大橋 鉱二

BACKGROUND: MicroRNAs (miRNAs) play crucial roles in the development of various diseases, including chronic kidney disease (CKD). Although previous studies in clinically severe patients have investigated associations between CKD and miRNAs, with particular attention on renal fibrosis, relationships in a general population have yet to be established. The aim of this study was to examine the relationship between expression level of circulating miRNAs and CKD in a middle-aged Japanese population. METHODS: A final total of 513 individuals (216 men and 297 women) who participated in the health check-up program in 2012 were included in our analysis. Quantitative real-time polymerase chain reaction was used to determine expression levels of 22 miRNAs. Estimated glomerular filtration rate (eGFR) was calculated based on serum creatinine level, sex, and age. Participants with eGFR <60 mL/min/1.73 m2 were defined as having CKD. RESULTS: Three different miRNAs (miR-17, miR-21, and miR-150) showed significant correlations with eGFR after Bonferroni correction and were selected for further analyses. Expression levels of miR-17, miR-21, and miR-150 miRNAs were positively associated with eGFR after adjusting for potential confounders (P = 0.004, 0.002, and 0.004, respectively). Logistic regression analyses showed significantly lower odds ratios for CKD (eGFR <60 mL/min/1.73 m2) in the highest tertile of all three miRNAs (miR-17, miR-21, and miR-150) compared with the lowest tertile (P = 0.003, 0.01, and 0.02, respectively). CONCLUSIONS: We found that three circulating miRNAs were significantly associated with CKD in a general Japanese population, which suggested that these miRNAs may be biomarkers for CKD among general adults.

BACKGROUND: Higher intake of fruits and vegetables is associated with reduced risk of specific types of cancer and of cardiovascular disease (CVD), but the protective role of the vitamins contained in fruits and vegetables on CVD is controversial. This discrepancy can raise the question of the effects of antioxidants in vitamins on CVD. Recently, we reported that higher vegetable intake was significantly associated with the decreased DNA methylation level of ATP-binding cassette transporter A1 (ABCA1), a gene associated with HDL-cholesterol metabolism. OBJECTIVE: We investigated whether ABCA1 DNA methylation mediates an effect of dietary vitamin intake on lipid profiles, an important risk factor for CVD, in a Japanese population. METHODS: A total of 225 individuals (108 men and 117 women) with no clinical history and no drug use for dyslipidemia participated in this cross-sectional study. We used the pyrosequencing method to measure the ABCA1 DNA methylation levels at 8 CpG sites, and we used mean DNA methylation level in statistical analysis. Dietary vitamin intake was assessed with the FFQ and adjusted for the residual method. RESULTS: In women, higher dietary vitamin intake [vitamin A, β-carotene, folic acid, vitamin C (VC), vitamin D, and vitamin E] was significantly associated with lower mean ABCA1 DNA methylation levels (P = 0.004, 0.03, 0.005, 0.001, 0.03, and 0.04, respectively). In addition, in women, we found a significant inverse association between mean ABCA1 DNA methylation and HDL cholesterol (P = 0.04) but not for other lipid indexes. Mediation analysis showed a significant indirect effect of VC intake on HDL cholesterol through ABCA1 DNA methylation level in women (P = 0.04). CONCLUSIONS: Although this study does not prove causality, the results suggest that ABCA1 DNA methylation mediates the protective effect of VC on HDL cholesterol in women, which could offer a novel biological mechanism in CVD prevention.

Fructose consumption is rising globally, but maternal high fructose intake might adversely affect offspring. Our previous report demonstrated that excess maternal fructose intake impairs hippocampal function in offspring, indicating that the hippocampi of offspring are highly sensitive to maternal fructose. Here, we examined the effect of maternal high fructose on mitochondrial physiology and uncoupling protein (UCP) expression. Rat dams received a 20% fructose solution during gestation and lactation. Immediately after weaning, offspring hippocampi were isolated. Maternal high fructose consumption attenuated the mitochondrial O2 consumption rate and stimulated lipid hydroperoxide production in the hippocampi of offspring. Reduced Ucp5 and mitochondrial transcription factor A (Tfam) mRNA levels were also observed after maternal exposure to fructose. We assessed the promoter regions of both genes and found that this treatment enhanced DNA methylation levels. In addition, luciferase assays showed that this DNA methylation could reduce the transcription of both genes. Chromatin immunoprecipitation analysis demonstrated that specificity protein 1 binding to the Ucp5 promoter regions was reduced by DNA methylation. In addition, Ucp5 knockdown induced the up-regulation of reactive oxygen species levels in a rat brain glioma cell line, whereas reduced O2 consumption was observed with Tfam knockdown. Maternal high fructose intake thus induces reduced O2 oxygen consumption and increases oxidative stress in offspring, at least partly through epigenetic mechanisms involving Ucp5 and Tfam.-Yamada, H., Munetsuna, E., Yamazaki, M., Mizuno, G., Sadamoto, N., Ando, Y., Fujii, R., Shiogama, K., Ishikawa, H., Suzuki, K., Shimono, Y., Ohashi, K., Hashimoto, S. Maternal fructose-induced oxidative stress occurs viaTfam and Ucp5 epigenetic regulation in offspring hippocampi.

OBJECTIVE: Dietary intake of vegetables is one of the key lifestyle factors associated with preventing cancer and cardiovascular disease (CVD). Although previous studies have provided evidence that dietary factors can alter global DNA methylation levels in humans, little work has been done on dietary factors influencing methylation levels of specific genes associated with CVD. The aim of this study was to examine whether dietary intake of vegetables was associated with adenosine triphosphate-binding membrane cassette transporter A1 (ABCA1) DNA methylation levels in leukocytes in a Japanese population. METHODS: This cross-sectional study included 279 Japanese adults (125 men, 154 women) without any clinical history of cancer, stroke, or ischemic heart disease. ABCA1 DNA methylation levels in leukocytes were measured using a pyrosequencing method. Information on dietary vegetable intake was obtained from the validated food frequency questionnaire. RESULTS: Mean ABCA1 DNA methylation levels in men and women were 35.6% ± 6.5% and 36.9% ± 6.7%, respectively. In women, multivariable linear regression analysis showed that the group with the highest dietary vegetable intake (carrot, broccoli, pumpkin, and all vegetables) showed significantly lower levels of ABCA1 DNA methylation than the lowest intake group (P = 0.04, <0.001, 0.001, and 0.02, respectively). No significant association was observed between dietary intake of vegetables and DNA methylation levels in men. CONCLUSIONS: High dietary intake of vegetables was associated with decreased ABCA1 DNA methylation levels in Japanese women. This may contribute to a better understanding of the protective effects of dietary vegetable intake on CVD.

© 2019 Elsevier B.V. Objectives: MicroRNAs (miRNAs) dysregulate gene expression by binding to target messenger RNAs, and play an important role in the pathogenesis of various diseases, including cancers, cardiovascular diseases and diabetes. Circulating miRNAs have increasingly been recognized as biomarkers for detecting and diagnosing those diseases. Few studies have investigated the association of circulating miRNA with the early stages of cognitive impairment, such as mild cognitive impairment, in the general population. The purpose of this study was to examine the association between cognitive function and several serum miRNAs levels related to amyloid precursor protein (APP) proteolysis in a Japanese general population who had never been diagnosed with dementia. Methods: We conducted a cross-sectional study of 337 Japanese subjects (144 men, 193 women) who attended a health examination. The short form of the Mini-Mental State Examination (SMMSE) was used to assess cognitive function. Serum levels of 6 miRNAs (let-7d, miR-17, miR-20a, miR-27a, miR-34a, miR-103a) were measured by quantitative real-time polymerase chain reaction. Results: Multivariable-adjusted odds ratios (ORs) for lower SMMSE score (SMMSE score < 28) were significantly increased in the lowest tertile of serum miR-20a (OR, 2.08; 95% confidence interval (CI), 1.09–4.04) and miR-103a (OR, 1.91; 95%CI, 1.00–3.69) compared to the highest tertile. Moreover, serum levels of miR-20a, -27a, and -103a were linearly and positively associated with SMMSE scores after adjustment for confounding factors. Conclusion: Low serum levels of miR-20a, -27a, and -103a are independently associated with cognitive impairment.

Global fructose consumption is on the rise; however, maternal high-fructose intake may have adverse effects on offspring. We previously demonstrated that excessive fructose intake by rat dams altered steroidogenic gene transcription in the hippocampus of offspring. Herein, we examined how maternal high-fructose intake influences the regulation of adrenal glucocorticoid levels in offspring. Rat dams received 20% fructose solution during gestation and lactation. After weaning, the offspring were provided normal water. Maternal high-fructose intake did not alter mRNA expression levels of adrenal corticosterone-synthesizing and corticosterone-inactivating proteins or the circulating adrenocorticotropic hormone levels of offspring at postnatal day (PD) 21; however, it increased circulating corticosterone levels and decreased mRNA and protein levels of adrenal 5α-reductase type 1 and 11β-hydroxysteroid dehydrogenase type 2 in offspring at PD160. Furthermore, maternal high-fructose intake enhanced DNA methylation of the adrenal 5α-reductase 1 promoter region in PD160 offspring. Thus, maternal high-fructose intake was found to affect adrenal steroid hormone clearance in adult offspring - at least in part - through epigenetic mechanisms.

Epidemiological studies have been demonstrated that fructose, which are used for beverages, are associated with the incidence of metabolic disorders. However, the pathological mechanism of fructose effect remains unclear. Recently, there are accumulating evidences that nutrition status may induce epigenetic modification, which lead to cause several diseases such as diabetes. Interestingly, it is becoming clear that the adverse effect of fructose is mediated by epigenetic modifications. Here, this chapter describes the epigenetic effect of high-fructose consumption.

The worldwide increase in fructose consumption may be associated with the incidence of several diseases such as insulin resistance, diabetes, hypertension, and obesity. It is becoming clear that the adverse effect of fructose is mediated by epigenetic modifications. This chapter describes the effects of fructose consumption from the viewpoint of epigenetics. Recently, it has become increasingly clear that maternal nutrition can strongly influence the susceptibility of adult offspring to metabolic disease. Excess maternal fructose consumption may induce DNA methylation, which may, in turn, cause metabolic disease. Fructose-induced epigenetic changes appear to be a novel mechanism of disease pathology and may induce epigenetic alterations in stem cells/fetal cells, resulting in adverse effects during fetal development and child health later in life.

日本臨床化学会栄養専門委員会は、我が国における血清総25(OH)D測定の標準化の状況を調査した。現在、基準測定操作法にLC-MS/MS、基準測定操作法による特性値のある常用参照標準物質にNIST SRM 972a、さらに、世界的にVDSPが実施されている。LC-MS/MS実施4施設はVDSPに参加していないが、SRM 972a(レベル1〜4)の25(OH)D3測定値の施設間CVは4.7%以下と収束が認められた。千葉大学のLC-MS/MSは25(OH)D2、25(OH)D3、3-epi-25(OH)D3を分画することができ、測定値と総25(OH)Dの計算値[25(OH)D2+25(OH)D3]は、SRM 972a(レベル1〜4)の特性値と±1・SDの範囲にあった。自動分析法6機種によるSRM 972aの総25(OH)D測定値は特性値の±3・SD範囲を超える機種もあり、患者血清の測定では千葉大学LC-MS/MS値との比較でバイアスがCLSIのDocument EP09-A3の評価法の許容範囲(±5%)を超える機種もある。自動分析法はVDSPで認証されているが、その測定値は正確度において改善が求められた。さらに、コンセンサス値との比較においても、許容範囲を超えるバイアス(低値偏りや高値偏り)が存在した。このことから、我が国における自動分析法の血清総25(OH)D測定の標準化は達成されていない現状と考える。千葉大学LC-MS/MS値とのPassing-Bablok回帰式を用いた補正により自動分析法の測定値はコンセンサス値に収束したが、補正はトレーサビリティ連鎖を崩すので実施せずに製造業者による改善が求められる。現状では、ビタミンD栄養の評価は欠乏症ならびに充足の判断値において自動分析法の測定値の差[欠乏症の判断値濃度(20ng/mL)で-4〜4ng/mL、充足の判断値濃度(30ng/mL)で-3〜4ng/mL]を考慮しなければならない。(著者抄録)

In this study, we examined whether compound 48/80 (C48/80), a mast cell degranulator, causes hepatic oxidative damage in rats. Serum and liver biochemical parameters were determined 0.5, 3 or 6 h after a single treatment with C48/80 (0.75 mg/kg). Serum histamine and serotonin levels increased 0.5 h after C48/80 treatment but diminished thereafter. Increases in serum vitamin C (VC) and transaminases and hepatic hydrogen peroxide, lipid peroxide, and myeloperoxidase levels and a decrease in hepatic reduced glutathione level occurred 0.5 h after C48/80 treatment and further proceeded at 3 h, but these changes diminished at 6 h. Serum lipid peroxide and hepatic VC levels increased 3 h after C48/80 treatment. Hepatic glycogen level decreased 0.5 h after C48/80 treatment and further decreased at 3 h. Pre-administered ketotifen diminished all these changes found at 3 h after treatment, while pre-administered. NPC 14686 diminished these changes except changes in serum histamine and serotonin levels. Hepatocellular apoptosis observed at 3 h after C48/80 treatment was attenuated by pre-administered ketotifen and NPC 14686. These results indicate that C48/80 causes oxidative damage by enhancing VC synthesis via reduced glutathione depletion-dependent glycogenolysis and lipid peroxidation through neutrophil infiltration following mast cell degranulation in rat livers.

The aim of this study was to clarify the effect of water-immersion restraint stress (WIRS) on tryptophan (Trp) catabolism through the kynurenine (Kyn) pathway in rat tissues. The tissues of rats subjected to 6 h of WIRS (+WIRS) had increased tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) activities and increased TDO and IDO1 (one of two IDO isozymes in mammals) mRNA expression levels, with decreased Trp and increased Kyn contents in the liver. +WIRS rats had unchanged TDO and IDO activities in the kidney, decreased TDO activity and unchanged IDO activity in the brain, and unchanged IDO activity in the lung and spleen, with increased Kyn content in all of these tissues. Pretreatment of stressed rats with RU486, a glucocorticoid antagonist, attenuated the increased TOD activity, but not the increased IDO activity, with partial recoveries of the decreased Trp and increased Kyn contents in the liver. These results indicate that WIRS enhances hepatic Trp catabolism by inducing both IDO1 and TDO in rats.

Background: Recently, several studies have shown that microRNAs are present in high-density lipoprotein, and high-density lipoprotein-microRNA may be a promising disease biomarker. We investigated the stability of high-density lipoprotein-microRNAs in different storage conditions as this is an important issue for its application to the field of clinical research. Methods: microRNAs were extracted from the high-density lipoprotein fraction that was purified from the serum. miR-135 a and miR-223, which are known to be present in high-density lipoprotein, were quantified by quantitative real-time PCR. The influence of preanalytical parameters on the analysis of high-density lipoprotein-miRNAs was examined by the effect of RNase, storage conditions, and freezing and thawing. Results: The concentrations of microRNA in high-density lipoprotein were not altered by RNase A treatment (0-100 U/mL). No significant change in these microRNAs was observed after storing serum at room temperature or 4 degrees C for 0-24 h, and there was a similar result in the cryopreservation for up to two weeks. Also, high-density lipoprotein-microRNAs were stable for, at least, up to five freeze-thaw cycles. Conclusions: These results demonstrated that high-density lipoprotein-microRNAs are relatively resistant to various storage conditions. This study provides new and important information on the stability of high-density lipoprotein-microRNAs.

Neurosteroids, steroidal hormones synthesized de novo from cholesterol within the brain, stimulate hippocampal functions such as neuron protection and synapse formation. Previously, we examined the effect of maternal fructose on the transcriptional regulation of neurosteroidogenic enzymes. We found that the mRNA expression level of the steroidogenic acute regulatory protein (StAR), peripheral benzodiazepine receptor (PBR), cytochrome P450(11), 11-hydroxysteroid dehydrogenase (HSD), and 17-HSD was altered. However, we could not determine whether maternal fructose intake played a role in the gestation or lactation period because the dam rats were fed fructose solution during both periods. Thus, in this study, we analyzed the hippocampi of the offspring of dams fed fructose during the gestation or lactation period. Maternal fructose consumption during either the gestation or lactation period did not affect the mRNA levels of StAR, P450(17), 11-HSD-2, and 17-HSD-1. PBR expression was down-regulated, even when rats consumed fructose during the lactation period only, while fructose consumption during gestation tended to activate the expression of P450(11)-2. We found that maternal fructose intake during gestation and lactation differentially affected the expression of hippocampal neurosteroidogenic enzymes in the offspring.

Aims: Fructose may play a crucial role in the pathogenesis of metabolic syndrome (MetS). However, the pathogenic mechanism of the fructose-induced MetS has not yet been investigated fully. Recently, several reports have investigated the association between mitochondrial DNA (mtDNA) and MetS. We examined the effect of fructose-rich diets on mtDNA content, transcription, and epigenetic changes. Main methods: Four-week-old male Sprague-Dawley rats were offered a 20% fructose solution for 14 weeks. We quantified mRNAs for hepatic mitochondrial genes and analyzed the mtDNA methylation (5-mC and 5-hmC) levels using ELISA kits. Key findings: Histological analysis revealed non-alcoholic fatty liver disease (NAFLD) in fructose-fed rats. Hepatic mtDNA content and transcription were higher in fructose-fed rats than in the control group. Global hypomethylation of mtDNA was also observed in fructose-fed rats. Significance: We showed that fructose consumption stimulates hepatic mtDNA-encoded gene expression. This phenomenon might be due to epigenetic changes in mtDNA. Fructose-induced mitochondrial epigenetic changes appear to be a novel mechanism underlying the pathology of MetS and NAFLD. (C) 2016 Elsevier Inc. All rights reserved.

Objectives: Circulating micro (mi)RNA profiles are influenced by various medical conditions, and miRNAs have been examined as potential biomarkers for cancer, cardiovascular disease, and diabetes. However, few studieshave investigated whether circulating miRNAs and cigarette smoking are correlated. Our aim was to determine theassociation between smoking status and expression of various miRNAs in a Japanese population sample.Methods: We conducted a cross-sectional analysis of 526 subjects (219 men and 307 women) aged 39 years and older who had undergone a health examination at a clinic in Yakumo, Hokkaido in August 2012. We used quantitative real-time polymerase chain reaction to determine serum miRNA expression. We then calculated the odds ratios for elevated serum miRNA levels according to smoking status using never-smokers as the reference group.Results: Expression of lethal (let)-7d, miRNA(miR)-150, miR-192, miR-197 and miR320 was significantly higher in current smokers than in never-smokers. Multivariate logistic regression analyses showed that while current smokers were significantly more likely to have elevated serum levels of miRNA let-7d, miR-21, miR-122, miR-146, miR-150, miR-192, miR-197, and miR320 than never-smokers, former smokers had significantly higher odds of having elevated miR-1, miR-146, miR-150, miR-195, and miR-320 levels in their sera.Conclusions: We found that cigarette smoking is associated with elevated expression of various serum miRNAs. Our results suggest that it is necessary to consider the confounding effect caused by smoking when evaluating expression of serum miRNAs for diagnosing pathological conditions.

Background: Circulating microRNAs (miRs) may be promising biomarkers for several diseases. We previously found that miR-122 can function as a biomarker for non-alcoholic fatty liver disease (NAFLD). However, little is known regarding the time course of circulating miR-122 levels during the development of NAFLD. Here, we examined circulating miR-122 levels using a rat model of NAFLD. Methods: To clarify changes in serum levels of miR-122 during development of NAFLD, experimental rats were fed a high-fat diet (HFD) for 2-10 weeks, while control rats received standard chow. Serum and liver tissue was collected from all animals at 2, 6, and 10 weeks of feeding. Clinical laboratory parameters (cholesterol, TG, AST, ALT, NEFA) were determined by biochemistry analyzer. Hepatic lipid accumulation was estimated by Oil red 0 staining. Circulating miR-122 levels were then measured by real-time polymerase chain reaction. Results: Over the 10 weeks of feeding, body weight, total liver lipids, and liver and serum triacylglycerol were increased in the HFD group compared to the control group. However, no significant changes in serum alanine aminotransferase activity were observed, suggesting that NAFLD status was mild. In contrast, we observed drastic up-regulation of circulating miR-122 levels. Our findings suggest that serum miR-122 level is indeed useful for assessing early NAFLD and might be superior to clinical markers traditionally used to monitor hepatic disease. (C) 2015 Elsevier B.V. All rights reserved.

We examined how dietary supplementation of vitamin E protects against liver oxidative damage in rats with water-immersion restraint stress (WIRS). Before WIRS exposure, rats received a normal diet (ND) or vitamin E-supplemented diet (VESD) (500 IU alpha-tocopherol/kg diet) at a mean dose of 15 g/animal/d for 4 wk. The two diet groups had serum transaminases and lactate dehydrogenase activities and adrenocorticotropic hormone, corticosterone, and glucose levels to a similar extent. VESD-fed rats had higher liver a-tocopherol concentrations and lower liver ascorbic acid, total coenzyme Q(9) (CoQ(9)), reduced CoQ(9), reduced CoQ(10), and lipid peroxide (LPO) concentrations than ND-fed rats. When the two diet groups were exposed to 6 h of WIRS, the serum liver cell damage index enzyme activities increased more greatly in ND-fed rats than in VESD-fed rats but the serum stress marker levels increased to a similar extent. The WIRS exposure caused no change in liver LPO concentration with the further increase in liver a-tocopherol concentration in VESD-fed rats but increased liver LPO concentration without changing liver alpha-tocopherol concentration in ND-fed rats. Upon the WIRS exposure, liver reduced glutathione concentration decreased with the further decrease in liver ascorbic acid concentration in VESD-fed rats and those concentrations decreased in ND-fed rats. The WIRS exposure recovered the decreased liver total CoQ(9) and reduced CoQ(9) concentrations in VESD-fed rats but decreased liver total CoQ(9), reduced CoQ(9), and reduced CoQ(10) concentrations in ND-fed rats. These results indicate that dietary vitamin E supplementation protects against liver oxidative damage without affecting the stress response in rats with WIRS.

The present study was conducted to clarify the protective effect of Brazilian propolis ethanol extract (BPEE) against stress-induced gastric mucosal lesions in rats. The protective effect of BPEE against gastric mucosal lesions in male Wistar rats exposed to water-immersion restraint stress (WIRS) for 6 h was compared between its repeated preadministration (50mg/kg/day, 7 days) and its single preadministration (50mg/kg). The repeated BPEE preadministration attenuated WIRS-induced gastric mucosal lesions and gastric mucosal oxidative stress more largely than the single BPEE preadministration. In addition, the repeated BPEE preadministration attenuated neutrophil infiltration in the gastric mucosa of rats exposed to WIRS. The protective effect of the repeated preadministration of BPEE againstWIRS-induced gastricmucosal lesions was similar to that of a single preadministration of vitamin E (250mg/kg) in terms of the extent and manner of protection. From these findings, it is concluded that BPEE preadministered in a repeated manner protects against gastricmucosal lesions in rats exposed to WIRS more effectively than BPEE preadministered in a single manner possibly through its antioxidant and anti-inflammatory actions.

Background: In many industrialized countries, non-alcoholic fatty liver disease (NAFLD) is recognized as an important disease that increases the risk of cardiovascular disease, type-2 diabetes, and metabolic syndrome. Most people with NAFLD are asymptomatic, and the disease is discovered incidentally during clinical laboratory tests. Present screening methods for NAFLD use ultrasound, and CT scans that are time-consuming, and a simple screening method for NAFLD is needed. In this study, we investigated whether serum miRNAs are associated with NAFLD and assessed the potential of serum miRNAs as a biomarker for NAFLD. Methods: We assessed intrahepatic fat by ultrasound scan, and the serum levels of five miRNAs (miR-21, miR-34a, miR-122, miR-145, and miR-451), which help regulate cholesterol and fatty acid homeostasis in liver tissue, by real-time PCR in a cross-sectional sample of 403 participants who attended health examinations. Results: Serum levels of miRNAs, miR-21, miR-34a, miR-122, and miR-451 were higher in participants with NAFLD. The serum level of miR-122 was correlated with the severity of liver steatosis. Conclusion: Serum levels of miRNAs, particularly miR-122, may be a useful biomarker for NAFLD. © 2013 Elsevier B.V.