Fermentable carbohydrate stimulates FFAR2-dependent colonic PYY cell expansion to increase satiety

Abstract

Objective: Dietary supplementation with fermentable carbohydrate protects against body weight gain. Fermentation by the resident gut microbiota produces short-chain fatty acids, which act at free fatty acid receptor 2 (FFAR2). Our aim was to test the hypothesis that FFAR2 is important in regulating the beneficial effects of fermentable carbohydrate on body weight and to understand the role of gut hormones PYY and GLP-1.

Methods: Wild-type or Ffar2^-/- mice were fed an inulin supplemented or control diet. Mice were metabolically characterized and gut hormone concentrations, enteroendocrine cell density measurements were carried out. Intestinal organoids and colonic cultures were utilized to substantiate the in vivo findings.

Results: We provide new mechanistic insight into how fermentable carbohydrate regulates metabolism. Using mice that lack FFAR2, we demonstrate that the fermentable carbohydrate inulin acts via this receptor to drive an 87% increase in the density of cells that produce the appetite-suppressing hormone peptide YY (PYY), reduce food intake, and prevent diet-induced obesity.

Conclusion: Our results demonstrate that FFAR2 is predominantly involved in regulating the effects of fermentable carbohydrate on metabolism and does so, in part, by enhancing PYY cell density and release. This highlights the potential for targeting enteroendocrine cell differentiation to treat obesity.

Keywords: Colon; Diet; Microbiota; Obesity; Peptide YY.

Figure 1

The effect of fermentable carbohydrate supplementation on metabolic parameters in high-fat fed WT and Ffar2^−/−mice. Percentage body weight change (n = 19–22) (A), food intake (B), energy expenditure (C), activity (D), subcutaneous adiposity at 11 weeks (E), hepatic triglyceride (F), plasma leptin (G), fecal energy loss (H), hypothalamic expression of Agrp (I), Npy (J), Pomc (K), and scapular brown adipose tissue Ucp1 expression (L) for male Ffar2^−/− and WT littermate C57BL/6 mice fed a high-fat diet supplemented with inulin (HFI) or cellulose (HFC) (control) n = 10–12 over a 14 week period. Data represent mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical differences determined by GEE (A–B) or two-way ANOVA with post-hoc Bonferroni comparisons (C–H).

Figure 2

The effect of fermentable carbohydrate supplementation on bacterial density or species composition in high-fat fed WT and Ffar2^−/−mice. Total bacteria (A), Bifidobacteria spp. (B), Lactobacillus spp. (C), for male Ffar2^−/− and WT littermates fed a high-fat diet supplemented with inulin (HFI) or cellulose (HFC) (control) n = 10–12 over a 10 week period. Data represent mean ± S.E.M. *p < 0.05, ***p < 0.001. Statistical differences determined by two-way ANOVA with post-hoc Bonferroni comparisons.

Figure 3

Gut hormone concentration following fermentable carbohydrate supplementation or incubation with SCFA in WT and Ffar2^−/−mice. Portal vein peptide YY (PYY) (A), portal vein Glucagon-like peptide-1 (GLP-1) (B), colonic PYY (C), and colonic GLP-1 (D) concentrations of male Ffar2^−/− and WT littermates fed a high-fat diet supplemented with inulin (HFI) or cellulose (HFC) for 14 weeks (A–B, n = 10–12) or 2 weeks (C–D n = 4–6). Percentage PYY (E) of GLP-1 (F) release from mixed colonic cultures taken from Ffar2^−/− and WT littermates controls following incubation with 50 mM SCFA (30 mM acetate, 12.5 mM propionate, 7.5 mM butyrate) (E). Data represent mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical differences determined by student's t-test or two-way ANOVA with post-hoc Bonferroni comparisons.

Figure 4

The effect of fermentable carbohydrate supplementation on L cell density in high-fat fed WT and Ffar2^−/−mice. PYY (A), GLP-1 (B), GLP-1/PYY co-localized cells per mm² (C) and the percentage of PYY+ cells which also stained positive for BrdU (D) within the proximal colon of male Ffar2^−/− and WT littermates fed a high-fat diet supplemented with inulin (HFI) or cellulose (HFC) for 2 weeks (n = 4–6). Representative images of PYY and GLP-1 staining are shown in E, F, G, and H. Representative images of PYY and BrdU staining are shown in I and J. Scale bars are equal to 100 μm. Data represent mean ± S.E.M. *p < 0.05, ***p < 0.001. Statistical differences determined by two-way ANOVA with post-hoc Bonferroni comparisons.

Figure 5

The effect of fermentable carbohydrate on colonic transcription factor expression in vivo. Gene expression relative to 18S was determined for Pax4 (A) Pax6 (B), Foxa1 (C), and Foxa2 (D) in the colon of male Ffar2^−/− and WT littermates fed a high-fat diet supplemented with inulin (HFI) or cellulose (HFC) for 2 weeks, n = 6–8. Data represent mean ± S.E.M. *p < 0.05. Statistical differences determined by two-way ANOVA with post-hoc Bonferroni comparisons.

Figure 6

Effect of CFMB and AR420626 on PYY and GLP-1 cell fate in intestinal organoids. Organoids from PYY-GFP mice were cultured in absence (A, D) or presence of 10 μM CFMB (B) and 10 μM AR420626 (E) for 96 h. Shown are Z-stack projections covering the whole organoid. Scale bars: 100 μm. (C, F) PYY cell numbers after 96 h continuous exposure with 10 μM CFMB (C) and 10 μM AR420626 (F). n = 30–60 organoids (2 platings). Number of GLP-1 +ve cells in organoids from PYY-GFP mice after 96-hour exposure to CFMB (G) and AR420626 (H). n = 100–300 organoids (3 platings). Expression of Pyy (I) and Gcg (J) following 96-hour exposure to CFMB in WT and Ffar2^−/− organoids. All data are presented as mean ± S.E.M, ****p < 0.0001, ***p < 0.001. *p < 0.05. Statistical differences determined by nonpaired 2-tailed Student's t test (C, F, G, H) or 2-way ANOVA (I–J).

Figure 7

Mechanism by which fermentable carbohydrate prevents body weight gain. Following consumption, fermentable carbohydrate (FCH) is fermented by gut microbiota to produce short chain fatty acids (SCFAs). SCFAs subsequently act at free fatty acid receptor 2 (FFAR2) within the colon to enhance PYY cell density, thereby augmenting the capacity of the gut to release PYY. PYY acts to reduce orexigenic drive via AgRP neurons within the arcuate nucleus and reduce food intake.