藤井 亮輔

BACKGROUND: Incidence of ischemic stroke increased after natural disasters. Therefore, it is important to establish a means of identifying high-risk populations for incident stroke. We performed a prospective cohort study to examine whether these three cardiovascular disease-related miRNAs (miR-126, miR-197, and miR-223) are associated with incident stroke among elderly survivors of the Great East Japan Earthquake. METHOD: This cohort study was conducted using the data of 1192 survivors of the Great East Japan Earthquake over 60-years old who underwent a health check-up in December 2011. We followed up participants to record stroke cases until the end of 2016. We measured serum miRNAs by quantitative real-time polymerase chain reaction. HRs for incident stroke were estimated by Cox proportional hazard regression analyses. RESULT: The serum miR-197 level was significantly associated with the incident stroke; the HR per one standard deviation change in the miR-197 level was 1.65 (95% confidence interval: 1.19 - 2.30). In contrast, the levels of miR-126 and miR-223 were not associated with the incident stroke. CONCLUSION: We found that a higher miR-197 level is associated with an increased risk of incident stroke; thus, miR-197 is expected to be useful as a predictive biomarker.

INTRODUCTION: Previous Mendelian randomization (MR) studies for the coffee-kidney association have reported inconsistent relationships in European populations and never examined mediators of this association. We aimed to evaluate this causal relationship using two-sample MR among both East Asian and European ancestries and to explore underlying mechanisms using plasma caffeine levels. METHODS: Among East Asians, the largest genome-wide association study (GWAS) results for coffee intake, plasma caffeine levels, and kidney outcomes were obtained from 152,634; 8940; and 47,070 Japanese adults. Among Europeans, summary statistics were acquired from European GWAS with 428,860; 7719; and 564,470 adults for each trait. We applied different MR methods (inverse-variance weighted [IVW] with random effects, weighted median, weighted mode, and MR-Egger). RESULTS: After excluding possible pleiotropic variants, among East Asian ancestry, drinking an extra coffee intake per week showed a protective association on serum creatinine-based estimated glomerular filtration rate (eGFRcre) (β = 0.077; 95% confidence interval [CI] = 0.003 to 0.150). Analysis in European ancestry also showed a causal relationship between drinking an extra coffee intake per day and eGFRcre (β = 0.052; 95% CI = 0.027 to 0.078). These results were consistent across different MR methods accounting for invalid instruments. Higher plasma caffeine levels were associated with lower eGFRcre among both East Asian (β = -0.071; 95% CI = -0.137 to -0.006) and European ancestries (β = -0.048; 95% CI = -0.057 to -0.040). CONCLUSIONS: Our cross-ancestry MR study found beneficial effects of coffee intake on eGFRcre. However, given the possible adverse effects of plasma caffeine levels on eGFRcre, interpretation of the results should be carefully considered and further investigations on noncaffeine and biological pathways are needed.

Thioredoxin-interacting protein (TXNIP) plays an important role in glucose metabolism, and its expression is regulated by DNA methylation (DNAm). Although the association between TXNIP DNAm and type 2 diabetes mellitus has been demonstrated in studies with a cross-sectional design, prospective studies are needed. We therefore examined the association between TXNIP DNAm levels and longitudinal changes in glycemic traits by conducting a longitudinal study involving 169 subjects who underwent two health checkups in 2015 and 2019. We used a pyrosequencing assay to determine TXNIP DNAm levels in leukocytes (cg19693031). Logistic regression analyses were performed to assess the associations between dichotomized TXNIP DNAm levels and marked increases in glycemic traits. At four years, the TXNIP DNA hypomethylation group had a higher percentage of changes in fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) compared to those in the hypermethylation group. The adjusted odds ratios for FPG and HbA1c levels were significantly higher in the TXNIP DNA hypomethylation group than in the hypermethylation group. We found that TXNIP DNA hypomethylation at baseline was associated with a marked increase in glycemic traits. Leukocyte TXNIP DNAm status could potentially be used as an early biomarker for impaired glucose homeostasis.

The renal angina index (RAI) is a validated scoring tool for predicting acute kidney injury (AKI). We investigated the efficacy of the RAI in 2436 heterogeneous patients (mean age, 70 years) treated in cardiac intensive care units (CICUs). The RAI was calculated from creatinine and patient condition scores. AKI was diagnosed by the Kidney Disease: Improving Global Outcome criteria. The primary and secondary endpoints were the development of severe AKI and all-cause mortality, respectively. Four hundred thirty-three patients developed AKI, 87 of them severe. In multivariate analyses, the RAI was a significant independent predictor of severe AKI. During the 12-month follow-up period, 210 patients suffered all-cause death. Elevated RAI was independently associated with all-cause mortality, as was NT-proBNP (p < 0.001). The RAI is a potent predictor not only of severe AKI but also of adverse outcomes and substantially improved the 12-month risk stratification of patients hospitalized in CICUs.

OBJECTIVES: The mitochondrial DNA (mtDNA) is unique and circular with multiple copies of the genome. The lower mtDNA copy number (mtDNA-CN) in leukocytes is associated with the risk of all-cause mortality. However, its long-term association is unknown. Thus, the study examined the association between mtDNA-CN and the risk of all-cause mortality in a long-term follow-up study in the Japanese population. DESIGN: This longitudinal study included the study cohort from an annual, population-based health checkup in the town of Yakumo, Hokkaido, Japan. SETTING AND PARTICIPANTS: 814 participants (baseline age range: 38-80 years, mean: 56.3 years) were included in this study in 1990. They were followed-up regarding mortality for about 30 years (median: 28.1 years) till 2019. MEASURES: The genomic DNA was extracted from peripheral blood mononuclear cells and the mtDNA-CN was measured using real-time polymerase chain reaction. The level of the mtDNA-CN was divided into tertiles (low, middle, and high). The participants were categorized based on their age into middle-aged (<60 years old) or old-aged (≥60 years old). Survival analysis was performed for tertile of mtDNA-CN and compared using the log-rank test. Univariate and multivariable Cox proportional hazard regression analyses were performed to assess the association between mtDNA-CN and all-cause mortality. The model adjusted with age, sex, body mass index, systolic blood pressure, smoking habit, alcohol consumption, exercise habit, and education level. RESULTS: The low levels of mtDNA-CN resulted in a significant decrease in cumulative survival rate (P <  0.05). The risk of mortality was significantly higher in the middle-aged cohort when mtDNA-CN levels were low (hazard ratios [95% confidence intervals]: 1.98 [1.10-3.56]). CONCLUSION: This study demonstrated that leukocyte mtDNA-CN is associated with future mortality risk. Our study findings may lead to further research on the early prediction of mortality and its underlying mechanisms.

OBJECTIVE: The association between knee osteoarthritis (OA) and miRNAs has been widely reported. However, the utility of miRNAs as predictors of knee osteoarthritis (KOA) progression in longitudinal studies has not been reported. We aimed to identify circulating miRNAs (c-miRNAs) associated with KOA progression in the general population and to examine their potential use as predictors of KOA progression. METHODS: In 2012 and 2018, 66 participants (128 knees) took part in a resident health check-up in the Yakumo study. If the KL classification progressed two or more levels, the patient was classified as having progressive OA. Quantitative real-time polymerase chain reaction was used to screen 21 c-miRNAs. The expression levels of those c-miRNAs were compared between the progressive OA group and non-progressive OA group using student-t-test. Logistic analysis was performed in c-miRNAs less than p < 0.10 in univariate analysis. RESULTS: The progressive OA group consisted of 78 knees. The results of the comparison between the progressive OA group and the non-progressive OA group showed that six c-miRNAs as follows; let7d (p = 0.030), c-miRNA-122 (p < 0.001), 150 (p = 0.070), 199 (p = 0.078), 21 (p = 0.016) and 320 (p = 0.093) were extracted as factors related to the progression of knee OA. In addition, logistic regression analysis identified c-miRNA-122 as an independent factor involved in the progression of knee osteoarthritis (odds ratio: 1.510, 95% confidence interval: 1.060-2.140, p = 0.023). The ROC curve showed by c-miRNA-122 for the progression of OA risk had an area under the curve of 0.702 (95% CI: 0.609-0.795). The threshold of c-miRNA-122 was -4.609. CONCLUSION: The expression level of c-miRNA-122 was associated with the risk of KOA progression in community dwelling Japanese people.

Background: Carotenoids have been reported to exert protective effects against age-related diseases via changes in DNA methylation. Although lower thioredoxin-interacting protein (TXNIP) DNA methylation is associated with age-related diseases, only a few studies have investigated the factors influencing TXNIP DNA methylation. Carotenoids may be a factor linking TXNIP to specific pathophysiological functions. The aim of this study was to examine whether serum carotenoid levels are associated with TXNIP DNA methylation levels. Methods: We conducted a cross-sectional study using 376 health examination participants (169 men). DNA methylation levels were determined using a pyrosequencing assay. Serum carotenoid levels were determined by high-performance liquid chromatography. Multivariable regression analyses were performed to examine the associations between TXNIP DNA methylation levels and serum carotenoid levels with adjustment for age, BMI, HbA1c, CRP, smoking habits, alcohol consumption, exercise habits, and percentage of neutrophils. Results: Multiple linear regression analyses showed that TXNIP DNA methylation levels were positively associated with serum levels of zeaxanthin/lutein (β [95%CI]: 1.935 [0.184, 3.685]), β-cryptoxanthin (1.447 [0.324, 2.570]), α-carotene (1.061 [0.044, 2.077]), β-carotene (1.272 [0.319, 2.226]), total carotenes (1.255 [0.040, 2.469]), total xanthophylls (2.133 [0.315, 3.951]), provitamin A (1.460 [0.402, 2.519]), and total carotenoids (1.972 [0.261, 3.683]) in men (all p<0.05). Of these, provitamin A showed the stronger association (standardized β=0.216). No significant association of TXNIP DNA methylation and serum carotenoid was observed in women. Conclusions: The findings of this study suggest that carotenoid intake may protect against age-related diseases by altering TXNIP DNA methylation status in men.

BACKGROUND: Epigenetic studies have reported relationships between dietary nutrient intake and methylation levels. However, genetic variants that may affect DNA methylation (DNAm) pattern, called methylation quantitative loci (mQTL), are usually overlooked in these analyses. We investigated whether mQTL change the relationship between dietary nutrient intake and leukocyte DNAm levels with an example of estimated fatty acid intake and ATP-binding cassette transporter A1 (ABCA1). METHODS: A cross-sectional study on 231 participants (108 men, mean age: 62.7 y) without clinical history of cancer and no prescriptions for dyslipidemia. We measured leukocyte DNAm levels of 8 CpG sites within ABCA1 gene by pyrosequencing method and used mean methylation levels for statistical analysis. TaqMan assay was used for genotyping a genetic variant of ABCA1 (rs1800976). Dietary fatty acid intake was estimated with a validated food frequency questionnaire and adjusted for total energy intake by using residual methods. RESULTS: Mean ABCA1 DNAm levels were 5% lower with the number of minor alleles in rs1800976 (CC, 40.6%; CG, 35.9%; GG, 30.6%). Higher dietary n-3 PUFA intake was associated with lower ABCA1 DNAm levels (1st (ref) vs. 4th, β [95% CI]: -2.52 [-4.77, -0.28]). After controlling for rs180076, the association between dietary n-3 PUFA intake and ABCA1 DNAm levels was attenuated, but still showed an independent association (1st (ref) vs. 4th, β [95% CI]: -2.00 [-3.84, -0.18]). The interaction of mQTL and dietary n-3 PUFA intake on DNAm levels was not significant. CONCLUSIONS: This result suggested that dietary n-3 PUFA intake would be an independent predictor of DNAm levels in ABCA1 gene after adjusting for individual genetic background. Considering mQTL need to broaden into other genes and nutrients for deeper understanding of DNA methylation, which can contribute to personalized nutritional intervention.

Background: The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) has become a global health problem. NAFLD has few initial symptoms and may be difficult to detect early, so there is need for a minimally invasive early detection marker. We hypothesized that miR-122 and miR-20a levels combined, as the miR-122/miR-20a ratio might detect NAFLD more sensitively. Methods: This study involved 167 participants with low alcohol intake. Those who had an increase in echogenicity of the liver parenchyma and hepato-renal contrast on ultrasonography were classified as the NAFLD group (n = 44), which was further classified into mild (n = 26) and severe (n = 18) groups based on echogenic intensity and hepatic vessel and diaphragm visualization. Participants without fatty liver were included in the normal group, except for those with an abnormal body mass index, glycated hemoglobin, and systolic blood pressure (n = 123) values. Serum miR-122 and miR-20a expression levels in participants were measured by real-time polymerase chain reaction, and the miR-122/miR-20a was calculated. Results: In the NAFLD group, miR-122 expression was significantly higher and the miR-20a was significantly lower than in the normal group, in agreement with previous studies. miR-122/miR-20a was also significantly higher in the NAFLD group. Receiver operating characteristic curve analysis was performed with miR-122/miR-20a as an NAFLD detection marker, and the area under the curve of miR-122/miR-20a was significantly larger than that of miR-122 or miR-20a alone. Conclusions: The miR-122/miR-20a ratio, combined with miR-122 and miR-20a levels, is a useful biomarker to detect NAFLD with high sensitivity.

OBJECTIVES: The adverse health effects of consuming sugar-sweetened beverages have been studied worldwide. However, no recent report on the actual sugar contents of Japanese sugar-sweetened beverages is available. Therefore, we analyzed the glucose, fructose, and sucrose contents of common Japanese beverages. METHODS: The glucose, fructose, and sucrose contents of 49 beverages (8 energy drinks, 11 sodas, 4 fruit juices, 7 probiotic drinks, 4 sports drinks, 5 coffee drinks, 6 green tea drinks, and 4 black tea drinks) were determined using enzymatic methods. RESULTS: Three zero calorie drinks, 2 sugarless coffee drinks, and 6 green tea drinks contained no sugar. Three coffee drinks contained only sucrose. The orders of median glucose, fructose, and sucrose contents in the categories of beverages containing sugars were as follows: for glucose, fruit juice > energy drink ≥ soda ≫ probiotic drink > black tea drink > sports drink; for fructose, probiotic drink ≥ energy drink > fruit juice > soda ≫ sports drink > black tea drink; and for sucrose, black tea drink > energy drink ≥ probiotic drink > fruit juice > soda > coffee drink ≫ sports drink. The total fructose as a percentage of the total sugar content in the 38 sugar-containing beverages was between 40% and 60%. The total sugar content analyzed was not always equivalent to the carbohydrate content indicated on the nutrition label. CONCLUSIONS: These results indicate that information on the actual sugar content of common Japanese beverages is necessary for the exact assessment of beverage-derived sugar intake.

BACKGROUND: Previous studies have proposed different formulas of estimating glomerular filtration rate (eGFR) among clinical patients. The comprehensive comparison of eGFR formulas is not well established in a Japanese population. We compared eGFR values and chronic kidney disease (CKD) classification of nine different eGFR in a Japanese general population sample. METHODS: We analyzed 469 Japanese community-dwelling adults (184 men) without any self-reported kidney disease. GFR estimated using the 4- and 6-parameter Modification of Diet in Renal Disease (MDRD) formulas (MDRD4 and MDRD6); the CKD-EPI formulas based on creatinine with (CKD-EPI-2009) and without race coefficient (CKD-EPI-2021), on cystatin C (CKD-EPI-Cys), on both (CKD-EPI-CreCys); the Japanese creatinine-based formula (JPN-Cre), cystatin C-based formula (JPN-Cys), and modified CKD-EPI formula (JPN-CKD-EPI). CKD stages were defined by KDIGO guidelines (eGFR < 60 ml/min/1.73 m2). RESULTS: eGFRJPN-Cre (mean = 71.2; SD = 14.3) were much lower than eGFRCKD-EPI-2021 (mean = 94.2; SD = 12.7), while eGFRJPN-Cys (mean = 102.8; SD = 24.2) was comparable to the MDRD and CKD-EPI formulas. The difference between eGFRCKD-EPI-2021 and eGFRJPN-Cre showed a V-shaped distribution across eGFR levels, indicating complex errors between these formulas. We observed very low agreement in CKD classification between eGFRJPN-Cre and the eGFRCKD-EPI-2021 (kappa = 0.13; 95% confidence interval: 0.06, 0.23). CONCLUSIONS: JPN-Cre was substantially different from the CKD-EPI formula without race term (CKD-EPI-2021), which means that it is impossible to recalibrate those with a simple coefficient. Although a comparison with measured GFR should be necessary, choice of the estimation method needs caution in clinical decision-making and academic research.

The consumption of high-fructose corn syrup (HFCS) has been increasing in recent decades, especially among children. Some reports suggest that children and adolescents are more sensitive to the adverse effects of fructose intake than adults. However, the underlying mechanism of the difference in vulnerability between adolescence and adulthood have not yet been elucidated. In this study, we attempted to elucidate the different effects of HFCS intake at different growth stages in rats: childhood and adolescence (postnatal day (PD) 21-60), young adulthood (PD60-100), and adulthood (PD100-140). Since alterations in hepatic glucocorticoid (GC) metabolism can cause diseases including insulin resistance, we focused on GC metabolizing enzymes such as 11 beta-hydroxysteroid dehydrogenase 1 and 2 (Hsd11b1 and Hsd11b2) and steroid 5 alpha-reductase 1 (Srd5a1). Western blotting showed an increase in Hsd11b1 expression and a decrease in Hsd11b2 expression in childhood and adolescence but not in adulthood. We also observed changes in Hsd11b1 and Hsd11b2 activities only in childhood and adolescence, consistent with the results of mRNA and protein expression analysis. The effect of high-fructose intake with regards to GC metabolism may therefore vary with developmental stage. This study provides insight into the adverse effects of fructose on GC metabolism in children in the context of increasing rates of HFCS consumption.

DNA methylation can be affected by numerous lifestyle factors, including diet. Tobacco smoking induces aryl hydrocarbon receptor repressor (AHRR) DNA hypomethylation, which increases the risk of lung and other cancers. However, no lifestyle habits that might increase or restore percentage of AHRR DNA methylation have been identified. We hypothesized that dietary intakes of vegetables/fruits and serum carotenoid concentrations are related to AHRR DNA methylation. A total of 813 individuals participated in this cross-sectional study. A food frequency questionnaire was used to assess dietary intake of vegetables and fruits. AHRR DNA methylation in peripheral blood mononuclear cells were measured using pyrosequencing method. In men, dietary fruit intake was significantly and positively associated with AHRR DNA methylation among current smokers (P for trend = .034). A significant positive association of serum provitamin A with AHRR DNA methylation was observed among current smokers (men: standardized β = 0.141 [0.045 to 0.237], women: standardized β = 0.570 [0.153 to 0.990]). However, compared with never smokers with low provitamin A concentrations, percentages of AHRR DNA methylation were much lower among current smokers, even those with high provitamin A concentrations (men: β = -19.1% [-33.8 to -19.8], women: β = -6.0% [-10.2 to -1.7]). Dietary intake of vegetables and fruits rich in provitamin A may increase percentage of AHRR DNA methylation in current smokers. However, although we found a beneficial effect of provitamin A on AHRR DNA methylation, this beneficial effect could not completely remove the effect of smoking on AHRR DNA demethylation.

Carotenoids are abundant pigments mainly contained in vegetables and fruits, and show antioxidant properties by quenching free radicals in human body. Few studies have investigated associations between serum carotenoid levels and premature mortality. The objective of this study was to investigate the association between serum carotenoid level and premature mortality in a Japanese population. This study included 446 Japanese adults (174 men, aged of 40-64) recruited as participants in the Japan Collaborative Cohort (JACC) Study. Serum carotenoid level was measured by high-performance liquid chromatography. Premature mortality was defined as death before 65 years old during the follow-up period. Premature mortality was ascertained in 60 men (34.5%) and 65 women (23.9%). In men, compared to the 1st tertile of serum β-cryptoxanthin and provitamin A, those who were in the 3rd tertile had lower risks of premature all-cause mortality (OR, 95% CI: 0.19, 0.07-0.47 for β-cryptoxanthin, and 0.24, 0.09-0.61 for provitamin A). In women, compared to the 1st tertile of serum β-cryptoxanthin, those who were in the 3rd tertile had higher risks of premature all-cause mortality (OR, 95% CI: 1.94, 1.00-4.03). These significant associations were observed in analyses for premature cancer mortality. We found significant associations between higher levels of serum β-cryptoxanthin and provitamin A and lower risks of premature mortality among Japanese men, while a different directional association was found in women. Although these findings suggest roles of serum carotenoids on premature mortality, further studies are needed to validate this association in other populations.

BACKGROUND: Smoking is well known to be a major risk factor for cancer, and to decrease the levels of aryl hydrocarbon receptor repressor (AHRR) DNA methylation. AHRR is a key regulator for AHR signaling, which is involved in chemical metabolism and cancer development. Therefore, smoking-induced AHRR DNA hypomethylation may be associated with cancer development. However, it has not been reported that association between AHHR DNA methylation and cancer mortality in Asian population. Hence, we examined whether AHRR DNA methylation levels were associated with cancer mortality in a Japanese population. METHODS: This study was conducted with 812 participants (aged 38-80 years) who received a health check-up in 1990, and did not have a clinical histories. We followed up the participants until the end of 2019 (median: 27.8 years), and 100 participants died from cancer. The AHRR DNA methylation levels in peripheral blood mononuclear cells (PBMCs) were measured by the pyrosequencing method. We calculated the hazard ratio (HR) and 95% confidence interval (CI) for cancer mortality according to the baseline levels of AHRR DNA methylation. RESULTS: We found that AHRR DNA hypomethylation was associated with a higher risk of all cancer mortality, especially smoking related cancers and lung cancer. (all cancer: HR, 1.28, 95% CI, 1.09-1.51; smoking-related cancers: HR, 1.35, 95% CI, 1.12-1.62; lung cancer: HR, 1.68, 95% CI, 1.24-2.26). CONCLUSIONS: Smoking-induced AHRR DNA hypomethylation in PBMCs was associated with the risk of cancer mortality in Japanese population; therefore, hypomethylation of AHRR may be a useful biomarker of cancer mortality risk.

AIMS: This study aimed to analyze differences in sensitivity to hepatic lipid metabolism at different ages, through DNA methylation, using an experimental rat model of high-fructose corn syrup (HFCS) intake. MAIN METHODS: The experimental was divided into three periods: childhood and adolescence (postnatal day (PD) 21-60), young adulthood (PD61-100), and adulthood (PD101-140). Rats in the different age groups were assigned to receive either water (C: control group) or 20% HFCS solution (H: HFCS-fed group). We measured hepatic mRNA levels of peroxisome proliferator-activated receptor alpha (Ppara), carnitine palmitoyltransferase 1A (Cpt1a), fatty acid synthase (Fasn), and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (Pgc1a) using real-time PCR. Additionally, we examined the DNA methylation levels of Ppara, Cpt1a, Fasn, and Pgc1a using pyrosequencing. KEY FINDINGS: Gene expressions of Cpt1a and Ppara in childhood and adolescence were significantly lower in the H group than in the C group. Conversely, Fasn and Pgc1a expressions were significantly higher in the H group than in the C group. Additionally, there was hypermethylation of Cpt1a and Ppara and hypomethylation of Fasn and Pgc1a in the H groups of childhood and adolescence. However, only one gene expression and methylation change was observed in young adulthood and adulthood groups. We found that HFCS intake in rats had stronger lipid metabolic effects in childhood and adolescence than in other generations, and that its mechanism involved epigenetic regulation. SIGNIFICANCE: We anticipate that these research findings will be a breakthrough for elucidating the varying effects of growth stage in the future.

BACKGROUND: Previous cross-sectional studies have shown that several circulating microRNA levels are associated with hypertension, but there are no prospective studies among general populations. OBJECTIVE: We evaluated the impact of circulating inflammatory- and oxidative stress-responsive microRNAs on changes in blood pressure and the development of hypertension in normotensive Japanese. METHOD: The study subjects were 84 normotensive participants (33 men and 51 women) who were given a health examination in both 2012 and 2017. In five years, 29 participants developed hypertension. Serum levels of miRNAs (miR-21, miR-27a, and miR-133a) were measured using qRT-PCR. Odds ratios (ORs) and 95% confidence intervals (CIs) for incident hypertension were estimated by logistic regression analysis. RESULTS: Serum miR-27a and -133a levels were lower in newly hypertensive subjects compared with normotensive subjects. With 1-unit lower serum miR-27a and -133a, the confounders adjusted ORs and 95% CI for incident hypertension were 0.84 (0.72-0.96) and 0.75 (0.58-0.91), respectively. The group with high levels of serum miR-27a and -133a had lower ORs than the group with low levels of these miRNAs (OR and 95% CI of miR-27a: 0.29, 0.08-0.91; miR-133a: 0.08, 0.01-0.37, respectively). CONCLUSIONS: Circulating miR-27a and -133a are potential biomarkers for the prediction and prevention of hypertension.

There are concerns about the negative effects of fructose intake during pregnancy on the next generation. We have previously reported that offspring from dams fed with fructose during gestation and lactation demonstrate abnormal lipid metabolism in the liver. In this study, we aimed to elucidate the molecular mechanism of the effects of maternal high-fructose corn syrup (HFCS) consumption on offspring. Pregnant Sprague-Dawley rats were fed with 20% HFCS water solution during gestation and lactation. Offspring were put on a normal diet after weaning, and the serum parameters and gene expression patterns were studied at predetermined intervals. Offsprings from pregnant rats fed with 20% HFCS (HFCS group) developed insulin resistance and hyperlipidemia at 60 d of age. RNA-seq analysis demonstrated that peroxisome proliferator-activated receptor α (PPARα) expression is downregulated by maternal HFCS intake. Hepatic Pparα expression in the HFCS group appeared to be suppressed by the enhanced DNA methylation of its promoter region. It is suggested that the development of insulin resistance and hyperlipidemia in the HFCS group may be attributable to aberrant Pparα methylation in the offspring liver. Pparα hypermethylation may be one of molecular mechanism underlying the toxicity of maternal fructose intake.

Background: Thioredoxin-interacting protein (TXNIP) controls the cellular redox balance by binding to and inhibiting the expression and function of thioredoxin. DNA methylation of the TXNIP gene is involved in the regulation of TXNIP mRNA expression. Changes in TXNIP DNA methylation levels are associated with the development of various diseases such as type 2 diabetes mellitus (T2DM). However, few studies have focused on the influence of lifestyle factors such as alcohol intake on TXNIP DNA methylation.Objectives: This research examines the association of drinking behaviors with TXNIP DNA methylation levels in the general Japanese population.Methods: We conducted a cross-sectional study of 404 subjects (176 males and 228 females) who were divided into non-, moderate and heavy drinkers based on self-reported drinking behaviors. TXNIP DNA methylation levels in leukocytes were determined using a pyrosequencing assay.Results: The mean TXNIP DNA methylation level in heavy drinkers (74.2%) was significantly lower than that in non- and moderate drinkers (non: 77.7%, p < .001; moderate: 76.6%, p = .011). Multivariable linear regression analysis showed that log-transformed values of daily (b = -1.34; p < .001) and cumulative (b = -1.06; p = .001) alcohol consumption were associated with decreased TXNIP DNA methylation levels.Conclusion: TXNIP DNA methylation levels in heavy drinkers was lower than in non- and- moderate drinkers. Decreased TXNIP DNA methylation level increases the expression of TXNIP and elevates the risk of developing of diseases such as T2DM. Therefore, decreasing alcohol use in heavy drinkers may lessen the likelihood of some alcohol-related illnesses moderated through TXNIP DNA methylation.

The increasing prevalence of nonalcoholic fatty liver disease (NAFLD) is a global health problem. In recent years, the inhibitory effect of brain-derived neurotrophic factor (BDNF) on diabetes mellitus and fatty liver has been clarified. The purpose of this study was to analyze the relationship between serum BDNF and NAFLD which caused by abnormal metabolism of glucose and lipids. This cross-sectional study involved 429 participants (mean age, 63.5 years: men, 38.5%) with low alcohol intake. Of the participants, those who had an increase in echogenicity of the liver parenchyma and hepato-renal contrast on ultrasonography were classified as the NAFLD group (n = 88), and the others were classified as the normal (n = 341) group. The NAFLD group was further classified into a mild group (n = 60) and a severe group (n = 28) based on the intensity of echogenicity and visualization of the hepatic vessels and diaphragm. Median BDNF levels were higher in the NAFLD group than the normal group (35.5 vs. 42.3 ng/mL, p < 0.01). Furthermore, BDNF levels tended to be associated with the severity of NAFLD (p < 0.01). In addition to the univariate analysis, in the sex- and age-adjusted model, there was a significant association between the BDNF levels and NAFLD severity (p < 0.01). The fully adjusted regression analysis also showed a positive association between the serum BDNF level and NAFLD (p < 0.01). These results suggest that NAFLD patients have a compensatory increase in circulating BDNF levels.

Metabolic syndrome (MetS) is cluster of metabolic diseases, including abdominal obesity, hyperglycemia, high blood pressure, and dyslipidemia, that directly escalate the risk of type 2 diabetes, heart disease, and stroke. Thioredoxin-interacting protein (TXNIP) is a binding protein for thioredoxin, a molecule that is a key inhibitor of cellular oxidation, and thus regulates the cellular redox state. Epigenetic alteration of the TXNIP-encoding locus has been associated with components of MetS. In the present study, we sought to determine whether the level of TXNIP methylation in blood is associated with MetS in the general Japanese population. DNA was extracted from the peripheral blood cells of 37 subjects with and 392 subjects without MetS. The level of TXNIP methylation at cg19693031 was assessed by the bisulfite-pyrosequencing method. We observed that TXNIP methylation levels were lower in MetS subjects (median 74.9%, range 71.7-78.4%) than in non-MetS subjects (median 77.7%, range 74.4-80.5%; p = 0.0024). Calculation of the confounding factor-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for hypomethylation revealed that subjects with MetS exhibited significantly higher ORs for hypomethylation than did those without MetS (OR, 2.92; 95% CI, 1.33-6.62; p = 0.009). Our findings indicated that lower levels of TXNIP methylation are associated with MetS in the general Japanese population. Altered levels of DNA methylation in TXNIP at cg19693031 might play an important role in the pathogenesis of MetS.

Consumption of fructose-containing beverages such as high-fructose corn syrup (HFCS) is increasing, raising concerns about the negative effects of excessive fructose intake. A recent report indicated that excess HFCS intake impairs hippocampal function. In this study, we focused on neurotrophic factors (NFs) in the hippocampus from the viewpoint of epigenetics to clarify the adverse effects of fructose. We analyzed the effects of HFCS intake on hippocampal function in three age categories: childhood and adolescence (postnatal day (PD) 21-60), young adulthood (PD60-100), and late adulthood (PD100-140). For the experiments, male Sprague-Dawley rats were divided into three age categories, the control group was received distilled water and the HFCS group was received 20% HFCS solution for 40 days in each period. We analyzed mRNA and protein levels for qPCR and western blotting, respectively, of a hippocampal NF, brain-derived neurotrophic factor (Bdnf). HFCS consumption reduced hippocampal Bdnf mRNA and protein expressions in childhood and adolescence. Moreover, pyrosequencing assays revealed increased DNA methylation at the Bdnf promoter in childhood and adolescence. This Bdnf levels reduction may be due to hypermethylation of the promoter regions. It should be noted that this phenomenon was observed only in childhood and adolescence fructose consumption. Our results indicate that the sensitivity of the hippocampus to fructose may vary with age. This study provides insight into the adverse effects of excessive HFCS consumption on the hippocampus in children.

Given that fructose consumption has increased by more than 10-fold in recent decades, it is possible that excess maternal fructose consumption causes harmful effects in the next generation. This study attempted to elucidate the mechanism of the harmful effects of excessive maternal fructose intake from the perspective of offspring liver function. Female rats during gestation and lactation were fed water containing fructose, and their offspring were fed normal water. We attempted to elucidate the mechanism of fructose-induced transgenerational toxicity by conducting a longitudinal study focusing on hepatic programming prior to disease onset. Impaired Insulin resistance and decreased high-density lipoprotein-cholesterol levels were observed at 160 days of age. However, metabolic disorders were not observed in 60-day-old offspring. Microarray analysis of 60-day-old offspring livers showed the reduction of hepatic insulin-like growth factor-1 (Igf1) mRNA expression. This reduction continued until the rats were aged 160 days and attenuated Igf1 signaling. Hepatic microRNA-29 (miR-29a) and miR-130a, which target Igf1 mRNA, were also found to be upregulated. Interestingly, these miRNAs were upregulated in the absence of metabolic disorder. In this study, we found that maternal fructose intake resulted in dysregulated expression of Igf1 and its target miRNAs in the offspring liver, and that these offspring were more likely to develop metabolic disorders. Abnormal hepatic programming induced by an imbalanced maternal nutritional environment is maintained throughout life, implying that it may contribute to metabolic disorders.

<title>Abstract</title><sec> <title>Background</title> Public perceptions and personal characteristics are heterogeneous between countries and subgroups, which may have different impacts on health-protective behaviors during the coronavirus disease 2019 (COVID-19) pandemic. To assess whether self-reported perceptions of COVID-19 and personal characteristics are associated with protective behaviors among general adults and to compare patterns in six different countries. </sec><sec> <title>Methods</title> This cross-sectional study uses the secondary data collected through an online survey between 15 and 23 April 2020 across six countries (China, Italy, Japan, Korea, the UK, and the USA). A total of 5945 adults aged 18 years or older were eligible for our analysis. A logistic regression model was used to estimate odds ratios (OR) and 95% confidence intervals (95%CI) of three recommended behaviors (wearing a mask, handwashing, and avoiding social gatherings). </sec><sec> <title>Results</title> In most countries except for China, the participants who perceived wearing a mask as being extremely effective to curtail the pandemic were more likely to wear a mask (OR, 95%CI: Italy: 4.14, 2.08–8.02; Japan: 3.59, 1.75–7.30; Korea: 7.89, 1.91–31.63: UK: 9.23, 5.14–17.31; USA: 4.81, 2.61–8.92). Those who perceived that handwashing was extremely effective had higher ORs of this preventive behavior (OR, 95%CI: Italy: 16.39, 3.56–70.18; Japan: 12.24, 4.03–37.35; Korea: 12.41, 2.02–76.39; UK: 18.04, 2.60–152.78; USA: 10.56, 2.21–44.32). The participants who perceived avoiding social gathering as being extremely effective to curtail the pandemic were more likely to take this type of preventive behavior (OR, 95%CI: China: 3.79, 1.28–10.23; Korea: 6.18, 1.77–20.60; UK: 4.45, 1.63–11.63; USA: 4.34, 1.84–9.95). The associations between personal characteristics, living environment, psychological status, and preventive behaviors varied across different countries. Individuals who changed their behavior because of recommendations from doctors/public health officials were more likely to take preventive behaviors in many countries. </sec><sec> <title>Conclusions</title> These findings suggest that higher perceived effectiveness may be a common factor to encourage preventive behaviors in response to the COVID-19 pandemic. These results may provide a better understanding of the homogeneity and heterogeneity of factors related to preventive behaviors and improve public health policies in various countries and groups. </sec>

Background: Although a number of microRNAs (miRNA) reflecting kidney function has been identified, prospective studies are now urgently needed to determine a clinical utility of these miRNAs among general populations. The purpose of this study was to examine the associations between serum miRNAs and kidney function in a population-based study. Methods: We conducted a five-year prospective study (2012–2017) of 169 individuals without chronic kidney disease (CKD) at the baseline survey (mean age, 62.5; 96 women). The real-time qPCR was used to measure serum levels of five previously reported miRNAs. Participants with eGFR < 60 mL/min/1.73 m2 were defined as having CKD. Changes in eGFR were defined as eGFR2017 − eGFR2012. Results: After adjusting for covariates including baseline eGFR, lower serum levels (1st tertile) of miR-126 were associated with a greater decline of eGFR (β [SE] = −3.18 [1.50]) and a higher odds ratio (OR) of CKD onset over five years (OR [95% CI] = 3.85 [1.01–16.8]), compared with the 3rd tertile. Conclusions: We found baseline serum miR-126 levels were associated with changes in eGFR and new CKD cases in a five-year prospective study. This result suggests that miR-126 may be a potential biomarker of CKD even among general populations.

DNA methylation plays an important role in the pathogenesis and progression of cardiovascular disease (CVD) but the prospective association of DNA methylation with CVD has not been evaluated. Here, we conducted a prospective study to examine whether long interspersed nuclear element-1 (LINE-1) DNA methylation is associated with CVD mortality in a Japanese population. We targeted 822 Japanese who participated in a health check-up in 1990 and had no clinical history of cancer, stroke or ischaemic heart disease. DNA was extracted from peripheral blood mononuclear cells and LINE-1 DNA methylation at three CpG sites was measured using a pyrosequencing method. We used propensity score (PS) matching to reduce the effect of potential confounding. During 18 118.7 persons-years of follow-up, there were 329 deaths from all-causes and 85 deaths from CVD. In PS-matched analysis, a significantly higher HR for CVD mortality was observed in the hypermethylation group than in the hypomethylation group for elderly participants (HR 2.77; 95% CI 1.55 to 4.93). No significant association between LINE-1 DNA methylation and CVD was observed for middle-aged participants. Based on this prospective study, we suggest that LINE-1 DNA hypermethylation is associated with increased CVD mortality risk in an elderly population.

DNA methylation (DNAm) is one of the most studied epigenetic modifications. DNAm has emerged as a key biological mechanism and biomarkers to test associations between environmental exposure and outcomes in epidemiological studies. Although previous studies have focused on associations between DNAm and either exposure/outcomes, it is useful to test for mediation of the association between exposure and outcome by DNAm. The purpose of this scoping review is to introduce the methodological essence of statistical mediation analysis and to examine emerging epidemiological research applying mediation analyses. We conducted this scoping review for published peer-reviewed journals on this topic using online databases (PubMed, Scopus, Cochrane, and CINAHL) ending in December 2020. We extracted a total of 219 articles by initial screening. After reviewing titles, abstracts, and full texts, a total of 69 articles were eligible for this review. The breakdown of studies assigned to each category was 13 for smoking (18.8%), 8 for dietary intake and famine (11.6%), 6 for other lifestyle factors (8.7%), 8 for clinical endpoints (11.6%), 22 for environmental chemical exposures (31.9%), 2 for socioeconomic status (SES) (2.9%), and 10 for genetic factors and race (14.5%). In this review, we provide an exposure-wide summary for the mediation analysis using DNAm levels. However, we found heterogenous methods and interpretations in mediation analysis with typical issues such as different cell compositions and tissue-specificity. Further accumulation of evidence with diverse exposures, populations and with rigorous methodology will be expected to provide further insight in the role of DNAm in disease susceptibility.