GCK, GCKR polymorphisms and risk of chronic kidney disease in Japanese individuals: data from the J-MICC Study

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 association of glucokinase and glucokinase regulatory protein (GCKR) polymorphisms with the risk of CKD in Japan, we examined this association among Japanese individuals using cross-sectional data. The subjects for this analysis were 3,314 consecutively selected participants from the Japan Multi-Institutional Collaborative Cohort Study. Age- and sex- adjusted odds ratios (aORs) of CKD stages 3-5 were calculated for each genotype by logistic regression and the effects of genotype on estimated glomerular filtration rate were evaluated by linear regression. Gene-environment interaction was also investigated based on questionnaire information. When subjects with GCKR rs780094 G/A and G/G, or GCKR rs1260326 T/C and C/C were combined together and compared with the references (GCKR rs780094 A/A or GCKR rs1260326 T/T), the aORs were 0.84 (0.69-1.02) or 0.81 (0.67-0.99) (p = 0.075 or 0.037), respectively. A significant OR for interaction between GCKR rs1260326 T/T and current smoking (OR = 1.79, p = 0.041) was also observed. The present study suggests a possible association of the T/T genotype of GCKR rs1260326 polymorphism with elevated risk of CKD and its interaction with current smoking, which may support the possibility of performing risk evaluation and prevention of this potentially life-threatening disease based on genetic traits in the near future.


A family history of diabetes is not associated with arterial stiffness in non-diabetic Japanese population


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

Previous studies reported that exposure to dioxins was associated with an increased risk of various diseases in general populations. The aim of this study was to examine the association between levels of dioxins in blood and allergic and other diseases. 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. 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. 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.

International Archives of Occupational and Environmental Health


Chronic academic stress increases a group of microRNAs in peripheral blood

MicroRNAs (miRNAs) play key roles in regulation of cellular processes in response to changes in environment. In this study, we examined alterations in miRNA profiles in peripheral blood from 25 male medical students two months and two days before the National Examination for Medical Practitioners. Blood obtained one month after the examination were used as baseline controls. Levels of seven miRNAs (miR-16, -20b, -26b, -29a, -126, -144 and -144*) were significantly elevated during the pre-examination period in association with significant down-regulation of their target mRNAs (WNT4, CCM2, MAK, and FGFR1 mRNAs) two days before the examination. State anxiety assessed two months before the examination was positively and negatively correlated with miR-16 and its target WNT4 mRNA levels, respectively. Fold changes in miR-16 levels from two days before to one month after the examination were inversely correlated with those in WNT4 mRNA levels over the same time points. We also confirmed the interaction between miR-16 and WNT4 3'UTR in HEK293T cells overexpressing FLAG-tagged WNT4 3'UTR and miR-16. Thus, a distinct group of miRNAs in periheral blood may participate in the integrated response to chronic academic stress in healthy young men.


Abundant daily non-sedentary activity is associated with reduced prevalence of metabolic syndrome and insulin resistance.

Background: Non-exercise activity thermogenesis (NEAT) has recently drawn attention because of its potential to prevent weight gain. Aim: This study evaluated the relationships between the duration of daily non-sedentary activities and the prevalence of metabolic syndrome and insulin resistance in the Japanese population. Material/Subjects and Methods: A total of 518 eligible subjects (380 men and 138 women) who attended the Tokushima Prefectural General Health Checkup Center and participated in the baseline survey of a cohort study conducted in Tokushima Prefecture, Japan were analyzed. Information about lifestyle characteristics including leisure-time exercise and daily non-exercise activities was obtained from a questionnaire. Logistic and multiple linear regression analyses were performed to evaluate the associations between the duration of daily non-exercise non-sedentary activities (beyond sitting) and prevalence of metabolic syndrome (and its components) and insulin resistance. Results: Subjects with longer duration of daily non-sedentary activities had significantly lower adjusted odds ratios for metabolic syndrome (P for trend = 0.024), abdominal obesity (P for trend = 0.023), and low HDL-cholesterol levels (P for trend = 0.002), after adjustment for sex, age, and other probable covariates including leisure-time exercise. Longer duration of daily non-sedentary activities was further associated with lower Homeostasis Model of Assessment-Insulin Resistance (HOMA-IR) values (P for trend = 0.009). Conclusions: Our results suggest that abundant daily non-sedentary activity might be associated with a lower prevalence of metabolic syndrome, especially for the components of central obesity and low HDL-cholesterol levels, and with a lower prevalence of insulin resistance, independent of leisure-time exercise.

Journal of Endocrinological Investigation


Consumption of coffee, not green tea, is inversely associated with arterial stiffness in Japanese men.

Background/Objectives:Studies on the associations between coffee and green tea consumption and arterial stiffness are rare. This study evaluated the possible relationships between coffee and green tea consumption and brachial-ankle pulse wave velocity (ba-PWV) values in Japanese men.Subjects/Methods:In total, 540 eligible men who enrolled in the baseline survey of a cohort study in Tokushima Prefecture, Japan, and who underwent ba-PWV measurement were analyzed. Information about lifestyle characteristics including coffee and green tea intake were obtained from a structural self-administered questionnaire. Multiple linear regression analyses were used to evaluate the associations between coffee and green tea consumption and ba-PWV.Results:Subjects with greater coffee consumption were younger and showed higher proportions of current smoking and alcohol consumption. Subjects with greater green tea consumption were older and showed lower proportions of current smoking and alcohol consumption. Greater coffee consumption was significantly inversely associated with ba-PWV after the adjustment for probable covariates, including serum low-density lipoprotein cholesterol and high-density lipoprotein cholesterol (P for trend =0.031). After additional adjustment for serum triglycerides, this inverse association persisted, but was somewhat attenuated (P for trend =0.050). In contrast, green tea consumption was not associated with ba-PWV.Conclusions:Coffee consumption was inversely associated with arterial stiffness independent of known atherosclerotic risk factors, and this association was partly mediated by reduced circulating triglycerides. Further prospective or interventional studies are needed to confirm the causal association.European Journal of Clinical Nutrition advance online publication, 17 July 2013; doi:10.1038/ejcn.2013.132.


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.


Consumption of seafood, serum liver enzymes, and blood levels of PFOS and PFOA in the Japanese population.

Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) have been shown to accumulate in the human body. The purpose of the present study was to examine the factors associated with the blood levels of PFOS and PFOA. A cross-sectional study was performed on 307 men and 301 women (aged 16-76 years) living in 15 prefectures in Japan. Blood levels of PFOS and PFOA were measured by liquid chromatography-mass spectrometry. Hepatic enzymes (-GTP, GOT, and GPT) and -3 polyunsaturated fatty acids (DHA and EPA) levels in serum were also measured. Associations between the levels of PFOS and PFOA in blood and the intake frequency of 41 kinds of dishes, foods and beverages and the serum levels of liver enzymes and -3 polyunsaturated fatty acids were examined using rank correlations. Frequency of intake of boiled fish in broth, sliced raw fish and coastal fish showed significant positive correlations with PFOS concentrations in blood after adjustments for potential confounders. Serum levels of GOT, GPT, DHA and EPA showed significant positive correlations with PFOS and PFOA in blood. There was also a significant regional difference in the blood levels of PFOS and 2013PFOA, with medians being highest in the Tokai/Hokuriku/Kinki region. These findings suggest that the concentrations of PFOS in blood were mainly associated with fish consumption and that the levels of PFOS and PFOA were associated with the serum levels of liver enzymes in Japanese populations. Further investigations are required to clarify the reason for the regional differences in blood levels of PFOS and PFOA in Japan.


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

It is unclear whether consumption of coffee and green tea is associated with metabolic syndrome. 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. 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. 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.


Relationship of dietary factors and habits with sleep-wake regularity.

The purpose of this study was to evaluate the associations between dietary factors and sleep-wake regularity in the Japanese population. We analyzed 1368 eligible subjects (931 men and 437 women) aged 35-69 years who had participated in the baseline survey of a cohort study in Tokushima Prefecture, Japan. Information on individual lifestyle characteristics, including dietary habits and sleep-wake regularity, was obtained by a selfadministrated questionnaire. Logistic regression analyses were performed to evaluate adjusted associations of the intake energy ratios of macronutrients, as well as intake frequency, and the amount of staple foods with sleepwake regularity. The lowest quartile of protein intake as well as the highest quartile of carbohydrates showed significantly higher multivariable-adjusted odds ratios of 2.1 (95% confidence interval, 1.3-3.3) and 2.1 (1.3-3.5), respectively, for poor sleep-wake regularity compared with the respective second quartile that is thought to be moderate intake. Regarding intake of staple foods, low weekly intake frequency at breakfast (<5 times/week), the lowest intake amount (<1 bowl or slice/roll) at breakfast, and the highest intake amount (>=2 bowls or slices/ rolls) at lunch and dinner exhibited significantly high adjusted odds ratios for poor sleep-wake regularity. Additionally adjusting for sleep duration, these results did not substantially alter. Our results suggested that low intake energy ratio of proteins and high intake energy ratio of carbohydrates, skipping intake of the staple foods at breakfast, and excessive intake amount of the staple foods at lunch and dinner may be associated with poor sleep-wake regularity.

Asia Pacific Journal of Clinical Nutrition


Evaluation of the Sugar Content in the Side Dishes Served in Elementary School Lunches

小学校給食の副食に含まれる糖質量の検討

Kuroda et al. proposed a simple method for calculating the carbohydrate content based on a food exchange list (Shokuhin Koukanhyou), with a sugar content of dietary staples of 40 % for rice, 50 % for bread, and 20 % for boiled noodles and a side dish containing 20 g sugar. This study focused on the sugar content in in school lunches, which have a nutrient composition that is different from diabetic meals. The total energy content of school lunches and the sugar content in their side dishes were investigated in 42 school lunches served in elementary school. The average energy intake in the total lunch increased depending on the school grade (670±44 to 752±50 kcal; means±standard deviation). On the other hand, the sugar content of side dishes increased from 29.7±7.5 g in the 1st-2nd grade to 31.2±8.1 g in the 5th-6th grade, thus it could be estimated to be 30 g regardless of the energy intake. Two hundred milliliters of milk containing approximately 10 g of carbohydrate was served in every school lunch, which accounted for the 10 g increase in comparison to hospital lunches. These results indicate that the sugar content in the side dishes of elementary school lunches can be estimated to be 30 g regardless of the energy intake.

「食品交換表に基づく新たなカーボカウント法」(黒田ら，糖尿病2010)に基づいて糖尿病食1食に含まれる糖質量を算出する場合，1食を主食と副食にわけ，副食は一律20 gと概算する．しかし，小児期は必要エネルギー量や栄養素バランスが食品交換表とは異なる．よって本研究では，小児期の副食糖質量を検討することを目的とした．学校給食摂取基準に基づく学校給食42食分を小学校の学年別に検討した．その結果，1食のエネルギーは670±44∼752±50(平均値±標準偏差)kcalと学年が上がるに従い増加したが，副食糖質量は29.7±7.5∼31.2±8.1 gであり，エネルギー量にかかわらず副食糖質量を約30 gに概算できた．学校給食では糖質約10 gの牛乳が毎食提供されるため糖尿病食や病院普通食に比較して副食糖質量が10 g増加すると考えられた．以上より，小学校給食では副食糖質量を30 gとしてカーボカウントを指導できることが明らかとなった．<br>

Journal of the The Japan Diabetes Society


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-, interleukin (IL)-1, and tumor necrosis factor-), Th2 cytokines (IL-4, IL-5, and IL-13), and -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)- 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- and/or TNF- 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.

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. 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 =0.05. 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)-1, IL-6, tumor necrosis factor-, 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-1 showed independently positive association with depressive mood (p<0.05). Interactions between these two cytokines and gender or BMI were not observed. Besides IL-1, circulating VEGF may be a potential biomarker for negative mood states in healthy young adults.

International Journal of Psychophysiology


Effects of chronic academic stress on mental state and expression of glucocorticoid receptor α and β 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 GRβ 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 GRβ gene in response to psychological stress.


A novel biomental tool for assessing psychological stress response

メンタルヘルスを支える新たなストレスバイオマーカー (特集 職場のメンタルストレスの新しい視点--ストレス社会を生き抜く)

Stress plays an important role in both mental and physical problems. Stressful life events initiate a coordinated physiological process that is produced by interactions between the hypothalamuspituitary- adrenal axis, sympathetic nervous system, and immune system. The response to psychological stress varies considerably and depends on a wide range of environmental and genetic factors. Establishment of a new biomental tool for objectively assessing stress response is required. We focus on high-throughput analysis of gene expression using microarray system to study the complex stress responses. Alternative splicing(AS)regulates the gene expression program in response to surrounding environment. However, acute psychological stress-initiated AS events have not been documented yet. Academic examinations are one of the brief naturalistic stressors and have been shown to change gene expression in peripheral leukocytes, which is postulated to be involved in the psychological response. Using the GeneChip human exon1．0ST array, AS events of 27 genes with splicing indices >1．0could be detected immediately after the examination among healthy university students. 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 p 53-dependent pathways and mRNA decay system. These results indicate that AS mediated regulation of gene expression in response to brief naturalistic stressors in peripheral leukocytes, and suggest the SMG -1 splice variant as a potential biomarker for acute 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.