Prebiotic Inulin and Sodium Butyrate Attenuate Obesity-Induced Intestinal Barrier Dysfunction by Induction of Antimicrobial Peptides

Abstract

Defects in the mucosal barrier have been associated with metabolic diseases such as obesity and non-alcoholic fatty liver disease (NAFLD). Mice fed a Western-style diet (WSD) develop obesity and are characterized by a diet-induced intestinal barrier dysfunction, bacterial endotoxin translocation and subsequent liver steatosis. To examine whether inulin or sodium butyrate could improve gut barrier dysfunction, C57BL/6 mice were fed a control diet or a WSD ± fructose supplemented with either 10% inulin or 5% sodium butyrate for 12 weeks respectively. Inulin and sodium butyrate attenuated hepatosteatitis in the WSD-induced obesity mouse model by reducing weight gain, liver weight, plasma and hepatic triglyceride level. Furthermore, supplementation with inulin or sodium butyrate induced expression of Paneth cell α-defensins and matrix metalloproteinase-7 (MMP7), which was impaired by the WSD and particularly the fructose-added WSD. Effects on antimicrobial peptide function in the ileum were accompanied by induction of β-defensin-1 and tight junction genes in the colon resulting in improved intestinal permeability and endotoxemia. Organoid culture of small intestinal crypts revealed that the short chain fatty acids (SCFA) butyrate, propionate and acetate, fermentation products of inulin, induce Paneth cell α-defensin expression in vitro, and that histone deacetylation and STAT3 might play a role in butyrate-mediated induction of α-defensins. In summary, inulin and sodium butyrate attenuate diet-induced barrier dysfunction and induce expression of Paneth cell antimicrobials. The administration of prebiotic fiber or sodium butyrate could be an interesting therapeutic approach to improve diet-induced obesity.

Keywords: NAFLD; antimicrobial peptides; butyrate; defensin; innate immunity; inulin; obesity.

Figure 1

Inulin and sodium butyrate reduce body weight and liver weight gain and attenuate hepatic steatosis in WDF-fed mice. C57BL/6 mice fed either a control diet (CD), a Western-style diet (WSD) or a WSD supplemented with 10% inulin (inu) or 5% sodium butyrate (but) with or without additional fructose (F) are shown. Weight development of mice fed diets without fructose (A) and with fructose (B) during the intervention of 12 weeks. Body weight change (C), liver weight (D), triglyceride concentration in plasma (E) and hepatic triglyceride level relative to protein concentration in liver tissue (F) after the 12-week intervention period. (G) Representative images of liver specimens stained with hematoxylin and eosin. Scale bar: 100 µm. Data are presented as means +/- standard error of the mean (SEM) (n = 6–8). Statistical analysis was performed by 2-way ANOVA with Tukey’s multiple comparisons test (A, B) or one-way ANOVA with Sidak´s post-test. Significant differences are indicated as *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.

Figure 2

Inulin and sodium butyrate induce Paneth cell antimicrobial expression which is decreased by high-fat and high-sugar diet. C57BL/6 mice fed different diets as described in Figure 1 are shown. Relative mRNA expression level of cryptdin-1 (A), cryptdin-4 (B), CRS1C (C), pan-cryptdin (D) and matrix metalloproteinase-7 (MMP7) (E) in ileal tissue determined by quantitative RT-PCR. Data are presented as means +/- standard error of the mean (SEM) (n = 6–8). Statistical analysis was performed by one-way ANOVA with Dunnett´s or Sidak´s post-test. Significant differences are indicated as *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.

Figure 3

Supplementation with inulin or sodium butyrate induces Defa5 protein expression in ileal Paneth cells. C57BL/6 mice fed different diets as described in Figure 1 are shown. (A) Representative images of Defa5 immunofluorescence staining in ileal tissue showing Defa5 expression in green and cell nuclei detected by DAPI in blue. Scale bar: 100 µm. (B) Quantification of Defa5 expression. Data are presented as means +/- standard error of the mean (SEM). Statistical analysis was performed by one-way ANOVA with Dunnett´s or Sidak´s post-test. Significant differences are indicated as **p-value < 0.01; ***p-value < 0.001.

Figure 4

Effects of inulin and sodium butyrate on antimicrobial peptide expression. C57BL/6 mice fed different diets as described in Figure 1 are shown. Relative mRNA expression level of lysozyme (A), Reg3γ (B), MyD88 (C) in the ileum and mBD-1 (D) in the colon determined by quantitative RT-PCR. Data are presented as means +/- standard error of the mean (SEM) (n= 6–8). Statistical analysis was performed by one-way ANOVA with Dunnett´s or Sidak´s post-test or by Kruskal-Wallis-test for non-parametric data with a Dunn’s post-test. Significant differences are indicated as *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.

Figure 5

Effects of inulin and sodium butyrate on small intestinal bacterial overgrowth (SIBO). C57BL/6 mice fed different diets as described in Figure 1 are shown. (A) Representative images of FISH staining in ileal tissue showing bacterial growth in red and cell nuclei detected by DAPI in blue. Scale bar: 100 µm. (B) Quantification of bacterial overgrowth. Data are presented as means +/- standard error of the mean (SEM). Statistical analysis was performed by one-way ANOVA with Kruskal-Wallis-test for non-parametric data with a Dunn’s post-test. Significant differences are indicated as ***p-value < 0.001.

Figure 6

Effect of inulin and sodium butyrate supplementation on intestinal tight junctions. C57BL/6 mice fed different diets as described in Figure 1 are shown. Relative mRNA expression level of occludin in the ileum (A) and in the colon (B), claudin-2 (C), claudin-5 (D) and ZO-1 (E) in the colon determined by quantitative RT-PCR. Data are presented as means +/- standard error of the mean (SEM) (n = 6–8). Statistical analysis was performed by one-way ANOVA with Dunnett´s or Sidak´s post-test or by Kruskal-Wallis-test for non-parametric data with a Dunn’s post-test. Significant differences are indicated as *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.

Figure 7

Supplementation with inulin and sodium butyrate improves intestinal permeability and inflammation. C57BL/6 mice fed different diets as described in Figure 1 . (A) Intestinal permeability was determined by claudin-3 level in the urine. (B) Endotoxin concentrations in the portal venous plasma. (C) PEG4000 recovery in urine in percent of orally administered PEG4000 by gavage. Relative mRNA expression level of TNF-α (D) and IL-6 (F) in the ileum and TNF-α (E) and IL-6 (G) in the colon determined by quantitative RT-PCR. Data are presented as means +/- standard error of the mean (SEM) (n = 6–10). Statistical analysis was performed by one-way ANOVA with Dunnett´s or Sidak´s post-test or by Kruskal-Wallis-test for non-parametric data with a Dunn’s post-test. Significant differences are indicated as *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001. PEG4000, polyethylene glycol 4000.

Figure 8

Sodium butyrate induces expression of antimicrobial peptides in murine organoids in vitro. Organoids were treated with 1mM and 2mM sodium butyrate for 30h. Relative mRNA expression level of cryptdin-1 (A), cryptdin-4 (B), pan-cryptdin (C), MMP7 (D), lysozyme (E) Reg3γ (F), mBD-1 (G) determined by quantitative RT-PCR derived in organoids from the small intestine of healthy C57BL/6 mice (n=6). Organoids were treated with sodium butyrate (1mM), sodium butyrate in combination with HJC0152 (5μM) or MS-275 (2μM) or HDAC8-IN-1 (0.5μM). Relative mRNA expression level of cryptdin-1 (H) and cryptdin-4 (I) determined by quantitative RT-PCR derived in organoids from the small intestine of healthy C57BL/6 mice (n=3-6). Data are presented as means ± SEM and were analyzed by unpaired t-test (A–G) or one-way ANOVA with Sidak´s post-test (H, I). Significant differences are indicated as *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.

Figure 9

Effect of sodium propionate and acetate on expression of antimicrobial peptides in murine organoids in vitro. Organoids were treated with sodium propionate (1mM) or sodium acetate (1mM) for 30h. Relative mRNA expression level of cryptdin-1 (A), cryptdin-4 (B), pan-cryptdin (C), MMP7 (D), lysozyme (E) Reg3γ (F), mBD-1 (G) determined by quantitative RT-PCR derived in organoids from the small intestine of healthy C57BL/6 mice (n=3-6). Data are presented as means ± SEM and were analyzed by unpaired t-test. Significant differences are indicated as *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.