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Comparative Study
. 2008 Apr 17;3(4):213-23.
doi: 10.1016/j.chom.2008.02.015.

Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome

Peter J Turnbaugh  1 , Fredrik Bäckhed, Lucinda Fulton, Jeffrey I Gordon
Affiliations
Comparative Study

Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome

Peter J Turnbaugh et al. Cell Host Microbe. .

Abstract

We have investigated the interrelationship between diet, gut microbial ecology, and energy balance using a mouse model of obesity produced by consumption of a prototypic Western diet. Diet-induced obesity (DIO) produced a bloom in a single uncultured clade within the Mollicutes class of the Firmicutes, which was diminished by subsequent dietary manipulations that limit weight gain. Microbiota transplantation from mice with DIO to lean germ-free recipients promoted greater fat deposition than transplants from lean donors. Metagenomic and biochemical analysis of the gut microbiome together with sequencing and metabolic reconstructions of a related human gut-associated Mollicute (Eubacterium dolichum) revealed features that may provide a competitive advantage to members of the bloom in the Western diet nutrient milieu, including import and processing of simple sugars. Our study illustrates how combining comparative metagenomics with gnotobiotic mouse models and specific dietary manipulations can disclose the niches of previously uncharacterized members of the gut microbiota.

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Figures

Figure 1
Figure 1. Experimental design
(A) Diet-induced obesity (DIO) in germ-free mice colonized with a complex microbial community. (B) Conventionally-raised (CONV-R) wild-type mice fed a Western or CHO diet. (C) Specific dietary shifts after two months on the Western diet. (D) Microbiota transplantation experiments from donor mice on multiple diets to lean germ-free CHO-fed recipients. Numbers in parentheses refer to the age of mice at each step in the protocol. Mouse diets are labeled Western, FAT-R, CARB-R, and CHO (see Tables S1 and S2).
Figure 2
Figure 2. Diet-induced obesity alters gut microbial ecology in conventionalized mice
Adult C57BL/6J conventionalized mice were fed a low-fat high-polysaccharide (CHO) or high-fat/high-sugar (Western) diet. 16S rRNA gene sequence-based surveys and UniFrac-based analyses were performed on the distal gut (cecal) contents of ten mice (n=5 mice/group) and the cecal contents from the donor mouse. Black boxes indicate nodes that were reproduced in >70% of all jackknife replications (n=96 sequences). Pie charts show the average relative abundance of bacterial lineages in the CHO diet versus Western diet cecal microbiota. The asterisk indicates that the sample was also analyzed based on whole community shotgun sequencing.
Figure 3
Figure 3. Diet-induced obesity (DIO) is linked to changes in gut microbial ecology, resulting in an increased capacity of the distal gut microbiota to promote host adiposity
(A) The relative abundance (% of total 16S rRNA gene sequences) of the Firmicutes and Bacteroidetes divisions in the distal gut (cecal) microbiota of conventionalized, wild-type C57BL/6J mice fed a standard low-fat high-polysaccharide chow diet (CHO; n=5) or a high-fat/high-sugar Western diet (n=5). (B) DIO is associated with a marked reduction in the overall diversity of the cecal bacterial community. The Shannon index of diversity was calculated at multiple phylotype cutoffs (defined by % identity of 16S rRNA gene sequences) for each individual cecal dataset using DOTUR (Schloss and Handlesman, 2005). The average diversity at each cutoff is plotted for mice fed the CHO and Western diets. (C) DIO is linked to a bloom of the Mollicutes class of bacteria within the Firmicutes division. The relative abundance of the Mollicutes is shown for conventionalized mice fed the CHO or Western diet. (D) Microbiota transplantation experiments reveal that the DIO community has an increased capacity to promote host fat deposition. Total body fat was measured using dual-energy x-ray absorptiometry (DEXA) before and after a two-week colonization of adult germ-free CHO-fed C57BL/6J wild-type mice with a cecal microbiota harvested from mice maintained on CHO or Western diet (n=14 mice/treatment group). Mean values±SEM are shown. Asterisks in panels A-D indicate that the differences are statistically significant (Student’s t-test, p<0.05), after using the Bonferroni correction to limit false positives.
Figure 4
Figure 4. Phylogeny of selected representatives from the Firmicutes division, including the Mollicute bloom and closely related human strains
16S rRNA gene sequences for previously sequenced Firmicute genomes and Mollicute strains isolated from the human gut were identified in the RDP database (Cole et al., 2005). All Mollicute sequences obtained from conventionalized C57BL/6J mice fed a CHO or Western diet (n=801 sequences) and from our previous survey of obese humans (length>1250 nucleotides; n=571 sequences; Ley et al., 2006b) were separately binned into phylotypes using DOTUR (99% identity; Schloss and Handlesman, 2005). One representative of each of the six dominant mouse phylotypes was chosen (together comprising 81% of the mouse Mollicute sequences) in addition to one representative of each of the ten dominant human phylotypes. Likelihood parameters were determined using Modeltest (Posada and Crandall, 1998) and a maximum-likelihood tree was generated using PAUP (Swofford, 2003). Bootstrap values represent nodes found in >70 of 100 repetitions. Phylotypes from the Mollicute bloom are shown in blue; wedge size is proportional to the indicated relative abundance (% of Mollicute 16S rRNA gene sequences). The Mollicute bloom and relatives are shaded in blue, previously sequenced Mollicutes (including the obligate parasites, Mycoplasma, and Mesoplasma florum) are shaded in yellow, and recently sequenced Firmicutes found in the ‘normal’ distal human gut microbiota are shaded in red. Akkermansia muciniphila, a Verrucomicrobia, was used to root the tree (shaded in green).
Figure 5
Figure 5. Metabolic reconstructions of the Eubacterium dolichum genome and the Western diet microbiome
Predicted gene presence calls for the Western diet microbiome and/or the E.dolichum genome are displayed in the upper right. Fermentation end-products and cellular biomass are highlighted in white ellipses. Note that culture-based studies of E.dolichum have demonstrated its ability to produce lactate, acetate, and butyrate (Moore et al., 1976), suggesting that the apparent gap in the pathway for generating butyrate reflects the draft nature of the genome assembly or the possibility that this organism uses novel enzymes to generate this end-product of anaerobic fermentation. Abbreviations for enzymes (in boldface): Pgi, phosphoglucose isomerase; Pfk, phosphofructokinase; Fba, fructose-1,6-bisphosphate aldolase; Tpi, triose-phosphate isomerase; Gap, glyceraldehyde-3-phosphate dehydrogenase; Pgk, phosphoglycerate kinase; Pgm, phosphoglycerate mutase; Eno, enolase; Pyk, pyruvate kinase; EI, PTS enzyme I; HPr, PTS protein HPr; EIIA/B/C, PTS proteins; DXPS, 1-deoxy-D-xylulose-5-phosphate synthase; DXPR, DXP-reductoisomerase; MEPC, MEP cytidylyltransferase; MEK, CDP-ME kinase; MECS, MECDP-synthase; MDPS, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase; MDPR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; Ldh, L-lactate dehydrogenase; Pfl, pyruvate formate-lyase; Pat, phosphate acetyltransferase; Ak, acetate kinase; Aca, acetyl-CoA C-acetyltransferase; Bhbd, 3-hydroxybutyryl-CoA dehydrogenase; Ech, enoyl-CoA hydratase; Bcd, butyryl-CoA dehydrogenase; Ptb, phosphotransbutyrylase; Bk, butyrate kinase; 1-Pfk, 1-phosphofructokinase; Npd, N-acetylglucosamine-6-phosphate deacetylase; Gpi, phosphoglucosamine isomerase; Fbf, fructan beta-fructosidase.
Figure 6
Figure 6. Metabolic and principal component analysis (PCA) of sequenced Firmicute genomes
(A) PCA analysis of 14 previously sequenced Mollicute genomes (mostly Mycoplasma) and draft genome assemblies of nine human gut-associated Firmicutes (http://genome.wustl.edu/pub/). MetaGene was used to predict proteins from each genome (Noguchi et al., 2006). Proteins were then assigned to KEGG orthologous groups based on homology (BLASTP e-value<10−5; KEGG version 40; Kanehisa et al., 2004). Genomes were clustered based on the relative abundance of KEGG metabolic pathways (number of assignments to a given pathway divided by total number of pathway assignments). Only pathways found at >0.6% relative abundance in at least two genomes were included. The first two components are shown, representing 17% and 8% of the variance respectively. Abbreviations: Mca, Mycoplasma capricolum; Mfl, Mesoplasma florum L1; Mga, Mycoplasma gallisepticum R, Mge, Mycoplasma genitalium G37; Mhy232, Mycoplasma hyopneumoniae 232; Mhy7448, Mycoplasma hyopneumoniae 7448; MhyJ, Mycoplasma hyopneumoniae J; Mmo, Mycoplasma mobile 163K; Mmy, Mycoplasma mycoides subsp. mycoides SC str. PG1; Mpe, Mycoplasma penetrans HF-2; Mpn, Mycoplasma pneumoniae M129; Mpu, Mycoplasma pulmonis UAB CTIP; Msy, Mycoplasma synoviae 53; Upa, Ureaplasma parvum; E.dolichum, Eubacterium dolichum; CL250, Clostridium sp. L2-50; C.symbiosum, Clostridium symbiosum; Dlo, Dorea longicatena; Eel, Eubacterium eligens; Ere, Eubacterium rectale; Eve, Eubacterium ventriosum; Rob, Ruminococcus obeum; and Rto, Ruminococcus torques. (B) KEGG pathway relative abundance has a significant correlation with genome size. A linear regression was performed comparing PCA1 to genome size (or draft assembly size). PCA1 has a significant correlation to genome size (R2=0.9, p<0.05). (C) Metabolic pathways in E.dolichum. Pathways are marked partial if most genes are present and absent if ≤2 genes are present.
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