Research Article

Gut Microbiota from Twins Discordant for Obesity Modulate Metabolism in Mice

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Science  06 Sep 2013:
Vol. 341, Issue 6150, 1241214
DOI: 10.1126/science.1241214

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  1. Fig. 1 Reliable replication of human donor microbiota in gnotobiotic mice

    (A) Features of the four discordant twin pairs. (B) Assembly of bacterial communities in mice that had received intact and uncultured fecal microbiota transplants from the obese and lean co-twins in DZ pair 1. Principal coordinates analysis plot of principal coordinate 1 (PC1) based on an unweighted UniFrac distance matrix and 97%ID OTUs present in sampled fecal communities. Circles correspond to a single fecal sample obtained at a given time point from a given mouse and are colored according to the experiment (n = 3 independent experiments). Note that assembly is reproducible within members of a group of mice that have received a given microbiota, as well as between experiments. (C) Body composition, defined by QMR, was performed 1 day and 15 dpc of each mouse in each recipient group. Mean values (± SEM) are plotted for the percent increase in fat mass and lean body mass at 15 dpc for all recipient mice of each of the four obese co-twins’ or lean co-twins’ fecal microbiota, normalized to the initial body mass of each recipient mouse. A two-way ANOVA indicated that there was a significant donor effect (P ≤ 0.05), driven by a significant difference in adiposity and total body mass between mice colonized with a lean or obese co-twin donor’s fecal microbiota (adjusted P ≤ 0.05; SEmbedded Imagedák’s multiple comparison test). (D) Mean values (± SEM) are plotted for the percent change in fat mass at 15 dpc for all recipient mice of each of the four obese co-twins’ or lean co-twins’ fecal microbiota. Data are normalized to initial fat mass (n = 3 to 12 animals per donor microbiota; 51 to 52 mice per BMI bin; total of 103 mice). ***P ≤ 0.001, as judged by a one-tailed unpaired Student’s t test. (E) More prolonged time course study for recipients of fecal microbiota from co-twins in discordant DZ pair 1 (mean values ± SEM plotted; n = 4 mice per donor microbiota). The difference between the gain in adiposity calculated relative to initial fat mass (1 dpc) between the two recipient groups of mice is statistically significant (P ≤ 0.001, two-way ANOVA).

  2. Fig. 2 Cohousing Obch and Lnch mice transforms the adiposity phenotype of cage mates harboring the obese co-twin’s culture collection to a lean-like state

    (A) Design of cohousing experiment: 8-week-old, male, germ-free C57BL/6J mice received culture collections from the lean (Ln) twin or the obese (Ob) co-twin in DZ twin pair 1. Five days after colonization, mice were cohoused in one of three configurations: Control groups consisted of dually housed Ob-Ob or Ln-Ln cage mates; the experimental group consisted of dually housed Obch-Lnch cage mates (data shown from five cages per experiment; two independent experiments) or Obch-Lnch- GFch-GFch cage mates (n = 3 cages per experiment). All mice were fed a LF-HPP diet. (B) Effects of cohousing on fat mass. Changes from the first day after cohousing to 10 days after cohousing were defined using whole-body QMR. *P ≤ 0.05, **P ≤ 0.01 compared with Ob-Ob controls, as defined by one-tailed unpaired Student’s t test. (C) Targeted GC-MS analysis of cecal short-chain fatty acids. Compared with Ob-Ob controls, the concentrations of propionate and butyrate were significantly higher in the ceca of Obch, Ln-Ln, Lnch, and GFch mice. (D) Nontargeted GC-MS analysis of cecal levels of cellobiose and “maltose or a similar disaccharide.” *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005. (E) Evidence that bacterial species from the Lnch microbiota invade the Obch microbiota. Shown are SourceTracker-based estimates of the proportion of bacterial taxa in a given community sampled from a cage mate. For Obch-Lnch cohousing experiments, Obch and Lnch microbiota were designated as sink communities, whereas the gut microbiota of Ob-Ob and Ln-Ln controls (at 5 dpc) were considered source communities. Red indicates species derived from the Lnch gut microbial community. Blue denotes species derived from the Obch microbiota. Black denotes unspecified source (i.e., both communities have this species), whereas orange indicates an uncertain classification by the SourceTracker algorithm. “Other” after a genus name means unclassified. An asterisk placed next to a species indicates that it is a successful invader as defined in the text. Average relative abundance (RA) in the fecal microbiota is shown before cohousing (b, at 5 dpc) and after cohousing (a, at 15 dpc). The average fold-change (fc) in relative abundance for a given taxon, for all time points before and after cohousing is shown (excluding the first 2 days immediately after gavage of the microbiota and immediately after initiation of cohousing). Note that only taxa with significant changes in relative abundance between samples collected before and samples collected after cohousing are shown. For a full accounting of all taxa, see table S8A.

  3. Fig. 3 Effect of cohousing on metabolic profiles in mice consuming the LF-HPP diet

    (A) Spearman’s correlation analysis of cecal metabolites and cecal bacterial species–level taxa in samples collected from Obch, Lnch, GFch, Ln39ch, and ObchLn39 cage mates and from Ob-Ob and Ln-Ln controls (correlations with P ≤ 0.0001 are shown). Taxonomic assignments were made using a modified taxonomy from the National Center for Biotechnology Information (of the U.S. National Institutes of Health) (23). Bacterial species and cecal metabolites enriched in animals colonized with either the Ln or Ob culture collections are colored red and blue, respectively. An asterisk in the colored box indicates that a taxon or metabolite is significantly enriched in mice colonized with Ln (red) or Ob (blue) culture collections. Bacterial species colored red denote significant invaders from Lnch mice into the gut microbiota of Obch cage mates. (B) Cecal bile acids measured by UPLC-MS. Note that levels are plotted as log-transformed spectral abundances. Significance of differences relative to Ob-Ob controls were defined using a two-way ANOVA with Holm-Šidák’s correction for multiple hypotheses; *P ≤ 0.05; **P ≤ 0.01. (C and D) QPCR assays of FXR and Fgf15 mRNA levels in the distal ileum. Data are normalized to Ln-Ln controls. (E) QPCR of hepatic Cyp7a1 mRNA, normalized to Ln-Ln controls. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.001 (defined by one-tailed, unpaired Student’s t test using Ob-Ob mice as reference controls). (F) Correlating cecal bile acid profiles with the FXR-Fgf15-Cyp7A signaling pathway in the different groups of mice. (Top) The dendrogram highlights the differences in the profiles of 37 bile acid species between Ob-Ob controls and the other three treatment groups (table S11). The dendrogram was calculated using the Bray-Curtis dissimilarity index and the average relative abundance of each bile acid species among all mice belonging to a given treatment group.

  4. Fig. 4

    Effects of NHANES-based LoSF-HiFV and HiSF-LoFV diets on bacterial invasion, body mass and metabolic phenotypes. (A and B) Mean ± SEM percent changes in total body mass (A) and body composition [fat and lean body mass, normalized to initial body mass on day 4 after gavage (B)] occurring between 4 and 14 days after colonization with culture collections from the Ln or Ob co-twin in DZ pair 1. Cohousing Ln and Ob mice prevents an increased body mass phenotype in Obch cage mates fed the representative LoSF-HiFV human diet (n = 3 to 5 cages per treatment group; 26 animals in total). **P ≤ 0.01, based on a one-way ANOVA after Fisher’s least significant difference test (also see table S14 for statistics). (C) Spearman’s correlation analysis between bacterial species–level taxa and metabolites in cecal samples collected from mice, colonized with culture collections from DZ twin pair 1 Ln and Ob co-twins and fed a LoSF-HiFV diet. Red and blue squares indicate metabolites or taxa that are significantly enriched in samples collected from dually housed Ln-Ln or Ob-Ob controls respectively. An asterisk in the colored box indicates that that a taxon or metabolite is significantly enriched in mice colonized with Ln (red) or Ob (blue) culture collections. (D and E) Mean ± SEM of changes in body mass and body composition in mice colonized with intact uncultured microbiota from DZ twin pair 2 and fed the representative HiSF-LoFV human diet. Ob-Ob controls have greater total and lean body mass than Ln-Ln controls, but this phenotype is not rescued in Obch animals (see table S14 for statistics). *P < 0.05, **P ≤ 0.01 based on a one-way ANOVA. Note that the HiSF-LoFV diet produces a significantly greater increase in body mass, specifically fat mass, in mice harboring the lean co-twins microbiota (Ln-Ln and Lnch) than when they are fed the LoSF-HiFV diet [see (A) versus (D), and (B) versus (E); two-way ANOVA with Holm-Šidák’s correction for multiple hypotheses].

  5. Fig. 5

    Invasion analysis of species-level taxa in Obch or Lnch mice fed the NHANES-based LoSF-HiFV diet. Red indicates species derived from the Lnch gut microbial community. Blue denotes species derived from the Obch microbiota. The mean relative abundance of each species-level taxon before (b: 3 and 4 dpc) and after (a: 8, 10 and 14 dpc) cohousing is noted. Average fold-change (fc) in relative abundance of taxa before and after colonization was defined as in the legend to Fig. 2E. An asterisk (*) denotes bacterial species that satisfy our criteria for classification as successful invaders (see text).

  6. Fig. 6 Acylcarnitine profile in the skeletal muscle of mice colonized with the Ob or Ln culture collections from DZ twin pair 1 and fed the LoSF-HiFV diet

    Each column represents a different animal and each row a different acylcarnitine. The identities and levels of these acylcarnitines were determined by targeted MS/MS (see table S15 for mean values ± SEM for each treatment group). A two-way ANOVA with Holm-Šidák’s correction was used to calculate whether the level of each acylcarnitine was significantly different between Ob-Ob and Ln-Ln, Lnch, or Obch animals. *P ≤ 0.05.

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