Grape proanthocyanidin-induced intestinal bloom of Akkermansia muciniphila is dependent on its baseline abundance and precedes activation of host genes related to metabolic health

Abstract

We previously showed that C57BL/6J mice fed high-fat diet (HFD) supplemented with 1% grape polyphenols (GP) for 12 weeks developed a bloom of Akkermansia muciniphila with attenuated metabolic syndrome symptoms. Here we investigated early timing of GP-induced effects and the responsible class of grape polyphenols. Mice were fed HFD, low-fat diet (LFD) or formulations supplemented with GP (HFD-GP, LFD-GP) for 14 days. Mice fed HFD-GP, but not LFD-GP, showed improved oral glucose tolerance compared to controls. A. muciniphila bloom occurred earlier in mice fed LFD-GP than HFD-GP; however, timing was dependent on baseline A. muciniphila levels rather than dietary fat. Mice gavaged for 10 days with GP extract (GPE) or grape proanthocyanidins (PACs), each delivering 360 mg PACs/kg body weight, induced a bloom of fecal and cecal A. muciniphila, the rate of which depended on initial A. muciniphila abundance. Grape PACs were sufficient to induce a bloom of A. muciniphila independent of specific intestinal gene expression changes. Gut microbial community analysis and in vitro inhibition of A. muciniphila by GPE or PACs suggest that the A. muciniphila bloom in vivo occurs via indirect mechanisms.

Keywords: Akkermansia; Grape; Gut; Microbes; Polyphenols; Proanthocyanidins.

Figure 1. Effect of 14 days of GP supplementation on oral glucose tolerance

A Oral glucose tolerance tests were performed at baseline (day 0) and after 14 days on diets. Blood glucose concentrations (mg/dL) expressed as mean ± SD (n= 11 – 12 mice per group) were measured at the indicated time points (0-120 min) following oral administration of 2 g/kg glucose. B. Area under the curve (AUC) representation of data in A. was determined for individual mice, horizontal bar represents mean for each group. Between-group difference by diet base was determined by unpaired t-test (* = p < 0.05), while difference across all four groups was determined by one-way ANOVA followed by Tukey's multiple comparisons test. Different letters indicate significant difference between groups (p < 0.05) and the same letter indicates no difference.

Figure 2. Effect of 14 days of GP supplementation on intestinal gene expression and cecal weights

Relative mRNA levels of selected genes expressed in A. jejunum, B. ileum, and C. colon tissues was determined by qPCR. Target mRNA was normalized to HMBS as endogenous control and data were analyzed according to the 2-^ΔCT method. Data are mean ± SD (n=8 samples per group) and the average of technical duplicates are used for each sample. Between-group difference was determined by unpaired, 2-tailed t-test: *p<0.05; **p<0.01; ***p<0.001. D. Cecal weights (g) of individual mice. Significant difference between diet groups in panels A - C is signified by letters a, b, or c; different letters indicate significant difference (p<0.05) between groups and the same letter indicates no difference. **** = p < 0.001.

Figure 3. GP altered microbial composition in both HFD and LFD conditions. A-B

Microbiota α diversity as calculated by (A) OTU richness and (B) Shannon index of fecal and cecal samples by diet and day of study. Asterisks represent significant differences between GP-supplemented groups and control groups determined by repeated measurement two-way ANOVA followed by Sidak's multiple comparisons tests (for fecal samples from Day 0-Day 14) or Mann-Whitney tests (for cecal samples from Day 14), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C. Bray-Curtis principal coordinate plots of gut microbial communities by day of study. ADONIS tests were performed to assess differential clustering caused by GP supplementation and diet base. R² and p values representing the percentages of overall variation explained by GP supplementation and corresponding significance are listed, while the effect of diet base and other analyses by diet base/supplementation can be found in Supplementary Table 4. D. Relative abundance of the five dominant bacterial phyla. Low-abundance phyla (< 0.3%) were combined into the Other category. In panels A-C, analyses were performed on 15,000 sequences per sample, while non-rarefied data was used in panel D. n = 6 mice per group.

Figure 4. Comparison of PAC vs. GPE treatment on A. muciniphila bloom and cecal weight

A qPCR quantification of A. muciniphila relative to total bacteria in fecal samples collected on days 0, 3, 7, and 10 during oral gavage with PAC, GPE, or vehicle as well as cecal content after euthanasia on day 10. Kruskal-Wallis test was used to detect differences between groups followed by pair wise comparisons using Dunn's multiple comparison test to detect differences between vehicle vs. GPE and vehicle vs. PAC groups. B. Comparison of cecal weights (g) of individual mice in each group. One-way ANOVA was performed to evaluate differences between the 3 groups followed by Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5. Grape polyphenols and proanthocyanidins inhibit the in vitro growth of A. muciniphila

GPE^cp and PAC standard were adjusted to pH 7.1, dilutions were prepared and added to molten agar to achieve 1.0, 0.33, 0.11, 0.03, or 0.012 mg of proanthocyanidin (B2 equivalents) per mL medium. A. muciniphila culture (OD = 0.875) was diluted 10^-3 and 10^-4 and 30 μL aliquots were spread on the surface of solid medium impregnated with A. GPE^cp or B. PAC standard. Photograph (representative of two independent experiments) shows 24-well plates incubated under anaerobic conditions at 37°C after 3 days.