Human Milk Oligosaccharide Supplementation Affects Intestinal Barrier Function and Microbial Composition in the Gastrointestinal Tract of Young Sprague Dawley Rats

Abstract

Human milk oligosaccharides (HMOs) are chief maternal milk constituents that feed the intestinal microbiota and drive maturation of the infant gut. Our objective was to determine whether supplementing individual HMOs to a weanling diet alters growth and gut health in rats. Healthy three-week-old Sprague Dawley rat pups were randomized to control, 2'-O-fucosyllactose (2'FL)- and 3'sialyllactose (3'SL)-fortified diets alone or in combination at physiological doses for eight weeks. Body composition, intestinal permeability, serum cytokines, fecal microbiota composition, and messenger RNA (mRNA) expression in the gastrointestinal tract were assessed. Males fed a control diet were 10% heavier and displayed elevated interleukin (IL-18) (p = 0.01) in serum compared to all HMO-fortified groups at week 11. No differences in body composition were detected between groups. In females, HMOs did not affect body weight but 2'FL + 3'SL significantly increased cecum weight. All female HMO-fortified groups displayed significant reductions in intestinal permeability compared to controls (p = 0.02). All HMO-fortified diets altered gut microbiota composition and mRNA expression in the gastrointestinal tract, albeit differently according to sex. Supplementation with a fraction of the HMOs found in breast milk has a complex sex-dependent risk/benefit profile. Further long-term investigation of gut microbial profiles and supplementation with other HMOs during early development is warranted.

Keywords: 2′-O-fucosyllactose; 3′sialyllactose; gut microbiota; human milk oligosaccharides; inflammation; intestinal permeability.

Figure 1

Body weight of (A) male and (B) female rats, as well as food intake for (C) male and (D) female rats fed the AIN-93 diet fortified with 3′sialyllactose (3′SL), 2′fucosyllactose (2′FL), both, or neither for eight weeks. Values are means ± standard error of the mean (SEM), n = 8–10. In the overall model, there was a significant sex effect for body weight (p = 0.001) and food intake (p = 0.001); therefore, subsequent analysis was performed in males and females separately. Within males and females, the superscripts a, b are used to depict differences between groups, where groups without a common superscript differ (p < 0.05). Control: AIN-93; 3′SL: AIN-93 + 3′sialyllactose; 2′FL: AIN-93 + 2′fucosyllactose; 3′SL + 2′FL: AIN-93 + 3′sialyllactose+ 2′fucosyllactose.

Figure 2

Oral glucose tolerance test (OGTT) in (A) male and (B) female rats; insulin tolerance test (ITT) in (C) male and (D) female rats fed the AIN-93 diet fortified with 3′SL, 2′FL, both, or neither for eight weeks. Values are means ± SEM, n = 8–10. In the overall model, there was a significant sex effect for OGTT (p = 0.001); therefore, subsequent analysis was performed in males and females separately. Control: AIN-93; 3′SL: AIN-93 + 3′sialyllactose; 2′FL: AIN-93 + 2′fucosyllactose; 3′SL + 2′FL: AIN-93 + 3′sialyllactose + 2′fucosyllactose. Significance was set at p < 0.05.

Figure 3

Intestinal permeability and jejunum mRNA levels. Plasma concentrations of fluorescein isothiocyanate (FITC) dextran 4000 (FD4) in (A) male and (B) female rats, as well as jejunum mRNA levels in (C) male zonula occluden-1 (ZO-1), (D) female ZO-1, (E) male occludin, and (F) female occludin in rats fed the AIN-93 diet fortified with 3′SL, 2′FL, both, or neither for eight weeks. Values are means ± SEM, n = 8–10. In the overall model, there was a significant sex effect for IPT (p = 0.03), ZO-1 (p = 0.001), and occludin (p = 0.0002); therefore, subsequent analysis was performed in males and females separately. Control: AIN-93; 3′SL: AIN-93 + 3′sialyllactose; 2′FL: AIN-93 + 2′fucosyllactose; 3′SL + 2′FL: AIN-93 + 3′sialyllactose + 2′fucosyllactose. Significance was set at p < 0.05.

Figure 4

Proximal colon messenger RNA (mRNA) levels of (A) matrix metalloproteinase 2 (MMP2), (C) MMP9, (E) mucin 2 (MUC2) (G) G-protein-coupled receptor 41 (GPR41), and (I) GPR43 in male and (B) MMP2, (D) MMP9, (F) MUC2, (H) GPR41, and (J) GPR43 in female rats fed the AIN-93 diet fortified with 3′SL, 2′FL, both, or neither for eight weeks. Values are means ± SEM, n = 8–10. Control: AIN-93; 3′SL: AIN-93 + 3′sialyllactose; 2′FL: AIN-93 + 2′fucosyllactose; 3′SL + 2′FL: AIN-93 + 3′sialyllactose + 2′fucosyllactose. Significance was set at p < 0.05.

Figure 5

Relative abundance plots of bacterial taxa using 16S rRNA sequencing in male and female rat feces at 11 weeks of age. Taxa were identified to the taxonomic level of family using the Silva reference database. Control: AIN-93; 3′SL: AIN-93 + 3′sialyllactose; 2′FL: AIN-93 + 2′fucosyllactose; 3′SL + 2′FL: AIN-93 + 3′sialyllactose + 2′fucosyllactose. * p < 0.05 (males); † p < 0.05 (females).