MTHFR, MTR and MTRR polymorphisms and risk of chronic kidney disease in Japanese: cross-sectional data from the J-MICC Study.

PURPOSE: Chronic kidney disease (CKD) is well known as a strong risk factor for both of end-stage renal disease and cardiovascular disease. To clarify the associations of MTHFR, MTR, and MTRR polymorphisms with the risk of CKD in Japanese, we examined this association among Japanese subjects using cross-sectional data. METHODS: The subjects for this analysis were 3,318 participants consecutively selected from the Japan Multi-institutional Collaborative Cohort (J-MICC) Study. The polymorphisms were genotyped by a multiplex polymerase chain reaction-based Invader assay. Age- and sex-adjusted odds ratio (aOR) of CKD with stage 3-5 was calculated for each genotype. RESULTS: When those with MTHFR C677T C/C were defined as references, those with MTHFR C677T C/T and T/T demonstrated the aORs for CKD of 1.14 (95 % CI 0.93-1.40) and 1.39 (1.06-1.82), respectively. Marginally significantly decreased risk of CKD with increasing number of MTR A2756G G allele (p = 0.058) was observed. Stratified analyses by plasma folate low (<7.4 ng/ml) or high (≥7.4 ng/ml) suggested significantly higher OR of CKD for those with MTHFR C677T T/T and low serum folate with the aOR of 2.07 (95 % CI 1.30-3.31) compared with that for those with MTHFR C677T T/T and high serum folate. CONCLUSIONS: The present study found a significant association between the subjects with the T/T genotype of MTHFR C677T polymorphism and the elevated risk of CKD, which may suggest the possibility of the risk evaluation and prevention of this potentially life-threatening disease based on genetic traits in the near future.

Significant interaction between RETN -420 G/G genotype and lower BMI on decreased risk of type 2 diabetes mellitus (T2DM) in Japanese--the J-MICC Study.

We examined the association of the RETN (resistin) -420 C>G polymorphism (rs1862513) with risk of diabetes mellitus (DM), considering lifestyle factors, in Japanese. Subjects were participants of J-MICC Study, where 2,651 participants aged 35-69 years provided their blood for genotyping and lifestyle data after informed consent. Odds ratio (OR) of DM for RETN-420 G/G genotype was estimated using unconditional logistic regression model. Statistically significant interaction on risk of DM was observed between RETN-420 G/G genotype and BMI<25 (OR for interaction = 0.12; P = 0.046), and when subjects with RETN-420 C/C+C/G and BMI ≥ 25 (n = 69 for DM and 544 for non-DM) were defined as the reference, the adjusted ORs for subjects with RETN-420 G/G genotype and BMI>25 (n = 10 for DM and 111 for non-DM), RETN-420 C/C+C/G and BMI<25 (n = 81 for DM and 1,605 for non-DM), and RETN-420 G/G and BMI<25 (n = 1 for DM and 230 for non-DM) were demonstrated to be 0.72 (95% confidence interval: 0.36-1.46), 0.40 (0.28-0.56) and 0.03 (0.005-0.25), respectively. The present study revealed the significant interaction of RETN-420 G/G genotype with lower BMI on the decreased risk of DM, but the direction was opposite to the reported ones in Japanese. We should be careful in interpretation of the present study results because of the limited sample sizes. Further investigation of this association as well as of the actual biological roles of RETN in the genesis of human metabolic disorders including DM will be required.

Inverse correlation between coffee consumption and prevalence of metabolic syndrome: baseline survey of the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study in Tokushima, Japan.

BACKGROUND: It is unclear whether consumption of coffee and green tea is associated with metabolic syndrome. METHODS: This cross-sectional study enrolled 554 adults who had participated in the baseline survey of the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study in Tokushima Prefecture, Japan. Consumption of coffee and green tea was assessed using a questionnaire. Metabolic syndrome was diagnosed using the criteria of the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) and the Japan Society for the Study of Obesity (JASSO). Logistic regression analysis was used to examine the association between consumption of coffee and green tea and prevalence of metabolic syndrome and its components. RESULTS: After adjustment for sex, age, and other potential confounders, greater coffee consumption was associated with a significantly lower prevalence of metabolic syndrome, as defined by NCEP ATP III criteria (P for trend = 0.03). Participants who drank more coffee had a lower odds ratio (OR) for high serum triglycerides (P for trend = 0.02), but not for increased waist circumference or high blood pressure. Using JASSO criteria, moderate coffee consumption (1.5 to <3 cups/day) was associated with a significantly lower OR for high plasma glucose (OR = 0.51, 95% CI 0.28-0.93). Green tea consumption was not associated with the prevalence of metabolic syndrome or any of its components. CONCLUSIONS: Coffee consumption was inversely correlated with metabolic syndrome diagnosed using NCEP ATP III criteria, mainly because it was associated with lower serum triglyceride levels. This association highlights the need for further prospective studies of the causality of these relationships.

Association between blood levels of PCDDs/PCDFs/dioxin-like PCBs and history of allergic and other diseases in the Japanese population.

BACKGROUND: Previous studies reported that exposure to dioxins was associated with an increased risk of various diseases in general populations. OBJECTIVES: The aim of this study was to examine the association between levels of dioxins in blood and allergic and other diseases. METHODS: We conducted a cross-sectional study on 1,063 men and 1,201 women (aged 15-76 years), who were living throughout Japan and not occupationally exposed to dioxins, during 2002-2010. In fasting blood samples, polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like PCBs (DL-PCBs) were analyzed by isotope dilution high-resolution gas chromatography/mass spectrometry. We obtained information on life style and self-reported history of diseases using a questionnaire. Blood pressure, blood levels of hemoglobin A1c, and serum lipids were also measured. Multiple logistic regression models were used to analyze the association between dioxin levels in blood and various diseases. RESULTS: Toxic equivalents of PCDDs/PCDFs and total dioxins showed significant inverse dose-response relationships with atopic dermatitis, after adjustments for potential confounders. The highest quartile for total dioxins had an adjusted odds ratio of 0.26 (95 % confidence interval 0.08-0.70) compared to the reference group (first quartile). The odds ratios for hypertension, diabetes mellitus, hyperlipidemia, gout in men, and gynecologic diseases in women significantly increased with increasing toxic equivalents of PCDDs/PCDFs, DL-PCBs, and total dioxins in blood. CONCLUSIONS: The present findings suggest that background exposure to dioxins was associated with reduced risk of atopic dermatitis. The results also support the idea that low-level exposure to dioxins is associated with an increased risk of diabetes, hypertension, and hyperlipidemia.

Circulating cytokine signatures in healthy medical students exposed to academic examination stress.

Stress-induced production of proinflammatory cytokines in the brain and periphery is associated with mental distress. In this study, we measured changes in levels of salivary cortisol and 50 circulating immune mediators in 28 4th-grade medical students (19 males and 9 females) 7 weeks before, 1 day before, immediately after, and 1 week after an authorized nationwide examination for promotion. Repeated measures ANOVA with multiple testing correction and post hoc tests revealed that the examination significantly increased levels of proinflammatory cytokines (granulocyte colony-stimulating factor, interferon-γ, interleukin (IL)-1ß, and tumor necrosis factor-α), Th2 cytokines (IL-4, IL-5, and IL-13), and ß-nerve growth factor in association with significant decreases in salivary cortisol levels and anxiety after the examination. These mediators may have a negative impact on the mental state of healthy young adults exposed to naturalistic stressors.

MicroRNAs miR-144/144* and miR-16 in peripheral blood are potential biomarkers for naturalistic stress in healthy Japanese medical students.

Non-coding microRNAs (miRNAs) are suggested to serve fundamental roles in cellular stress responses and in coping with sudden environmental changes in experimental animals. We examined whether naturalistic stressor-responsive miRNAs were detectable in whole blood. Blood and saliva were collected between 16:00 and 17:00 from 10 healthy medical students (5 males and 5 females; aged 22.4±0.8 years, mean±SD) 7 weeks before, one day before, immediately after, and one week after a nationally administered examination for academic promotion. Samples obtained one week after the examination were used as baseline controls. State anxiety and salivary cortisol levels reached maximum levels the day before the examination. Eleven candidate miRNAs (miR-144, -144*, -16, -15a, -19a, -19b, -26b, -30b, -106b, -126, and -142-3p) were extracted using a human miRNA microarray, and quantitative real-time reverse transcription PCR confirmed significant elevation of miR-144/144* and miR-16 levels immediately after finishing the examination. miR-16 levels in individual students were positively correlated with those of serum tumor necrosis factor (TNF)-α measured immediately after the examination. Percentage changes in miR-144* and miR-16 levels from immediately after to one week after the examination were significantly correlated with percentage changes in circulating interferon-γ and/or TNF-α levels over the same time points. Our results suggest that miR-144/144* and miR-16 may constitute a part of an integrated response to naturalistic stressors in healthy young adults.

Autism-associated gene expression in peripheral leucocytes commonly observed between subjects with autism and healthy women having autistic children.

Autism spectrum disorder (ASD) is a severe neuropsychiatric disorder which has complex pathobiology with profound influences of genetic factors in its development. Although the numerous autism susceptible genes were identified, the etiology of autism is not fully explained. Using DNA microarray, we examined gene expression profiling in peripheral blood from 21 individuals in each of the four groups; young adults with ASD, age- and gender-matched healthy subjects (ASD control), healthy mothers having children with ASD (asdMO), and asdMO control. There was no blood relationship between ASD and asdMO. Comparing the ASD group with control, 19 genes were found to be significantly changed. These genes were mainly involved in cell morphology, cellular assembly and organization, and nerve system development and function. In addition, the asdMO group possessed a unique gene expression signature shown as significant alterations of protein synthesis despite of their nonautistic diagnostic status. Moreover, an ASD-associated gene expression signature was commonly observed in both individuals with ASD and asdMO. This unique gene expression profiling detected in peripheral leukocytes from affected subjects with ASD and unaffected mothers having ASD children suggest that a genetic predisposition to ASD may be detectable even in peripheral cells. Altered expression of several autism candidate genes such as FMR-1 and MECP2, could be detected in leukocytes. Taken together, these findings suggest that the ASD-associated genes identified in leukocytes are informative to explore the genetic, epigenetic, and environmental background of ASD and might become potential tools to assess the crucial factors related to the clinical onset of the disorder.

Circulating vascular endothelial growth factor is independently and negatively associated with trait anxiety and depressive mood in healthy Japanese university students.

OBJECTIVE: Anxiety and depressive mood are sometimes accompanied by modulation of neuroendocrine and immune functions. The aim of this study was to identify circulating immune mediators reflecting anxiety and depressive mood in healthy young adults. METHODS: Anxiety and depressive mood in 209 healthy medical students (125 males and 84 females, aged 20.7±2.7years (mean±SD)) were assessed by the Spielberger state-trait anxiety inventory (STAI) and the Zung self-rating depression scale (Zung-SDS), respectively. Cortisol and chromogranin A (CgA) levels in saliva were measured using enzyme immunoassay kits, and 50 different mediators in sera were measured by a multiplex-suspension array system. The level of statistical significance was set at α=0.05. RESULTS: Forty-four mediators were measurable in sera, and each mediator showed substantial individual variations. After determining Pearson correlation coefficients, we selected candidate cytokines whose levels were associated with STAI-state (2 cytokines), STAI-trait (8 cytokines), or SDS scores (8 cytokines). The candidate cytokines plus interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and macrophage migration inhibitory factor were then subjected to multiple regression analysis adjusted for gender, BMI, and salivary concentrations of cortisol and CgA. Vascular endothelial growth factor (VEGF) was independently and negatively associated with both trait anxiety (p<0.05) and depressive mood (p<0.01). IL-1ß showed independently positive association with depressive mood (p<0.05). Interactions between these two cytokines and gender or BMI were not observed. CONCLUSION: Besides IL-1ß, circulating VEGF may be a potential biomarker for negative mood states in healthy young adults.

Effects of chronic academic stress on mental state and expression of glucocorticoid receptor α and ß isoforms in healthy Japanese medical students.

Chronic academic stress responses were assessed by measuring mental state, salivary cortisol levels, and the glucocorticoid receptor (GR) gene expression in healthy Japanese medical students challenging the national medical license examination. Mental states of 17 male and 9 female medical undergraduates, aged 25.0 ± 1.2 years (mean ± SD), were assessed by the State and Trait Anxiety Inventory (STAI) and the Self-Rating Depression Scale (SDS) 2 months before, 2 days before, and 1 month after the examination. At the same time points, saliva and blood were collected. STAI-state scores peaked 2 days before the examination. Scores on STAI-trait and SDS, and salivary cortisol levels were consistently higher during the pre-examination period. One month after the examination, all these measures had significantly decreased to baseline levels. Real-time reverse transcription PCR showed that this chronic anxious state did not change the expression of the functional GRα mRNA isoform in peripheral leukocytes, while it resulted in reduced expression of the GRß isoform 2 days before the examination. Our results replicate and extend a significant impact of chronic academic stressors on the mental state of healthy Japanese medical students and suggest a possible association of GRß gene in response to psychological stress.

Interleukin-18 deficiency reduces neuropeptide gene expressions in the mouse amygdala related with behavioral change.

In this study, we examined the effects of IL-18 deficiency on behaviors and gene expression profiles in 6 brain regions. IL-18(-/-) mice reduced depressive-like behavior and changed gene expressions predominantly in the amygdala compared with wild-type mice. Pathway analysis of the differentially expressed genes ranked behavior as the top-scored biological function. Of note, the absence of IL-18 decreased Avp, Hcrt, Oxt, and Pmch mRNA levels and the number of arginine vasopressin- and oxytocin-positive cells in the amygdala, but not in the hypothalamus. Our results suggest that IL-18-dependent vasopressinergic and oxytocinergic circuitry in the amygdala may regulate depressive-like behaviors in mice.

Brief naturalistic stress induces an alternative splice variant of SMG-1 lacking exon 63 in peripheral leukocytes.

Alternative splicing (AS) not only regulates the gene expression program in response to surrounding environment, but also produces protein isoforms with unique properties under stressful conditions. However, acute psychological stress-initiated AS events have not been documented in human studies. After assessments of changes in salivary cortisol levels and anxiety among 28 fourth-grade medical students 7 weeks prior to, 1 day before, immediately after, and 1 week after an examination for promotion, we selected 5 male students, who showed a typical stress response, and screened AS events in their circulating leukocytes using the GeneChip human exon 1.0 ST array. AS events of 27 genes with splicing indices >1.0 could be detected between immediately after and either 7 weeks before, 1 day before, or 1 week after the examination. The examination stress preferentially caused skipping rather than inclusion: 21 out of the 27 pre-mRNAs underwent skipping of exons, and skipping in 3'UTR was observed in 8 genes. Among the candidate genes, real-time reverse transcription PCR validated the stress-initiated skipping of exon 63 of SMG-1 that encodes a phosphatidylinositol 3-kinase-related protein kinase crucial for activations of p53-dependent pathways and nonsense-mediated mRNA decay. Our results indicate a significant impact of brief naturalistic stressors on AS-mediated regulation of gene expression in peripheral leukocytes, and suggest the SMG-1 splice variant as a potential biomarker for acute psychological stress.

High-throughput screening of brief naturalistic stress-responsive cytokines in university students taking examinations.

This study was designed to prospectively examine the impact of a brief naturalistic stressor (academic examination) on salivary/serum cortisol, measures of anxiety and depressive mood, and 50 circulating immune mediators assessed 7 days before, the first day of, and 2 days after the first term examination period (5 days) among 20 male and 6 female medical students (19.7+/-3.1 years, mean+/-SD). Of 42 serum factors detected, repeated measures ANOVA and Bonferroni post hoc testing indicated that concentrations of macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein (MCP)-3, and beta-nerve growth factor (beta-NGF) were significantly decreased 2 days after finishing examinations, compared with the levels on the first day of examinations (p<0.05) in association with a concomitant post-examination decreases (p<0.05) in anxiety and salivary cortisol levels. In contrast, interleukin (IL)-16 was reciprocally increased between the two time points (p<0.05). However, after correction for multiple comparisons, only changes in MIF were significant (p<0.05/42=0.00119), and MIF levels peaked on the first day of examinations was significantly higher than those measured both 7 days before and 2 days after the examination. The present high-throughput analysis with multiplex cytokine panels reconfirms the impact of brief naturalistic stressors on immune outcomes, and suggests a potential role of MIF in the acute stress response.

beta-Carotene and beta-cryptoxanthin but not lutein evoke redox and immune changes in RAW264 murine macrophages.

The mechanism of immunological benefits induced by carotenoids has not been fully elucidated. Here, we investigated some of the immunity-related properties of beta-carotene and two other carotenoids, beta-cryptoxanthin, and lutein, on the murine macrophages cell line RAW264. beta-Carotene added to the culture medium accumulated in the cells in a time- and dose-dependent manner. The accumulation was positively correlated with cellular lipid peroxidation, demonstrating the pro-oxidative activity of beta-carotene, and also with the synthesis of glutathione, an intracellular antioxidant. Conversely, accumulation of beta-carotene was negatively correlated with the transcription of immune-active molecules, such as IL-1beta, IL-6, and IL-12 p40, in cells stimulated by LPS and INF-gamma. The transcription of the pro-inflammatory cytokines IL-1beta and IL-6 was more sensitive to the accumulation of beta-carotene than was IL-12 p40. The accumulation of beta-cryptoxanthin in cells resulted in effects similar to those of beta-carotene. However, lutein accumulated minimally and did not significantly affect the cells. These results demonstrate that beta-carotene, and beta-cryptoxanthin as well, can accumulate in RAW264 cells and induce changes in intracellular redox status, which in turn regulate the immune function of macrophages.

Changes in behavior and gene expression induced by caloric restriction in C57BL/6 mice.

Caloric restriction (CR) is an effective method for prevention of age-associated diseases as well as overweight and obesity; however, there is controversy regarding the effects of dieting regimens on behavior. In this study, we investigated two different dieting regimens: repeated fasting and refeeding (RFR) and daily feeding of half the amount of food consumed by RFR mice (CR). CR and RFR mice had an approximate 20% reduction in food intake compared with control mice. Open field, light-dark transition, elevated plus maze, and forced swimming tests indicated that CR, but not RFR, reduced anxiety- and depressive-like behaviors, with a reduction peak on day 8. Using a mouse whole genome microarray, we analyzed gene expression in the prefrontal cortex, amygdala, and hypothalamus. In addition to the CR-responsive genes commonly modified by RFR and CR, each regimen differentially changed the expression of distinct genes in each region. The most profound change was observed in the amygdalas of CR mice: 884 genes were specifically upregulated. Ingenuity pathway analysis revealed that these 884 genes significantly modified nine canonical pathways in the amygdala. alpha-Adrenergic and dopamine receptor signalings were the two top-scoring pathways. Quantitative RT-PCR confirmed the upregulation of six genes in these pathways. Western blotting confirmed that CR specifically increased dopamine- and cAMP-regulated phosphoprotein (Darpp-32), a key regulator of dopamine receptor signaling, in the amygdala. Our results suggest that CR may change behavior through altered gene expression.