Overall structural changes of gut microbiota in life-long CR

To determine whether the CR-mediated protection of mice against obesity-associated metabolic syndrome and promotion of healthy aging are associated with alteration of gut microbiota structure, we first profiled the overall structural changes of gut microbiota from all available animals at 62, 83 and 141 weeks of age by bar-coded pyrosequencing of the V3 region of 16S rRNA genes. Of 293,557 valid reads from 288 samples with an average of 1,019 reads per sample (±205 s.d.), 4,613 species-level operational taxonomic units (OTUs) were delineated using 97% as a homology cut-off value (Supplementary Fig. S1).

β-Diversity analysis can indicate the extent of similarity between microbial communities by measuring the degree to which membership or structure is shared between communities23. Based on the data matrix of the weighted UniFrac distance, unweighted pair-group method using arithmetic averages and principal coordinate analysis showed both age-dependent and diet-responsive structural rearrangement of gut microbiota (Fig. 1 and Supplementary Fig. S2). Although no significant age-related shift of gut microbiota was observed around mid-life ages (between 62 and 83 weeks), the gut microbiota from all groups of mice alive at the late-life age of 141 weeks displayed the same trend of moving into an ‘aging space.’ Conversely, separated microbiota clusters were observed in high-fat diet-fed mice relative to low-fat diet-fed mice at the two mid-life ages. Moreover, in parallel with its profound effects on health improvement and longevity, CR showed more prominent impact on the overall architecture of gut microbiota than exercise, particularly with unique microbiota clusters detected in the LFD+CR group both at mid-life and late-life ages. The differences between the gut microbiota of animals with or without voluntary exercise were not significant in the present study. These results suggest a possible correlation of the clustering pattern of gut microbiota with the health conditions in response to life-long nutritional intervention.

Figure 1: Age-dependent and diet-responsive alteration trajectories of global gut microbiota structures. (a) Unweighted pair-group method using arithmetic average based on the weighted UniFrac distance of gut microbiota from the six groups of mice at 62, 83 and 141 weeks (wk) of age. The average relative abundance (% of total 16S rRNA gene V3 region sequences) of bacterial lineages of the gut microbiota within each group of mice is displayed as pie charts at the phylum level. Weighted UniFrac principal coordinate analysis of animals at (b) 62 (LFD, n=21; LFD+CR, n=29; LFD+Ex, n=22; HFD, n=28; HFD+CR, n=29; and HFD+Ex, n=23), (c) 83 (LFD, n=16; LFD+CR, n=22; LFD+Ex, n=19; HFD, n=12; HFD+CR, n=14; and HFD+Ex, n=15) and (d) 141 (LFD, n=6; LFD+CR, n=15; LFD+Ex, n=6; HFD, n=0; HFD+CR, n=10; and HFD+Ex, n=1) weeks of age. Full size image

Specific phylotypes modulated by life-long CR

As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe)24 was employed to identify specific phylotypes responding to life-long CR at both mid-life (62 weeks of age) and late life (141 weeks of age). We did not analyse data at 83 weeks of age because there is no significant age-related shift of gut microbiota between 62 and 83 weeks.

In mid-life, 34 phylotypes at the OTU level were discovered as high-dimensional biomarkers for separating gut microbiota between LFD and LFD+CR mice (Fig. 2a and Supplementary Table S1). Sixteen of these OTUs were higher, and eighteen were lower in the CR than in the ad libitum group. For example, the abundances of these selected phylotypes in Streptococcaceae (OTU65 belonging to Lactococcus) and TM7 (OTU98) were lower in CR animals. Interestingly, OTU45 in the genus Lactobacillus was one of the most predominant phylotypes in bacterial communities of LFD+CR mice but was notably low in LFD mice (12.4% versus 0.05%, respectively; P<0.001, one-way ANOVA).

Figure 2: Key phylotypes of gut microbiota responding to life-long CR identified using LEfSe. (a) LFD (n=21) versus LFD+CR (n=29) mice at 62 weeks. (b) LFD (n=6) versus LFD+CR (n=15) mice at 141 weeks. (c) HFD (n=28) versus HFD+CR (n=29) mice at 62 weeks. The left histogram shows the LDA scores computed for features (on the OTU level) differentially abundant between the ab libitum and CR mice. The right heat map shows the relative abundance (log 10 transformation) of OTUs. Full size image

At the late-life age of 141 weeks, 27 OTUs were higher and 27 were lower in the LFD+CR group than in the LFD group (Fig. 2b and Supplementary Table S2). Ten of these OTUs were also significantly different between the two treatment groups in mid-life. For example, although OTU45 in the genus Lactobacillus was not the predominant phylotype in bacterial communities of LFD+CR mice, the relative abundance of this OTU was still higher in LFD+CR mice than in LFD mice (1.7% versus 0.024%, respectively; P<0.001, one-way ANOVA). Different from mid-life, the OTUs belonging to Bifidobacterium were higher in LFD+CR mice, but the OTU469 of Desulfovibrionaceae was lower in LFD+CR mice. Some members in the genus Bifidobacterium are well-known probiotic strains25, and some in the family Desulfovibrionaceae have previously been found to be positively associated with obesity and inflammation13.

The mice had a significantly different gut microbiota structure between the LFD and HFD groups (Supplementary Fig. S3), confirming results of previous studies13,18. CR also shifted the gut microbiota in mice fed with high-fat diet but not as dramatic compared with their low-fat diet companions (Fig. 1b and Supplementary Fig. S4). In mid-life, 30 phylotypes were selected as key variables for separating the gut microbiota under different food intake conditions (Fig. 2c and Supplementary Table S3); 18 of them were higher and 12 were lower in the HFD+CR group than in the HFD group. All the phylotypes in Porphyromonadaceae were higher in the HFD+CR than in the HFD group. Most of the OTUs responding to CR in HFD+CR mice were not found in LFD+CR mice. Only three OTUs (in Lactococcus (OTU65), Bacteroidales (OTU366) and Peptostreptococcaceae (OTU37), respectively) were reduced, and three OTUs in Tannerella (OTU119, 155 and 267) were increased by CR both with mice fed with high-fat diet and low-fat diet. Because all the HFD mice had died before 141 weeks, we could not obtain any data regarding gut microbiota responding to restriction of high-fat diet intake at the late-life stage of mice.

We also employed partial least square discriminate analysis to confirm the results, and the identified specific phylotypes responding to life-long CR were similar to those from LEfSe (Supplementary Figs S5 and S6).

We also identified a few OTUs that were different between the mice in the exercise group and their ad libitum companions by LEfSe; however, the relative abundance of these OTUs was very low (Supplementary Fig. S7). Conversely, efforts to classify groups with or without voluntary exercise on the same diet with partial least square discriminate analysis did not establish validated models, indicating that the differences between the gut microbiota of animals with or without voluntary exercise were not significant in the present study.

Structural modulation of gut microbiota during aging

Further analysis suggested that CR significantly affected the succession of gut microbiota during aging. Division-level analysis showed that the Firmicutes/Bacteroidetes ratio of gut microbiota in all the mouse groups decreased from 62 to 141 weeks (Fig. 1a). Using LEfSe, we compared the gut microbiota of mice in each group between mid-life and late life to identify the specific phylotypes with OTU levels associated with aging.

In LFD mice, the phylotypes mainly responsible for decrease of the phylum Firmicutes during aging were in the genus Allobaculum (12 OTUs; 28.1% at 62 weeks versus 0.20% at 141 weeks) (Fig. 3a and Supplementary Table S4). In LFD+CR mice, not only OTUs in the genus Allobaculum (29.7% at 62 weeks versus 7.2% at 141 weeks) but also OTUs in the genus Lactobacillus (21.0% at 62 weeks versus 2.0% at 141 weeks) made a significant contribution to the decrease of Firmicutes during aging (Fig. 3b and Supplementary Table S5).

Figure 3: Key phylotypes of gut microbiota responding to aging. (a) LFD+CR mice (n=15). (b) LFD mice (n=6). The left histogram shows the LDA scores computed for features (OTU level) differentially abundant between 62 and 141 weeks. The right heat map shows the relative abundance (log 10 transformed) of OTUs. Full size image

Conversely, the increase of the phylum Bacteroidetes during aging in LFD mice was due to the increase of OTUs in the genus Bacteroides (family Bacteroidaceae; 0.84% at 62 weeks versus 26.7% at 141 weeks). However, in LFD+CR mice, OTUs in the family Porphyromonadeceae (9.4% at 62 weeks versus 28.2% at 141 weeks), instead of bacteria in Bacteroides, were largely responsible for the increase of Bacteroidetes with age. Thus, the apparent phylum level changes associated with aging were actually mediated by different phylogenetic groups in animals with or without CR treatment.

Correlation of mid-life gut microbiota with lifespan

We next used the Kendall tau rank correlation coefficient to directly measure the correlation between the phylotypes of gut microbiota in mid-life and the lifespan based on two types of diet. We identified that 45 OTUs significantly correlated with lifespan in mice fed on low-fat diet. Except for one Bacteroidales OTU, the remaining 15 OTUs significantly positively correlated with lifespan belonged to Firmicutes. Particularly, eight OTUs in Lactobacillus showed strong correlation with lifespan. The 30 phylotypes negatively correlated with lifespan were distributed in the five Phyla of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and TM7 (Fig. 4a and Supplementary Table S6). In the mice on the high-fat diet, 20 OTUs were positively correlated, and 18 OTUs were negatively correlated with lifespan, most of which were in Firmicutes and Bacteroidetes, except for two Actinobacteria OTUs (Fig. 4b and Supplementary Table S7). Because of the strong impact of different diet backgrounds on the gut microbiota, only three OTUs in Lactococcus (OTU65), Bacteroidales (OTU366) and Peptostreptococcaceae (OTU37) showed the same behaviour both in mice on low-fat diet and high-fat diet. These three OTUs were negatively correlated with lifespan. We also found that the OTUs belonging to the same family or genus could have opposite correlation with lifespan. For example, in the mice fed with low-fat diet, there were three OTUs in Lachnospiraceae positively correlated with lifespan, but the other four OTUs in the same family were negatively correlated with lifespan.

Figure 4: Correlation of mid-life gut microbiota with lifespan. The phylotypes significantly correlated with lifespan in mid-life (62 weeks) gut microbiota of animals on (a) low-fat diet (LFD, n=15; LFD+CR, n=22; and LFD+Ex, n=17) and (b) high-fat diet (HFD, n=21; HFD+CR, n=21; and HFD+Ex, n=18). The left heat map shows the correlation coefficient between these OTUs and physiological parameters of mid-life. The right heat map shows the relative abundance (log 10 transformed) of OTUs. The bottom bar shows the lifespan of each mouse. Full size image

Mid-life metabolic phenotypes, such as food intake, body weight and fat content, were highly correlated with lifespan, a finding that has been reported by Zhou et al22. Most of the OTUs significantly positively correlated with lifespan showed strong negative correlation with food intake, body weight and fat content, and vice versa (Fig. 4a).

CR reduces antigen load to the hosts from the gut microbiota

Through long-term CR, the relative abundance of the OTUs negatively correlated with lifespan was significantly reduced, and the relative abundance of OTUs positively correlated with lifespan was increased in mice both on low-fat diet and high-fat diet (Fig. 5a and Supplementary Figs S8 and S9). The serum levels of lipopolysaccharide (LPS)-binding protein (LBP) from all available animals at mid-life were measured to determine the antigen load from the gut microbiota. Compared with their ab libitum companions, LBP levels were lower in mice from the two CR intervention groups (Fig. 5c). Conversely, exercise showed no significant impact on both the relative abundance of OTUs correlated with lifespan and the serum level of LBP. These results suggest that modulation of the gut microbiota by CR could significantly reduce the antigen load to the host, contributing to CR-induced lifespan extension.