Prebiotic oligofructose protects against high-fat diet-induced obesity by changing the gut microbiota, intestinal mucus production, glycosylation and secretion

Abstract

Obesity is a major risk factor for the development of type 2 diabetes and cardiovascular diseases, and gut microbiota plays a key role in influencing the host energy homeostasis. Moreover, obese mice have a different gut microbiota composition, associated with an alteration of the intestinal mucus layer, which represents the interface between the bacteria and the host. We previously demonstrated that prebiotic treatment with oligofructose (FOS) counteracted the effects of diet-induced obesity, together with changes in the gut microbiota composition, but it is not known if the intestinal mucus layer could be involved. In this study, we found that, in addition to preventing high-fat diet (HFD) induced obesity in mice, the treatment with FOS increased the expression of numerous genes involved in mucus production, glycosylation and secretion, the expression of both secreted and transmembrane mucins, and the differentiation and number of goblet cells. These results were associated with significant changes in the gut microbiota composition, with FOS significantly increasing the relative and absolute abundance of the bacterial genera Odoribacter, Akkermansia, two unknown Muribaculaceae and an unknown Ruminococcaceae. Interestingly, all these bacterial genera had a negative association with metabolic parameters and a positive association with markers of the mucus layer. Our study shows that FOS treatment is able to prevent HFD-induced metabolic disorders, at least in part, by acting on all the processes of the mucus production. These data suggest that targeting the mucus and the gut microbiota by using prebiotics could help to prevent or mitigate obesity and related disorders.

Keywords: glycosyltransferases; goblet cells; gut barrier; gut microbiota; high-fat diet; mucins; mucus; obesity; oligosaccharides; type 2 diabetes.

Figure 1.

Oligofructose supplementation prevents diet-induced obesity and glucose intolerance. (a) Body weight gain evolution and (c) fat mass gain evolution in grams during 6 weeks of treatment; (b) Final body weight gain and (d) final fat mass gain in grams after 6 weeks of treatment. (e) Adipose tissue weights of subcutaneous (SAT), epididymal (EAT), visceral (VAT) and brown (BAT) adipose tissue in grams after 6 weeks of treatment (n = 10–12/group). (f) Plasma glucose (mg dL⁻¹) profile before and after 2 g/kg glucose oral challenge measured during the oral glucose tolerance test (OGTT) and (g) the mean area under the curve (AUC) (mg dL⁻¹ min⁻¹) (n = 10–12/group). (h) Plasma insulin (µg L⁻¹) measured 30 minutes before and 15 minutes after the glucose administration during the OGTT. (i) Insulin resistance index determined by multiplying the area under the curve (from −30 to 15 min) of blood glucose and plasma insulin obtained during the OGTT (n = 8-9/group). Data are means ± s.e.m. Data with different subscript letters are significantly different (P < 0.05), according to one-way ANOVA followed by Tukey post hoc test for b,d,e,g−i and according to two-way ANOVA followed by Bonferroni post hoc test for a,c,f.

Figure 2.

Oligofructose increases microbiota fermentation, intestinal cell proliferation and markers of the gut barrier. (a) Full cecum, (b) empty cecum and (c) cecal content weight in grams after 6 weeks of treatment. (d) Representative images of the cecum taken immediately after sacrificing the mice. (e) Jejunum length in centimeters. (f-k) mRNA relative expression of markers of the gut barrier function measured in the jejunum, ileum, cecum and colon: antimicrobial peptides mRNA expression: (f) Lysozyme C (Lyz1), (g) Regenerating islet-derived 3-gamma (Reg3g), (h) Phospholipase A2 group II (Pla2g2); (i) Intectin; (j) Trefoil factor 3 (Tff3); (k) Proglucagon. Data are means ± s.e.m. (n = 9–12/group). Data were analyzed using one-way ANOVA followed by Tukey post hoc test. Data with different subscript letters are significantly different (P < 0.05). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3.

Oligofructose increases goblet cells differentiation and number and markers of mucins production. (a-e) mRNA relative expression of transcriptional factors involved in the goblet cells differentiation, in the jejunum, ileum, cecum and colon: atonal bHLH transcription factor 1 (Math1), SAM pointed domain containing ETS transcription factor (Spdef), E74-like ETS transcription factor 3 (Elf3), kruppel-like factor 4 (Klf4), hes family bHLH transcription factor 1 (Hes1) (n = 8-12/group). (f,g) Percentage of blue area on the total mucosal area in the proximal colon and representative images for each group (n = 9-11/group). (h-m) mRNA relative expression of markers involved in mucin production, in the jejunum, ileum, cecum and colon: (h) anterior gradient 2 (Agr2), (i) mucin 2 (Muc2), (j-m) mucin 1/3/4/13 (Muc1, Muc3, Muc4, Muc13) (n = 8-12/group). Data are means ± s.e.m. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4.

Oligofructose increases the expression of glycosyltransferases involved in mucin glycosylation. mRNA relative expression of glycosyltransferases in the jejunum, ileum, cecum and colon: (a) glucosaminyl (N-acetyl) transferase 1 (Gcnt1), (b) glucosaminyl (N-acetyl) transferase 4 (Gcnt4), (c) UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 6 (B3gnt6), (d) core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 (C1galt1), (e) C1GALT1 specific chaperone 1 (C1galt1c1), (f-h) fucosyltransferase 1/2/8 (Fut1, Fut2, Fut8), (i-l) ST3 beta-galactoside alpha-2,3-sialyltransferase 1/3/4/6 (St3gal1, St3gal3, St4gal4, St3gal6), (m) ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 2 (St6galnac2). Data are means ± s.e.m. (n = 7–12/group). Data were analyzed using one-way ANOVA followed by Tukey post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ND = Not Detectable.

Figure 5.

Oligofructose increases markers of mucus secretion. (a-e) mRNA relative expression of markers involved in the secretion of the mucus layer: (a) resistin-like beta (Retnlb), (b,c) autophagy protein 5/7 (Atg5, Atg7), (d) NOD‐like receptor family pyrin domain containing 6 (Nlrp6), (e) Fc gamma binding protein (Fcgbp). (f) Weight of the mucus in the colon in milligrams after scraping (n = 9-12/group). (g) Mucus thickness measured in micrometer in the proximal colon by ImageJ and (h) representative images for each group (n = 6-8/group). Data are means ± s.e.m. (n = 10–12/group). Data were analyzed using one-way ANOVA followed by Tukey post hoc test. Data with different subscript letters are significantly different (P < .05). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 6.

Summary figure of 35 markers measured in jejunum, ileum, cecum and colon. Markers involved in gut barrier function and mucins production, glycosylation and secretion measured by RT-qPCR. Markers are enclosed in small gray boxes. Green arrows indicate the ones that significantly increased due to the supplementation with oligofructose in the colon.

Figure 7.

Oligofructose induces changes in the fecal and cecal gut microbiota composition. (a-c) Principal coordinates analysis (PCoA) plot of the gut microbiota based on Bray-Curtis distances in (a,b) feces (before and at the end of the treatment) and in (c) the cecum, from mice grouped by treatment (control diet (CT), high fat diet (HFD) and high fat diet plus 10% oligofructose (HFD+FOS)) (n = 10–12/group). (d-g) Bar graphs showing grouped taxonomic profiles of the gut bacteria at a genus level: (d,e) relative and absolute abundance in the feces, before and at the end of the treatment; (f,g) relative and absolute abundance in the cecum, at the end of the treatment (n = 10–12/group). Only the bacterial genera that have a relative abundance > 1% are shown; the rest are included in “Others (<1%)”.

Figure 8.

Associations between gut bacterial genera and variables of metabolism, gut barrier and mucus layer. (a,b) Heat map displaying the most significant and numerous baseline associations between gut bacterial genera (absolute abundance in the feces at the end of the treatment) and variables of metabolism, gut barrier, goblet cells and mucus production, glycosylation and secretion (in the colon) (n = 10–12/group). (a) Heat map created considering all the samples from all the groups (CT, HFD and HFD+FOS) (n = 10–12/group). (b) Heat map created considering only the samples from the group HFD+FOS (n = 10). Statistically significant FDR-adjusted p-values are noted with asterisks (*P ≤ 0.05, **P ≤0.01, ***P ≤ 0.001, ****P ≤ 0.0001).