Barley β-glucan improves metabolic condition via short-chain fatty acids produced by gut microbial fermentation in high fat diet fed mice

<div><p>Dietary intake of barley β-glucan (BG) is known to affect energy metabolism. However, its underlying mechanism remains poorly understood because studies have presented inconsistent results, with both positive and negative effects reported in terms of satiety, energy intake, weight loss, and glycemic control. The objective of this study was to clarify the physiological role underlying the metabolic benefits of barley BG using a mouse model of high fat diet (HFD)-induced obesity. Male 4-wk-old C57BL/6J mice were fed an HFD with 20% barley flour containing either high BG (HBG; 2% BG) or low BG (LBG; 0.6% BG) levels under conventional and germ-free (GF) conditions for 12 wks. In addition, mice were fed either an HFD with 5% cellulose (HFC; high fiber cellulose) or 5% barley BG (HFB; high fiber β-glucan) for 12 wks. Then, metabolic parameters, gut microbial compositions, and the production of fecal short-chain fatty acids (SCFAs) were analyzed. The weight gain and fat mass of HBG-fed mice were lower than those of control mice at 16-wk-old. Moreover, the secretion of the gut hormones PYY and GLP-1 increased in HBG-fed mice, thereby reducing food intake and improving insulin sensitivity by changing the gut microbiota and increasing SCFAs (especially, butyrate) under conventional condition. These effects in HBG-fed mice were abolished under GF conditions. Moreover, the HFB diets also increased PYY and GLP-1 secretion, and decreased food intake compared with that in HFC-fed mice. These results suggest that the beneficial metabolic effects of barley BG are primary due to the suppression of appetite and improvement of insulin sensitivity, which are induced by gut hormone secretion promoted via gut microbiota-produced SCFAs.</p></div

<i>Bifidobacterium longum</i> Alleviates Dextran Sulfate Sodium-Induced Colitis by Suppressing IL-17A Response: Involvement of Intestinal Epithelial Costimulatory Molecules

<div><p>Although some bacterial strains show potential to prevent colitis, their mechanisms are not fully understood. Here, we investigated the anti-colitic mechanisms of <i>Bifidobacterium longum</i> subsp. <i>infantis</i> JCM 1222^T, focusing on the relationship between interleukin (IL)-17A secreting CD4⁺ T cells and intestinal epithelial costimulatory molecules in mice. Oral administration of JCM 1222^T to mice alleviated dextran sulfate sodium (DSS)-induced acute colitis. The expression of type 1 helper T (Th1)- and IL-17 producing helper T (Th17)-specific cytokines and transcriptional factors was suppressed by JCM 1222^T treatment. Intestinal epithelial cells (IECs) from colitic mice induced IL-17A production from CD4⁺ T cells in a cell-cell contact-dependent manner, and this was suppressed by oral treatment with JCM 1222^T. Using blocking antibodies for costimulatory molecules, we revealed that epithelial costimulatory molecules including CD80 and CD40, which were highly expressed in IECs from colitic mice, were involved in IEC-induced IL-17A response. Treatment of mice and intestinal epithelial cell line Colon-26 cells with JCM 1222^T decreased the expression of CD80 and CD40. Collectively, these data indicate that JCM 1222^T negatively regulate epithelial costimulatory molecules, and this effect might be attributed, at least in part, to suppression of IL-17A in DSS-induced colitis.</p> </div

IECs from DSS mice induce IL-17A response via CD80/CD86 and CD40 dependent costimulation.

<p>(a) IECs from DSS mice were co-cultured with CD4⁺ T cells from control mice in the presence of anti-CD3 antibody. In the indicated groups, IECs were pretreated with CD80 or CD86 blocking antibodies, and CD4⁺ T cells were pretreated with CD40L blocking antibody before co-culturing. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). ^**<i>p</i><0.01 versus CD4⁺ T cells alone (CD4⁺T); ^##<i>p</i><0.01 versus co-culture of CD4⁺ T cells and IECs from DSS-treated mice (DSS-IEC). (b) IECs were cultured in the absence (gray bars) and presence (black bars) of CD40 agonist antibody for 24 h. IL-6 concentration in the supernatant was measured by ELISA. The data are representative of three experiments. (c) CD4⁺ T cells were cultured for 3 days in fresh medium or supernatant from IECs (IECs sup) cultured as described in (b) in the presence of anti-CD3/CD28 antibodies. The IL-17A concentration in the supernatant was measured by ELISA. The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). ^**<i>p</i><0.01 versus the supernatant from IECs stimulated with CD40 agonist antibody (CD40 agonist Ab). (d) CD4⁺ T cells were cultured with fresh medium or supernatant from CD40-stumulated IEC of DSS mice (DSS-IEC sup) as described in (c) in the presence of IL-6-neutralizing antibody (anti-IL-6) or IgG control antibody (isotype). The IL-17A concentration in the supernatant was measured by ELISA. Results are expressed as means ± standard error (n = 6). ^**<i>p</i><0.01 versus fresh medium; ^#<i>p</i><0.05 versus the supernatant from CD40-stumulated IEC of DSS mice (DSS-IEC sup).</p

Oral treatment of <i>B. longum</i> JCM 1222^T (B.l) alleviates DSS-induced acute colitis.

<p>Mice were monitored daily for weight loss (a) and DAI (b). On day 5, the entire colon was removed (c), and the length was measured (d). The data are representative of four experiments. Colonic tissue sections were stained with hematoxylin-eosin for histological examination (e). The data are representative of two experiments. Scale bars represent 100 μm. The data are Results are expressed as means ± standard error (n = 4). ^**<i>p</i><0.01 versus control mice (Control); ^#<i>p</i><0.05 and ^##<i>p</i><0.01 versus DSS-treated mice (DSS). </p

Oral treatment of <i>B. longum</i> JCM 1222^T (B.l) suppresses the expression of costimulatory molecules in IEC.

<p>(a) mRNA expression of costimulatory molecules in IECs was analyzed by quantitative PCR. Levels of mRNA were normalized to β-actin mRNA, and expressed relative to control mice. The data are representative of three experiments. Results are expressed as means ± standard error (n = 6 (Control) or 7 (DSS, and DSS+<i>B</i>.<i>l</i>)). (b) IECs were stained for cytokeratin and CD80 or CD40 and analyzed by flow cytometry. Debris was gated out by forward and side scatter. Representative plots (upper panel) and the mean and standard error values of the percentage of cytokeratin/CD80 or CD40 positive cells (lower panel) are shown (n = 4). The data are representative of two experiments. (c) Cryosections of colonic tissue were labeled for CD40 (green) and E-cadherin (red). The data are representative of two experiments. Scale bars represent 50 μm. ^**<i>p</i><0.01 versus control mice (Control); ^#<i>p</i><0.05 and ^##<i>p</i><0.01 versus DSS-treated mice (DSS). </p

HBG barley flour suppresses HFD-induced obesity.

<p>Body weight changes (A), fat mass (B), blood glucose (C), and plasma triglyceride (D) were measured in male mice fed Co, HBG, or LBG diets for 12 weeks. Values are means ± S.E.M. n = 8–13. *, <i>P</i> < 0.05, compared with Co (Tukey-Kramer test). Co, control; Epi, epididymal; HBG, β-glucan rich barley; LBG, general barley; Peri, perirenal; Sub, subcutaneous; WAT, white adipose tissue.</p

Metabolic benefits of HBG barley flour are abolished under GF conditions.

<p>Fecal short chain fatty acids in germ-free and conventional mice were measured by GC/MS (A). Body weight changes (B), fat mass (C), plasma PYY (D), and GLP-1 levels (E) were measured in male germ-free mice fed a Co or HBG diets for 12 weeks. Daily food intakes were measured at 8 to 10 weeks of age (F). Values are means ± S.E.M. n = 5. **, <i>P</i> < 0.01, compared with Co (Tukey-Kramer test for A) (student’s t-test for B-F). Co, Control; Conv-Co, Conventional mice fed a Co diet; Epi, epididymal; GF-Co, Germ-Free mice fed a Co; GF-HBG, Germ-Free mice fed a HBG; GLP-1, glucagon-like peptide 1; HBG, β-glucan rich barley; Peri, perirenal; PYY, peptide YY; Sub, subcutaneous; WAT, white adipose tissue.</p

HBG barley flour produces SCFAs and changes the gut microbial composition.

<p>Fecal short chain fatty acids were measured in male mice fed Co, HBG, or LBG diets for 12 weeks (A). <i>Firmicutes</i> / <i>Bacteroidetes</i> ratio (B), and <i>Actinobacteria</i> (C) in feces were measured by quantitative real-time PCR for Co, HBG, or LBG diets for 2 weeks. Values are means ± S.E.M. n = 4–8. *, <i>P</i> < 0.05, **, <i>P</i> < 0.01, and ***, <i>P</i> < 0.001, compared with Co. Co (Tukey-Kramer test), control; HBG, β-glucan rich barley; LBG, general barley.</p

Oral treatment of <i>B. longum</i> JCM 1222^T (<i>B.l</i>) suppresses Th17-specific cytokines and transcription factors.

<p>(a) Cytokine production from colonic tissue culture was measured by ELISA. (b) mRNA expression of transcription factors in LPL was analyzed by quantitative PCR. Levels of mRNA were normalized to β-actin mRNA, and expressed relative to control mice. (c) LPL was analyzed for cytokine-expression profiles by intracellular cytokine staining. The frequency of CD4⁺ T cells expressing the indicated cytokines is shown (n = 3-6). The data are representative of two experiments. Results are expressed as means ± standard error (n = 4). ^*<i>p</i><0.05 and ^**<i>p</i><0.01 versus control mice (Control); ^#<i>p</i><0.05 versus DSS-treated mice (DSS). </p

<i>B. longum</i> JCM 1222^T (B.l) suppresses the expression of costimulatory molecules in Colon-26 cells.

<p>(a) mRNA expression of CD80 and CD40 was analyzed by quantitative real-time PCR in Colon-26 cells after stimulation with IFN-γ. Levels of mRNA were normalized to β-actin mRNA, and expressed relative to before stimulation (0 h). Results are expressed as means ± standard error (n = 4). ^*<i>p</i><0.05 and ^**<i>p</i><0.01 versus before stimulation (0 h). (b) Colon-26 cells were pre-incubated with <i>B. longum</i> JCM 1222^T, and then treated with penicillin and streptomycin and incubated with IFN-γ. Messenger RNA expression of CD80 and CD40 was analyzed after stimulation with IFN-γ for 6 and 72 h, respectively. The data are representative of three experiments. (c) Colon-26 cells were incubated with <i>B. longum</i> JCM 1222^T and IFN-γ. Cell surface protein expression of CD80 and CD40 was analyzed by flow cytometry after stimulation with IFN-γ. Debris was gated out by forward and side scatter. Histograms show a representative experiment (specific antibody stains are shown in filled histograms, and isotype control antibody stains are shown in open histograms) and bar figures are representative of two experiments. ^**<i>p</i><0.01 versus non-treated cells (Control); ^#<i>p</i><0.05 and ^##<i>p</i><0.01 versus IFN-γ-stimulated cells.</p