Introduction

Obesity is associated with several metabolic alterations like type 2 diabetes, cardiovascular diseases and changes in the gut microbiota composition and gut barrier disruption.1 Among the components of the gut barrier, it has been shown that the mucus layer is altered when the mice are fed a high-fat diet (HFD), western-style diet (WSD) or low-fibre diet and in ob/ob mice, as well as in patients with dysglycaemia. Among the alterations, it has been observed a reduced thickness, increased penetrability and altered mucin glycan composition.2–9 The mucus exerts important roles in gut barrier protection and represents the interface of communication between bacteria and host. It is produced by the goblet cells (GCs) and constituted of glycoproteins called mucins, among which the main component is the secreted Muc2. The transmembrane mucins, involved in glycocalyx formation, are other important components of the gut barrier, conferring cell protection and mediating host–microbe interactions.10 Mucins are glycosylated thanks to glycosyltransferases and mucin glycans supply attachment sites and allow bacterial growth and colonisation. Indeed, bacteria are able to produce glycosyl hydrolases (GHs) to degrade mucin glycans and use them as energy source.10

α−1,2-fucosyltransferase, encoded by the FUT2 gene, is one of the glycosyltransferases responsible for the presence of histo-blood group antigens on multiple organs and on the gastrointestinal mucosa.11 In recent years, genome-wide association studies have underlined the importance of FUT2 biology and showed that different polymorphisms may result in distinct secretor status, associated with the development of pathophysiology such as intestinal inflammation.12 Furthermore, FUT2 has been shown to have significant effects on the intestinal bacterial community composition.12–15 One of the major prototypical secretor-type oligosaccharides is the human milk oligosaccharide (HMO) 2’-fucosyllactose (2’FL).16 17 In vivo and in vitro studies showed that 2’FL exerts biological properties as prebiotic, antibacterial, antiviral and immunomodulating effects and modifies the host’s epithelial cell-surface glycome.18 This has prompted an increased interest in 2’FL as HMO source in infant formula and, more recently, 2’FL is also being investigated in pathological contexts.19 For example, in mice fed HFD, it was observed that 2’FL reduced body weight and fat mass gain.20 21 In addition, 2’FL protected against gut barrier disruptions induced by inflammatory stimuli, by increasing GCs number and Muc2 expression.22 23 Further in vivo studies exploring the role of 2’FL on the mucus layer in the context of obesity induced by HFD feeding are still lacking.

To fill this gap, we designed a study aimed at deciphering whether the impact of 2’FL on metabolism could be linked to changes in the intestinal mucus production, glycosylation, secretion and degradation. In addition, we explored whether the effects on mucus layer and metabolism might be associated with modifications in gut microbiota composition, faecal proteome and endocannabinoid (eCB) system.

We believe that a comprehensive investigation into the intricate mechanisms of the mucus layer, including its biosynthesis, turnover and degradation, may offer novel insights into developing efficacious interventions for mitigating or preventing obesity and related metabolic disorders.

Results2’FL counteracts metabolic alterations induced by HFD

Mice fed an HFD diet supplemented with 2’FL showed significantly lower body weight and fat mass gain (subcutaneous, epididymal, visceral and brown adipose tissues) compared with mice fed HFD alone (figure 1A–E). This could not be explained by food intake or lean/muscle mass since there were no differences between HFD and HFD+2’FL groups (online supplemental figure 1A-C). Additionally, 2’FL supplementation reduced glucose intolerance, as evidenced by the shape of the glycaemia curve during the oral glucose tolerance test and by the lower insulin levels in fasting state (figure 1F–I). These effects coincide with changes in hormones involved in metabolic pathways, since 2’FL significantly increased the concentration of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) and decreased leptin and glucagon (for the latter not significantly) while ghrelin was significantly reduced by the HFD, with no effects of 2’FL (figure 1J–N).

Figure 1

2’FL supplementation counteracts diet-induced obesity and glucose intolerance. (A) Body weight gain evolution and (C) fat mass gain evolution. (B) Final body weight gain and (D) fat mass gain. (E) Adipose tissue weights of subcutaneous (SAT), epidydimal (EAT), visceral (VAT) and brown (BAT) adipose tissue. (F) Plasma glucose (mg/dL) profile before and after 2 g/kg of glucose oral challenge measured during the oral glucose tolerance test (OGTT) and (G) the mean area under the curve (AUC) (mg/dL×min). (H) Plasma insulin (µg/L) measured 30 min before and 15 min 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. (J–N) Plasma levels from the portal vein of glucagon-like peptide-1 (GLP-1), peptide YY (PYY), ghrelin, leptin and glucagon. Data are means±SEM (n=7–10/group). One-way ANOVA followed by Tukey post hoc test was applied to figure B, D, E, G–K, N while Kruskal-Wallis followed by Dunn’s test was applied to figure L,M, based on data distribution. Two-way ANOVA followed by Tukey post hoc test was applied to figure A, C, F. Data with different subscript letters are significantly different (p<0.05). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. 2’FL, 2’-fucosyllactose; ANOVA, analysis of variance; HFD, high-fat diet.

2’FL increases intestinal cells proliferation and markers involved in gut barrier function

2’FL supplementation significantly increased full caecum and its content weight by about 80% and 150% compared with control and HFD, respectively (figure 2A–C). 2’FL supplementation also increased the length of the jejunum by almost 15% compared with CT and HFD (figure 2D).

Figure 2

2’FL increases microbiota fermentation, intestinal cell proliferation and markers of the gut barrier. (A) Full caecum, (B) empty caecum and (C) caecal content weight. (D) Jejunum length. (E–J) mRNA relative expression of markers of the gut barrier function measured in the jejunum, ileum, caecum and colon. Antimicrobial peptides mRNA expression: (E) Lysozyme C (Lyz1), (F) Regenerating islet-derived 3-gamma (Reg3g), (G) Phospholipase A2 group II (Pla2g2a); (H) Proglucagon; (I) Trefoil factor 3 (Tff3); (J) Intectin. Data are means±SEM (n=7–12/group). Data were analysed using one-way ANOVA followed by Tukey post hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. 2’FL, 2’-fucosyllactose; ANOVA, analysis of variance; ND, not detectable.

Analysing the expression of genes involved in gut barrier function by qPCR, we found that 2’FL significantly increased the antimicrobial peptides Lyz1 and Reg3g in the caecum, and proglucagon in the caecum and colon while it induced the expression of Reg3g in the jejunum and colon, Pla2g2a in the colon and intectin in the ileum, without reaching significance (figure 2E–J).

2’FL affects GCs differentiation and mucus production and secretion

We next determined whether the effects of 2’FL supplementation on metabolism and gut barrier function were linked to changes in intestinal mucus. We showed that 2’FL significantly affected the expression of genes involved in GCs differentiation at different sites, with increased expression of Elf3 in caecum and Hes1 in colon, and decreased Math1 and Spdef in caecum (figure 3A–E). In order to determine if the mucus inside the GCs was affected by the dietary treatments, we measured the proportion of the blue area (representing the mucins) over the total mucosal area, in histological sections using an Alcian blue staining. We found 22% more blue area in HFD+2’FL compared with HFD, though this difference did not reach significance (figure 3F,G).

Figure 3

2’FL supplementation impacts on goblet cells and mucins production. (A–E) mRNA relative expression of transcriptional factors involved in the goblet cells differentiation, in the jejunum, ileum, caecum and colon: (A) atonal bHLH transcription factor 1 (Math1), (B) SAM pointed domain containing ETS transcription factor (Spdef), (C) E74 like ETS transcription factor 3 (Elf3), (D) kruppel like factor 4 (Klf4), hes family basic helix-loop-helix (bHLH) transcription factor 1 (Hes1). (F) Percentage of blue area on the total mucosal area in the proximal colon and (G) representative images for each group. (H–M) mRNA relative expression of markers involved in mucin production, in the jejunum, ileum, caecum and colon: (H) anterior gradient 2 (Agr2), (I) mucin 2 (Muc2), (J–M) mucin 1/4/13/17 (Muc1, Muc4, Muc13, Muc17). Data are means±SEM (n=6–12/group). One-way ANOVA followed by Tukey post hoc test or Kruskal-Wallis followed by Dunn’s test were applied based on data distribution. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. 2’FL, 2’-fucosyllactose; ANOVA, analysis of variance; HFD, high-fat diet.

Next, we set out to assess whether 2’FL treatment impacts intestinal mucins. We found that 2’FL significantly affected Agr2 expression, required for the post-transcriptional synthesis and secretion of Muc2, which was decreased in caecum and increased in colon. In accordance with this observation, we also found a significant increase in Muc2 expression (figure 3H,I). With regard to transmembrane mucins, 2’FL supplementation led to increased Muc4 in jejunum, caecum and colon, Muc13 in jejunum and caecum, and Muc17 in caecum and colon (figure 3J–M). Furthermore, Muc1 and Muc13 expressions in the colon were negatively correlated with body weight and fat mass gain (online supplemental figure 2A,B).

Finally, we observed that dietary treatments differentially affected the expression of genes involved in intestinal mucus secretion and stabilisation. In particular, 2’FL supplementation tended to increase Retnlb in jejunum but decreased in the other intestinal segments. 2’FL supplementation increased two other key markers, Nlrp6 in caecum and colon and Fcgbp in colon while slightly counteracting the effects of the HFD on the expression of Atg5 and Atg7 (figure 4A–E).

Figure 4

2’FL 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) autophagy protein 5 (Atg5), (C) autophagy protein 7 (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 after scraping in milligrams. (G) Mucus thickness in the proximal colon measured by ImageJ (in micrometre) and (H) representative images for each group. Data are means±SEM (n=6–12/group). One-way ANOVA followed by Tukey post hoc test or Kruskal-Wallis followed by Dunn’s test were applied based on data distribution. *p<0.05, **p<0.01, ***p<0.001. 2’FL, 2’-fucosyllactose; ANOVA, analysis of variance; HFD, high-fat diet.

Although there was no difference in mucus thickness as assessed on histological sections (figure 4G,H), we found a significant higher weight of the mucus collected by scraping the colon in mice supplemented with 2’FL, suggesting a potential increase in mucus production (figure 4F).

Using a fluorescence in situ hybridisation (FISH) approach against 16S RNA to detect bacteria combined with a Muc2C3-specific staining of the mucus on colon sections, we observed an abrupt change from the inner to outer mucus layer with bacterial concentrations jumping from almost virtually free of bacteria to a high density without any perceptible gradient. The bacterial front was found to be morphologically intact in all groups. The thickness of the bacteria-free mucus was not statistically different between CT and HFD groups, though we observed a significant increase in the 2’FL treated group compared with the control group (p=0,01 Kruskal-Wallis test) (figure 5A).

Figure 5

Pictures representative of the bacterial penetration assessed by measuring the distance between the bacterial front and the epithelial cells (A, B) and the bacterial density in the inner mucus layer (C, D). (A) Mouse distal colon section in which Muc2C3 immunostaining shows the Muc2-positive mucus layer on the epithelium. The inner mucus layer (M) is almost completely devoid of bacteria, which are visualised by a FISH approach using a general bacterial probe conjugated with C3 (red), whereas the outer mucus layer contains large concentrations of bacteria with a clearly delineated bacterial front (BF). The sections are counterstained with DAPI to visualise nuclei (blue). Epithelial cells (EC) emit some autofluorescence making them visible (Scale bar: 50 µm). (B) Quantitative measurement of the spatial separation between the epithelial cells and the bacterial front. (C) Magnification (×20) of the inner mucus layer and of penetrating bacteria. Epithelial cells are on the left, while the bacterial front is on the right (scale bar: 10 µm). (D) Quantification of the bacterial density in the inner mucus layer (number of bacterial cells counted divided by the surface area of mucus). Data are means±SEM (n=7–8/group). Arrow heads show bacteria in red. Data were analysed using Kruskal-Wallis test followed by Dunn’s test. **p<0.01. FISH, fluorescence in situ hybridisation; HFD, high-fat diet.

When focusing on the apparent virtually free of bacteria inner mucus layer, we found that some bacteria, though very few, were able to penetrate it. We quantified the density by counting these cells and normalising to the area of mucus, but we found no differences between groups (figure 5B).

2’FL affects mucin glycan profile

To determine whether HFD and 2’FL supplementation affected mucin glycosylation, we first measured the expression of glycosyltransferases involved in elongation, branching and termination of the mucin glycan chain. We found that 2’FL significantly increased Gcnt4, B3gnt6 and C1galt1 in colon, C1galt1c1 in caecum and colon, Fut1 in jejunum and colon, Fut8 and St3gal1 in colon, St3gal3 in jejunum and colon and St3gal6 in colon (figure 6A–M). Interestingly, Fut2 was decreased by the HFD in caecum and colon, but not affected by 2’FL supplementation. All the data from the mRNA expression described in the colon are schematised in figure 7.

Figure 6

2’FL increases the expression of glycosyltransferases involved in mucin glycosylation. mRNA relative expression of glycosyltransferases in the jejunum, ileum, caecum 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 one 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–M) ST3 b-galactoside a-2,3-sialyltransferase 1/3/4/6 (St3gal1, St3gal3, St4gal4, St3gal6), (O) ST6 N-acetylgalactosaminide a-2,6-sialyltransferase 2 (St6galnac2). Data are means±SEM. (n=7–12/group). Data were analysed using one-way ANOVA followed by Tukey post hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. 2’FL, 2’-fucosyllactose; ANOVA, analysis of variance; ND, not detectable.

Figure 7

Schematic figure summarising the expression analysis of 35 genes in the jejunum, ileum, caecum and colon. Markers involved in gut barrier function and mucins production, glycosylation and secretion measured by RT-qPCR. Markers are enclosed in small grey boxes. Purple arrows indicate those that significantly changed due to 2’FL supplementation in the colon. 2’FL, 2’-fucosyllactose.

We next analysed mucin glycosylation by tandem mass spectrometry (MS/MS) and found that two of them were significantly higher in HFD, compared with the CT and/or HFD+2’FL group (figure 8A–D). Figure 8E shows that 10 glycans were present in all the mice, 8 had a lower prevalence in the HFD group only or were restored following supplementation with 2’FL, and 3 were less prevalent in the HFD+2’FL group.

Figure 8

High-fat diet and 2’FL supplementation affects mucin glycans composition in the colon. (A) Glycan relative abundance in percentage; relative abundance of (B) sialylated glycans, (C) fucosylated glycans and (D) sulfated glycans. (E) Glycan prevalence calculated by dividing the number of mice for which the glycan was present for the total number of mice in the group. Only glycans present in at least 3 mice and in at least one group are shown. Data are means±SEM (n=4–5/group). Data were analysed using Kruskal-Wallis followed by Dunn’s test. *p<0.05. 2’FL, 2’-fucosyllactose; HFD, high-fat diet; ND, not detectable.

2’FL affects the endocannabinoid system

We previously discovered that different bioactive lipids belonging to the eCB system are able to exert control over the gut microbiota and the gut barrier function.2 24–27 Hence, we measured caecal levels of eCBs (arachidonoylglycerol (AG) and anandamide (NAE 20:4)) and related N-acylethanolamines, and found that HFD+2’FL mice had significant lower levels of NAEs (16:1, 18:3, 20:0), LEA, OEA, PEA, DHEA and HEA, compared with CT and/or HFD mice. While, they had significant higher levels of mono-oleoylglycerol (OG) and mono-palmitoylglycerol (PG) (figure 9A). 2’FL affected the expression of genes involved in the biosynthesis and degradation of eCBs, by significantly upregulating Daglb and Abdh6, and downregulating Abdh4, Faah and Mgl (figure 9B).

Figure 9

Different caecal eCBome tone in 2’FL supplemented mice. (A) Concentrations of the eCBome-related mediators in the caecal tissue (pmol/g wet tissue weight) measured by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). (B) mRNA relative expression of receptors and metabolic enzymes for monoacylglycerols and N-acylethanolamines measured by RT-qPCR. Data are means±SEM (n=9–10/group). Data were analysed using one-way ANOVA followed by Tukey post hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. 2’FL, 2’-fucosyllactose; Abdh4, alpha/beta-hydrolase 4; Abdh6, α/β-Hydrolase domain-containing 6; AEA, N-arachidonoylethanolamine; AG, 2-arachidonoylglycerol; ANOVA, analysis of variance; Cb1/Cb2, cannabinoid type 1/2 receptors; DEA, N-docosatetraenylethanolamine; Dagla, diacylglycerol lipase-alpha; Daglb, diacylglycerol lipase beta; DHEA, N-docosahexaenoylethanolamine; Faah, fatty-acid amide hydrolase; Gpr119, G-protein-coupled receptor 119; HEA, N-homo-linolenylethanolamine; LEA, N-linoleylethanolamine; Mgl, monoacylglycerol lipase; Naaa, N-acylethanolamine acid amidase; NAE, N-acylethanolamine; Napepld, N-acyl phosphatidylethanolamine phospholipase D; Nat1, N-acetyltransferase 1; OEA, N-oleoylethanolamine; OG, mono-oleoylglycerol; PEA, N-palmitoylethanolamine; PG, mono-palmitoylglycerol; Pparg, peroxisome proliferator-activated receptor gamma SEA, N-stearoylethanolamine.

2’FL changes gut microbiota composition

Before the treatment all mice shared a similar faecal microbiota composition, while in the end both the faecal and caecal microbiota profiles were significantly clustered based on the diets (figure 10A–C). The results shown below refer to changes observed in both relative and absolute abundance.

Figure 10

2’FL induces changes in the caecal and faecal gut microbiota composition. Principal coordinates analysis (PCoA) plot of the gut microbiota, in which mice are grouped by treatment, based on the Bray-Curtis dissimilarity in (A) faeces before the treatment (B) faeces at the end of the treatment and (C) caecum at the end of the treatment (n=9–10/group). (D–G) Bar graphs showing grouped taxonomic profiles of the gut bacteria at the genus level: (D, E) relative and absolute abundance in the caecum, at the end of the treatment; (F, G) relative and absolute abundance in the faeces, before and at the end of the treatment (n=9–10/group). Only the bacterial genera with >1% relative abundance are shown; the rest are indicated as ‘others (<1%)’. 2’FL, 2’-fucosyllactose; HFD, high-fat diet.

At the phylum level, the caecal gut microbiota of CT and HFD groups was dominated by Desulfobacterota while HFD+2’FL by Bacteroidota and Verrucomicrobiota. In the faeces, the CT group was dominated by Bacteroidota, HFD by Desulfobacterota and HFD+2’FL by Bacteroidota and Verrucomicrobiota (online supplemental figure 3A–D and online supplemental tables 1 and 2).

At the genus level, the caecal gut microbiota was enriched in uncultured Desulfovibrionaceae in the CT and HFD groups (35.1 and 51.7%, respectively), whereas Akkermansia and Bacteroides were the dominant genera in the HFD+2’FL group (39% and 24.8%, respectively) (figure 10D,E). Similarly, the faecal gut microbiota was dominated by uncultured Desulfovibrionaceae, Rikenellaceae RC9 gut group and Akkermansia in the CT group (19.4%, 17.3% and 18.4%, respectively), by uncultured Desulfovibrionaceae in the HFD group (38.8%), and Bacteroides and Akkermansia in the HFD+2’FL group (37.1% and 29.8%, respectively) (figure 10F,G) .

Notably, HFD-fed mice had significant lower levels of Akkermansia, Parasutterella, unclassified Tannerellaceae, Muribaculaceae and Rikenellaceae RC9 gut group compared with CT mice while 2’FL treatment significantly increased Akkermansia, Parasutterella, unclassified Tannerellaceae and Bacteroides compared with HFD only, in the faeces (figure 11A–C, online supplemental table 3).

Figure 11

Bacterial genera significantly differed in absolute abundance (FDR-corrected p<0.05) in (A) HFD compared with CT (log2 fold change values calculated relative to CT), (B) HFD+2’FL compared with CT (log2 fold change values calculated relative to CT) and (C) HFD+2’FL compared with HFD (log2 fold change values calculated relative to HFD). Bar colour and bottom legend denote family-level taxonomic classification. See online supplemental table 3 for full results. 2’FL, 2’-fucosyllactose; HFD, high-fat diet.

2’FL affects bacterial glycosidases and faecal proteome

To evaluate the mucus degradation by the gut microbiota, we investigated bacterial GHs alpha-L-fucosidase and alpha-D-galactosidase by in-gel fluorescent activity-based probes (ABP) labelling.28 29 We found ABP-labelling for alpha-L-fucosidase only in the HFD+2’FL group, with mice within this group displaying different profiles. While, alpha-D-galactosidase labelling was present in CT and HFD+2’FL, without any signals in HFD (figure 12A).

Figure 12

High-fat diet and 2’FL supplementation affects faecal proteome. (A) Cy5-ABP-labelling of alpha-L-fucosidase and alpha-D-galactosidase from mouse faecal extract (1 µg of proteins; 1 µM α-L-fucosidase and 0.5 µM α-galactosidase). Principal component analysis (PCA) of (B) faecal mouse proteins and GHs together, of (C) mouse proteins only and of (D) GHs separately. (E–G) Volcano plot comparing the different groups together, including mouse proteins and GHs. PCA and volcano plot were done with MetaboAnalyst (n=9–10/group). 2’FL, 2’-fucosyllactose; GHs, glycosyl hydrolases; HFD, high-fat diet.

To further confirm the presence of GHs, we analysed the total faecal proteome, using a bespoke database containing mouse proteins and GHs involved in mucin glycan degradation: fucosidases, galactosidases, hexosaminidases and sialidases. The principal component analysis (PCA) showed different clustering between HFD and HFD+2’FL (figure 12B–D). Particularly, when taking only GHs into account, CT and HFD displayed overlapping clusters, while HFD+2’FL cluster was completely separated. The volcano plot showed that HFD feeding significantly changed the abundance of 17 proteins, while 2’FL supplementation changed 12 proteins compared with CT and 30 compared with HFD (figure 12E–G).

Interestingly, 2’FL supplementation significantly upregulated beta-galactosidase, alpha-L-fucosidase, beta-hexosaminidase and beta-N-acetylhexosaminidase, belonging to Bacteroidales and Lachnospiraceae bacterial families (figure 13A–H).