1. Introduction
A diet that includes barley has several beneficial effects, such as lowering the postprandial glucose rise [
1,
2,
3] and improved blood cholesterol concentration [
4,
5,
6]. Barley is rich in β-glucan, which comprises linear homopolysaccharides of β-(1→4) and β-(1→3) linkages and high molecular weight viscous polysaccharides [
6]. Barley β-glucan is a soluble dietary fiber that is fermented in human and animal colon to produce short-chain fatty acids (SCFAs) [
7,
8,
9]. Thus, the fermentability of β-glucans modifies the balance and diversity of the microbiota. These prebiotic actions may be the main mechanism for the beneficial effects of barley intake. However, the mechanism for the observed improvement of glucose tolerance following barley intake remains controversial.
In a previous report, we showed that high β-glucan barley (HGB) reduced postprandial glucose rise in healthy subjects [
10,
11], serum cholesterol levels in hypercholesterolemic subjects [
12], and abdominal fat area in moderately obese subjects [
13], and regulated the calorie intake and satiety index of healthy subjects [
14]. It is speculated these beneficial effects were caused by either the viscosity of β-glucan or prebiotic effects such as SCFA production.
With regard to the improvement of glucose tolerance, barley intake is reported to reduce postprandial glucose rise and the second meal effect [
15]. Moreover, it is suggested that the second meal effect is the result of colonic fermentation [
16]. The second meal effect [
17,
18] refers to the ability of foods to reduce postprandial glucose rise not only after the first meal but also after the next meal of the day. It is reported that boiled barley kernels as an evening meal decreased incremental blood glucose area at the following breakfast and increased plasma glucagon-like peptide-1 (GLP-1) at fasting and during the day by comparison with the consumption of white wheat bread [
15].
G protein-coupled receptors, which sense SCFAs, are thought to contribute to the regulation of glucose metabolism [
19]. Indeed, rats fed a high fiber diet had a higher plasma GLP-1 level after oral glucose administration than those fed a low fiber diet. GLP-1 is secreted from L cells, which are predominantly located in the ileum and colon [
20,
21]. A previous study involving rats reported that fermentable dietary fibers increase the contents of the distal digestive tract, augment GLP-1 secretion, and boost proglucagon mRNA expression [
22,
23,
24].
Our previous report showed that barley intake increased the biosynthesis of short-chain fatty acids (SCFAs) by the gut microbiota, but we did not measure gut hormone levels [
25]. Given that high β-glucan barley stimulates SCFA production, we hypothesized that a high β-glucan barley diet might result in greater GLP-1 secretion by comparison to a cellulose based diet. A recent study showed that the secretion of GLP-1 increased in mice fed high β-glucan barley and improved insulin sensitivity through modification to the gut microbiota and elevated levels of SCFAs [
26]. However, another report showed that barley β-glucan promoted fermentation in cecum but did not alter glucose tolerance or insulin secretion [
27].
The main aim of this study was to examine the mechanism for improvement of glucose tolerance by increases in GLP-1 secretion in mice given a diet rich in β-glucan barley. We quantified the number of L cells, mRNA expression related to L cell functions in the ileum, and GLP-1 levels in the portal vein and cecum contents of mice fed a fatty diet with and without supplementation of high β-glucan barley. An improvement of glucose tolerance mediated by GLP-1 secretion in mice fed a diet of high β-glucan barley might provide useful information for a therapeutic approach to diabetes and obesity.
2. Materials and Methods
2.1. Chemical Composition of High β-Glucan Barley
These experiments were performed using a new two-rowed, waxy, hull-less high β-glucan barley cultivar Kirari-mochi. HGB flour pearled to 60% yield was obtained from NARO (Tsukuba, Japan). Total dietary fiber was quantified using a previously published protocol [
28]. The β-glucan content was determined by the McCleary procedure [
29]. Protein and lipid content in HGB were analyzed by the Kjeldahl and acid hydrolysis method, respectively. The overall nutritional make-up of HGB is summarized in
Table 1.
2.2. Experimental Design
Male C57BL/6J mice at 5 weeks of age were obtained from Charles River Laboratories Japan, Inc. (Yokohama, Japan). The mice were kept in the same holding room under a 12 h light/12 h dark regime with continual air exchange at a temperature of 22 °C (±1 °C) and humidity of 50% (± 5%). The mice were given commercial chow (NMF; Oriental Yeast Co., Ltd., Shiga, Japan) for 1 week before being divided into 2 groups matched for body mass (
n = 8 per group). Each mouse was individually held in a plastic cage and fed the experimental diet (
Table 2). The modified fatty AIN-93G diet (fat energy ratio 50%) was prepared by adding lard. Cellulose and HGB flour were included in the control and HGB diets, respectively, to give 5% total dietary fiber (
Table 2). Mice were allowed ad libitum access to water and food throughout the study (83 days). Animal experiments were performed twice; the first experiment analyzed cecal GLP-1 pool size and the second experiment (
n = 8 for the new control and experimental groups) evaluated cecal short-chain fatty acid (SCFA) pool size. Both food intake and body weight for each mouse were recorded 3 times per week. Body weight measurements were always performed at the same time of the day. On the final week, an oral glucose tolerance test (OGTT) was carried out. Mice were fasted for 6 h and then glucose (1.5 g/kg body) was orally administered. Blood glucose concentrations were analyzed from the tail using a blood glucose meter (Sanwa Kagaku Kenkyusho Co., Ltd., Aichi, Japan) at 5 timepoints (0, 15, 30, 60, and 120 min). At the end of the study, the mice were sacrificed by isoflurane/CO
2 anesthesia after fasting for 6 h. Blood was withdrawn from portal vein (the first experiment only) and the heart and, following centrifugation, the resultant serum was stored at −80 °C. Liver, cecum, and adipose tissues were weighed. Samples of ileum were quickly saturated in RNAprotect Tissue Reagent (Qiagen, Hilden, Germany) to facilitate stabilization of RNA. The samples of cecum were stored at −40 °C until further analysis. All animal experiments were conducted with the approval of the Animal Research Committee of Otsuma Women’s University (Tokyo, Japan) (no. 12001, 16006).
2.3. Biochemical Analyses of the Serum
Serum cholesterol (TC), triglyceride (TG), and non-esterified fatty acids (NEFAs) were determined using a Hitachi 7180 automatic analyzer (Hitachi Ltd., Tokyo, Japan). Enzyme-linked immunosorbent assays (ELISAs) were performed to quantify serum insulin and leptin levels. GLP-1 levels in plasma derived from the portal vein were determined. Dipeptidyl peptidase IV inhibitor (Millipore, Billerica, MA, USA) was immediately added to the blood to prevent degradation of GLP-1. Quantification of GLP-1 was performed using a commercial ELISA kit (GLP-1 Active; Shibayagi Corp., Gunma, Japan).
2.4. Analysis of Short-Chain Fatty Acids in Cecal Digesta
Cecal SCFA content was measured using a previously described method [
30]. A 7890B GC system (Agilent, Tokyo, Japan) equipped with a 5977A MSD (Agilent) was used for analysis of SCFAs. A DB-5MS capillary column (30 m × 0.53 mm) (Agilent) was used to separate the SCFAs. SCFA concentrations (expressed as μmol/cecum) were calculated by comparing their peak areas with an internal standard (crotonic acid).
2.5. Measurement of Total GLP-1 Level in the Cecum
Cecal tissues were extracted using Kenny’s method [
31]. In brief, frozen tissue samples were extracted using an acidic ethanol solution (5 mL/g wet weight tissue). The tissue samples were subsequently homogenized and then incubated for 24 h prior to clarification by centrifuging. All extraction procedures were performed at 4 °C. The supernatant was decanted, and total GLP-1 level was measured using a total GLP-1 ELISA kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
2.6. mRNA Expression Analysis in the Ileum
Oligonucleotide primer sequences are shown in
Table S1. mRNA levels were quantified by real-time PCR (QuantStudio3 RT-PCR system; Applied Biosystems, Foster City, CA, USA). Complementary DNA was used to quantify the mRNA expression levels according to the 2
−ΔΔCT method with a commercial PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Data were assessed from threshold cycle (Ct) values, which indicate the cycle number at which the fluorescence signal reaches a fixed threshold that is well above background. ΔCT is the difference in Ct values for genes of interest compared to transcription factor II B (TFII B), which served as a reference. The ΔΔCT is the difference between the ΔCT for the control group and the ΔCT for the HGB group. Relative expression levels are shown as fold differences compared to the control group (arbitrary units).
2.7. Measurement of the Number of L Cells in the Ileum
After collecting the ileum, samples were immersed in 4% paraformaldehyde/phosphate buffer and fixed, then dehydrated, degreased, paraffin-penetrated, paraffin-embedded, sliced, spread, and dried at the Institute of Nutrition and Pathology, Inc. (Kyoto, Japan). Deparaffinization and rehydration were performed with xylene, anhydrous ethanol, 99% ethanol, and 70% ethanol. Endogenous peroxidase inhibition was achieved with 30% (v/v) H2O2 and methanol. Then, each ileal section was blocked using diluted 5% (v/v) goat serum (Normal) (DakoCytomation, Glostrup, Denmark) and 0.5% (w/v) BSA/PBS (bovine serum albumin/phosphate buffered saline). Anti-GLP-1 (7–36) -NH2; rabbit Ab (Yanaihara Institute Inc., Shizuoka, Japan) was used as the primary antibody. For the secondary antibody, Histofine Simple Stain Mouse MAX-PO (Rabbit) (Nichirei Bioscience Inc., Tokyo, Japan) with 1% (v/v) mouse serum (Normal) (DakoCytomation, Glostrup, Denmark) was used. After the antigen–antibody reaction, samples were colored with the ENVISION kit/HRP (horseradish peroxidase) (DAB; 3,3’-Diaminobenzidine) (DakoCytomation, Glostrup, Denmark). In addition, nuclear staining was performed with hematoxylin and the samples were enclosed with Mount Quick (Daido Sangyo Corporation, Saitama, Japan). Each ileal section was photographed under a light microscope. From images of the ileum, we measured the number of cells per unit area (1 mm2) using WinROOF (Version 6.0.1; Mitani Corporation, Fukui, Japan).
2.8. Statistical Analyses
Data are given as the mean ± standard error of the mean (SE). Comparisons of data between two groups were performed by unpaired Student’s t-test (JMP Version 14.2.0; SAS Institute Inc., Cary, NC, USA). Time-dependent changes in the blood glucose levels during the OGTT were analyzed by two-way ANOVA (diet × time). A p-value of <0.05 was considered statistically significant.
4. Discussion
Herein, we investigated the mechanism for improvement of glucose tolerance in mice fed a high-fat diet containing HGB. Results from this study indicated that HGB intake improved glucose tolerance in OGTT. GLP-1 levels in the portal vein and cecal pool size were significantly increased in the HGB group. These findings suggest that the effect of HGB on glucose metabolism was mediated by increased GLP-1 secretion. Previous studies in human [
15] and animal experiments [
26] showed that the serum GLP-1 concentrations were increased by the barley intake. However, effects of HGB intake on the L cell function related to the GLP-1 secretion were not observed in the previous experiments. This is the first evidence to show an increase in the number of ileal L cells causing an increase in intestinal GLP-1 pool size and GLP-1 secretion without the alteration of mRNA expression related to GLP-1 secretion.
These results, together with those from our previous report [
25], suggest that augmentation in the number of L cells and the subsequent elevation in the level of GLP-1 were induced by production of SCFAs. Thus, a new mechanism for increasing GLP-1 secretion by HGB intake is proposed. It has been generally recognized that the preventive effect of barley intake on postprandial blood glucose rise is triggered by delayed gastric emptying time due to the viscous properties of barley β-glucan and the suppression of digestion and absorption of starch in barley [
32,
33]. However, our previous report showed that improvement of glucose tolerance became more pronounced when HGB with partially hydrolyzed β-glucan was supplemented into the test diet [
34]. It is possible that a prebiotic effect, such as intestinal fermentation, contributed more to the improved glucose tolerance than the physicochemical effect, such as increased viscosity. Furthermore, HGB imparts a second meal effect, which is induced through intestinal fermentation [
15]. In a human intervention study, it was reported that the postprandial blood glucose rise was suppressed when a standard diet without barley was consumed at lunch after the intake of a HGB diet at breakfast [
35,
36].
Our experiments showed no difference in serum insulin concentration between the two groups, but the active GLP-1 level in the portal vein of the HGB group was significantly elevated by comparison to the control group. The total GLP-1 pool size of the cecal contents in the HGB group was also significantly elevated compared to the control group. GLP-1 is produced by prohormone convertase 1/3 from the precursor proglucagon in L cells. However, changes in the expression levels of peroxisome proliferator-activated receptor β/δ (PPARβ/δ), proglucagon, and prohormone convertase 1/3 related to GLP-1 secretion in the ileum were not observed. The expression levels of NeuroD, which is thought to reflect the number of L cells, increased significantly in the HGB group. These results indicated that the improvement of glucose tolerance following HGB intake is fostered by an increase in GLP-1 secretion accompanying the increased number of L cells.
Several studies propose secretion of GLP-1 and gastric inhibitory peptide (GIP) may be coupled with changes to the microflora of the lower gut as well as glucose metabolism [
37,
38,
39,
40]. In 1996, the first published report appeared, suggesting fermentation in the lower digestive tract promoted an increase of GLP-1 secretion [
22]. It was also reported that a prebiotic oligofructose led to an increase in total cecal GLP-1 concentration [
41]. Similar effects were observed with resistant starch, which increased the concentration of plasma GLP-1 [
42,
43]. Our study indicated prebiotic barley β-glucan is one of the dietary fibers that promotes GLP-1 secretion.
Previous studies report that inert dietary fibers did not enhance colonic proliferation of epithelial cells, although fermentable soluble fibers were able to promote proliferation in the lower gut, which is associated with increased enteroglucagon secretion [
44,
45]. Enteroglucagon, which regulates intestinal epithelial cell proliferation and acts as a trophic factor for the intestinal mucosa, is synthesized in the L cells of the ileum and colon. It is speculated that increases in the number of L cells is mediated by the trophic action of enteroglucagon. Luminal infusion of acetate and butyrate was reported to significantly increase colonic GLP-1 secretion, whereas propionate had no such effect [
46]. Moreover, SCFAs may increase the release of GLP-1 via the SCFA receptor GPR43, which is expressed on the L cells [
47].
A major limitation of this study was the lack of data regarding serum insulin and GLP-1 concentrations during the OGTT. Future studies are needed to further elucidate the relationship between GLP-1 release and insulin response by HGB intake.