Methylation of histone H3 at lysine 4 and expression of the maltase-glucoamylase gene are reduced by dietary resistant starch

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Journal of Nutritional Biochemistry 24 (2013) 606 – 612

Methylation of histone H3 at lysine 4 and expression of the maltase-glucoamylase gene are reduced by dietary resistant starch Masaya Shimada a, b , Kazuki Mochizuki b, Toshinao Goda b,⁎ b

a Department of Nutrition, Faculty of Health Sciences, Chiba Prefectural University of Health Sciences, Japan Laboratory of Nutritional Physiology, The University of Shizuoka, Graduate School of Nutritional and Environmental Sciences and Global COE, Japan

Received 9 April 2010; received in revised form 15 October 2010; accepted 1 March 2012

Abstract Methylated histone H3 at lysine 4 (K4) is associated with euchromatin and is involved in the transactivation of genes. However, it is unknown whether histone methylation is involved with changes in gene expression induced by nutrients. In this study, we examined whether methylations of histone H3 at K4 on maltase-glucoamylase (Mgam), which is responsible for the digestion of starch in the small intestine, as well as Mgam expression were altered by feeding rats an indigestible starch (resistant starch, RS). The mRNA and protein levels and the activities of MGAM were reduced in rats fed an RS diet compared with those fed a regular starch diet. Furthermore, we found that decreases in di- and tri-methylation of histone H3 at K4, as well as reduced acetylation of histones H3 and H4 on the Mgam gene were associated with a reduction of Mgam gene expression. These results suggest that the reductions of jejunal MGAM levels and activities caused by the RS diet are regulated at the mRNA level through a decrease in methylation of histone H3 at K4 and reduced acetylation of histones H3 and H4 on the Mgam gene. © 2013 Elsevier Inc. All rights reserved. Keywords: Resistant starch; Methylated histone H3 at K4; Chromatin; Maltase-glucoamylase; Small intestine

1. Introduction α-Limit dextrin and maltotriose are pancreatic α-amylase digestion products of dietary starches that are further digested to glucose by disaccharidases, such as the maltase-glucoamylase complex and the sucrase-isomaltase complex, which are products of single genes, maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si), respectively. This digestion occurs in the brush border membrane of the small intestine. In particular, maltase and glucoamylase contribute greatly to malto-oligosaccharide digestion, and it is known that maltase prefers shorter malto-oligosaccharide substrates and glucoamylase prefers longer ones [1–4]. Recently, we demonstrated that levels of Mgam mRNA and MGAM activity in the mouse jejunum are enhanced by feeding a high starch/low fat-diet for 7 days [5]. In addition, our earlier study has shown that feeding rats a diet containing high amylose/low amylopectin-starch, which is less digestible in the small intestine than low amylose/high amylopectin-starch (regular starch), for 14 days leads to a decrease in the activities of maltase and other disaccharidases (α-glucosidases), such as sucrase and isomaltase, in the upper jejunum [6]. Further⁎ Corresponding author. Laboratory of Nutritional Physiology, School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Surugaku, Shizuoka-shi, Shizuoka 422–8526, Japan. Tel.: + 81 54 264 5533; fax: + 81 54 264 5565. E-mail address: [email protected] (T. Goda). 0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2012.03.006

more, feeding normal and streptozotocin-induced diabetic rats a diet containing an α-glucosidase inhibitor, acarbose, for 12 days leads to decreases in maltase activity, and reduced protein levels and activities of sucrase and isomaltase in the jejunum [7]. These results indicate that expression of Mgam and other α-glucosidases, and their protein and activity levels in the jejunum are associated with the inflow of available carbohydrate, namely glucose. Recent gene targeting studies have demonstrated that deficiency of the Mgam gene reduces jejunal maltase activity in both heterozygous and homozygous mice, and represses postprandial hyperinsulinemia in homozygous mice [4,8]. Furthermore, treatment with α-glucosidase inhibitors, such as acarbose, voglibose and miglitol, reduces postprandial hyperglycemia in diabetic patients [9,10]. These results suggest that changes in the expression of Mgam and in the activity of MGAM in the jejunum are involved in the elevation of postprandial hyperglycemia. Thus, it is important to understand the regulation of Mgam gene expression in the small intestine, to prevent metabolic diseases related to postprandial hyperglycemia, such as diabetes and obesity. The Si gene is regulated by transcription factors such as caudal type homeobox 2 (CDX2) and hepatic nuclear factor 1 (HNF1), which bind to cis-regulatory elements of Si [11,12]; therefore, Mgam is also likely to be regulated by transcription factors. However, our recent studies using rodents have demonstrated that induction of jejunal Mgam gene expression by intake of a high starch/low fat-diet is associated with an increase in the acetylation of histones H3 and H4 [5], which are histone modifications known to be important for

M. Shimada et al. / Journal of Nutritional Biochemistry 24 (2013) 606–612

transactivation [13–15]. These changes in acetylation are greater than any changes in CDX2 or HNF1 binding on Mgam [5]. Furthermore, reduced upper jejunal expression of solute carrier family 5, member 1 (Slc5a1), also termed sodium glucose cotransporter 1 (SGLT1), which participates in the transport of glucose from the lumen to enterocytes, by intake of a diet rich in an indigestible starch, resistant starch (RS), which is abundant in amylose and has the characteristics of dietary fiber, is associated with a decrease in histone H3 acetylation rather than binding of HNF1 [16]. Acetylation of histones leads to the recruitment of mRNA transcriptional complexes to the promoter/enhancer regions of genes and to the recruitment of mRNA elongational complexes to the transcribed regions close to the transcription initiation site. This recruitment occurs through the binding of bromodomain-containing proteins to acetylated histones [17–19]. Indeed, one of the bromodomain-containing proteins, CREB binding protein (CREBBP), which functions in the acetylation of histones and in the recruitment of transcriptional complexes, binds to the Mgam gene [5]. Thus, these results suggest that the induction of genes related to carbohydrate digestion/absorption in the small intestine through dietary carbohydrates is associated with alterations in histone modification and in recruitment of mRNA transcriptional/elongational complexes. Recent studies have shown that methylation of histone H3 at lysine 4 (K4) is more important for transcriptional initiation than histone acetylation because methylation is induced prior to acetylation and recruits many proteins related to transactivation [20,21]. In addition, methylation of histone H3 at K4 occurs through the binding of proteins containing plant homeodomains (PHD), chromodomains, Tudor domains or WD40 repeat domains to the methylated histones [22,23]. Therefore, it is likely that methylation of histone H3 at K4 is involved in the regulation of the expression of genes related to carbohydrate digestion/absorption in the jejunum in response to dietary carbohydrate intake. In this study, we hypothesized that the jejunal expression of Mgam is regulated by the methylation of histone H3 at K4, and we examined whether decreases in glucose inflow, by feeding a diet rich in RS, alter Mgam gene expression, methylation of histone H3 at K4, and acetylation of histones H3 and H4 at the Mgam locus. 2. Materials and methods 2.1. Animals and diets Seven week-old male Wistar rats (Japan SLC, Shizuoka, Japan) were divided into two groups, control and RS. The control group received a diet rich in α-cornstarch (Oriental Yeast, Tokyo, Japan), which contains approximately 25% amylose. The RS group received the same diet as the control group except that the α-cornstarch was replaced with the same amount of RS. As RS, we used Hi-maize 1043 (Nippon NSC, Tokyo, Japan) which is a heat-moisture treated high amylose (approximately 70%) cornstarch (HACS) and is classified as RS type 2 (RS2). The starch made by this procedure contains approximately 64.5% RS [24,25]. Details of the diet compositions are shown in Table 1. Animals were allowed free access to the diets and water for 7 days. At the end of the 7 day feeding period, a 6 h-fasting blood sample was obtained from the tail vein to determine plasma glucose and triacylglycerol concentrations. Rats were then killed by decapitation between 10:00 am and 11:00 am. The experimental procedures used in the present study conformed to the guidelines of the Animal Usage Committee of the University of Shizuoka.

607

Table 1 Diet compositions g/100g

Cornstarch Hi-maize ⁎ Casein Lard Corn oil Cellulose AIN93 mineral mixture AIN93 vitamin mixture L-cystine Choline bitartrate

Control

RS

55.0 20.0 10.0 5.0 5.0 3.5 1.0 0.3 0.2

55.0 20.0 10.0 5.0 5.0 3.5 1.0 0.3 0.2

⁎ Hi-maize contains approximately 60% resistant starch.

2.3. Real-time reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted by the acidified guanidine thiocyanate method, as described by Chomczynski and Sacchi [26] and stored at -80°C before use. The total RNA samples (2.5 μg) were converted into cDNA by RT using SuperScript III reverse transcriptase (Invitrogen, Tokyo, Japan) according to the manufacturer's instructions. To quantitatively estimate the mRNA levels of Mgam and of the general transcription factor IIB (Gtf2b), PCR amplification was performed on a LightCycler 480 instrument system (Roche, Tokyo, Japan). Real-time RT-PCR reactions were carried out in a total volume of 10 μl containing 400 nM of each gene specific primer, cDNA and SYBR Green I (Roche). The cycle threshold (CT ) values of Mgam and Gtf2b were converted to signal intensities by the delta-delta method [27], which calculates the signal intensities to be twice the difference between the CT of the test gene (Mgam) and that of a gene for normalization (Gtf2b). The formula is [2-(CT Mgam-CT Gtf2b)]. The sequences of the PCR primer pairs and the fragment sizes are shown in Table 2. 2.4. Western blot analysis Jejunal segments for protein extraction were homogenized in RIPA buffer [1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl and protease inhibitor tablets (Complete Mini; Roche)] and centrifuged at 20,000g for 30 min at 4°C. Supernatants were collected and the total protein concentrations were normalized using the Lowry method [28] before being stored at -80°C. The tissue extracts were separated by 8% SDS–PAGE and transferred to Immobilon paper (Millipore, Tokyo, Japan) at 80 V for 120 min in Tris/glycine/methanol transfer buffer. The membranes were blocked for 1 h in 10% skimmed milk in phosphate-buffered saline (PBS) with 0.05% Tween 20 and 0.5 M NaCl, pH 7.4 (PBS-Tween-NaCl) at room temperature. They were then incubated in 10% skimmed milk in PBS-Tween-NaCl with primary antibody at 4°C for N12 h, using MGAM (Santa Cruz Biotechnology, CA, USA) and α-tubulin (TUBA) (Cell Signaling, MA, USA) primary antibodies. After washing in PBS-Tween-NaCl, the membranes were incubated with secondary antibodies, anti-rabbit/mouse IgG conjugated to biotin (GE Healthcare, Tokyo, Japan), in 3% skimmed milk in PBSTween-NaCl. After washing in PBS-Tween-NaCl and incubating with horseradish peroxidase-conjugated anti-biotin as a third antibody (Cell Signaling), signals were detected by chemiluminescence (ECL Plus; GE Healthcare), according to the manufacturer's instructions. 2.5. Enzyme assays Jejunal homogenates in 10 mM potassium-phosphate buffer (pH 7.0) were incubated with 28 mM maltose for 15 min (maltase activity) and with 1% soluble starch for 30 min (glucoamylase activity), as described by Dahlqvist [29]. The reactions were terminated by adding Tris-HCl (pH 8.0) and concentrations of glucose produced by maltase or glucoamylase in the jejunal homogenates were determined using the Glucose CII test (Wako Pure Chemical Industries, Osaka, Japan), as described previously [5]. Protein was measured according to the method of Lowry et al. [28].

2.2. Preparation of intestinal samples

2.6. Chromatin immunoprecipitation (ChIP) assay

The jejunoileum, which is the entire small intestine except for the duodenum attached to the pancreas, was collected and the proximal one-third of the jejunoileum, which is defined as the jejunum, was used in this study. The jejunum was flushed twice with ice-cold 0.9% NaCl solution. Three-centimeter segments (300 mg each) were excised from the middle region of the jejunal loop, divided into three equal length segments (100 mg each), and immediately stored at -80°C. These samples were used for real-time reverse transcription polymerase chain reaction (RT-PCR), western blotting and enzyme activity assays. The remaining part of the jejunal loop was used for chromatin immunoprecipitation (ChIP) assays.

The mucosa was removed from the jejunum and then incubated in fixation solution (1% formaldehyde, 4.5 mM Hepes pH 8.0, 9 mM NaCl, 0.09 mM EDTA, 0.04 mM ethylene glycol tetraacetic acid) in PBS for 30 min at 37°C. The reaction was terminated by the addition of glycine to a final concentration of 150 mM. After being washed in FACS solution (1 x PBS , 2% bovine serum, 0.05% NaN3), the samples were sonicated in SDS lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS, 0.5 mM phenylmethanesulfonyl fluoride) containing one protease inhibitor tablet (Complete Mini; Roche)/10ml SDS lysis buffer. Sonication produced DNA fragments of 200-500 bp. ChIP assays, using 1 μg anti-acetyl histone H3 at K9/14 antibody

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Table 2 Sequences of the oligonucleotide primers used in this study Fragment size

Sequence

Mgam mRNA

65

Gtf2b mRNA

139

5’-GAACAGCAATGCCATGGAG-3’ 5’-CCCCAGTTGTGCGGTAAGTA-3’ 5’-TACAGGAGCGGCAAGTTTTGA-3’ 5’-ATTGCGTGGGAGGTTGATTCT-3’

Region on the Mgam gene ⁎ Mgam -4,000 Mgam -1,800 Mgam -500 Mgam 100 Mgam 1,200 Mgam 5,400 Mgam 49,900 Mgam over 11,500

-4,065 ~ -4,044 -3,967 ~ -3,947 -1,866 ~ -1,846 -1,738 ~ -1,718 -500 ~ -480 -433 ~ -412 101 ~ 121 161 ~ 181 1,152 ~ 1,172 1,225 ~ 1,244 5,304 ~ 5,325 5,381 ~ 5,401 49,869 ~ 49,890 49,988 ~ 50,008 101,801 ~ 101,822 101,911 ~ 101,931

119 149 89 81 94 98 140 131

5’-GTGGCTTCATAGAGGAATTTGG-3’ 5’-CAGATGGGTTCAGTGGAGAAT-3’ 5’-CAGCTTTGGAAGAAGAGCTTG-3’ 5’-CGTCCTGTGTGGAAGATGAAT-3’ 5’-AGGCTGTGGACTTGTAAGCAA-3’ 5’-CCTGACTTAACAGAGGTGACCA-3’ 5’-GTGCCAGAGCTGAACCATATT-3’ 5’-TTCAGAACCAACCATCAGCTC-3’ 5’-GCTGCACAATCATGAAGACCT-3’ 5’-ATCAGGGATGTGGCTACAGAT-3’ 5’-AAGTCATTGAGGAGTCCCTGTT-3’ 5’-ATGGCAGAGTCAGGAGTTTCA-3’ 5’-ATGGTCACTCTAAGGCTGTGCT-3’ 5’-ACTCAGGAGAATGGCACAGTT-3’ 5’-ACACATGTATGGACGGTAGCAG-3’ 5’-AATCAGTGGCCAGCAAGTCTA-3’

⁎ The region is denoted relative to the transcription initiation site.

(Millipore, Tokyo, Japan), anti-acetyl histone H4 at K5/8/12/16 antibody (Millipore), anti-mono-methyl-histone H3 at K4 antibody (Millipore), anti-di-methyl-histone H3 at K4 antibody (Millipore), anti-tri-methyl-histone H3 at K4 antibody (Millipore) or normal rabbit IgG were performed as described previously [30]. The precipitated DNA was subjected to real-time PCR using primers that corresponded with indicated sites in the promoter/enhancer and transcribed regions. The CT values of the ChIP signals detected by real-time PCR were converted to the percentage of the input DNA as ChIP signal, which was calculated by the delta-delta method [27], using the formula 100×[2-(CT IP sample-CT input)]. The sequences of the primers used in ChIP assays are indicated in Table 2. 2.7. Other assays Body weight, food intake and fecal weight were measured once every 2 or 3 days. Plasma glucose and triacylglycerol levels were enzymatically determined with commercial kits (Glucose CII-Test Wako and Triglyceride E-Test Wako; Wako Pure Chemical Industries, Osaka, Japan). 2.8. Statistical analysis Each value is expressed as the mean±SEM. Significance between two groups was evaluated by Student's t-test. Pb 0.05 was considered to indicate statistical significance.

3. Results 3.1. Effect of dietary RS on body weight, tissue weight, food intake, fecal weight and plasma parameters of rats No differences in body weight or food intake were seen between control and RS groups, while weights of liver and mesentery adipose tissue were significantly lower (Pb.05) in rats fed the RS diet compared with control rats. Fecal weight was significantly higher (Pb.05) in rats fed the RS diet compared with control rats. No diarrhea was observed in either group. Plasma glucose level did not differ between the two groups, while the plasma triacylglycerol level was significantly lower (Pb.05) in animals fed the RS diet compared with control animals (Table 3). 3.2. The effects of an RS diet on jejunal Mgam gene expression and protein levels, and on maltase and glucoamylase activities Jejunal expression of the Mgam gene (Fig. 1A) and levels of MGAM protein (Fig. 1B) were 35% and 58% lower, respectively, in rats fed an RS diet compared with rats fed a control diet (Pb.01). Jejunal maltase (Fig. 1C) and glucoamylase (Fig. 1D) activities were also 43% and 37%

lower, respectively, in rats fed the RS diet compared with rats fed the control diet (Pb.01). 3.3. The effect of an RS diet on the acetylation of histones H3 and H4 in the promoter/enhancer and transcribed regions of the Mgam gene To investigate whether acetylation of histones H3 and H4 on the Mgam gene is associated with a reduction of Mgam gene expression caused by an RS diet, we performed ChIP assays using two antibodies, one for acetylated histone H3 at K9/14 and the other for acetylated histone H4 at K5/8/12/16 on rats fed a control diet or an RS diet for 7 days. The ChIP signals of normal rabbit IgG were b0.014% per input. The ChIP signals for acetylated histone H3 at K9/14 were significantly lower in both promoter/enhancer (-1,800 and -500 bp, Pb.01) and transcribed (100 and 1,200 bp, Pb.01; 5,400 bp, Pb.05) regions of the Mgam gene in rats fed the RS diet compared with rats fed the control diet (Fig. 2B). The ChIP signals for acetylated histone H4 at K5/8/12/16 were also significantly lower in both promoter/enhancer (-1,800 bp, Pb.01) and transcribed (100 and 1,200 bp, Pb.05) regions of the gene in rats fed the RS diet compared with rats fed the control diet (Fig. 2C). 3.4. The effects of an RS diet on the methylation of histone H3 at lysine 4 in the promoter/enhancer and transcribed regions of the Mgam gene To investigate whether methylation of histone H3 at K4 on the Mgam gene is associated with a reduction of Mgam gene expression caused by an RS diet, we performed ChIP assays using three antibodies for mono-, di- and tri-methylated histone H3 at K4. The ChIP signals for mono-methylated histone H3 at K4 were not significantly different in both promoter/enhancer and transcribed regions of the Mgam gene between the two groups (Fig. 3B). The ChIP signals for di-methylated histone H3 at K4 were significantly lower in both promoter/enhancer (-1,800 and -500 bp, Pb.01) and transcribed (100 and 1,200 bp, Pb.01) regions of the gene in the rats fed the RS diet compared with rats fed the control diet (Fig. 3C). The ChIP signals for tri-methylated histone H3 at K4 were also significantly lower in both promoter/enhancer (-1,800 and -500 bp, Pb.01) and transcribed (100 bp, Pb.05) regions of the gene in the rats fed the RS diet compared with rats fed the control diet (Fig. 3D).

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Table 3 Effect of dietary RS on body weight, tissue weight, food intake, fecal weight and plasma parameters of rats

Initial body weight (g) Final body weight (g) Liver weight (g) Mesentery adipose weight (g) Food intake (g/d) Fecal weight (g/d) Plasma glucose (mg/100mL) Plasma triacylglycerol (mg/100mL)

Control

RS

158±3 192±2 7.13±0.25 2.06±0.03 16.1±0.5 1.60±0.04 115±2 34.2±3.4

160±4 186±3 6.03±0.30 ⁎ 1.18±0.27 ⁎

by feeding rats a diet containing RS, which is an indigestible starch, compared with a diet containing the same amount of regular starch, and that the reduction of the mRNA level was dosedependent [0% RS (control diet), 27.5% RS and 55% RS (RS diet), P = 0.0027 by Jonckheere-Terpstra's Test, data not shown]. Indeed, the increased RS intake was accompanied by increased amounts of undigested starch, as estimated from the weight of feces (Table 3). Furthermore, we showed that acetylation of histone H3 at K9/14 and acetylation of histone H4 at K5/8/12/16 on the Mgam gene were decreased by the RS diet in broad regions ranging from the promoter/enhancer to the transcribed regions (from -1,800 to 5,400 bp) close to the transcription initiation site (Fig. 2B-C). This is the first report showing that the amount of carbohydrate digestion in the small intestine affects jejunal Mgam gene expression and histone acetylation. It has been demonstrated that histone acetylation in the transcribed regions leads to recruitment of mRNA elongation complexes, whereas histone acetylation in the promoter/enhancer regions is associated with enhanced recruitment of mRNA transcriptional complexes [17–19]. Therefore, in rats fed the RS diet, it is likely that the reduction of histones H3 and H4 acetylation in the promoter/enhancer and transcribed regions leads to the repression of mRNA transcription initiation and mRNA elongation. However, this hypothesis requires further investigation. We also found that feeding rats the RS diet strongly (Pb.01) reduced di-methylation of histone H3 at K4 from -1,800 to 1,200 bp

14.1±0.8 4.29±0.55 ⁎ 122±2 20.3±1.3 ⁎

Values are expressed as means±SEM for five animals. ⁎ Pb.05; Significantly different from control (student's t-test).

4. Discussion Our recent studies have demonstrated that feeding mice a high starch/low fat-diet induces expression of Mgam and Si genes and also increases the acetylation of histones H3 and H4 in both the promoter/enhancer and transcribed regions of the genes [5,30]. It is still unclear whether the amount of carbohydrate in the jejunum alters the expression of Mgam and Si genes and the acetylation of histones on these genes, because our studies have shown that a high ratio of starch to fat in the diet induces expression of Mgam and Si genes and the acetylation of histones on these genes. In this study, we found that the jejunal Mgam mRNA level was reduced

A

609

B

Control

RS 335KDa 285KDa

MGAM

1.0

**

Relative protein level

0.5

TUBA

**

0.5

0.0

0.0

C

µmol/h/mg protein

50KDa

1.0

D 20

8

15

6

**

10

5

0

Control

RS

µmol/h/mg protein

Relative mRNA level

210KDa

** 4

2

0

Control

RS

Fig. 1. Mgam mRNA and MGAM protein levels and maltase and glucoamylase activities in the jejunum of rats fed a control diet or an RS diet for 7 days. (A) Mgam mRNA level. (B) MGAM protein level. (C) Maltase activity. (D) Glucoamylase activity. Means±SEM of five animals are shown. **Pb.01, RS diet vs. control diet (Student's t-test).

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A

-4,000

-1,800

-500

100

5,400

1,200

49,900

over 11,500 bp

Mgam transcribed region

B Acethylated histone H3 K9/14

Relative to input (%)

0.80

Control RS

0.60

0.40

** 0.20

**

**

** *

0.00

C Acethylated histone H4 K5/8/12/16

Relative to input (%)

0.25 0.20 0.15

*

0.10

*

** 0.05 0.00

-4,000

-1,800

-500

100

1,200

5,400

49,900

over 11,500

bp Fig. 2. Acetylation of histones H3 and H4 on the Mgam gene in the jejunum of rats fed a control diet or an RS diet for 7 days. (A) Primer pairs used for PCR in ChIP assays. (B) Acetylated histone H3 at K9/14. (C) Acetylated histone H4 at K5/8/12/16. Means±SEM of five animals are shown. *Pb.05, **Pb.01, RS diet vs. control diet (Student's t-test).

and tri-methylation of histone H3 at K4 from -1,800 to -500 bp; regions that are close to the transcription initiation site of the Mgam gene (Fig. 3C-D). The reduction of di-/tri-methylation of histone H3 at K4 on Mgam was closely associated with the reduction of histone acetylation, as well as reduced expression. Recent studies have demonstrated that methylation of histone H3 at K4 is more important for the initial activation of transcription than acetylation of histones because di-/tri-methylation of histone H3 at K4 induces acetylation of histones [20,21]. Thus, it is very likely that decreased histone acetylation is caused by the decreased methylation of histone H3 at K4. Further studies are needed to examine whether the binding of histone acetyltransferase to proteins on methylated histone H3 at K4 of the Mgam gene is reduced by feeding rats an RS diet. It remains unknown which signals alter methylation of histone H3 at K4 and acetylation of histones H3 and H4 on the Mgam gene, and Mgam gene expression along the jejunum-ileum in rats fed the RS diet. It seems likely that the amount of glucose available for absorption that flows into each jejunoileal segment would be involved in eliciting the signal. It has been reported that a diet containing an inhibitor of disaccharidases, acrabose, repressed activities of disaccharidases, including maltase and glucoamylase, and reduced glucose uptake in the proximal small intestine [7,31]. Thus, a diet containing RS possibly leads to decreased levels of available carbohydrate in the jejunum. We have already shown that histone H3 acetylation on the Mgam gene, as well as Mgam

expression, are enhanced in the jejunum of mice fed a high regular cornstarch/fat diet [5]. Taken together, it is likely that the glucose available for uptake by the absorptive cells in the jejunum would be responsible for the induction of methylation of histone H3 at K4 and acetylation of histones H3 and H4 on the Mgam gene, as well as for induced Mgam expression. The RS group produced feces of increased weight (Table 3). Our previous study also demonstrated that feeding rats an RS diet increases the cecal content [32]. Phillips et al. reported that intake of RS increased fecal weight and the amount of starch excreted in the feces [33]. Thus, undigested RS in the feces can be increased by feeding rats an RS diet. In addition, previous studies have suggested that undigested RS is used as energy by bacterial flora; bacteria produce short-chain fatty acids from RS, and the hosts use the short-chain fatty acids as energy [34]. These characteristics of RS lead to a decreased energy value of the diet. In addition, the present and previous studies by us have demonstrated that increased RS intake reduces the activities and protein levels of α-glucosidases, including maltase and glucoamylase in the jejunum [32]. These reductions could subsequently decrease postprandial hyperglycemia, hyperinsulinemia and hyperlipidemia. Thus, the reduced weights of liver and adipose tissues, and the decreased level of plasma triacylglycerol in the RS-fed rats could be caused by these combined effects. In conclusion, we have demonstrated for the first time that the reduction of Mgam gene expression by an RS diet in rats is associated

M. Shimada et al. / Journal of Nutritional Biochemistry 24 (2013) 606–612

A

-4,000

-1,800

-500

100

1,200

5,400

49,900

611

over 11,500 bp

Mgam transcribed region

B Mono-methylated histone H3K4 Relative to input (%)

0.30

Control RS 0.20

0.10

0.00

C Di-methylated histone H3K4 Relative to input (%)

10

5

**

** ** **

0

D Tri-methylated histone H3K4 Relative to input (%)

0.15

0.10

0.05

0.00

-4,000

**

**

-1,800

-500

*

100

1,200

5,400

49,900

over 11,500

bp Fig. 3. Methylation of histone H3 at K4 on the Mgam gene in the jejunum of rats fed a control diet or an RS diet for 7 days. (A) Primer pairs used PCR in ChIP assays. (B) Monomethylated histone H3 at K4. (C) Di-methylated histone H3 at K4. (D) Tri-methylated histone H3 at K4. Means±SEM of five animals are shown. *Pb.05, **Pb.01, RS diet vs. control diet (Student's t-test).

with a decrease in di-/tri-methylation of histone H3 at K4, as well as decreased acetylation of histones H3 and H4. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (20590233), a grant -in-Aid for Young Scientists (22680054) from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Global COE Program, the Center of Excellence for Evolutionary Human Health Sciences, the

Ministry of Education, Culture, Sports, Science and Technology of Japan and The Naito Foundation.

References [1] Gray GM. Starch digestion and absorption in nonruminants. J Nutr 1992;122(1): 172–7. [2] Nichols BL, Avery S, Sen P, Swallow DM, Hahn D, Sterchi E. The maltaseglucoamylase gene: common ancestry to sucrase-isomaltase with complementary starch digestion activities. Proc Natl Acad Sci USA 2003;100(3): 1432–7.

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