levels of hepatic lipid and carbohydrate metabolism-related mRNAs that increase lipogenesis. We did not observe similar changes in the levels of the transcription factors regulating the expression of these genes. This suggested that the excess sucrose-induced, circadian rhythm-dependent amplification of lipogenesis is post-transcriptionally regulated via the stability of metabolic gene transcripts. Of note, our findings also provide evidence that fructose causes some of the HSD-induced, circadian rhythm-dependent alterations in lipogenic gene expression. Our discovery of HSD-induced circadian rhythm-dependent alterations in lipogenesis at the post-transcriptional level may inform future studies investigating the complex relationships among sucrose uptake, circadian rhythm, and metabolic enzyme expression. Our findings could contribute to the design of chrono-nutritional interventions to prevent or manage the development of fatty liver and hyperlipidemia in sucrose-induced metabolic syndrome.

Helicobacter pylori infection modulates endogenous hydrogen sulfide production in gastric cancer AGS cells.

BACKGROUND: Persistent Helicobacter pylori infection induces gastric mucosal atrophy, which is a precancerous condition. Hydrogen sulfide (H2 S), a gaseous biological transmitter, has been implicated in both the physiological functions of the gastrointestinal tract and its diseases. To understand gastric epithelial cell response against H pylori infection, we investigated the metabolic changes of gastric cancer cells co-cultured with H pylori and observed the modulation of endogenous H2 S production. MATERIALS AND METHODS: Gastric cancer AGS cells were co-cultured with an H pylori standard strain possessing bacterial virulence factor CagA (ATCC 43504) and a strain without CagA (ATCC 51932). Three hours after inoculation, the cells were subjected to metabolomics analysis using gas chromatography-tandem mass spectrometry (GC-MS/MS). Orthogonal projections to latent structures discriminant analysis (OPLS-DA) and pathway analysis were performed. In addition, intracellular H2 S levels were measured by using HSip-1 fluorescent probe. RESULTS: Results of OPLS-DA showed a significant difference between the metabolism of untreated control cells and cells inoculated with the H pylori strains ATCC 51932 or ATCC 43504, mainly due to 45 metabolites. Pathway analysis with the selected metabolites indicated that methionine metabolism, which is related to H2 S production, was the most frequently altered pathway. H pylori-inoculated cells produced more endogenous H2 S than control cells. Moreover, ATCC 43504-inoculated cells produced less H2 S than ATCC 51932-inoculated cells. CONCLUSIONS: H pylori infection modulates endogenous H2 S production in AGS cells, suggesting that H2 S might be one of the bioactive molecules involved in the biological mechanisms of gastric mucosal disease including mucosal atrophy.

Oral administration of antibiotics results in fecal occult bleeding due to metabolic disorders and defective proliferation of the gut epithelial cell in mice.

Antibiotics sometimes exert adverse effects on the pathogenesis of colitis due to the dysbiosis resulting from the disruption of gut homeostasis. However, the precise mechanisms underlying colitogenic effects of antibiotic-induced colitis are largely unknown. Here, we show a novel murine fecal occult bleeding model induced by the combinatorial treatment of ampicillin and vancomycin, which is accompanied by an enlarged cecum, upregulation of pro-inflammatory cytokines IL-6 and IL-12, a reduction in Ki-67-positive epithelial cell number and an increase in the apoptotic cell number in the colon. Moreover, gas chromatography-tandem mass analysis showed that various kinds of metabolites, including glutamic acid and butyric acid, were significantly decreased in the cecal contents. In addition, abundance of butyric acid producer Clostridiales was dramatically reduced in the enlarged cecum. Interestingly, supplementation of monosodium glutamate or its precursor glutamine suppressed colonic IL-6 and IL-12, protected from cell apoptosis and prevented fecal occult blood indicating that the reduced level of glutamic acid is a possible mechanism of antibiotic-induced fecal occult bleeding. Our data showed a novel mechanism of antibiotic-induced fecal occult bleeding providing a new insight into the clinical application of glutamic acid for the treatment of antibiotic-induced colitis.

Orexigenic action of oral zinc: metabolomic analysis in the rat hypothalamus.

We previously reported an orexigenic action of oral zinc administration in male Sprague-Dawley (SD) rats during an early stage of feeding with a zinc-deficient diet, without decreased zinc concentrations in tissues. The overall conclusion was that orally but not intraperitoneally administered zinc stimulates food intake in short-term zinc-deficient-diet fed rats. We here investigate the mechanism of the orexigenic action of zinc using GC-MS/MS-targeted metabolomic analysis in the rat hypothalamus. Four-week-old, male SD/Slc rats were used, and after 2 days of feeding with a zinc-deficient diet, 3 mg of ZnSO4 in 5 mL saline solution were administered to each rat either orally or intraperitoneally. Three hours after administration, the rats were sacrificed and the hypothalamus were excised and analyzed. We found that the oral administration group showed increased concentrations of 3-aminopropanoic acid (ß-alanine), hypotaurine, dopamine, and biotin. In light of metabolomic analysis of these results, we indicate directions for further research.

[Stressors and the Sense of Coherence Related to the Mental Health of Nurses Assuming the Roles of Wives and/or Mothers - Investigation into the Effects of Leaving Jobs Because of Marriage, Childbirth, and Childrearing].

Many female nurses leave their jobs because of major life events. However, the mental health status and related factors among nurses who assume the roles of wives and/or mothers have been insufficiently examined. Therefore we examined the mental health levels and related factors among such nurse. We conducted a questionnaire survey on 763 female nurses working at general hospitals with over 200 beds in Fukuoka Prefecture. Of 402 responses, 108 were divided into two groups: nurses who had left because of marriage, childbirth, or childrearing (leaving group), and those who had not (non-leaving group). The following were assessed: work satisfaction level, the Brief Job Stress Questionnaire, The General Health Questionnaire (GHQ) 28, and the Sense of Coherence (SOC) scale. Results showed that nurses who had assumed the roles of wives and/or mothers had lower mental health status than general women, and nurses who retained their jobs had higher mental health status and sense of comprehensibility on the SOC scale than those who left. Multiple regression analyses using the total GHQ score as an objective variable showed that only the sense of comprehensibility on the SOC scale correlated with mental health status in the non-leaving group. For the leaving group, having support, high work and life satisfaction levels, and several work stressors were correlated. These findings strongly suggest that to maintain and improve the mental health of nurses who assume the role of wives and/or mothers, greater support, higher satisfaction, reduced stressors, and maintenance and improvement of the sense of comprehensibility are required.

Effect of perioperative oral care on postoperative infections in patients with cancer: A systematic review and meta-analysis.

AIM: This systematic review aimed to assess the effect of non-pharmacologic perioperative oral hygiene care on reduced incidence of postoperative pneumonia (PP), surgical site infection (SSI), and the length of hospital stay in patients with cancer, and to describe the details of oral hygiene care. METHODS: We searched seven databases. Eligibility criteria were based on perioperative oral hygiene care provided by healthcare professionals to patients aged ≥18 years who were surgically treated under general anesthesia and were evaluated for the incidence of PP and SSI. We reported risk ratios (RR) for dichotomous outcomes for PP and SSI using a fixed-effects model of meta-analysis. RESULTS: The search resulted in 850 articles, among which two were randomized controlled trials (RCTs) and 21 were observational studies. Most studies indicated that dentists and medical care providers performed a combination of oral cleaning, and oral hygiene instructions. In RCTs, perioperative oral hygiene care significantly reduced the incidence of PP (RR, 0.86; p = .60), while in observational studies, perioperative oral hygiene care significantly reduced the incidence of PP (RR, 0.55; p < .001) and SSI (RR, 0.47; p < .001). The length of hospital stay was also significantly reduced (p < .05). However, the effectiveness of nursing intervention was not clear. CONCLUSIONS: Perioperative oral hygiene care implemented by healthcare professionals prevented PP and SSI and reduced length of hospital stays for patients after cancer surgery. As daily perioperative oral hygiene care is performed by nurses, it is necessary to research the effects of oral hygiene by nurses in the future.

Metabolome Characteristics of Liver Autophagy Deficiency under Starvation Conditions in Infancy.

The liver function is essential for metabolism, detoxification, and bile synthesis, even in the neonatal period. Autophagy plays significance roles in THE adult liver, whereas the role of liver autophagy in the early neonatal period largely remains unclear. To clarify the importance of liver autophagy in the neonatal starvation period, we generated liver-specific autophagy-deficient (Atg5flox/flox; Albumin-Cre) mice and investigated under starvation conditions comparing with control (Atg5flox/+; Albumin-Cre) mice, focusing on serum metabolites and liver histopathology. As a result, autophagy in the liver was found to unessential for the survival under postnatal starvation. A metabolomics analysis of serum metabolites by gas chromatography-tandem mass spectrometry showed a significant difference between the groups, especially after 12-h starvation, suggesting the synergistical adaption of metabolic pathways, such as the "malate-aspartate shuttle", "aspartate metabolism", "urea cycle", and "glycine and serine metabolism". Liver-specific autophagy-deficiency under postnatal starvation conditions can cause a characteristic metabolic alteration suggesting a change of the mitochondrial function. Neonates seemed to maintain ketone production under starvation conditions, even in the autophagy-deficient liver, through a change in the mitochondrial function, which may be an adaptive mechanism for avoiding fatal starvation.

A novel fractionation method of the rough ER integral membrane proteins; resident proteins versus exported proteins?

We treated the high salt-washed canine pancreatic rough ER (KRM) with 0.18% Triton X-100, separated the extract from the residual membrane (0.18%Tx KRM), and processed the extract with SM-2 beads to recover membrane proteins in proteoliposomes. To focus on integral membrane proteins, KRM, 0.18%Tx KRM and proteoliposomes were subjected to sodium carbonate treatment, and analyzed by 2-D gel electrophoresis. Consequently we found that a distinct group of integral membrane protein of KRM preferentially extracted from the membrane and recovered in proteoliposomes did exist, while majority of KRM integral membrane proteins were fractionated in 0.18%Tx KRM, which retained the basic structure and functions of KRM. Protein identification showed that the former group was enriched with proteins exported from the ER and the latter group comprised mostly of ER resident proteins. This result will potentially affect the prevailing view of the ER membrane structure as well as protein sorting from the ER.

Efficacy and safety of ipragliflozin in Japanese patients with type 2 diabetes receiving conventional therapy: clinical implication of the importance of exercise habits during treatment with ipragliflozin.

AIMS: To evaluate the efficacy and safety of ipragliflozin in Japanese patients with type 2 diabetes mellitus and inadequate glycemic control and investigate the impact of maintaining exercise habits during treatment. MATERIALS AND METHODS: A total of 20 patients were enrolled. Patients aged 20-70 years with type 2 diabetes mellitus were administered 50 mg of ipragliflozin once daily for 12 weeks. The primary endpoint was the change in glycated hemoglobin levels from baseline to week 12. Key secondary endpoints included changes in total body weight, body composition, serum lipid levels, and diabetes therapy-related quality of life from baseline to week 12. Adverse events were recorded throughout the study. RESULTS: The patients' glycated hemoglobin levels were 0.69% lower at week 12 versus baseline (adjusted mean difference from baseline; P < 0.01, n = 18). Mean total body weight, body composition, and serum lipid levels also improved significantly from baseline. Of note, stratification analysis by physical activity level revealed slight but significant reductions in skeletal muscle mass and muscle mass, but not body fat mass, in the minimal exercise group compared to the data for the moderate exercise group. One of the subdomain structures in diabetes therapy-related quality of life questionnaire, "Anxiety and dissatisfaction with treatment," was significantly improved. Although no major hypoglycemic episodes occurred, two adverse events were reported. CONCLUSIONS: Daily treatment with ipragliflozin was associated with marked improvements in glycemic control and body composition without major side effects, and this improvement was affected by exercise habits. This study was registered with UMIN CTR (no. UMIN000014388).

Effects of zinc ion on type A monoamine oxidase in monkey brain mitochondria.

The effects of ZnSO(4) on types A and B monoamine oxidase (MAO) isozymes in monkey brain mitochondria were investigated, in vitro. Type A MAO activity in monkey brain decreased to about 50% with 1 microM ZnSO(4) using serotonin as a substrate, and this inhibition was proportional to the concentration of ZnSO(4). ZnSO(4) had no effect, however, on type B MAO activity in monkey brain using beta-phenylethylamine as a substrate. The inhibition by ZnSO(4) of type A MAO activity was competitive and reversible. ZnSO(4) did not inhibit either type A or type B MAO activity in rat brain mitochondria. Almost similar results were also obtained when ZnCl(2) was used, in vitro. These results indicate that the inhibiting action of zinc ion differs depending on animal species and organ. Type A MAO in monkey brain mitochondria was highly sensitive to zinc ion, while type B activity was less sensitive.

Zinc benzoate, a contaminating environmental compound derived from polystyrene resin inhibits A-type monoamine oxidase.

The contaminants in deionized and distilled water (DDI water) boiled with polystyrene resin inhibited A-type monoamine oxidase (MAO, MAO-A preferentially deaminates serotonin and norepinephrine and regulates these amines concentration) activity in monkey brain mitochondria. To identify these contaminants, we attempted measurements by HPLC, FT-IR and NMR. The compound inhibiting MAO-A activity was zinc benzoate. Although it potently inhibited MAO-A activity, zinc benzoate did not effect MAO-B in monkey brain mitochondria. It also reversibly and competitively inhibited MAO-A activity in a dose-dependent manner. Zinc benzoate, however, did not inhibit either MAO-A or -B activities in rat brain mitochondria. These results indicate that zinc benzoate, which inhibits MAO-A activity, is easily incorporated in DDI water by boiling polystyrene and also may be a contaminating environmental chemical compound that alters the levels of serotonin and norepinephrine in the central nervous system.

Calcium disodium edetate enhances type A monoamine oxidase activity in monkey brain.

The effects of metal chelators on monoamine oxidase (MAO) isozymes, MAO-A and MAO-B, in monkey brain mitochondria were investigated in vitro. MAO-A activity increased to about 40% with 0.1 microM calcium disodium edetate (CaNa2EDTA) using serotonin as a substrate, and this activation was proportional to the concentration of CaNa2EDTA. On the other hand, MAO-A activities were decreased gradually with an increasing concentration of o-phenanthroline and diethyldithiocarbamic acid, but these metal chelators had no effect on MAO-B activity in monkey brain. The activation of MAO-A activity by CaNa2EDTA was reversible. CaNa2EDTA did not activate both MAO-A and MAO-B activities in rat brain mitochondria. Zn and Fe ions were found in the mitochondria of monkey brain. Zn ions potently inhibited MAO-A activity, but Fe ions did not inhibit either MAO-A or MAO-B activity in monkey brain mitochondria. These results indicate that the activating action of CaNa2EDTA on MAO-A was the result of the chelating of Zn ions contained in mitochondria by CaNa2EDTA. These results also indicate the possibility that Zn ions may regulate physiologically the level of serotonin and norepinephrine content in brain by inhibiting a MAO-A activity.

Isolation and characterization of 13 microsatellite markers for the viviparous surfperch Ditrema temmincki (Embiotocidae) and cross-species amplification.

Thirteen microsatellite loci were isolated from a size-selected genomic library of the surfperch (Ditrema temmincki Bleeker). All loci displayed a high degree of length polymorphism, as observed in the total number of alleles per locus (two to 23) and a high degree of estimated heterozygosity, ranging from 0.080 to 0.893. The primers developed for D. temmincki were also tested for their ability to amplify homologous sequences in D. viride and Neoditrema ransonetii. Distinct differences were observed among three species of surfperches, in both genetic variability and the frequency distribution of the alleles.

A novel approach for N-glycosylation studies using detergent extracted microsomes.

Recently, it has become apparent that asparagine-linked (N-linked) oligosaccharide at an early stage of processing can play an important role in quality control of the secretory pathway. Here, we have developed a system for better understanding of the N-glycosylation machinery and its involvement in quality control in the endoplasmic reticulum (ER). Rough microsomes (RM) treated with 0.18% Tx-100 (TxRM) preserved translocation activities to a similar extent detected in RM. TxRM were depleted of many soluble proteins including glucosidase II, BiP and Erp72, but maintained approximately 80% of calnexin, a membrane protein. More importantly, TxRM revealed insufficient glycosylation of T cell receptor-alpha (TCR-alpha), suggesting that a factor or factors extracted with 0.18% Tx-100 is responsible for facilitating the transfer of oligosaccharides to the protein. In addition, the top band of TCR-alpha translated in TxRM migrated slower than that in RM, but faster than that in RM treated with castanospermine (CST), an inhibitor of glucosidase I/II. This suggests that the trimming of the inner two glucose sugars is impaired by the loss of glucosidase II. Furthermore, we demonstrated that TCR-alpha coprecipitated with calnexin migrated between unglucosylated and diglucosylated forms on SDS-PAGE. Thus, the treatment of RM with low concentration of detergent is a very powerful method for elucidating not only N-glycosylation processes but also other biological functions such as quality control in the ER.

Inhibition by Zn(2+) of A-form monoamine oxidase in monkey brain mitochondria.

The effects of ZnSO(4) on mitochondrial monoamine oxidase (MAO) activity in monkey brain were compared with those in rat and rabbit, in vitro. After preincubation at 25 degrees C for 20 min with 1 microM ZnSO(4), MAO-A activity in monkey brain was about 50% using serotonin (5-HT) as a substrate, and the inhibition was proportional to the concentration of ZnSO(4). However, ZnSO(4) had no effect on MAO-B activity in monkey brain using beta-phenylethylamine (beta-PEA) as a substrate. The inhibition by ZnSO(4) of MAO-A activity was competitive and reversible. CdSO(4) also inhibits MAO-A, but not MAO-B in monkey brain mitochondria. ZnSO(4) did not inhibit either MAO-A or MAO-B activity in rat and rabbit brain mitochondria. These results indicate that the inhibiting action of Zn(2+) differs depending on animal species. In monkey brain mitochondria, MAO-A was highly sensitive to Zn(2+) and MAO-B was less sensitive. These results also suggest that Zn(2+) may regulate the level of catecholamine content in monkey brain.

The transcription factor CCAAT-binding factor CBF/NF-Y regulates the proximal promoter activity in the human alpha 1(XI) collagen gene (COL11A1).

We have characterized the proximal promoter region of the human COL11A1 gene. Transient transfection assays indicate that the segment from -199 to +1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated that the ATTGG sequence, within the -147 to -121 fragment, is critical to bind nuclear proteins in the proximal COL11A1 promoter. We demonstrated that the CCAAT binding factor (CBF/NF-Y) bound to this region using an interference assay with consensus oligonucleotides and a supershift assay with specific antibodies in an EMSA. In a chromatin immunoprecipitation assay and EMSA using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We also showed that four tandem copies of the CBF/NF-Y-binding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a CBF/NF-Y-binding fragment suppressed the COL11A1 promoter activity. Furthermore, overexpression of a dominant-negative CBF-B/NF-YA subunit significantly inhibited promoter activity in both transient and stable cells. These results indicate that the CBF/NF-Y proteins regulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter region.