An orally active carbon monoxide-releasing molecule enhances beneficial gut microbial species to combat obesity in mice

Abstract

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, β-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction of inflammatory markers in colon and cecum and restoration of metabolic profiles in these tissues. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

Graphical abstract

Fig. 1

CO delivered by CORM-401 to different body compartments preventsbodyweight gain and metabolic dysfunction in mice on HFD. Mice were maintained for 14 weeks either on a standard (SD), high fat diet (HFD), HFD plus CORM-401 (HFD-CO) or HFD plus the negative control MnS (HFD-MnS). CORM-401 (0.09 mmol/kg) or MnS (0.09 mmol/kg) was administered orally three times a week. (A) Body weight of animals in the four groups studied as recorded on a weekly basis (n = 10). (B) Body weight gain as percentage of the initial weight. (C) Fasting glycemia measured at week 13. (D) Plasma lactate levels detected by untargeted metabolomics at week 14. (E) Experimental design for the quantification of CO in blood and tissues. Blood was collected at different times after a single administration of CORM-401 in SD or HFD fed mice. Tissues, cecum content and faeces were collected at 1, 3, 6, 24 and 48 h after a single treatment with CORM-401 or DPBS (CON) in 8-week old mice (n = 3–5). (F) Blood carboxyhemoglobin (COHb) levels before and after oral gavage with CORM-401. (G) Total CO content in organs and faeces calculated from the area under the curve (AUC) obtained over a 48 h period following treatment with CORM-401 (green) versus untreated control (CON) mice (black). (H–K) Time course of CO accumulation in cecum tissue, cecum content, colon and faeces over time. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group (CON), ^#P < 0.05 and ^##P < 0.01 vs. HFD group. n.s., non-significant. Student's t-test, one-way or two-way ANOVA with Bonferroni test for multiple comparisons were used. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article)

Fig. 2

CORM-401 modulates the gut microbiota composition during HFD-induced obesity. Metagenomics analysis of bacterial DNA extracted from fecal samples at week 14. (A-B) Principal Coordinate Analysis (Bray-Curtis distance method) plots of gut microbiota analyzed in fecal samples collected from mice under SD, HFD, HFD + MnS or HFD + CORM-401 (HFD-CO) after the 14 weeks protocol (n = 6). The plots show a clear clustering of bacterial species between SD and HFD groups (A) and a separation between HFD and HFD-CO (B). (C) Stacked taxonomic bar plots at the phylum level (top 10 phyla) in fecal samples from the four groups. The Metagenome-Atlas was used for annotation, the MicrobiotaProcess package was used for graphs generation. (D) The Firmicutes/Bacteroidetes ratio is significantly increased in HFD vs. SD and markedly diminished in HFD-CO. (E) Rare abundance of bacterial phyla in the four groups calculated from the total number of sequence reads (MicrobiotaProcess). Data represent the mean ± SEM. *P < 0.05, ***P < 0.001, vs. SD, ^#P < 0.05 vs. HFD group. n.s., non-significant. One-way ANOVA with Bonferroni test for multiple comparisons was used.

Fig. 3

CORM-401 enriches the abundance of the beneficial bacteria A. muciniphila. Volcano plots showing the differences of gut microbiota species at metagenomics (A) and metatrascriptomics (B) levels, respectively, in HFD vs. HFD-COs. The dot colour corresponds to the different bacterial phyla as reported on the top left hand side of the square and denotes significantly enriched species in HFD or HFD-CO. Grey dots represent non-significant species. (C) DNA and RNA relative counts of A. muciniphila species at week 14 in fecal samples from animals fed SD, HFD, HFD-MnS and HFD-CO (n = 5–6). (D) Relative gene abundance of A. muciniphila confirmed by qPCR in the same samples (n = 3). (E) Spearman test to assess a significant correlation between A. muciniphila relative abundance and body weight in the 4 groups. The Spearman coefficient (r) and p values are reported. One-way ANOVA with Bonferroni test for multiple comparisons between groups was used. Data represent the mean ± SEM. *P < 0.05 and **P < 0.01 vs. SD, ^#P < 0.05 vs. HFD group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

CORM-401 alters selective metabolic pathways in gut microbiota during HFD. Functional analysis of the abundance of KEGG modules of gut microbiota's metabolic pathways inferred from annotation of CMMG genome from metatranscriptomic sequencing data of fecal samples from mice under SD, HFD, HFD-MnS and HFD-CO (n = 6). (A) Heatmap of the predictive metabolic pathways that were significantly different (P < 0.05) between HFD and HFD-CO. The abundance profiles were transformed into Z scores (scale shown in color bar) computed from the relative abundances of the selected metabolic pathways across different groups. (B–C) Relative abundance of the citrate cycle and the microbiota phyla and families, respectively, contributing to the enrichment of this pathway. (D-E) Relative abundance of the beta-oxidation pathway and the microbiota phyla and families, respectively, contributing to the enrichment of this pathway. The results show that in HFD-CO the Akkermansiaceae family from the Verrucomicrobiota phylum promotes the enrichment of the citrate cycle pathway, while the increase in the beta-oxidation pathway is ascribable to a great extent to the Rickenellaceae and a small increase in the Muribaculaceae families from the Bacteroidota phylum. Box plots were analyzed by Wilcoxon non parametric test. *P < 0.05 and **P < 0.01 between two groups. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Alterations of fecal metabolites induced by HFD are markedly reversed by treatment with CORM-401. Untargeted metabolomics analysis was used to detect fecal metabolites at week 14 in samples from mice fed a SD, HFD, HFD-MnS or HFD-CO. Partial least squares discriminant analysis (PLS-DA) score plots showing the distribution of positively (A) and negatively (B) charged fecal metabolites in HFD versus HFD-CO (n = 10). Parameters showing the goodness fit (pR2Y) and predictability of the model (pQ2) are shown underneath the plots. (C) Heatmap of the top 30 fecal metabolites significantly different between all groups. The name of the specific metabolite is in blue on the right hand side of the heatmap and the main metabolic pathways to which the metabolites belong are reported in different colors on the left of the heatmap. Significantly abundant metabolites (P ≤ 0.05) between HFD and HFD-CO groups were selected using the Wilcoxon non-parametric test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

CORM-401 normalizes the levels of key fecal amino acids and plasma tryptophan-derived bacterial metabolites altered by obesity. Untargeted metabolomics analysis was used to detect metabolites at week 14 in samples from mice fed a SD, HFD, HFD-MnS and HFD-CO (n = 10). (A to F) Levels of selected amino acids and derivatives in faeces expressed as relative peak areas. These amino acids were significantly increased by HFD and diminished to normal levels by CORM-401. (G to I) Levels of selected microbial metabolites in plasma from the same groups. It is shown that 4-hydroxindol and cholic acid were markedly decreased by HFD and restored to basal levels by CORM-401, while the uremic toxin guanidinosuccinic acid was increased by HFD and normalized by CORM-401. Data represent the mean ± SEM. *P < 0.05, **P < 0.01 vs. SD. ^#P < 0.05 vs. HFD group. One-way ANOVA with Bonferroni test for multiple comparisons between groups was used. (J) Relative abundance of the urea cycle pathway in bacteria, which was significantly increased by HFD and normalized by CORM-401 (n = 6). Box plots were analyzed by Wilcoxon non-parametric test. *P < 0.05 and **P < 0.01 between two groups. (K) The results show that the relative abundance of the microbiota Oscillospiraceae family from the Firmicutes phylum contributing to the urea cycle pathway is increased in HFD and normalized by CORM-401.

Fig. 7

CORM-401 reestablishes a normal hypoxic phenotype and glycolysis gene expression in the colon while restoring bacterial glycolysis in HFD-fed mice. (A) Representative images of colonic sections from mice under SD, HFD or HFD-CO stained with pimonidazole to visualize hypoxia at 14 weeks after treatments. (B) Quantification of colonic hypoxic areas calculated based on pimonidazole staining using ImageJ (n = 3–4). (C and D) HIF-1α and HIF-2α relative mRNA expression levels measured by RT-QPCR at week 14 in colon of mice fed a SD, HFD, HFD-MnS and HFD-CO (n = 10). (E) mRNA expression of glycolytic genes in the colon (n = 8–10). (F) Untargeted metabolomic profile of lactate in faeces collected at week 14 (n = 9–10). Data represent the mean ± SEM. *P < 0.05, **P < 0.01 vs. SD. ^#P < 0.05 vs. HFD group. One-way ANOVA with Bonferroni test for multiple comparisons between groups was used. (G) Relative abundance of the glycolysis pathway in bacteria, which was significantly decreased by HFD and normalized by CORM-401 (n = 6). Box plots were analyzed by Wilcoxon non parametric test. **P < 0.01 between two groups. (H) The results show that several families contribute to the recovery of bacterial glycolysis in the HFD-CO group, with strong relative abundance of Atopobiaceae family from the Actinobacteriota phylum and Erysipelotrichaceae from the Firmicutes phylum.