13C-Stable isotope resolved metabolomics uncovers dynamic biochemical landscape of gut microbiome-host organ communications in mice – PubMed Black Hawk Supplements

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CONCLUSIONS: Multicompartmental SIRM analyses provided new insights into the current understanding of dynamic interorgan metabolite transport between the gut microbiome and host at the whole-body level in mice. Moreover, this study singled out microbiota-derived metabolites that are potentially involved in the gut-liver, gut-brain, and gut-skeletal muscle axes. Video Abstract.
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13C-Stable isotope resolved metabolomics uncovers dynamic biochemical landscape of gut microbiome-host organ communications in mice - PubMed

13C-Stable isotope resolved metabolomics uncovers dynamic biochemical landscape of gut microbiome-host organ communications in mice

Xia Xiao et al. Microbiome. .

Abstract

Background: Gut microbiome metabolites are important modulators of host health and disease. However, the overall metabolic potential of the gut microbiome and interactions with the host organs have been underexplored.

Results: Using stable isotope resolved metabolomics (SIRM) in mice orally gavaged with 13C-inulin (a tracer), we first observed dynamic enrichment of 13C-metabolites in cecum contents in the amino acids and short-chain fatty acid metabolism pathways. 13C labeled metabolites were subsequently profiled comparatively in plasma, liver, brain, and skeletal muscle collected at 6, 12, and 24 h after the tracer administration. Organ-specific and time-dependent 13C metabolite enrichments were observed. Carbons from the gut microbiome were preferably incorporated into choline metabolism and the glutamine-glutamate/GABA cycle in the liver and brain, respectively. A sex difference in 13C-lactate enrichment was observed in skeletal muscle, which highlights the sex effect on the interplay between gut microbiome and host organs. Choline was identified as an interorgan metabolite derived from the gut microbiome and fed the lipogenesis of phosphatidylcholine and lysophosphatidylcholine in host organs. In vitro and in silico studies revealed the de novo synthesis of choline in the human gut microbiome via the ethanolamine pathway, and Enterococcus faecalis was identified as a major choline synthesis species. These results revealed a previously underappreciated role for gut microorganisms in choline biosynthesis.

Conclusions: Multicompartmental SIRM analyses provided new insights into the current understanding of dynamic interorgan metabolite transport between the gut microbiome and host at the whole-body level in mice. Moreover, this study singled out microbiota-derived metabolites that are potentially involved in the gut-liver, gut-brain, and gut-skeletal muscle axes. Video Abstract.

Keywords: Inulin; Metabolite; Metabolomics; Microbiome; Stable isotope.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1

Time-depended changes of the mouse cecum content metabolome after [U-13C]-inulin administration. a Experimental in vivo characterization of the mouse metabolome after inulin administration. Male and female mice were orally administered [U-13C]-inulin or 12C-inulin (n = 3/time point/sex) at 5 mg/g. Biological samples were collected at 6, 12, and 24 h after [U-13C]-inulin administration and at 24 h after 12C-inulin administration. b Multivariate unsupervised principal component analysis shows the discrimination of biochemicals between [U-13C]-inulin (circles, red: T6h, green: T12h, blue: T24h) and 12C-inulin (triangles, blue: T24h) treated mice. c Heatmap indicates the apparent time-dependent effects of [U-13C]-inulin on the cecum content metabolome. d Multivariate empirical Bayes time-series analysis of the cecum content metabolome after [U-13C]-inulin administration. Biochemicals with high Hotelling’s T2 values (> 400) comprise those with the number of 13C ranged from 4 to 11. e Pathway analysis of metabolites that were inversely correlated with time, regardless of sex, revealed that these metabolites were mainly enriched in the amino acid and short-chain fatty acid metabolism pathways

Fig. 2
Fig. 2

13C fractional enrichment of metabolites in cecum contents after [U-13C]-inulin administration. Mice of each sex (F: female, M: male) were orally administered [U-13C]-inulin (n = 3/time point/gender) at 5 mg/g, and cecum contents were collected at 6, 12, and 24 h after administration. The samples were processed and analyzed by LC‒HRMS as described in the “Methods” section. Tracing of inulin carbon through the a central carbon metabolism pathway and b amino acid metabolism pathway. The circles indicate the carbon on each metabolite. The values shown are the mean ± SEM (n = 3)

Fig. 3
Fig. 3

The changes in mouse plasma, liver, brain, and skeletal muscle metabolome after [U-13C]-inulin (T6h, T12h, and T24h) and 12C-inulin (T24h) administration. Mice of each sex (F: female, M: male) were orally administered [U-13C]-inulin or inulin (n = 3/time point/gender) at 5 mg/g. Biological samples were collected at 6, 12, and 24 h after [U-13C]-inulin administration and at 24 h after inulin administration. a–d PCA shows the changes in the mouse metabolome after [U-13C]-inulin (T6h, T12h, and T24h) and 12C-inulin (T24h) administration. e,f Heatmap analysis shows the time-dependent changes in the mouse metabolome after [U-13C]-inulin administration. In plasma samples, the differentially expressed biochemicals were found mainly in clusters #1 and #2

Fig. 4
Fig. 4

13C differently incorporated into mouse liver, brain, and skeletal muscle tissues. a Time-dependent analysis shows the number of differentially expressed biochemicals in the mouse liver, brain, and skeletal muscle after [U-13C]-inulin administration. The blue bar indicates the non-labeled features (12C), and the orange bar represents the labeled features (13C). b Relative distribution of 13C isotopologues for differentially expressed biochemicals (time-dependent analysis) detected in the liver, brain, and skeletal muscle. A total number of 31, 33, and 17 differentially expressed biochemicals were 13C isotopologues, with the number of 13C ranging from 1 to 6 in the liver, and from 1 to 3 in the brain and skeletal muscle. c Treatment-dependent analysis shows the number of differentially expressed biochemicals in the mouse liver, brain, and skeletal muscle by comparing [U-13C]-inulin treated and 12C-inulin treated groups. d Distribution of 13C isotopologues of differentially expressed biochemicals (treatment-dependent analysis) detected in the liver, brain, and skeletal muscle. A total number of 8, 10, and 3 differentially expressed biochemicals were 13C isotopologues, with the number of 13C ranging from 1 to 6 in the liver, and from 1 to 5 in the brain and skeletal muscle

Fig. 5
Fig. 5

The 13C enrichment profile and time-dependent changes of 13C-labeled choline and lactate in different biological samples after oral administration of [U-13C]-inulin to male and female mice. a 13C fractional enrichments in the isotopologues of choline detected at different time points (6, 12, and 24 h) in cecum content, plasma, liver, brain, and skeletal muscle, respectively. The x-axis denotes the number of 13C atoms present in each compound. b Time-dependent changes of 13C5-choline in different samples. c 13C fractional enrichments in the isotopologues of lactate detected in different samples. d Time-dependent changes of ∑13Cn-lactate (n = 1–3) in different samples. The values shown are the mean ± SEM (n = 3). * 0.01 < p < 0.05, two-tailed t-test (see “Methods”)

Fig. 6
Fig. 6

GABA in the brain can partially originate from the gut microbiome. a 13C fractional enrichments in the isotopologues of glutamine, glutamate, and GABA detected at different time points (6 h, 12 h, 24 h) in the brain, cecum content, and liver, respectively. The values shown are the mean ± SEM (n = 3). F: female; M: male. b Representative full scan mass spectra of GABA and its isotopologues (13C1–13C4) detected in the mouse brain, cecum content, and liver after oral administration of 12C-inulin or 13C-inulin. Enlarged spectra in the m/z range of 106–108 demonstrated the presence of a high abundance of 13C4-GABA (m/z 108.0843) in the brain and cecum contents, suggesting that gut microbiome-derived GABA partially contributes to the brain pool of GABA

Fig. 7
Fig. 7

Phosphatidylcholine (PC 34:2) and lysophosphatidylcholine (LPC 16:0) are labeled with 13C in mice treated with [U-13C]-inulin. a 13C fractional enrichments in the isotopologues (13C1–13C10) of PC 34:2 detected at different time points (6, 12, and 24 h) in cecum content, plasma, liver, brain, and skeletal muscle, respectively. b 13C fractional enrichments in the isotopologues (13C1–13C10) of LPC 16:0. The average values of three biological replicates are shown

Fig. 8
Fig. 8

In silico and in vitro identification of gut microbial species that contribute to the degradation of inulin and biosynthesis of 13C5-choline. a Comparative-analysis of metabolic pathways in seven representative strains that are capable of metabolizing carbohydrates. The total number of pathway classes associated with carbohydrate degradation in each species was compared using MetaCyc and calculated using Excel. b The biochemical potential of gut microbiome-related metabolic models in the HMP database was examined for the capability to produce choline. The top 20 genera with the highest number of models are shown. c The production of ethanolamine and choline in the human fecal microbiome, B. fragilis, B. thetaiotaomicron, and E. faecalis. 13C fractional enrichments of choline and its precursor ethanolamine were showed. Detection of 13C-labeled choline in culture media indicates that choline synthesized in the gut microbiome can be exported to the extracellular pool and contribute to the host phospholipid biosynthesis. Red circle: 13C

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13C-Stable isotope resolved metabolomics uncovers dynamic biochemical landscape of gut microbiome-host organ communications in mice – PubMed