Metagenomic and metabolomic analysis showing the adverse risk-benefit trade-off of the ketogenic diet

Abstract

Background: Ketogenic diets are increasingly popular for addressing obesity, but their impacts on the gut microbiota and metabolome remain unclear. This paper aimed to investigate how a ketogenic diet affects intestinal microorganisms and metabolites in obesity.

Methods: Male mice were provided with one of the following dietary regimens: normal chow, high-fat diet, ketogenic diet, or high-fat diet converted to ketogenic diet. Body weight and fat mass were measured weekly using high-precision electronic balances and minispec body composition analyzers. Metagenomics and non-targeted metabolomics data were used to analyze differences in intestinal contents.

Results: Obese mice on the ketogenic diet exhibited notable improvements in weight and body fat. However, these were accompanied by a significant decrease in intestinal microbial diversity, as well as an increase in Firmicutes abundance and a 247% increase in the Firmicutes/Bacteroidetes ratio. The ketogenic diet also altered multiple metabolic pathways in the gut, including glucose, lipid, energy, carbohydrate, amino acid, ketone body, butanoate, and methane pathways, as well as bacterial secretion and colonization pathways. These changes were associated with increased intestinal inflammation and dysbiosis in obese mice. Furthermore, the ketogenic diet enhanced the secretion of bile and the synthesis of aminoglycoside antibiotics in obese mice, which may impair the gut microbiota and be associated with intestinal inflammation and immunity.

Conclusions: The study suggest that the ketogenic diet had an unfavorable risk-benefit trade-off and may compromise metabolic homeostasis in obese mice.

Keywords: Gut microbiota; High-fat diet; Ketogenic diet; Metabolome; Metagenome; Obesity.

Fig. 1

Changes in mice’s body weight, fat mass, and fat/weight% under different feeding conditions. A-C depicts the time-dependent changes in body weight, fat mass, and fat/weight% of mice over 20 weeks. D-F illustrates the body weight, fat mass, and fat/weight% of mice at week 20. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not statistically significant; ND, normal diet; HFD, high-fat diet; KD, ketogenic diet; HK, HFD converted to a ketogenic diet; n = 8. One-way ANOVA was used to decide significant differences between groups, data were presented as mean ± SEM

Fig. 2

Alpha Diversity, species and abundance of microorganisms. A, observed species at the community level; B, Chao 1 index at the community level; C, the number of intestinal species; D, the mean abundance of gut bacteria at the phylum level. *P < 0.05; **P < 0.01; ns, not statistically significant; n = 3. One-way ANOVA was used to decide significant differences between groups, data were presented as mean ± SEM

Fig. 3

LEfSe generated cladogram to identify specific bacterial species in each group. A, specific bacterial species in ND and HFD group (n = 3); B, specific bacterial species in HK and HFD group (n = 3); C, specific bacterial species in KD and HFD group (n = 3)

Fig. 4

Enrichment of GO function and KEGG pathway of differentially expressed genes. A, the functions of differentially expressed genes between HFD and ND groups (n = 3); B, the altered metabolic pathways between HFD and ND groups (n = 3); C, the functions of differentially expressed genes between HFD and HK groups (n = 3); D, the altered metabolic pathways between HFD and HK groups (n = 3). E, the functions of differentially expressed genes between HFD and KD groups (n = 3); F, the altered metabolic pathways between HFD and KD groups (n = 3)

Fig. 5

Carbohydrate-related enzymes abundance and dominant bacterias association maps between each group. A, Carbohydrate-related enzymes relative abundance between each group (n = 3); B, the correlation between dominant bacteria in four mice groups (n = 3); pos, positive correlation; neg, negative correlation. GH, Glycoside Hydrolases; GT, Glycosyl Transferases; CBM, Carbohydrate-Binding Module; PL, Polysaccharide Lyases; Data were calculated by the abundance and change relationship of different species in each mice group

Fig. 6

Differential metabolites and metabolic pathways between mice groups. A, metabolic pathways differed between the HFD and ND group (n = 6); B, heatmap showing the levels of the main metabolites associated with the metabolic pathways shown in A; C, metabolic pathways differed between the HK and HFD group (n = 6); D, heatmap showing the levels of the main metabolites associated with the metabolic pathways shown in C; E, metabolic pathways differed between the HFD and KD group (n = 6); F heatmap showing the levels of the main metabolites associated with the metabolic pathways shown in E