Influence of exercise and/or genistein on gut microbiota after 12 weeks of Western diet in C57BL/6 adult mice

Diet and exercise have been studied independently as potential treatments to revert the negative effects of Western diets on host health through the modification of gut microbiota. The potential synergic effect of diet and exercise has not been well studied. This study aimed to analyze the impact of exercise (Exe), genistein (Gen), and their combined effect on gut microbiota after 12 wks of a high‐fat, high‐sugar diet (HFD). Methods Eighty‐three C57BL/6 mice (male n=41, female n=42) aged 4–5 wks were randomized to one of the following groups for a 12‐wk period: control (CON), HFD, HFD + Exe, HFD + Gen, or HFD + Exe + Gen. The HFD diet contained 20% carbohydrate, 20% protein, and 60% fat with sugar‐supplemented drinking water (42 g sugar/L). Gen containing diets had 600 mg Gen/kg of diet. Exe groups underwent moderate‐intensity aerobic exercise 30 min/d for 5 d/wk (wk 1 at 10 m/min for 15 min, wk 2 at 10 m/min increased to 30 min, wks 3–12 12 m/min for 30 min). Microbial DNA was extracted from fecal pellets collected at 12 wks and sequenced on the Illumina miSeq platform using primers for the V4 region of the 16S rRNA gene. The QIIME 2019.7 pipeline was used to align genomic sequences (DADA2) and assign taxonomy (Greengenes 13_8 database). a‐ (Shannon, Observed Operational Taxonomical Units (Ob OTUs), Faith PD, Pielou’s evenness) and b‐diversity (Jaccard, Bray‐Curtis, weighted and unweighted UniFrac) metrics were compared (Kruskal‐Wallis (KW) sum‐rank test and pairwise permutational multivariate analysis of variance, respectively) by treatment group. All‐against‐all LEfSe analysis was used to identify differential microbial abundance group differences (KW sum‐rank test). Results Microbial abundance (Shannon, Pielou’s Evenness) did not differ by study group but the CON group had a higher species richness (Ob OTUs, Faith PD; p<0.001) after 12 wks when compared to all other groups. HFD + Exe, and HFD + Gen + Exe groups also had a higher microbial richness (Ob OTUs, Faith PD; p<0.01) when compared to HFD + Gen suggesting that exercise may increase the number of unique microbes present in the community. All b‐diversity metrics suggested that the microbial community structure differed significantly (p<0.001), such that Exe, Gen, and Gen + Exe treatments each had unique effects on microbial diversity. LEfSe analysis (LDA score > 2; p<0.05) identified that the genus Ruminococcus was more abundant in both CON and HFD + Exe + Gen groups, when compared to all other groups. Conclusion These data suggest that exercise may have a greater impact on species richness than genistein but that each treatment elicits unique effects on the overall microbial community structure. Abundance of Ruminococcus , a lactic acid producer, common among CON mice was rescued only in HFD + Exe + Gen mice. The lack of an effect with Exe or Gen alone is interesting given that Ruminococcus has independently been shown to convert Gen to active secondary metabolites and has also been positively associated with Exe. This may suggest that a synergistic approach is required to overcome the negative gut microbial effects of a HFD. Further study is needed to understand how gut microbiome changes relate to metabolic health outcomes. Support or Funding Information Midwestern Arizona Alzheimer’s Consortium (AZ0049) Alpha diversity boxplot for Observed OTUs. (*) denotate significant group differences (p<0.05) after Benjamini‐Hochberg correction for multiple comparisons. Treatment groups are as follows: (*) Control differed to HFD and HFD + Gen; (**) HFD vs HFD + Exe; (***) HFD vs HFD + Exe + Gen. High Fat Diet (HFD); (****) HFD+ Exe vs HFD + Gen; (*****) HFD + Exe + Gen vs HFD+ gen. Exercise (Exe); Genistein (gen). Figure 1 Effect size of differentially abundant genera of Rominococcus all‐against‐all Linear discriminate analysis of effect size (LEfSe) between a) Control, High‐Fat Diet (HFD), HFD + Exercise (Exe), HFD +Genistein (Gen), and HFD + Exe + Gen groups b) between High‐Fat Diet (HFD), HFD + Exercise (Exe), HFD +Gcnistein (Gen), and HFD + Exe + Gen groups The phylogenetic features were tested by LEfSe to reveal the genera which significantly differed between the different groups. Figure 2