Blautia Coccoides is a Newly Identified Bacterium Increased by Leucine Deprivation and has a Novel Function in Improving Metabolic Disorders

Abstract

Gut microbiota is linked to human metabolic diseases. The previous work showed that leucine deprivation improved metabolic dysfunction, but whether leucine deprivation alters certain specific species of bacterium that brings these benefits remains unclear. Here, this work finds that leucine deprivation alters gut microbiota composition, which is sufficient and necessary for the metabolic improvements induced by leucine deprivation. Among all the affected bacteria, B. coccoides is markedly increased in the feces of leucine-deprived mice. Moreover, gavage with B. coccoides improves insulin sensitivity and reduces body fat in high-fat diet (HFD) mice, and singly colonization of B. coccoides increases insulin sensitivity in gnotobiotic mice. The effects of B. coccoides are mediated by metabolizing tryptophan into indole-3-acetic acid (I3AA) that activates the aryl hydrocarbon receptor (AhR) in the liver. Finally, this work reveals that reduced fecal B. coccoides and I3AA levels are associated with the clinical metabolic syndrome. These findings suggest that B. coccoides is a newly identified bacterium increased by leucine deprivation, which improves metabolic disorders via metabolizing tryptophan into I3AA.

Keywords: aryl hydrocarbon receptor; blautia coccoides; gut microbiota; indole‐3‐acetic acid; leucine deprivation.

Figure 1

The gut microbiota modified by leucine deprivation is sufficient and necessary for the metabolic improvements. A–J) HFD mice were given with antibiotic cocktail (termed as Abx) in drinking water. After 4 weeks, the Abx mice were transplanted with the fecal microbiota from control diet (Cont‐FMT) or leucine‐deprived diet ((‐) Leu‐FMT) donors daily for 30 days (n = 5–7 biological replicates per group). A) Experimental procedures. B) Fed and fasting blood glucose levels. C) Fed and fasting serum insulin levels assayed by ELISA. D) HOMA‐IR index. E) Glucose tolerance tests. The right panel is the area under curve (AUC). F) Insulin tolerance tests (1 U kg⁻¹). The right panel is the AUC. G) Total fat mass. H) The H&E staining of subcutaneous white adipose tissue (sWAT). Scale bars, 50 µm. The right panel is the frequency distribution of adipocyte cell size in sWAT and the box plot is the average adipocyte diameter. I) Real‐time PCR analysis of browning related genes (Ucp1, Pgc1α, Prdm16, and Cidea) in sWAT. J) Western blot analysis of UCP1 protein levels in sWAT. The bottom panel is the densitometry analysis of UCP1 protein levels. A.U.: arbitrary units. K–P) 10‐week‐old male C57BL/6J wild‐type (WT) mice were given Abx in drinking water or given autoclaved water (Con). After 4 weeks, the Con or Abx mice were treated with the (‐) Leu or Cont diet for 7 days, respectively (n = 5–8 biological replicates per group). K) Experimental procedures. L) Fed and fasting blood glucose levels. M) Fed and fasting serum insulin levels assayed by ELISA. N) HOMA‐IR index. O) Glucose tolerance tests. The right panel is the AUC. P) Insulin tolerance tests (0.5 U kg⁻¹). The right panel is the AUC. All values are expressed as the mean ± SEM. Statistical comparisons were carried out by unpaired two‐tailed Student's t‐test or two‐way ANOVA; * p < 0.05, ** p < 0.01, and n.s.: no significance.

Figure 2

Leucine deprivation alters microbiota composition and increases B. coccoides abundance in mice. The cecal microbiota from 12‐week‐old male C57BL/6J WT mice treated with control diet (Cont) or leucine‐deprived diet ((‐) Leu) were used for bacterial 16S rDNA (A–C, F, and G, n = 6 biological replicates per group) and metagenomic sequencing (D, E, n = 3 biological replicates per group). A) PCoA of the gut microbiota structure based on Bray‐Curtis distance. B) Bacterial taxonomic profiling in the top 20 bacterial genus levels. C) Spearman's correlation analysis of 18 genus changed by (‐) Leu with metabolic parameters. D) The sequencing reads of bacterial species from genus Blautia. E) LEfSe analysis of metagenomic sequencing data. F) Spearman's correlation analysis of the relative abundance of B. coccoides with HOMA‐IR index. G) Spearman's correlation analysis of the relative abundance of B. coccoides with body fat. All values are expressed as the mean ± SEM. Statistical comparisons were carried out by nonparametric Mann‐Whitney U test; * p < 0.05, **p < 0.01.

Figure 3

B. coccoides improves metabolic disorders in HFD mice. NCD and HFD mice were either orally gavage with PBS, heat‐killed B. coccoides GA1 (KBC), or live B. coccoides GA1 (LBC) for 8 weeks (n = 6–8 biological replicates per group). A) Experimental procedures. B) Fed and fasting blood glucose levels. C) Fed and fasting serum insulin levels assayed by ELISA. D) HOMA‐IR index. E) Glucose tolerance tests. The right panel is the AUC. F) Insulin tolerance tests (0.75 U kg⁻¹). The right panel is the AUC. G) sWAT weight. H) The H&E staining of sWAT. Scale bars, 50 µm. The bottom panel is the frequency distribution of adipocyte cell size in sWAT and the box plot is average adipocyte diameter. I) Real‐time PCR analysis of browning related genes (Ucp1, Pgc1α, Prdm16, and Cidea) in sWAT. J) Western blot analysis of UCP1 protein levels in sWAT. The right panel is the densitometry analysis of UCP1 protein levels. A.U.: arbitrary units. All values are expressed as the mean ± SEM. Statistical comparisons were carried out by one‐way ANOVA; *p < 0.05 and **p < 0.01.

Figure 4

B. coccoides increases the circulating I3AA via metabolizing tryptophan. A–E) HFD mice were either orally gavage with PBS or live B. coccoides (LBC) for 4 weeks. Serum was collected for untargeted metabolomics profiling (n = 5–7 biological replicates per group). A) Partial least squares discriminant analysis (PLS‐DA) of metabolite composition. B) Volcano chart of detected metabolites. The indicate plot was methyl indole‐3‐acetate and I3AA. C) Heat map of tryptophan metabolites. D,E) The levels of I3AA, tryptophan (Trp), and its indole metabolites in cecal feces (D) and sera (E) detected by LC‐MS/MS analysis, respectively. F,G) Incubated the B. coccoides GA1 strain with Trp for the indicated time. I3AA (F) and Trp (G) concentrations in the supernatants were detected by LC‐MS/MS analysis, respectively. H) Incubated the B. coccoides DSM935 strain with Trp for 8 h. I3AA levels in the supernatants were detected by LC‐MS/MS. I) The probable metabolic pathway for generating I3AA. ArAT, aromatic amino acid aminotransferase; iorA/B, Indolepyruvate ferredoxin oxidoreductase subunit alpha/beta; AO, aldehyde oxidase. All values are expressed as the mean ± SEM. Statistical comparisons were carried out by unpaired two‐tailed Student's t‐test; *p < 0.05 and **p < 0.01.

Figure 5

I3AA has beneficial effects on metabolic improvements in vitro and in vivo. A) Primary hepatocytes were incubated with indicate dose of I3AA for 48 h and then stimulated with 100 nM insulin for 20 min (n = 6 replicates per group). Western blot analysis of p‐IR, p‐AKT, and p‐GSK3β levels. The right panel is the densitometry analysis of the relative abundance of phosphorylated proteins normalized to their total protein levels. A.U.: arbitrary units. B–J) HFD mice were orally gavage with PBS or 10 mg kg⁻¹ I3AA for 4 weeks (n = 6–7 biological replicates per group). B) Fed and fasting blood glucose levels. C) Fed and fasting serum insulin levels assayed by ELISA. D) HOMA‐IR index. E) Glucose tolerance tests. The right panel is the AUC. F) Insulin tolerance tests (0.5 U kg⁻¹). The right panel is the AUC. G) Total fat mass. H) The H&E staining of sWAT. Scale bars, 50 µm. The bottom panel is the frequency distribution of adipocyte cell size in sWAT and the box plot is average adipocyte diameter. I) Real‐time PCR analysis of browning related genes (Ucp1, Pgc1α, Prdm16, and Cidea) in sWAT. J) Western blot analysis of UCP1 protein levels. The bottom panel is the densitometry analysis of UCP1 protein levels. All values are expressed as the mean ± SEM. Statistical comparisons were carried out by unpaired two‐tailed Student's t‐test; *p < 0.05 and **p < 0.01.

Figure 6

Liver AhR is essential for I3AA induced metabolic improvements. HFD mice were injected with control adenovirus (Ad‐NC) or adenovirus expressing small‐hairpin RNA specific for mouse AhR (Ad‐shAhR) via the tail vein, respectively. The Ad‐NC or Ad‐shAhR mice were either orally gavage with PBS or 10 mg kg⁻¹ I3AA daily for 10 days (n = 5–6 biological replicates per group). A) Western blot analysis of AhR protein levels in the liver. The right panel is the densitometry analysis of AhR protein levels. A.U.: arbitrary units. B) Fed and fasting blood glucose levels. C) Fed and fasting serum insulin levels assayed by ELISA. D) HOMA‐IR index. E) Glucose tolerance tests. The right panel is the AUC. F) Insulin tolerance tests (0.5 U kg⁻¹). The right panel is the AUC. G) Body weight. H) Total fat mass. I) The H&E staining of sWAT. Scale bars, 50 µm. The right panel is the frequency distribution of adipocyte cell size in sWAT and the box plot is average adipocyte diameter. J) Real‐time PCR analysis of the gene expression related to sWAT browning, including Ucp1, Pgc1α, Prdm16, and Cidea. K) Western blot analysis of UCP1 protein levels. The right panel is the densitometry analysis of UCP1 protein levels. All values are expressed as the mean ± SEM. Statistical comparisons were carried out by two‐way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and n.s.: no significance.

Figure 7

Fecal B. coccoides and I3AA levels are correlated with human metabolic syndrome. A) The absolute abundance of B. coccoides in feces from subjects with metabolic syndrome (MS; n = 78) and healthy controls (HC; n = 43). B,C) Spearman's correlation analysis of the B. coccoides abundance with HOMA‐IR (B) or BMI (C), respectively. D) The concentrations of fecal I3AA in MS (n = 78) and health individuals (n = 43). E,F) Spearman's correlation analysis of the fecal I3AA levels with HOMA‐IR (E) or BMI (F), respectively. Statistical comparisons were carried out by the nonparametric Mann–Whitney U test; **p < 0.01.

Figure 8

Graphic abstract of the function and underlying mechanism of B. coccoides in ameliorating metabolic disorders. Leucine deprivation increases the abundance of B. coccoides, which improves insulin sensitivity, induces sWAT browning, and reduces body fat in HFD mice by metabolizing tryptophan into I3AA that activates AhR in the liver.