Structural modulation of gut microbiota in life-long calorie-restricted mice methods
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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) 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.
Specific phylotypes modulated by life-long CR
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 ( and ). 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 strains, and some in the family Desulfovibrionaceae have previously been found to be positively associated with obesity and inflammation.
Specific phylotypes modulated by life-long CR
The mice had a significantly different gut microbiota structure between the LFD and HFD groups ( ), confirming results of previous studies. CR also shifted the gut microbiota in mice fed with high-fat diet but not as dramatic compared with their low-fat diet companions ( and ). In mid-life, 30 phylotypes were selected as key variables for separating the gut microbiota under different food intake conditions ( and ); 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...
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 ( ). 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.
Structural modulation of gut microbiota during 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) ( and ). 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 ( and ).
Structural modulation of gut microbiota during aging
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 ( and ). 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 ( and ). Becau...
Methods
The animal experimental procedures, approved by the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, CAS, were described previously by Zhou et al. Male C57BL/6 J mice at 5 weeks of age were randomly assigned to one of the six groups ( n =30 for each group) and individually caged for a life-long trial: (1) low-fat diet with sedentary activity (LFD), (2) low-fat diet with 30% CR and sedentary activity (LFD+CR), (3) low-fat diet with voluntary running exercise (LFD+Ex), (4) high-fat diet with sedentary activity (HFD), (5) high-fat die with 30% CR and sedentary activity (HFD+CR) and (6) high-fat diet with voluntary running exercise (HFD+Ex). All faecal and serum samples for the current study were collected from this animal trial.
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As an algorithm to robustly identify features that are statistically different among biological classes, linear discriminant analysis (LDA) effect size (LEfSe) 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.
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 ( and ). 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 strains, and some in the family Desulfovibrionaceae have previously been found to be positively associated with obesity and inflammation.
The mice had a significantly different gut microbiota structure between the LFD and HFD groups ( ), confirming results of previous studies. CR also shifted the gut microbiota in mice fed with high-fat diet but not as dramatic compared with their low-fat diet companions ( and ). In mid-life, 30 phylotypes were selected as key variables for separating the gut microbiota under different food intake conditions ( and ); 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.
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 ( ). 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) ( and ). 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 ( and ).
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.
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 ( and ). 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 ( and ). 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 lif...
The animal experimental procedures, approved by the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, CAS, were described previously by Zhou et al. Male C57BL/6 J mice at 5 weeks of age were randomly assigned to one of the six groups ( n =30 for each group) and individually caged for a life-long trial: (1) low-fat diet with sedentary activity (LFD), (2) low-fat diet with 30% CR and sedentary activity (LFD+CR), (3) low-fat diet with voluntary running exercise (LFD+Ex), (4) high-fat diet with sedentary activity (HFD), (5) high-fat die with 30% CR and sedentary activity (HFD+CR) and (6) high-fat diet with voluntary running exercise (HFD+Ex). All faecal and serum samples for the current study were collected from this animal trial.
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