2-hydroxybutyric acid (2HB) serves as an important regulatory factor in a variety of diseases. The circulating level of 2HB in serum is significantly higher in multiple diseases, such as cancer and type 2 diabetes (T2D). However, there is currently no systematic study on 2HB-producing bacteria that demonstrates whether gut bacteria contribute to the circulating 2HB pool. To address this question, we used BLASTP to reveal the taxonomic profiling of 2HB-producing bacteria in the human microbiome, which are mainly distributed in the phylum Proteobacteria and Firmicutes. In vitro experiments showed that most gut bacteria (21/32) have at least one path to produce 2HB, which includes Aspartic acid, methionine, threonine, and 2-aminobutyric acid. Particularly, Fusobacterium nucleatum has the strongest ability to synthesize 2HB, which is sufficient to alter colon 2HB concentration in mice. Nevertheless, neither antibiotic (ABX) nor Fusobacterium nucleatum gavage significantly affected mouse serum 2HB levels during the time course of this study. Taken together, our study presents the profiles of 2HB-producing bacteria and demonstrates that gut microbiota was a major contributor to 2HB concentration in the intestinal lumen but a relatively minor contributor to serum 2HB concentration.

In our previous multicenter study, we delineated the inherent metabolic features of colorectal cancer (CRC). Therein, we identified a member of the ectonucleotide pyrophosphatase/ phosphodiesterase family (ENPP2) as a significant differential metabolite of CRC. In this study, the role of ENPP2 in CRC has been demonstrated using established in vitro and in vivo models including ENPP2 gene knockdown, and use of the ENPP2 inhibitor, GLPG1690. We found that CRC proliferation was decreased after either ENPP2 gene knockdown or use of ENPP2 inhibitors. We further evaluated the role of GLPG1690 in AOM/DSS-induced CRC mice via intestinal barrier function, macrophage polarization, inflammatory response and microbial homeostasis. Results of immunofluorescence staining and Western blotting showed that GLPG1690 can restore gut-barrier function by increasing the expression of tight junction proteins, claudin-1, occludin and ZO-1. M2 tumor-associated macrophage polarization and colonic inflammation were attenuated after treatment with GLPG1690 using the Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) model. Moreover, 16 S rDNA pyrosequencing and metagenomic analysis showed that GLPG1690 could alleviate gut dysbiosis in mice. Furthermore, administration of GLPG1690 with antibiotics as well as fecal microbiota transplantation assays demonstrated a close link between the efficacy of GLPG1690 and the gut microbiota composition. Finally, results of metabolomic analysis implicated mainly the gut microbiota-derived metabolites of aromatic amino acids in CRC progression. These findings may provide novel insights into the development of small-molecule ENPP2 inhibitors for the treatment of CRC.

Specific dietary patterns are crucial in the incidence and progression of colorectal cancer. Although there is evidence indicating that the ketogenic diet may inhibit colorectal cancer and potentially act as a therapeutic approach, the mechanisms by which it influences gut microbiota and modifies metabolic pathways remain poorly understood. This study aimed to explore the therapeutic effects of a ketogenic diet on colorectal cancer in AOM/DSS model through targeted bile acid metabolomics, transcriptomics, and 16S rDNA gene sequencing analysis. Our results demonstrate that the ketogenic diet provides both preventive and therapeutic benefits against colorectal cancer by reducing inflammatory responses and restoring intestinal barrier integrity. Additionally, serum metabolomic analysis indicated a reprogramming of bile acid metabolism. Furthermore, 16S rDNA sequencing and targeted bile acid metabolomics revealed that the ketogenic diet significantly altered gut microbiota composition and bile acid metabolism, leading to a unique serum bile acid profile characterized by increased primary bile acids and decreased secondary bile acids. Moreover, the combination of a ketogenic diet with antibiotics and fecal microbiota transplantation consistently resulted in higher abundances of Muribaculaceae, Akkermansia, and Lachnospiraceae NK4A136, while levels of Parasutterella decreased following ketogenic diet intervention. The concentrations of CA, CDCA, DCA, and 12-keto LCA in colorectal tissues or feces were consistent with those observed in ketogenic diet-treated subjects. An upregulation of FXR expression in colorectal tissues was noted, which modulated NF-κB metabolic pathways and contributed to a reduction in inflammatory responses. Our findings underscore that the ketogenic diet suppresses colorectal cancer by reshaping gut microbiota and modulating the intestinal FXR/NF-κB signaling pathway