Systemic gut microbial modulation of bile acid metabolism in host tissue compartments

Abstract

We elucidate the detailed effects of gut microbial depletion on the bile acid sub-metabolome of multiple body compartments (liver, kidney, heart, and blood plasma) in rats. We use a targeted ultra-performance liquid chromatography with time of flight mass-spectrometry assay to characterize the differential primary and secondary bile acid profiles in each tissue and show a major increase in the proportion of taurine-conjugated bile acids in germ-free (GF) and antibiotic (streptomycin/penicillin)-treated rats. Although conjugated bile acids dominate the hepatic profile (97.0 ± 1.5%) of conventional animals, unconjugated bile acids comprise the largest proportion of the total measured bile acid profile in kidney (60.0 ± 10.4%) and heart (53.0 ± 18.5%) tissues. In contrast, in the GF animal, taurine-conjugated bile acids (especially taurocholic acid and tauro-β-muricholic acid) dominated the bile acid profiles (liver: 96.0 ± 14.5%; kidney: 96 ± 1%; heart: 93 ± 1%; plasma: 93.0 ± 2.3%), with unconjugated and glycine-conjugated species representing a small proportion of the profile. Higher free taurine levels were found in GF livers compared with the conventional liver (5.1-fold; P < 0.001). Bile acid diversity was also lower in GF and antibiotic-treated tissues compared with conventional animals. Because bile acids perform important signaling functions, it is clear that these chemical communication networks are strongly influenced by microbial activities or modulation, as evidenced by farnesoid X receptor-regulated pathway transcripts. The presence of specific microbial bile acid co-metabolite patterns in peripheral tissues (including heart and kidney) implies a broader signaling role for these compounds and emphasizes the extent of symbiotic microbial influences in mammalian homeostasis.

Fig. 1.

Relative bile acid profiles of the liver, plasma, kidney, and heart of conventional rats. Values are mean intensity values + SEM (scaled log_e) measured using UPLC-MS. Abbreviations for the measured bile acids with additional structural information are given in the table. The prefixes G and T indicate conjugation with either glycine or taurine, respectively. For the bile acids labeled with the following scheme, C₂₄O_x-yΔ, x is the number of oxygens on the bile acid skeleton, and y is the number of double bonds in the structure. ²²Δ indicates a double bond on carbon 22.

Fig. 2.

Effects of microbial absence on bile acid profiles. (A) PCA scores plot of the bile acid signatures in all sample matrices of GF and CV animals. (B) Total relative signals of the bile acids based on conjugation state are compared for the GF and CV animals in the liver, kidney, heart, and plasma. Free taurine is also compared. Values are means of the scaled (log_e) data + SEM. Significant difference by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Intensity values for the measured bile acid species in all sample matrices of CV and GF rats. Values are mean intensities + SEM measured using UPLC-MS.

Fig. 3.

Effects of microbial absence and antibiotic treatment on bile acid profiles. (A) Intensity differences of individual bile acid species in GF rats in relation to CV rats in the liver, plasma, kidney, and heart. Bile acid species shown are those found to be statistically different (using a Student t test; P < 0.05) from those measured in CV rats; (B) Bile acid species found to be statistically different between control rats and those treated with antibiotics in the liver, kidney, and heart. Also shown is the relative intensity from the free taurine in the liver. Values are means of the scaled (log_e) data ± SEM. Statistical significance was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Hepatic metabolic and signaling pathways modulated by the gut microbiota. (A) Significantly regulated canonical gene expression pathways in GF rats (blue bars) and pathways common to AB rats (red bars). Scale is a log-transformed P value calculated by Fisher's exact test that a pathway is over-represented within the altered genes. Dotted red lines indicate threshold value (P < 0.05) for pathway change to be considered statistically significant. (B) Relationship between FXR/RXR-regulated genes and bile acid metabolic pathways. Purple shapes indicate up-regulated genes, and yellow shapes indicate down-regulated genes in GF rats with respect to conventional rats. Purple stars denote up-regulated genes in AB rats with respect to control rats. White shapes indicate genes that were present on the microarray but were unchanged in GF rats. Values indicate fold change in gene transcription in GF rats with respect to conventional rats and in AB rats with respect to control rats. ApoB, apolipoprotein B; G6PC, glucose-6-phosphatase; HNF, hepatocyte nuclear factor; LRH-1, liver receptor homolog-1; LXR, liver X receptor; PEPCK, phosphoenolpyruvate carboxykinase; PKLR, pyruvate kinase; PPARα, peroxisome proliferator-activated receptor α; RXR, retinoid X receptor; SHP, short heterodimer partner; SREBP-1, sterol regulatory element binding protein-1.