Background: Postprandial hyperglycemia induces expression of inflammatory cytokines including tumor necrosis factor (TNF), which promotes the onset of type 2 diabetes and cardiovascular diseases. In this study, we investigated whether a transient high-glucose culture enhanced sustained expression of TNF, or whether the induction is associated with histone acetylation, and bromodomain protein containing protein 4 (BRD4), which binds acetylated histone, in human juvenile macrophage-like THP-1 cells. Methods: THP-1 cells were cultured in medium with high-glucose in the presence or absence of (+)-JQ1, an inhibitor of bromodomain and extra-terminal domain family, for 24 h (day 0). Thereafter, the cells were returned to a low-glucose medium without (+)-JQ1 and cultured for 2 or 4 days and samples were collected. mRNA expression of inflammation genes, and histone H3 K9/14 acetylation and binding of BRD4 and RNA polymerase II around the TNF gene were measured by RT-qPCR and chromatin immunoprecipitation, respectively. Results: TNF mRNA levels, histone H3 K9/14 acetylation, and bindings of BRD4 and RNA polymerase II to the TNF gene were higher in cells exposed to high-glucose culture for 24 h and subsequently cultured in low-glucose medium for 2-4 days, compared with cells cultured in a low-glucose medium. The addition of (+)-JQ1 to the high-glucose medium for 24 h reduced histone H3 K9/14 acetylation, and BRD4 and RNA polymerase II bindings around TNF gene, and the mRNA levels. Conclusions: Histone H3 K9/14 acetylation and BRD4 binding are associated with the sustained expression of TNF mRNA induced by temporal high-glucose exposure in juvenile macrophage-like THP-1 cells.

BACKGROUND:Interleukin (IL)-1β is considered a major factor inducing metabolic disease development, which is related to poor dietary habits, by activating inflammatory responses. The relationship between IL-1β and intakes of foods and their nutrients, particularly in the Japanese population, is unclear. Our objective was to determine whether plasma IL-1β concentrations are positively associated with poor dietary habits in middle-aged Japanese men not receiving treatment for metabolic diseases. METHODS:This cross-sectional study included 398 healthy middle-aged men. Clinical and laboratory data were collected, and the plasma IL-1β levels for all participants were assessed by enzyme-linked immunosorbent assay (ELISA). Odds ratios (ORs) were calculated using IL-1β concentrations as tertile values and food group classification, food, and nutrient intake as explanatory variables, with the first tertile values as controls. RESULTS:The OR for the highest IL-1β concentration was lower in the highest intake of vegetables food group (OR = 0.63, 95% confidence interval [CI] = 0.45-0.89) and also lower in the highest intake of protein energy ratio (OR = 0.69, 95% CI = 0.49-0.98) and total dietary fiber (OR = 0.65, 95% CI = 0.45-0.86) in the nutrient category. However, the OR for the highest IL-1β concentration was higher in the highest intake of rice/cereal ratio food group (OR = 1.43, 95% CI = 1.05-1.97). CONCLUSION:Middle-aged Japanese men not receiving treatment for metabolic diseases but with higher plasma IL-1β concentration had poor dietary styles, including higher carbohydrate intakes and lower light-colored vegetables, protein, fat, and dietary fiber intakes.

AIM:We aimed to elucidate the effect of a healthy diet containing adequate amounts of protein and vegetables on metabolic indices. METHODS:In this randomized crossover study, twenty-two healthy Japanese participants ingested two different test meals: fish diet (F) or fish diet with adequate vegetable content (FV). Each 5-day diet load test was separated by a washout period of at least seven days. Metabolic indices were measured in fasting blood and 24-h urine samples. RESULTS:The delta (Δ) plasma glucose and Δserum low-density lipoprotein (LDL) cholesterol concentrations were significantly larger in the participants in group FV than in group F (p=0.042, p=0.013, respectively). The urinary pH in participants in group F on day 6 was significantly lower than on day 1 (p=0.008), and the Δurinary pH and Δnet gastrointestinal absorption of alkali of participants in group FV tended to be smaller than in group F (p=0.070, p=0.075, respectively). CONCLUSIONS:This study showed that a healthy diet containing adequate protein and vegetables reduced the dietary acid load and improved plasma glucose and serum LDL concentrations in healthy Japanese participants.

Objective: Heat shock proteins (HSPs) are molecular chaperones which play a critical role in maintaining cellular function via regulation of protein folding. Moreover, HSPs play to protect cells from damage induced by a variety of stress including heat shock and physical stress such as inflammation and oxidative stress. We aimed to reveal the combined effect of genetic variations in the genes for HSP90/HSP70 families and lifestyle factors (i.e., physical activity, sleep duration and dietary intake) on determination of metabolic parameters. Methods: A total of 824 Japanese males were randomly selected from the participants who visited a medical center for routine medical check-ups. The genotype for each single nucleotide polymorphism (SNP) was determined for each subject by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Result: We found the associations between the genotypes of HSP90B1 (rs2070908) and serum glucose levels, as well as the genotypes of HSPA1L (rs2227956) and blood pressure levels. On the other hand, the differences in glucose levels due to genotypes of HSP90B1 were only observed in the inactive group, and the differences in blood pressure levels due to genotypes of HSPA1L were only observed in the high Na/K intake ratio group. Conclusion: Our findings indicate that the combined effect of genetic variations in the genes for HSP families and lifestyle factors including physical activity or Na/K intake ratio are important for determining of serum glucose levels or blood pressure levels.

Being in a prolonged depressed state increases the risk of developing depression. To investigate whether green tea intake is effective in improving depression-like moods, we used an experimental animal model of depression with lipopolysaccharide (LPS) and clarified the effects of green tea on the biological stress response and inflammation in the brain. Regarding the stress reduction effect of green tea, we found that the sum of caffeine (C) and epigallocatechin gallate (E) relative to the sum of theanine (T) and arginine (A), the major components of green tea, or the CE/TA ratio, is important. The results showed that depression-like behavior, adrenal hypertrophy as a typical stress response, and brain inflammation were suppressed in mice fed green tea components with CE/TA ratios of 2 to 8. In addition, the expression of Npas4, which is reduced in anxiety and depression, was maintained at the same level as controls in mice that consumed green tea with a CE/TA ratio of 4. In clinical human trials, the consumption of green tea with CE/TA ratios of 3.9 and 4.7 reduced susceptibility to subjective depression. These results suggest that the daily consumption of green tea with a CE/TA ratio of 4–5 is beneficial to improving depressed mood.

Introduction: In mammals, circadian rhythms regulate many behavioral and physiological processes. Genetic and epidemiological studies have shown that dysregulation of the circadian rhythm induces chronic metabolic diseases, such as obesity, diabetes, and dyslipidemia. We aimed to know the interactions of genetic variations of seven core circadian clock genes with lifestyle factors on the determination of metabolic parameters. Methods: We have analyzed the impacts of genotype of seven core circadian clock genes (i.e., CLOCK, BMAL1, PER1, PER2, PER3, CRY1, and CRY2) and lifestyle factors (i.e., physical activity and sleep duration) in 575 Japanese males on the determination of metabolic parameters (i.e., body mass index [BMI], serum glucose, glycated hemoglobin [HbA1c], low-density lipoprotein cholesterol [LDL-C], and high-density lipoprotein cholesterol [HDL-C] levels). Results: We have detected the associations between genotypes of PER3 and serum HbA1c level and genotypes of CRY1 and serum LDL-C level. Additionally, the interactions of the genotypes of PER1 and PER3 with physical activity for determining BMI, the genotypes of CLOCK with physical activity for determining serum HbA1c levels were observed. Furthermore, for determining serum HDL-C levels, the interactions of the genotypes of CRY2 with physical activity or sleep duration were observed. Discussion/Conclusion: Our findings indicate that the interactions of genotypes for core circadian clock genes and lifestyle factors (i.e., physical activity and sleep duration) are important for determining metabolic parameters.

Studies indicate that induction of metabolic gene expression by nutrient intake, and in response to subsequently secreted hormones, is regulated by transcription factors binding to cis-elements and associated changes of epigenetic memories (histone modifications and DNA methylation) located in promoter and enhancer regions. Carbohydrate intake-mediated induction of metabolic gene expression is regulated by histone acetylation and the histone acetylation reader bromodomain-containing protein 4 (BRD4) on the gene body region, which corresponds to the transcribed region of the gene. In this review, we introduce carbohydrate-responsive metabolic gene regulation by (i) transcription factors and epigenetic memory in promoter/enhancer regions (promoter/enhancer-based epigenetics), and (ii) histone acetylation and BRD4 in the gene body region (gene body-based epigenetics). Expression of carbohydrate-responsive metabolic genes related to nutrient digestion and absorption, fat synthesis, inflammation in the small intestine, liver and white adipose tissue, and in monocytic/macrophage-like cells are regulated by various transcription factors. The expression of these metabolic genes are also regulated by transcription elongation via histone acetylation and BRD4 in the gene body region. Additionally, the expression of genes related to fat synthesis, and the levels of acetylated histones and BRD4 in fat synthesis-related genes, are downregulated in white adipocytes under insulin resistant and/or diabetic conditions. In contrast, expression of carbohydrate-responsive metabolic genes and/or histone acetylation and BRD4 binding in the gene body region of these genes, are upregulated in the small intestine, liver, and peripheral leukocytes (innate leukocytes) under insulin resistant and/or diabetic conditions. In conclusion, histone acetylation and BRD4 binding in the gene body region as well as transcription factor binding in promoter/enhancer regions regulate the expression of carbohydrate-responsive metabolic genes in many metabolic organs. Insulin resistant and diabetic conditions induce the development of metabolic diseases, including type 2 diabetes, by reducing the expression of BRD4-targeted carbohydrate-responsive metabolic genes in white adipose tissue and by inducing the expression of BRD4-targeted carbohydrate-responsive metabolic genes in the liver, small intestine, and innate leukocytes including monocytes/macrophages and neutrophils.

Introduction: Flavonoids have a variety of functions, such as antioxidant activity, and are expected to have a disease prevention effect. In order to verify the disease risk reduction effect of flavonoids, we carried out a crossover trial in seven adult men of normal or light overweight who ingested flavonoid-rich meals, with a diverse combination of basic foodstuffs, and low-flavonoid meals and compared blood disease-related inflammatory markers.Methods: On the first two days of the study, seven male volunteers were provided with low-flavonoid meals (flavonoid content below the detection limit of HPLC: less than 0.24 mg/meal) three times a day as a washout. For the next seven days, they were fed flavonoid-rich meals (46.9 ± 8.1 mg/meal) or low-flavonoid meals. Blood samples were collected from all the volunteers before breakfast on the third day, after the washout and before breakfast on the tenth day. The test was consisted of one cycle from the first day to the tenth day, and the participants carried out two cycles. Flavonoid concentrations in plasma and gene expression of inflammatory cytokine (interleukin 1 beta, interleukin 6, interleukin 18, and tumor necrosis factor-α) in whole blood cells were compared before and after the intervention. Gene expression in whole blood cells was measured using real time RT-PCR.Results: We found a significant increase in plasma flavonoid concentration (quercetin, kaempferol, daidzein, and genistein) upon intervention with flavonoid-rich meals (p < 0.05). In addition, the inflammatory cytokine gene expression was reduced in the subjects with a body mass index of more than, but not less than, 25 kg/m2 compared with that observed after the intake of low-flavonoid meals.Conclusion: These results suggest that flavonoid-rich meals have an anti-inflammatory effect in obese persons who are likely to have chronic inflammation.Keywords: Flavonoids, inflammatory cytokines, flavonoid-rich meal, human study

Hyperglycemia activates innate leukocytes such as monocytes and induces pro-inflammatory cytokine expression, resulting in increased monocyte adhesion to aortic endothelial cells. In this study, we investigated whether high glucose and/or tumor necrosis factor (TNF) would enhance pro-inflammatory cytokine expression of tumor necrosis factor (TNF) and interleukin (IL)-1β (IL1B) by altering histone modifications in U937, a juvenile macrophage cell line. The mRNA levels of TNF and IL1B in U937 cells were significantly affected by glucose concentration and TNF treatment. Mono-methylated histone H3K4 signals around TNF and IL1B were lower in cells treated with high glucose compared with low glucose. Conversely, tri-methylated histone H3K4 and H3K36 signals were higher in cells treated with high glucose compared with low glucose. TNF treatment of U937 cells cultured in high glucose enhanced histone H3K36 tri-methylation, particularly around the gene regions of TNF and IL1B. Histone acetylation was induced by treatment with TNF in high-glucose medium. The induction of acetylation and tri-methylation of K4 and K36 of histone H3 around TNF and IL1B by treatment with high glucose and/or TNF was positively associated with the induction of these genes in juvenile macrophage U937 cells.

Resistant starch (RS) consumption has beneficial effects on health, such as reduced postprandial blood glucose levels. In this study, we evaluated the effect of a 14-day diet containing RS on α-glucosidase activity and the expression of genes related to carbohydrate digestion/absorption in rats. We examined whether the effects of RS persist when the rats were shifted to a control diet. The results suggest that RS consumption reduces α-glucosidase activity and Mgam, Si and Sglt1 mRNA levels in the proximal jejunum. In addition, RS consumption appeared to influence the serum GIP level, up to 2 days after the animals were shifted to a control diet. To our knowledge, this is the first report that RS has a sustained effect on gut hormone expression and the expression of genes related to carbohydrate digestion/absorption in the proximal jejunum.

BACKGROUND:Rapid influx of energy caused by fasting/refeeding repeatedly enhances fatty acid synthesis leading to triacylglycerol accumulation and production of reactive oxygen species (ROS), increasing the risk of non-alcoholic steatohepatitis (NASH). Previous studies have reported that the ingestion of butyrate is effective at preventing hepatic disorders, which are accompanied by fat accumulation and inflammation. The aim of this study is to reveal the mechanism of action of butyrate, and thus we investigated the effects of dietary butyrate on the expressions of antioxidant enzymes in the livers of rats during refeeding following fasting. METHODS:Thirty-seven male rats were divided into six groups (6-7 animals per group): non-fasting, fasting, refeeding with a high sucrose diet as control for 12 or 24 h, and refeeding with a high sucrose diet containing 5% sodium butyrate (NaB) for 12 or 24 h. All groups except the non-fasting group were fasted for 72 h before refeeding. Statistical analysis was conducted among 4 refeeding groups (refeeding with the control diet for 12 or 24 h, and refeeding with a diet containing NaB for 12 or 24 h). RESULTS:Supplementation with NaB significantly reduced (p < 0.05) fatty acid synthase (Fas) gene expression and increased the expression of the carnitine palmitoyltransferase 1α (Cpt1a) gene, resulting in reduced triacylglycerol content in the livers of rats refed the NaB diet compared with controls at 24 h after the start of refeeding. The mRNA levels of the genes related to glutathione synthesis were significantly higher (p < 0.05) in the livers of the butyrate group than the control group. In addition, the mRNA level of Foxo3a, a transcription factor that regulates the expressions of antioxidant enzymes, was higher in the butyrate group than controls. The acetylation levels of histone H4 around the Foxo3a gene tended to be increased (p = 0.055) by refeeding with the NaB diet. CONCLUSION:NaB supplementation in the diet for refeeding reduced the rate of lipid synthesis and stimulated fatty acid oxidation in the liver, which inhibited fat accumulation and the risk of NASH. The transcriptional regulation of Foxo3a involves histone acetylation around the gene.

Our previous study demonstrated that the transfection of a short hairpin (sh)RNA targeting bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) family of proteins, into 3T3-L1 cells, a white adipocyte-like cell line, reduced the expression of insulin sensitivity genes, such as Adipoq, Fabp4, Lpl, S1c2a4 and Dgat1, and that BRD4 directly bound to the Adipoq, S1c2a4 and LP genes. In the present study, we aimed to identify other target genes of BRD4 by microarray analysis of Brd4 shRNA- and control shRNA-transfected cells. We found that the expression of many genes related to fat metabolism, and particularly those involved in fat accumulation in the glycolytic pathway, tricarboxylic acid cycle, and triacylglycerol synthesis, such as Dgat2, Gpd1, Acsl1, Pnpla2, Pgkfb3, Pcx, Fasn, Acacb and Cidec, was reduced by Brd4 shRNA transfection 2 and 8 days after the end of adipocyte differentiation. The binding of BRD4 at the 2-day and histone acetylation at the 8-day time point, in the vicinity of the Dgat2, Gpd1, Acsl1 and Cidec genes, was also reduced by Brd4 shRNA transduction. Treatment with low doses (10-100 nM) of the BET family inhibitor (+)-JQ-1 for 2, 4 or 8 days also reduced the expression of Dgat2, Gpd1, Fasn, Acab, Acsl1, Pnpla2 and Cidec in 3T3-L1 white adipocytelike cells. These results indicate that BRD4 regulates the expression of numerous genes involved in lipid accumulation at the transcriptional level in a white adipocyte-like cell line.

Background: As flavonoids have a variety of functions, such as antioxidant activity, there is growing interest in the development of flavonoid supplements. However, there have been reports of DNA damage due to exposure to flavonoids at high concentrations in rats, which could suggest that a habitual intake of flavonoid supplements may cause toxicity. Therefore, we considered that ingesting flavonoids from a typical meal combined basic foodstuffs are safe because of unlikely to result high concentrations like supplements, and focused on the intake of flavonoids from a typical meal. Thus, this study investigated the absorption of flavonoids in humans after the consumption of a typical meal. Methods: On the first 2 days of the study, seven healthy volunteers were provided with low-flavonoid meals (flavonoid content below the detection limit by HPLC: less than 0.24 mg/meal) three times a day as a washout. A flavonoid-rich meal (40.44 ± 1.49 mg/meal) was then provided for breakfast on the third day. Blood was collected from all volunteers 0, 2, 3, 7, 8, and 9 h after the flavonoid-rich meal was consumed. After enzyme hydrolysis of the plasma, the plasma concentrations of flavonoids aglycone of quercetin, daidzein and genistein were measured using LC-MS. Urine was also collected and pooled 24 h after the flavonoid-rich meal was consumed. Thereafter, the urine was treated with enzyme hydrolysis, and the measurement of urinary flavonoids was performed. Results: Plasma flavonoid peaks were observed 8 h after consumption of the flavonoid-rich meal (quercetin: 4.29 ± 1.46 μM, daidzein: 0.51 ± 0.41 μM, genistein: 0.91 ± 0.73 μM). Furthermore, flavonoids were confirmed to be present in plasma even at 9 h after the intake meal. The urinary recovery of flavonoids was 3.43 ± 1.50% for quercetin, 13.87 ± 6.68% for daidzein, and 16.89 ± 11.40% for genistein. Conclusion: These results suggest that consuming a typical meal that combines a variety of basic foodstuffs delays attainment of the plasma flavonoid peak compared with consuming a single type of food or supplements as previously reported. In addition, the flavonoid urinary recovery were also reduced compared with those previously reported.

Lipoprotein lipase (LPL) is the rate-controlling enzyme for the accumulation of triacylglycerol into adipocytes, which acts by digesting it into glycerol and fatty acids. In this study, we found that treatment with (+)-JQ1, an inhibitor of the bromodomain and extra-terminal (BET) family proteins, for 4 days from the end of stimulation to induce adipocyte differentiation reduced binding of BRD4, a BET family member, within the gene body of Lpl. This eventually downregulated the expression of Lpl in 3T3-L1 adipocytes. Longer treatment for 8 days reduced the acetylation of histones H3 and H4 within the gene body of Lpl and subsequent Lpl expression. Lpl expression in mesenteric adipose tissues was lower in Brd4+/- heterozygous mice at 14 days after birth than in wild-type mice at the same age. Furthermore, treatment with an inducer of insulin resistance, tumor necrosis factor-α, reduced BRD4 binding and histone acetylation in the gene body of Lpl and its expression. These results indicate that transcriptional elongation of Lpl controlled by BRD4 may be associated with adipocyte differentiation, and that its suppression is potentially associated with insulin resistance of adipocytes.

Epigallocatechin-3-gallate (EGCg), a major catechin in green tea, eliminates reactive oxygen species and development of lifestyle-related diseases.However, excessive EGCg intake could induce adverse effects, particularly liver injury.We examined whether optimal dietary doses of EGCg reduces the risk of liver injuries in non-obese type 2 diabetic Goto-Kakizaki (GK) rats by examining gene expression of the proinflammatory cytokines interleukin (IL)-1β, IL-18 and tumor necrosis factor-α (TNF-α) and fibrosis-related matrix metalloproteinases (MMPs) in the liver.GK rats at 9 weeks of age were fed a control high-fat diet or a high-fat diet containing 0.1%, 0.2% or 0.5% EGCg (w/w) for 25 weeks.Expression of mRNA and proteins related to inflammation were determined by qRT-PCR and western blot analysis, respectively.IL-1β and IL-18 mRNA in the liver were reduced by EGCg supplementation at concentrations of 0.1% and 0.1%-0.2%,respectively, but not at concentrations of 0.2% and 0.5% (IL-1β) or 0.5% (IL-18) EGCg.TNF-α mRNA in the liver was reduced by supplementation with EGCg at concentrations of 0.1%-0.5%.Expression of MMP2 in the liver was reduced by EGCg supplementation at a concentration of 0.2%, but not 0.1% or 0.5%.Importantly, IL-18 protein levels in the liver and serum were reduced by 0.1% EGCg, but not by 0.5%.EGCg supplementation at concentrations from 0.1% to 0.5% did not induce increases in the expression of liver injury marker genes, while low doses (0.1%-0.2%) of EGCg in GK rats reduced expression of injury-associated genes in the liver.

Impaired glucose tolerance (IGT) induces chronic inflammation and subsequent development of complications triggered by arteriosclerosis. Moreover, undernutrition in pregnant rodents can induce IGT in their offspring. Here, we assessed whether undernutrition in pregnant rats would induce chronic inflammation in their offspring by measuring the expression levels of inflammation-related genes in peripheral blood leukocytes. Pregnant Wistar rats were divided into two groups: the control group received an American Institute of Nutrition Rodent diet (AIN-93G) ad libitum, and the undernutrition group had their diet restricted by 50% (w/w) compared with the control group from day 10 of pregnancy until birth of the offspring. Subsequently, mothers and pups were allowed to access the AIN-93G diet freely. At day 35 after birth, male pups were fasted for 4 h and subsequently orally administered with glucose solution (2 g/kg body weight). Blood glucose area under the curve (AUC) after glucose loading was significantly greater in the undernutrition group than the control group. The mRNA levels for inflammatory cytokines were increased by glucose loading especially in the undernutrition group. Expressions of genes encoding S100A9 and cell adhesion molecule CD11b were increased by glucose loading in the undernutrition group. Thus, undernutrition of pregnant rats during mid to late gestation induced the expression of inflammation-related genes in peripheral blood leukocytes of their offspring, with the development of IGT and impaired insulin secretion.