Antibiotic treatment extensively depletes commensal gut microbiota

Following the schematic as described in Fig. 1A, we investigated four groups of mice in the experiment. In the control group (CTRL), the mice were continuously fed normal chow ad libitum. The calorie-restricted group (CR) was fed with a 70% of normal chow based on the food intake of CTRL group. Microbiota was depleted by antibiotic treatment in mice continuously fed ad libitum (AB) or fed with a 30% calorie-restriction diet (AB + CR). We started treating mice in AB and AB + CR groups with four nonabsorbable broad-spectrum antibiotics at the onset of calorie restriction. Fecal bacterial loads were examined by cultivation of anaerobic microbes using serial dilutions of resuspended fecal pellets on brain heart infusion (BHI) agar plates. Antibiotic treatment remarkably reduced culturable gut microbes by about one million-fold (Fig. 1B). Significant reduction of community diversity and richness of gut microbiota by the antibiotics were reflected by analyses of shannon index (Fig. 1C) and sobs index (Fig. 1D) via 16S rRNA gene sequencing, further indicating the effectiveness of antibiotic treatment.

Figure 1 Depletion of gut microbiota after antibiotic treatment. (A) Schematic design of the experiment. AL, ad libitum; Abx, antibiotic treatment. (B) Fecal bacterial loads in all groups of mice. Experiments were repeated 3 times. (C,D) Effects of antibiotic treatment on diversity and richness of fecal microbiota revealed respectively by shannon index (C) and sobs index (D). Data are expressed as means ± s.e.m, n = 9–10 per group. Full size image

Microbiota-depleted mice are resistant to CR-induced body weight loss

Upon antibiotic treatment and calorie restriction, the control mice and the microbiota-depleted mice responded differently to calorie restriction. As expected, CR was able to reduce body weight starting from the second week of application of CR diet (Fig. 2A). Since then, the mice without gut microbiota (AB + CR) experienced less body weight loss than the CR group (Fig. 2A), although the two groups of mice had equal levels of food intake (Fig. 2B). The significant discrepancy of body weight between the two groups stayed until the end of the experiment (Fig. 2A). In addition, at the end of the experiment, AB group gained more weight than the CTRL group (Fig. 2A), accompanied by an increase in food intake in the last few weeks (Fig. 2B). These results, therefore, indicated that gut microbiota plays an important role in CR-induced loss of body weight.

Figure 2 Body weight and metabolic alterations upon CR and gut microbiota depletion. (A) Body weight curves of the mice (n = 9–10 per group). **p < 0.01 between CR and AB + CR groups, #p < 0.05 and ###p < 0.001 between CTRL and AB groups. (B) Food intake (n = 9–10 per group). *p < 0.05 between AB and CTRL groups. (C,D) Quantification of body fat mass (C) and lean mass (D) by MRI scans (n = 9–10 per group). (E–H) Analyses with metabolic chamber to quantitate O 2 consumption (E), CO 2 production (F), respiratory exchange ratio (RER) (G) and energy expenditure (EE) (H) (n = 4 for each group). Data are expressed as means ± s.e.m. Full size image

Analyses of metabolic and blood parameters

We analyzed a few metabolic parameters of the mice. As compared to the mice without antibiotic treatment, microbiota-depleted mice were characterized by a significant increase in total body fat and a decrease in lean mass as determined by MRI (Fig. 2C,D). CR could slightly reduce the ratio of lean mass (Fig. 2D). However, the AB + CR group lost more lean mass than the CR group (Fig. 2D). In terms of metabolic rate or energy expenditure (EE) represented by O 2 consumption, CO 2 production and energy expenditure, the AB + CR group had the lowest metabolic rate among the four experimental groups, especially in the dark phase (Figs 2E–G and S1). In addition, CR was able to reduce metabolic rate during the dark period (Fig. 2E–G). Respiratory exchange ratio (RER) was reduced by either calorie restriction or antibiotic administration in light phase (Fig. 2H). In dark phase, calorie restriction was more effective to reduce RER in the mice treated with antibiotics (Fig. 2H). As decreased energy expenditure was commonly associated with weight gain and obesity29,30, our data suggested that the observed abrogation of CR-mediated body weight loss by microbiota deletion might be caused by a decrease in metabolic rate upon antibiotics administration. In other words, gut microbiota is likely important for the mice to maintain a relatively high level of metabolic rate so that depletion of the microbiota would result in a significant reduction of metabolic rate.

We also determined the fasting blood glucose and plasma levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Antibiotic treatment elevated fasting blood glucose level and plasma TC level in the mice (Fig. 3A,B), with mild alteration on plasma LDL-C level (Fig. 3C). These data indicated that gut microbiota depletion is able to increase the risk of metabolic dysregulation as the elevations of blood glucose and cholesterol levels are considered as the hallmarks of metabolic syndrome31,32. On the other hand, the four groups of mice had minimal change in plasma TG and HDL-C (Fig. 3D,E). In addition, the AB + CR mice had a significant elevation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as compared to other groups (Fig. 3F,G), indicating that liver damage might occur in response to calorie restriction in the absence of gut microbiota.

Figure 3 Impacts of CR and gut microbiota depletion on blood parameters. (A) Fasting blood glucose level in different groups of mice. (B–G) Blood levels of TC (B), LDL-C (C), TG (D), HDL-C (E), AST (F) and ALT (G) of the mice at the end of the experiment. Data are expressed as means ± s.e.m, n = 9–10 per group. Full size image

We also analyzed the histological and morphological changes of the mice. Antibiotic-treated mice were characterized by a significant increase in the length of the small intestine (Fig. S2A) and decreased liver/body weight ratio (Fig. S2B) but without apparent changes in the histology of the liver (figure not shown). Furthermore, under microscopical examination, hematoxylin-eosin staining of the jejunum sections in the antibiotic-treated mice revealed longer and thinner intestinal villi than the mice without antibiotic treatment (Fig. S2C).

Gut microbiota depletion and calorie restriction alters metabolism-modulating hormones

Interestingly, AB mice exhibited hyperphagia characterized by a significant increase in food intake (Fig. 2B). We thus assumed that the gut microbiota might be involved in the secretion of hormones that regulate body weight and appetite33,34. To verify our hypothesis, we determined the plasma levels of insulin, leptin, gastric inhibitory polypeptide (GIP) and peptide YY (PYY). The overall levels of these four hormones in all four groups were visualized by a heatmap shown in Fig. 4A. Leptin is a hormone that helps to regulate energy balance by suppressing hunger35 and leptin deficiency is commonly associated with obesity36. Calorie restriction could lower the plasma level of leptin, the effect of which was enhanced when the mice were exposed to antibiotic treatment (Fig. 4B). Insulin is known to regulate the metabolism of carbohydrates, lipids and proteins by aiding the body to store the glucose. Notably, similar to leptin, insulin is also an acute appetite suppressant37. Antibiotic treatment significantly decreased the plasma insulin level (Fig. 4C), consistent with the observed elevation of fasting blood glucose level upon antibiotics administration (Fig. 3A). GIP belongs to the family of incretins that are released by nutrients from the gastrointestinal tract to amply insulin secretion38. Besides its effects to induce insulin secretion upon glucose administration and to regulate fatty acid metabolism, GIP was recently found to be an obesity-promoting factor by acting on adipocytes39,40. Intriguingly, AB + CR mice had the highest level of GIP level among the four groups of mice (Fig. 4D). PYY is a hormone produced in the small intestine and helps to reduce appetite and limit food intake41. The plasma level of PYY in the AB + CR group was significantly higher than that of the CTRL group (Fig. 4E). Collectively, these data indicated that gut microbiota plays an important role in modulating hormones that regulate metabolism in the mice.

Figure 4 Role of CR and gut microbiota depletion on metabolic hormones. (A) Changes in plasma levels of related hormones shown by heatmap. (B–E) Blood levels of leptin (B), insulin (C), GIP (D) and PYY (E). Data are expressed as means ± s.e.m, n = 9–10 per group. Full size image

Calorie restriction alters the composition of gut microbiota

We then performed 16S rRNA gene sequencing with the mice feces collected before and after administration of calorie restriction and/or antibiotic treatment, aiming to find how gut microbiota responds to calorie restriction and mediates the changes of body weight and metabolism. By significance tests for differences in α diversity, the gut microbiota of CR mice featured a markedly increased shannon index (Fig. 5A) and sobs index (Fig. 5B). As shown by respective rarefaction curves (Fig. S3), these curves became much flatter to the right, indicating that a reasonable number of sequences were taken and the α diversity of the sampled community was sufficiently extrapolated. Collectively, these data indicate that calorie restriction could render the gut microbiota a more balanced and diversified ecosystem.

Figure 5 Structural rearrangement of gut microbiota in calorie-restricted mice. (A,B) Shannon index (A) and sobs index (B) of gut microbes as response to CR. (C) Composition of gut microbiota in CTRL and CR groups as shown in pie chart. (D) Partial least squares discriminant analysis (PLS-DA) of all the groups. (E) Generic differences between CTRL and CR groups with p-value < 0.1 in order of abundance. (F–H) Generic differences in Lactobacillus (F), Bifidobacterium (G) and Helicobacter (H) in the CTRL and CR groups at the 4th and 10th week of the experiment. Data are expressed as means ± s.e.m, n = 4–5 per group. *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

The calorie restriction-induced difference in microbiota composition was illustrated in Fig. 5C. After calorie restriction, the bacterial phyla Bacteroidetes and Actinobacteria were slightly increased, while Firmicutes and Verrucomicrobia were slightly decreased. Also, a structural rearrangement of gut microbiota occurred after calorie restriction as illustrated by a supervised partial least squares discriminant analysis (PLS-DA) and hierarchical clustering (Figs 5D and S4). Notably, the antibiotic-treated mice exhibited no significant structural modulation under a calorie-restricted diet (Fig. 5D). Actually, based on the analysis of β diversity distance matrix, the bacterial structures in AB and AB + CR groups were more alike to each other after the experimental treatment (Fig. S5), as antibiotics extensively depleted resident gut microbiota.

We next performed linear discriminant analysis (LDA) effect size (LEfSe) analysis and identified a few bacterial genera that were significantly different between CTRL and CR groups (Figs 5E and S6). Lactobacillus and Bifidobacterium are widely approved probiotic genera with extensive health-promoting and immunomodulatory properties42,43,44. Compared to the CTRL group, the proportion of Lactobacillus and Bifidobacterium was increased in the CR group (p < 0.05 and p = 0.09 respectively, Fig. 5F,G). Helicobacter is a bacterial genus living mostly in the upper gastrointestinal tract which was often considered to be infectious and pathogenic45. We found that Helicobacter genus was significantly reduced by calorie restriction (Fig. 5H). In addition, a few other genera were altered by calorie restriction such as Lachnospiraceae NK4A136, Parasutterella, Clostridiales vadinBB60, Lachnoclostridium, Oscillibaster, Roseburia and Gordonibacter (Figs 5E and S6). Together, these data suggested that the composition and architecture of gut microbiota are altered by calorie-restricted diet, likely contributing to the metabolic benefits of CR.

Gut microbiota contribute to CR-mediated metabolic improvement

To further investigate whether gut microbiota alteration during CR is causally associated with metabolic improvement of the mice, we performed fecal microbiota transplantation (FMT) in diet-induced obesity (DIO) mice using microbiota collected respectively from mice fed normal chow ad libitum (AL) or 30% CR (Fig. 6A). Upon transplantation, the microbiota of the recipient mice resembled their corresponding donor groups to certain degrees as evaluated by OTU level based on 16s rRNA gene sequencing (Fig. S7). Notably, compared to the mice undergoing FMT from the control AL mice (HAL group), the mice that received FMT from the CR mice (HCR group) exhibited reduced body weight gain (Fig. 6B), accompanied by a decrease in body fat mass and an increase in lean mass (Fig. 6C,D). In addition, there was a slight improvement in glucose tolerance together with a significant reduction of fasting blood glucose level in the HCR group as compared to the HAL group (Figs 6E and S8A). In addition, HCR mice exhibited a significant decrease in blood leptin level (Fig. S8B), which was largely consistent with our observation with the CR mice (Fig. 4B). We also determined the level of plasma insulin level but no significant difference was found among the three HFD groups (Fig. S8C). This result was also concordant with our previous data that plasma insulin level had minimal change between CTRL and CR groups (Fig. 4C). Besides, some other metabolic and blood parameters such as plasma AST, ALT, TG, TC, HDL-C, LDL-C and hepatic TC, showed no significant changes among these groups of mice (Fig. S9). Furthermore, we examined histological changes of the liver by hematoxylin-eosin staining. As compared to the HBL group, mice in the HAL group exhibited similar morphology shown as a similar degree of hepatosteatosis induced by HFD (Fig. 6F). However, the HFD-induced hepatic steatosis appeared to be significantly alleviated in the HCR group (Fig. 6F). Consistently, the hepatic triglyceride level of the HCR group was significantly lower than those of HBL and HAL groups (Fig. 6G).

Figure 6 FMT from CR mice attenuated diet-induced obesity and partially ameliorated metabolic disturbances. (A) Schematic design of the FMT experiment. NC, normal chow; HFD, high fat diet. (B) Body weight curves of mice fed HFD (n = 6–8 per group). (C,D) Quantification of body fat mass (C) and lean mass (D) by MRI scans (n = 6–8 per group). (E) Oral glucose tolerance test of mice received FMT (n = 6–8 per group) with area under curve (AUC) shown in the inlet. (F) Representative H&E staining of the liver. Scale bar, 50 μm. (G) Hepatic triglyceride levels of the mice fed HFD (n = 6–8 per group). Data are expressed as means ± s.e.m, *p < 0.05, **p < 0.01. Full size image

We also analyzed the microbial composition of the three groups of mice fed with HFD. Compared to the HBL group, FMT elevated the richness of microbiota reflected as sobs index (Fig. 7A). The diversity of microbial community shown by shannon index among the three groups had minimal changes (Fig. 7B). However, the HAL and HCR groups did have a structural discrepancy in the gut microbiota as illustrated by principal co-ordinates analysis (PCoA) and hierarchical clustering (Figs 7C and S10). We also applied LEfSe analysis and identified a few altered bacteria between the HAL and HCR groups (Fig. 7D,E). Compared to the HAL group, the microbial composition in the HCR group had increases in the abundances of Firmicutes (59.75% vs. 71.68%, p < 0.05) and Actinobacteria (1.25% vs. 4.13%, p = 0.06), together with a decrease in the abundance of Bacteroidetes (35.35% vs. 21.46%, p < 0.05). At genus level, the HCR group had a significant increase in genus Faecalibaculum, which significantly contributed to the overall differences (Fig. 7E). Besides, abundances of some other genera were different between the HAL and HCR groups such as Bacteroidales S24-7, Gordonibacter, Rikenella, Coriobacteriaceae UCG-002 and Coprococcus 1 (Figs. 7D,E).