石川 浩章

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.

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.

© 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.

BACKGROUND: MicroRNAs are present not only in exosomes but also in high-density lipoprotein (HDL) and have the potential as biomarkers for various diseases. Various purification methods have been developed to quantify HDL-miRNAs; however, they are unsuitable for clinical applications. Therefore, we aimed to establish a simpler analytical method to quantify HDL-miRNAs for clinical applications. METHODS: We purified HDL fraction from pooled plasma using a three-step protocol consisting of ultracentrifugation, phosphotungstic acid/MgCl2 precipitation and desalting/buffer exchange followed by the quantification of HDL-miRNAs by quantitative real-time PCR. In order to establish a method to quantify HDL-miRNAs by quantitative real-time PCR, we prepared standard curves for miR-223 and miR-92. The HDL-miRNAs of 10 volunteers were assessed. RESULTS: Exosomes and LDL were not detected in the purified HDL fraction. Furthermore, we confirmed that only HDL was purified and that the HDL recovery rate of our method was at least approximately 50%. The threshold cycle values of miR-223, miR-92, miR-146a and miR-150 in the same subject were 32.11 ± 0.58, 32.50 ± 0.35, 34.30 ± 0.70 and 34.91 ± 0.77, respectively ( n = 10). The coefficient of variation values for these miRNAs were 1.08-2.21%. In addition, the standard curve for the quantitative analysis of miRNAs showed high linearity (30-30,000 copies/ μL) with a correlation coefficient of >0.99. The concentrations of HDL-miR-223 and HDL-miR-92 in the plasma of 10 subjects were 1.98 ± 0.32 and 0.90 ± 0.14 copies/mL (×104). CONCLUSIONS: We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

Purpose It has been demonstrated that circulating microRNA profiles are affected by physiological conditions. Several studies have demonstrated that microRNAs play important roles in the regulation of adiposity. However, few have investigated the relationship between circulating microRNAs and obesity, which has become a major public health problem worldwide. This study investigated the association between circulating microRNAs and obesity in a Japanese population. Methods Obesity parameters, such as subcutaneous and visceral fat adipose tissue, body fat percentage, and body mass index were assessed in a cross-sectional sample of 526 participants who attended health examinations in Yakumo, Japan. In addition, five circulating microRNAs (miR-20a, -21, -27a, -103a, and -320), which are involved in adipocyte proliferation and differentiation, were quantified using real-time polymerase chain reaction amplification. Results We compared the circulating microRNA concentrations in a percentile greater than 75th (high) with below the value (low) of subcutaneous adipose tissue, visceral fat adipose tissue, body mass index, and per cent body fat. For visceral fat adipose tissue, significant decrease in miR-320 expression was observed in high group. Also, for body mass index, significant change of miR-20a, -27a, 103a, and 320 expression level was observed in high group. Multiple linear regression analysis demonstrated that circulating levels of some microRNA such as miR-27a were significantly associated with subcutaneous adipose tissue, visceral fat adipose tissue, and body mass index. Conclusions Our findings support the need for further studies to determine whether such changes are consistent across different populations and whether the identified microRNAs may represent novel biomarkers to predict the susceptibility and progression of obesity-related disorders.

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.

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.

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.

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.

Background Serum ornithine carbarnoyltransferase is a diagnostic marker of hepatic disorders due to its localization in periportal mitochondria. Methods We have developed a new method for the determination of serum ornithine carbarnoyltransferase. It is based on the reverse reaction of ornithine carbamoyltransferase, using ornithine-ketoacid aminotransferase, Delta(1)-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase, which together convert citrulline through ornithine to glutamate. The glutamate is then quantitatively measured using glutamate oxidase and Trinder's reagent. Results The results obtained by this method agreed well with those obtained using the diacetylmonoxime method as a gold standard [correlation coefficient (r) = 0.973 P&lt;0-001]. The endogenous amino acids sensitive to this method in serum (glutamate, ornithine and Delta(1)-pyrroline-5-carboxylate) were eliminated by the initial futile reaction. The new method appears to be more accurate at low levels of ornithine carbamoyltransferase activity than the diacetylmonoxime method. Conclusions Here we report a new method for serum ornithine carbamoyltransferase assay which might be useful for clinical diagnosis of hepatic disorders, including hepatic cancer.