Sustained effects of resistant starch on the expression of genes related to carbohydrate digestion/absorption in the small intestine.

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.

Serum gamma-glutamyltransferase is inversely associated with dietary total and coffee-derived polyphenol intakes in apparently healthy Japanese men.

PURPOSE: Serum γ-glutamyltransferase (GGT) has been proposed as a marker of oxidative stress. Here, we examined the association between serum GGT and the dietary intake of polyphenols, which have antioxidant properties. METHODS: A cross-sectional survey including 7960 apparently healthy Japanese men (aged 22-86 years) who participated in health checkups was conducted in Shizuoka, Japan. We analyzed these subjects' clinical serum parameters and lifestyle factors, including dietary polyphenol intake, which was evaluated by a self-administered questionnaire and by matching the subjects' food consumption data with our original polyphenol content database. RESULTS: The average intake of polyphenols was 1157 ± 471 mg/day, and green tea was the largest source of polyphenols at 40%, followed by coffee at 36%. Dividing the population according to quintiles of total polyphenol intake, the difference in polyphenol intake from coffee between the groups was much greater than the difference in polyphenol intake from green tea. The analysis of the association between polyphenol intake and biological parameters showed a significant negative association between polyphenol intake and the levels of systolic and diastolic blood pressure (SBP and DBP), GGT, and alanine aminotransferase (ALT) after adjusting for age, smoking habit, energy intake and alcohol intake. The GGT levels were inversely associated with the polyphenol intake from coffee, but not with that from green tea. Multivariable linear regression analyses demonstrated that the subjects' GGT levels were negatively and independently associated with their polyphenol intake. CONCLUSIONS: The intake of total polyphenol including coffee as a major contributor is inversely associated with the serum GGT concentration in Japanese males.

Molecular Regulations of Mucosal Maltase Expressions.

Two major α-glucosidase (maltase) genes, sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM), respectively, are expressed in the small intestine. In this review, we have summarized whether jejunal expression of these maltase genes is regulated by dietary manipulations, which may affect carbohydrate availability from the luminal side, through changes in the binding of transcription factors and/or histone code on these genes. Studies using a model of mice fed either a low-starch or a high-starch diet for 7 days, found the mRNA levels of SI, MGAM, and Na-glucose cotransporter (SGLT1) genes in the jejunum to be increased in parallel by feeding a high-starch diet. Chromatin immunoprecipitation assays, using jejunal tissue of mice and rats fed a high-starch diet, revealed that the diet increased the acetylations of histones H3 and H4, bindings of coactivators, including general control of amino acid synthesis (GCN5) and the transcriptional factors, including caudal-related homeobox 2 (CDX2), and hepatocyte nuclear factor 1 (HNF1), not only in the promoter/enhancer regions, but also in the transcribed regions of SI and MGAM genes. Feeding rats a diet rich in resistant starch led to a concomitant reduction of mRNA levels of the MGAM gene and histone H3 modifications (acetylations and di-/tri-methylations) in the jejunum. These data suggest that a signal elicited by available glucose in the jejunal mucosa is associated with SI and MGAM gene expressions through a histone code, such as acetylation and di-/tri-methylations of histone H3 in the promoter/enhancer and transcribed regions of SI and MGAM genes.

Regulation of the circadian rhythmic expression of Sglt1 in the mouse small intestine through histone acetylation and the mRNA elongation factor, BRD4-P-TEFb.

Jejunal sodium/glucose co-transporter (Sglt1) displays circadian expression. The jejunum was collected every 4 h from mice, and we examined histone acetylation and binding of bromodomain-containing protein-4 (BRD4) around of the gene. Histone acetylation increased in the transcribed region of Sglt1 prior to induction of the gene. Furthermore, the binding of mRNA elongation factor around the gene showed circadian rhythm.

Fasting for 3 days during the suckling-weaning transient period in male rats induces metabolic abnormalities in the liver and is associated with impaired glucose tolerance in adulthood.

PURPOSE: Recent studies suggest that nutritional status during developmental periods is associated with subsequent development of metabolic abnormalities. In this study, we examined whether malnutrition by fasting for 3 days during the suckling-weaning transient period induces subsequent development of metabolic abnormalities in rats. METHODS: Male Sprague-Dawley rats were fasted for 3 days during the suckling-weaning transient period. They are subsequently fed a high-fat, high-sucrose (HF) or low-fat, high-starch (LF) diet for 14 weeks from 17 weeks of age, and the liver and blood samples were collected for measuring mRNA and protein levels of metabolic genes and blood concentrations of glucose and insulin, respectively. RESULTS: Fasting for 3 days during the suckling-weaning transient period induced impaired glucose tolerance in rats fed the LF diet in adulthood. Liver triglycerides in rats fed the HF diet in adulthood increased to 140 % in rats fasted for 3 days during the suckling-weaning transient period compared with those non-fasted. Furthermore, liver expression of FBP1 and ACCα genes in adult rats fed the LF diet increased to 125 and 145 %, respectively, in rats fasted for 3 days during the suckling-weaning transient period compared to non-fasted rats. PEPCK1 protein expression levels in rats fed the LF diet were higher in rats fasted for 3 days during the suckling-weaning transient period than in non-fasted rats. CONCLUSION: Fasting for 3 days in rats during the suckling-weaning transient period enhances metabolic abnormalities in animals fed a HF or LF diet in adulthood by confounding metabolism of lipid and sugar in the liver.

Histone code of genes induced by co-treatment with a glucocorticoid hormone agonist and a p44/42 MAPK inhibitor in human small intestinal Caco-2 cells.

BACKGROUND: Inactivation of glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) is thought to be important in small intestinal maturation and expression of genes related to intestinal differentiation and functions. METHODS: We investigated target genes induced by co-treatment for 48h with a glucocorticoid hormone agonist, dexamethasone (Dex), and a p44/42 MAPK inhibitor, PD98059 (PD), in a small intestine-like cell line (Caco-2) using microarray analysis. We also investigated whether expression changes of the target genes induced by the co-treatment are associated with histone modifications around these genes. RESULTS: Co-treatment of Caco-2 cells with Dex and PD enhanced several genes related to intestinal differentiation and functions such as SCNN1A, FXYD3, LCT and LOX. Induction of the SCNN1A gene was associated with increased presence of acetylated histone H3 and H4 and di-methylated histone H3 at lysine (K) 4 around the transcribed region of the gene, and induction of the FXYD3 gene was associated with increased presence of acetylated histones H3 and H4 from the promoter/enhancer to the transcribed region of the gene. Induction of LCT and LOX genes was associated with increased presence of acetylated histone H4 on the promoter/enhancer region of the genes. CONCLUSIONS: Histone acetylation and/or histone H3 K4 methylation around the promoter/enhancer or/and transcribed regions of target genes are associated with induction of the genes by co-treatment with Dex and PD in Caco-2 cells. GENERAL SIGNIFICANCE: The histone code is specific to each gene with respect to induction by glucocorticoid hormone and inhibition of p44/42 MAPK in Caco-2 cells.

Polymorphism in microRNA-binding site in HNF1B influences the susceptibility of type 2 diabetes mellitus: a population based case-control study.

BACKGROUND: Recent genome-wide association studies (GWAS) have identified many SNPs associated with type 2 diabetes mellitus (T2DM). However, the functional roles for most of the SNPs have not been elucidated. MicroRNAs (miRNAs) are key regulators of gene expression involved in the development and progression of various diseases including T2DM. In this study, we investigated whether commonly occurring SNPs modulate miRNA-directed regulation of gene expression, and whether such SNPs in miRNA-binding sites are associated with the susceptibility for T2DM. METHODS: Genotypes of eleven 3' untranslated region (UTR) SNPs of seven susceptibility genes for T2DM were determined in 353 T2DM patients and 448 control subjects. In addition, the interactions of miRNAs with the 3'UTR in the hepatocyte nuclear factor 1ß (HNF1B) gene were investigated using luciferase reporter assays. RESULTS: One 3'UTR SNP (rs2229295) in the HNF1B gene was significantly associated with T2DM, and the frequency of an A allele (rs2229295) in T2DM patients was decreased compared with that in controls. Luciferase reporter assays showed that the SNP (rs2229295) altered the binding of two miRNAs (hsa-miR-214-5p and hsa-miR-550a-5p). CONCLUSIONS: We have detected the interactions of hsa-miR-214-5p/hsa-miR-550a-5p and the 3'UTR SNP of the HNF1B gene by in vitro luciferase reporter assays, and propose that the binding of such miRNAs regulates the expression of the HNF1B gene and the susceptibility of T2DM.

Self-reported faster eating is positively associated with accumulation of visceral fat in middle-aged apparently healthy Japanese men.

PURPOSE: Faster eating is positively associated with body mass index in apparently healthy Japanese populations. In the present study, we examined the associations between self-reported rate of eating and visceral and subcutaneous fat areas in apparently healthy middle-aged Japanese men. METHODS: We conducted a cross-sectional study of men who participated in health checkups in Japan. We removed participants who were diagnosed with metabolic diseases by the time of their health checkups. A total of 320 subjects aged 30-64 years (mean ± standard deviation, 47.4 ± 8.6 years) were selected. We compared the associations between rate of eating and various clinical parameters including visceral and subcutaneous fat areas, using analysis of covariance (ANCOVA), which was adjusted by age and lifestyle factors such as alcohol intake, energy intake, smoking, and physical activity. Multivariate logistic regression analyses (MLRA) were performed with visceral fat area (cm(2)) as the dependent variable and independent variables that included self-reported rate of eating. RESULTS: Tukey's multiple test following ANCOVA showed that self-reported rate of eating was positively associated with visceral fat area (cm(2)), but not with subcutaneous fat area (cm(2)). MLRA showed that the odds ratio of rate of eating for visceral fat area in tertile (T) 3 (>100 cm(2)) compared with T1 (≤70 cm(2)) was 1.99 (95% CI 1.40-2.90, P < 0.01), and the association remained after adjustment for the subcutaneous fat area. CONCLUSIONS: The present results show that self-reported faster eating is positively associated with visceral fat accumulation, independently of subcutaneous fat accumulation, in apparently healthy Japanese men.

Re-feeding rats a high-sucrose diet after 3 days of starvation enhances histone H3 acetylation in transcribed region and expression of jejunal GLUT5 gene.

Fasting for 3 days leads to reduction in the expression of GLUT5 and SGLT1 genes in jejunum. Re-feeding a high-sucrose diet in fasted rats enhanced mRNA levels and histone H3 acetylation on transcribed region of GLUT5 gene within 24 h, but not in SGLT1. Responsiveness of jejunal GLUT5 gene is associated with changes in histone H3 acetylation on transcribed region.

Practical application of flavonoid-poor menu meals to the study of the bioavailability of bilberry anthocyanins in human subjects.

Practical application of flavonoid-poor menus was evaluated on the bioavailability of anthocyanins as model flavonoids. Detectable amounts of flavonoids were not found in plasma and urine collected from 13 participants, who took the menus. After ingesting bilberry anthocyanins (919 µmol), average plasma AUC0-6h, Cmax, Tmax values and urinary recovery were 386.0 nmol h/mL, 139.1 nM, 1.31 h and 0.21%, respectively.

Principal component 1 score calculated from metabolic syndrome diagnostic parameters is a possible marker for the development of metabolic syndrome in middle-aged Japanese men without treatment for metabolic diseases.

PURPOSE: The risk of metabolic syndrome (MetS) is assessed based on the presence of risk factors that include dyslipidemia, hyperglycemia, hypertension and obesity. In this study, we assessed the risk of MetS using principle component (PC) analysis of MetS diagnostic parameters and examined whether the resulting eigenvalues are associated with the circulating concentrations of inflammatory cytokines [interleukin (IL)-1ß and IL-6] and a marker for insulin sensitivity (adiponectin) in middle-aged Japanese men without treatment for metabolic diseases. MATERIALS: We conducted a cross-sectional study of 308 Japanese men without treatment for metabolic diseases aged 40-69 years who participated in health checkups in Japan. We calculated the PC1 score from the following MetS diagnostic parameters: body mass index (BMI), fasting blood glucose, diastolic blood pressure, triacylglycerol and high-density lipoprotein cholesterol. We compared the relationship between PC1 scores and other clinical parameters, including IL-1ß, IL-6 and adiponectin, by Spearman's rank correlation coefficient analyses and Jonckheere-Terpstra test. RESULTS: The associations for most clinical parameters were higher with the PC1 score than with other MetS diagnostic parameters. Homeostasis model assessment-insulin resistance, an index of insulin resistance, showed stronger associations with PC1 score than with MetS diagnostic parameters. Significant associations for IL-1ß, IL-6 and adiponectin were observed with the PC1 score, BMI and triacylglycerol; these associations were higher with the PC1 score than with BMI and triacylglycerol. CONCLUSIONS: The present results show that the PC1 score is closely associated with parameters of MetS, inflammation and insulin resistance.

Self-reported rate of eating is associated with higher circulating ALT activity in middle-aged apparently healthy Japanese men.

PURPOSE: Elevated circulating activities of alanine aminotransferase (ALT), a marker for liver injury, and the lifestyle of a higher rate of eating in healthy and preclinical subjects are associated with increased risk of obesity and diabetes. In this study, we examined the associations between self-reported rate of eating and circulating ALT activity in middle-aged apparently healthy Japanese men. METHODS: We conducted a cross-sectional study of 3,929 apparently healthy men aged 40-59 years (mean ± SD age, 49.2 ± 5.8 years; BMI, 23.5 ± 2.8 kg/m²) who participated in health checkups in Japan. We analyzed their clinical serum parameters and lifestyle factors, including self-reported rate of eating. Associations between self-reported rate of eating and liver injury markers [ALT, γ-glutamyl transpeptidase (GTP), and aspartate aminotransferase (AST)], other clinical parameters or lifestyle factors were determined using analysis of variance followed by Tukey's test. Multivariate logistic regression analyses (MLRA) were performed with ALT activity as the dependent variable and independent variables that included self-reported rate of eating. RESULTS: MLRA showed that ALT activity showed trends for higher self-reported rate of eating after adjustment for age, energy intake, and smoking status. The association between ALT activity and self-reported rate of eating disappeared after adjustment for BMI. CONCLUSION: The results of this study show that ALT activity is positively associated with self-reported rate of eating in middle-aged apparently healthy Japanese men.

Induction by fructose force-feeding of histone H3 and H4 acetylation at their lysine residues around the Slc2a5 gene and its expression in mice.

It has been reported that fructose force-feeding rapidly induced jejunal Slc2a5 gene expression in rodents. We demonstrate in this study that acetylation at lysine (K) 9 of histone H3 and acetylation at K5 and K16 of histone H4 were more enhanced in the promoter/enhancer to transcribed regions of the Slc2a5 gene in fructose force-fed mice than in glucose force-fed mice. However, fructose force-feeding did not induce acetylation at K14 of histone H3, or at K8 and K12 of histone H4 around the Slc2a5 gene. These results suggest that fructose force-feeding induced selective histone acetylation, particularly of H3 and H4, around the jejunal Slc2a5 gene in mice.

Inhibition of postprandial hyperglycemia by either an insulin-dependent or -independent drug reduces the expression of genes related to inflammation in peripheral leukocytes of OLETF rats.

Treatment with the dipeptidyl peptidase-4 inhibitor, anagliptin, or with the α-glucosidase inhibitor, miglitol, reduced the oral sucrose load-inducible expression of interleukin (IL)-1ß, IL-18, tumor necrosis factor-α, S100a8, S100a9, S100a11, IL-1R2, IL-1Rn and tumor necrosis factor receptor 2 genes in peripheral leukocytes of Otsuka Long-Evans Tokushima fatty (OLETF) rats at the stage of impaired glucose tolerance. Inhibiting postprandial hyperglycemia reduced the expression of genes related to inflammation in peripheral leukocytes of OLETF rats.

The combined effects of genetic variation in the SIRT1 gene and dietary intake of n-3 and n-6 polyunsaturated fatty acids on serum LDL-C and HDL-C levels: a population based study.

BACKGROUND: Dyslipidemia due to high total cholesterol, LDL-cholesterol, triglycerides, or low HDL-cholesterol is an important risk factor for coronary heart disease (CHD). Both SIRT1 and PUFAs can influence the expression of genes for nuclear receptors and transcription factors related to lipid metabolism such as LXRα, LXRß, PPARα, SREBP-1c. METHODS: A total of 707 Japanese males and 723 females were randomly selected from the participants who visited a medical center for routine medical check-ups. We analyzed the combined effects of the genotype/haplotype of the SIRT1 gene and dietary n-6/n-3 PUFA intake ratio on the determination of serum lipid levels. RESULTS: We found that the SIRT1 gene marked with haplotype 2 was associated with decreased serum LDL-cholesterol and increased HDL-cholesterol levels. In addition, the associations between the SIRT1 haplotype 2 and decreased LDL-C and increased HDL-C levels were only observed in the low n-6/n-3 PUFA intake ratio group, but not in the high n-6/n-3 PUFA intake ratio group. CONCLUSIONS: Our findings indicate that the combination of genetic variation in the SIRT1 gene and dietary n-6 and/or n-3 PUFA intake influence the determination of inter-individual variations of serum levels of LDL-C and HDL-C.

RNA polymerase II phosphorylation at serine 2 and histone H3 tri-methylation at lysine 36 are key steps for thyroid hormone receptor ß gene activation by thyroid hormone in Rana catesbeiana tadpole liver.

Thyroid hormone (TH) is essential for amphibian metamorphosis, during which the expression of many genes is controlled directly or indirectly through TH receptors (TRs). Thyroid hormone binding to TRs induces coregulator switching on regulatory regions of TH-inducible genes: corepressors complexed with unliganded TRs are replaced by coactivators complexed with liganded TR resulting in transcriptionally active states. The coregulator switching is linked to histone acetylation. In our study, we have investigated the acetylation and methylation states of histones H3 and H4 using chromatin immunoprecipitation (ChIP) assays on the 5' coding region of the TRß gene, a primary TH-response gene, in the liver from Rana catesbeiana tadpoles either treated with or not treated with 3,3',5-triiodothyronine (T3). 3,3',5-Triiodothyronine treatment for 3 days increased the amount of TRß transcript by 19-fold. This increase was associated with increases in the acetylation of histone H4 and lysine 9 in histone H3 (H3-K9), and tri-methylation of lysine 36 in histone H3 (H3-K36). In addition, the amounts of RNA polymerase II (PolII) and serine 2 phosphorylation in PolII (PolII-S2) increased. These results suggest that T3 treatment enhances the elongation activity of PolII on the TRß gene in the liver by increasing histone H3-K36 tri-methylation through PolII-S2 phosphorylation.

Gene expression profile in the liver of Rana catesbeiana tadpoles exposed to low temperature in the presence of thyroid hormone.

Amphibian metamorphosis, which is controlled by thyroid hormone (TH), is highly temperature-sensitive. Using real-time PCR, we investigated the gene expression profile in the liver of Rana catesbeiana tadpoles kept at 28 and 4 °C and treated with 5 nM 3,3',5-triiodothyronine (T3). Out of the 48 genes tested, 12 were up-regulated at 4 °C in T3-treated or untreated tadpoles. These included genes involved in energy metabolism, transcription, and translation. Four TH-response genes, including TH receptor ß (TRß) gene, showed no response to T3 at 4 °C. Deiodinase III was the only gene down-regulated at 4 °C. Chromatin immunoprecipitation assays showed that CCAAT/enhancer-binding protein 2 gene activation by cold exposure was associated with an increase in the acetylation of histone H3 at lysine 9, whereas TRß gene activation by T3 at 28 °C was associated with an increase in the methylation of histone H3 at lysine 36 with no T3-dependent changes in methylation states on cold exposure. Our results suggest that the transfer of TH signal to chromatin modifications on a primary early TH response gene was specifically blocked by exposure to cold.

Trimethylation of histone H3K4 is associated with the induction of fructose-inducible genes in rat jejunum.

We previously reported that fructose force-feeding rapidly induces jejunal Slc2a5 gene expression in rats. In this study, we conducted microarray analyses using total RNA to identify genes upregulated in rat jejunum by fructose force-feeding. Rats were force-fed fructose, glucose or distilled water for 6h. Genes such as Slc2a5, Cdkn1c, Cabp2, Ranbp3, Vwce and Gcgr were induced by force-feeding with fructose compared with glucose or distilled water. Chromatin immunoprecipitation assays revealed that trimethylation of histone H3K4, and acetylation of histones H3 and H4, on the transcribed region of these fructose-inducible genes were enhanced by force-feeding of fructose, but not glucose or distilled water. These results suggest that the induction of genes in the rat jejunum by fructose force-feeding is coordinately regulated by histone modifications, particularly trimethylation of histone H3K4.

The combined effect of the T2DM susceptibility genes is an important risk factor for T2DM in non-obese Japanese: a population based case-control study.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a complex endocrine and metabolic disorder. Recently, several genome-wide association studies (GWAS) have identified many novel susceptibility loci for T2DM, and indicated that there are common genetic causes contributing to the susceptibility to T2DM in multiple populations worldwide. In addition, clinical and epidemiological studies have indicated that obesity is a major risk factor for T2DM. However, the prevalence of obesity varies among the various ethnic groups. We aimed to determine the combined effects of these susceptibility loci and obesity/overweight for development of T2DM in the Japanese. METHODS: Single nucleotide polymorphisms (SNPs) in or near 17 susceptibility loci for T2DM, identified through GWAS in Caucasian and Asian populations, were genotyped in 333 cases with T2DM and 417 control subjects. RESULTS: We confirmed that the cumulative number of risk alleles based on 17 susceptibility loci for T2DM was an important risk factor in the development of T2DM in Japanese population (P<0.0001), although the effect of each risk allele was relatively small. In addition, the significant association between an increased number of risk alleles and an increased risk of T2DM was observed in the non-obese group (P<0.0001 for trend), but not in the obese/overweight group (P=0.88 for trend). CONCLUSIONS: Our findings indicate that there is an etiological heterogeneity of T2DM between obese/overweight and non-obese subjects.

Analysis of N(ε) -ethyllysine in human plasma proteins by gas chromatography-negative ion chemical ionization/mass spectrometry as a biomarker for exposure to acetaldehyde and alcohol.

BACKGROUND: N(ε) -ethyllysine (NEL) is a major stable adduct formed by the reaction of acetaldehyde (AA) with lysine residues in proteins. However, its occurrence and levels in biological specimens and its relationship with AA/alcohol exposure-associated disorders have not been fully elucidated. In this study, we have developed a sensitive and specific method to quantitate NEL levels in human plasma proteins. METHODS: The method consists of (1) purification of the protein fraction of interest by Sephadex G-15 to remove low molecular substances, (2) hydrolysis of proteins with Pronase E in the presence of stable isotope-labeled internal standards, (3) derivatization of amino acids with pentafluorobenzyl (PFB) bromide, and (4) quantification of the PFB derivatives of NEL and l-lysine using gas chromatography-negative ion chemical ionization/mass spectrometry in a selected ion monitoring mode. RESULTS: Using the above method, the NEL levels in human plasma proteins obtained from 10 each of control subjects and alcoholic patients were measured. NEL was detected in all samples analyzed, the average level of NEL in the plasma proteins of alcoholic patients (1.17 ± 0.36 NEL/1,000 l-lysine) being significantly higher than that of control subjects (0.26 ± 0.07 NEL/1,000 l-lysine). CONCLUSIONS: The method could be applied to molecular epidemiological studies to investigate possible associations between the NEL levels in human tissue proteins and human diseases associated with exposure to AA and alcohol.