High-fat diet determines the composition of the murine gut microbiome independently of obesity

Abstract

Background & aims: The composition of the gut microbiome is affected by host phenotype, genotype, immune function, and diet. Here, we used the phenotype of RELMbeta knockout (KO) mice to assess the influence of these factors.

Methods: Both wild-type and RELMbeta KO mice were lean on a standard chow diet, but, upon switching to a high-fat diet, wild-type mice became obese, whereas RELMbeta KO mice remained comparatively lean. To investigate the influence of diet, genotype, and obesity on microbiome composition, we used deep sequencing to characterize 25,790 16S rDNA sequences from uncultured bacterial communities from both genotypes on both diets.

Results: We found large alterations associated with switching to the high-fat diet, including a decrease in Bacteroidetes and an increase in both Firmicutes and Proteobacteria. This was seen for both genotypes (ie, in the presence and absence of obesity), indicating that the high-fat diet itself, and not the obese state, mainly accounted for the observed changes in the gut microbiota. The RELMbeta genotype also modestly influenced microbiome composition independently of diet. Metagenomic analysis of 537,604 sequence reads documented extensive changes in gene content because of a high-fat diet, including an increase in transporters and 2-component sensor responders as well as a general decrease in metabolic genes. Unexpectedly, we found a substantial amount of murine DNA in our samples that increased in proportion on a high-fat diet.

Conclusions: These results demonstrate the importance of diet as a determinant of gut microbiome composition and suggest the need to control for dietary variation when evaluating the composition of the human gut microbiome.

Figure 1

Induction of RELMβ expression in the stool and colon is dependent upon gut bacteria. A) RELMβ immunoblot using proteins isolated from fecal pellets collected from wild-type mice fed a standard chow diet for 13 weeks and again after 21 weeks on a high fat diet; B) Quantitative RT-PCR of colonic mRNA for RELMβ in mice fed a standard chow diet and a high fat diet with and without the administration of oral antibiotics, Mean±SEM, N=5 mice per group.

Figure 2

RELMβ KO mice remain comparatively lean on a high fat diet compared to wild-type littermate controls. A) Body weight of female RELMβ wild-type (WT) and Knockout (KO) mice at 13 weeks of age on a standard chow diet or after 21 weeks on a high fat diet, Mean±SEM, N=4-5 mice per group; *p<0.05; B) MRI body composition analysis after 8 weeks on the high fat diet, Mean±SEM, N=4-5 mice per genotype, *p=0.004; C) Daily food intake in RELMβ KO and wild-type mice fed a high fat diet for 4 weeks, Mean±SEM, N=4-5 mice per genotype; D) Percent dietary fat absorbed by RELMβ KO and wild-type mice fed a high fat diet for 5 weeks, Mean±SEM, N=4-5 mice per genotype; E) Rectal temperatures of RELMβ KO and wild-type mice fed a high fat diet for 8 weeks, Mean±SEM, N=4-5 mice per genotype; F) Oxygen consumption (VO₂) measured via indirect calorimetry over 4 hours (light cycle) in RELMβ KO and wild-type mice after 21 weeks on a high fat diet, Mean±SEM, N=4 mice per group, *p<0.0001.

Figure 3

Analysis of gut bacterial communities by 16S rDNA analysis from mice on the standard chow and high fat diets. A) The figure shows the percentages of each community contributed by the indicated Phyla. Diet and genotype are indicated below the figure; B) UniFrac analysis of the bacterial communities studied. Each point corresponds to a community from a single mouse. Samples from the Standard chow and High Fat Diet are from the same mouse, as indicated by the color code. Open symbols indicate high fat diet, closed symbols the standard chow. Circles indicate the knockouts, squares the wild-type controls. Colors indicated individual mice.

Figure 4

Metagenomic analysis of community composition. Samples were pooled for wild-type mice from standard chow (blue) and high fat (yellow) diets; A) Comparison of contributions of bacterial and murine DNA for the two communities. Groups were assigned using MEGAN. The great majority of sequences from “Eukaryota” were murine; B) Metagenomic analysis of bacterial taxa and gene types. The bacterial phyla are indicated along the top of the figure for the most abundant four bacterial phyla (95% of total), the functional categories in the column to the right. The colored tiles in the body of the figure indicate the changes in gene content within each Phylum (that is, changes in gene types are compared between diets considering only sequences from each Phylum). Functional classes were assigned using KEGG and SEED annotation. The color scale (bottom) reports the odds ratio (set to the conservative edge of the confidence interval; if the interval included 1 the value was set to 1).