Gut microbiota-bile acid crosstalk contributes to the rebound weight gain after calorie restriction in mice

Abstract

Calorie restriction (CR) and fasting are common approaches to weight reduction, but the maintenance is difficult after resuming food consumption. Meanwhile, the gut microbiome associated with energy harvest alters dramatically in response to nutrient deprivation. Here, we reported that CR and high-fat diet (HFD) both remodeled the gut microbiota with similar microbial composition, Parabacteroides distasonis was most significantly decreased after CR or HFD. CR altered microbiota and reprogramed metabolism, resulting in a distinct serum bile acid profile characterized by depleting the proportion of non-12α-hydroxylated bile acids, ursodeoxycholic acid and lithocholic acid. Downregulation of UCP1 expression in brown adipose tissue and decreased serum GLP-1 were observed in the weight-rebound mice. Moreover, treatment with Parabacteroides distasonis or non-12α-hydroxylated bile acids ameliorated weight regain via increased thermogenesis. Our results highlighted the gut microbiota-bile acid crosstalk in rebound weight gain and Parabacteroides distasonis as a potential probiotic to prevent rapid post-CR weight gain.

Fig. 1. The reinstatement of chow diet or HFD after fasting and CR resulted in rapid weight gain and obesogenic metabolic phenotypes.

a The experimental workflow of the fasting experiment. The stars indicated the time of the samples collection. b Body weight in the fasting experiment. (*p < 0.05) c The AUC of OGTT. d Fasting blood glucose level. e Liver index level. f Workflow of the diets changing experiment. The stars indicated the time of the samples collection. g Body weight during the diets changing experiment (n = 8 per group in the 16-week experiment and n = 6 per group for the observation of weight regain in 20 weeks. Differences were compared with the CD + HF group, *p < 0.05). h Raw energy expenditures and raw average energy expenditures in the period of light and dark, n = 4 per group. The hypothesis tests result of analysis-of-covariance with timely body mass as a covariate was shown in the left panel. i AUC of the OGTT. j AUC of the ITT. k Liver, WAT, and BAT weights fold change after change to HFD, the differences were compared between the pre and post changing diets. l Liver index level. n = 9 per group in the fasting experiment, n = 8 per group in the diets changing experiment. Data are expressed as means $\pm$ SEM (c, i, j). p-values in figures were calculated by the two-tailed unpaired T-test in the GraphPad software. All box and whiskers plots showed the box (from the 25th to 75th percentiles), the median value (in the transverse line), and the whiskers (go down to the smallest value and up to the largest). Source data are provided as a Source data file. Oral glucose tolerance test (OGTT); insulin tolerance tests (ITT); area under the curve (AUC); high-fat diet (HFD); epididymal white adipose tissue (WAT); brown adipose tissue (BAT).

Fig. 2. Remodeling of gut microbiota under CR or HFD.

a PCoA plot based on the Canberra similarity of cecal microbial composition. PCoA1 axis level of the three groups. b Hierarchical clustering based on the Canberra similarity among three groups. c The ratio of Firmicutes to Bacteroidetes among three groups. d LDA effect size method was performed to compare enriched taxa (levels of genus) in each group. The bar plot listed the significantly differential taxa (score >3.5) in HF, CR, and CD groups. e Fold change of the species enriched in the CD group with the most abundance. f Heatmap of correlation coefficients between the KEGG L3 pathway and phenotypes of the individuals with similar body weight before 4-week HFD by Spearman correlation. Each correlation coefficient smaller than −0.8 or bigger than 0.8 was included in the plot, the p-values were adjusted by the method of Bonferroni. The color of each spot in the heatmap corresponds to the r value. g Relative abundance of the 7α-HSDH (K00076) pathway. h Relative abundance of the BSH (K01442) pathway. n = 5 per group. Data are expressed as mean ± SEM in the bar plots. All p-values in figures were calculated by the two-tailed unpaired T-test in the GraphPad software. Source data are provided as a Source data file. Oral glucose tolerance test (OGTT); insulin tolerance tests (ITT); area under the curve (AUC); epididymal white adipose tissue (WAT); brown adipose tissue (BAT); bile salt hydrolase (BSH); 7α-hydroxysteroid dehydrogenase (7α-HSDH); principal coordinate analysis (PCoA); linear discriminant analysis (LDA).

Fig. 3. The depletion of Parabacteroides distasonis and non-12OH BAs in CR.

a Concentration of total BAs in contents of cecum, liver, and serum among three groups. Three groups variation was assessed by the Kruskal–Wallis test. b The bar plots show the mean percentage of (non-)12OH and (un)conjugated BAs in contents of cecum, liver, and serum among three groups. c The BAs concentration percentage profiles in the serum of three groups. d Dysregulated non-12OH unconjugated BAs (LCA and UDCA) composition in the serum of three groups. e Dysregulated non-12OH unconjugated BAs (LCA and UDCA) composition in the cecal contents of three groups. f Heatmap of GRaMM’s correlation coefficients between the cecal BAs and the species significantly enriched in the CD group, the color of each spot in the heatmap corresponds to the r value, *p < 0.05; ^#p < 0.0001. n = 8 per group. Differences in the BAs concentration percentage data were assessed by the two-tailed multiple T-test in the GraphPad software, p-values were adjusted by the FDR’s method. Data are expressed as mean ± SEM in (d, e). All box and whiskers plots showed the box (from the 25th to 75th percentiles), the median value (in the transverse line), and the whiskers (go down to the smallest value and up to the largest). Source data are provided as a Source data file. 12α-hydroxylated bile acids (12OH BAs); non-12α-hydroxylated bile acids (non-12OH BAs); T-conjugated (taurine-conjugated); cholic acid (CA); deoxycholic acid (DCA); chenodeoxycholic acid (CDCA); muricholic acid (MCA); lithocholic acid (LCA); ursodeoxycholic acid (UDCA); 3-ketocholic acid (3-ketoCA); 7-ketodexycholic acid (7-ketoDCA); 23-nordeoxycholic acid (23-norDCA); α-muricholic acid (αMCA); 3β-ursodeoxycholic acid (βUDCA); β-muricholic acid (βMCA); ω-muricholic acid (ωMCA); glycocholic acid (GCA); glycodehydrocholic acid (GDCA); taurocholic acid (TCA); taurodeoxycholic acid (TDCA); taurochenodeoxycholic acid (TCDCA); tauro α-muricholic acid (TαMCA); tauro β-muricholic acid (TβMCA); taurolithocholic acid (TLCA); tauroursodeoxycholic acid (TUDCA); taurohyocholic acid (THCA); taurohyodeoxycholic acid (THDCA); taurodehydrocholic acid (TDHCA); tauro ω-muricholic acid (TωMCA).

Fig. 4. The depletion of Parabacteroides distasonis and non-12OH BAs after fasting.

a PLS-DA plot of the cecal microbiome in four groups, n = 7 per group. b The species rank of VIP scores of the PLS-DA analysis in chow diet. c Relative abundance of Parabacteroides distasonis among four groups. d The bar plots of the mean percentage of (non-)12OH and (un)conjugated BAs in the serum. e The BAs composition profiles in serum. p-values were adjusted by the FDR’s method. f UDCA was significantly reduced after fasting in the chow diet, data are expressed as means $\pm$ SEM. n = 9 per group. Differences were assessed by two-tailed multiple T-test in the GraphPad software. All box and whiskers plots showed the box (from the 25th to 75th percentiles), the median value (in the transverse line), and the whiskers (go down to the smallest value and up to the largest). Source data are provided as a Source data file. Partial least-squares discriminant analysis (PLS-DA); variable important in projection (VIP); cholic acid (CA); deoxycholic acid (DCA); chenodeoxycholic acid (CDCA); muricholic acid (MCA); lithocholic acid (LCA); ursodeoxycholic acid (UDCA); 12α-hydroxylated bile acids (12OH BAs); non-12α-hydroxylated bile acids (non-12OH BAs); T-conjugated (taurine-conjugated).

Fig. 5. The depletion of Parabacteroides distasonis and non-12OH BAs resulted in reduced energy expenditure in the weight-rebound mice.

a Relative abundance of Parabacteroides distasonis in the contents of cecum among three groups. b The (un)conjugated (non-)12OH BAs percentage in the serum. c The BAs composition profiles in the serum. p-values were adjusted by the FDR’s method. d The composition level of LCA and UDCA in the serum. p-values were adjusted by the FDR’s method. e Relative mRNA expression of Tgr5 and Gcg genes in the distal ileum, n = 6 per group. f Active GLP-1 level in serum. g Relative mRNA expression of thermogenesis-related genes in the BAT. h UCP1 protein expression in the BAT, n = 3 per group. n = 8 per group in the experiments. Differences were assessed by a two-tailed multiple T-test in the GraphPad software. Data were expressed as means $\pm$ SEM in the bar plots. All box and whiskers plots showed the box (from the 25th to 75th percentiles), the median value (in the transverse line), and the whiskers (go down to the smallest value and up to the largest). Source data are provided as a Source data file. 12α-hydroxylated bile acids (12OH BAs); non-12α-hydroxylated bile acids (non-12OH BAs); T-conjugated (taurine-conjugated); cholic acid (CA); deoxycholic acid (DCA); chenodeoxycholic acid (CDCA); muricholic acid (MCA); lithocholic acid (LCA); ursodeoxycholic acid (UDCA); Takeda G protein-coupled receptor 5 (Tgr5); glucagon-like peptide-1 (GLP-1); brown adipose tissue (BAT); uncoupling protein 1 (Ucp1); peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc1α); elongation of very long-chain fatty acids 3 (Elovl3); elongation of very long-chain fatty acids 6 (Elovl6).

Fig. 6. The treatment with Parabacteroides distasonis attenuated weight rebound after CR diet.

a Body weight (*p < 0.05. Differences between the CR + HF + PD group and the CR + HF + Vehicle group are marked in light-gray asterisks, differences between the CR + HF + PD group and the CR + HF + HKPD group are marked in dark-gray asterisks.) and the weight gain during the 4-week HFD. b Average energy intake during the first week of changing to HFD. c Raw average energy expenditures per hour during the period of light and dark, n = 4 per group. d Fasting blood glucose at the end of the experiment. e Fat mass and lean mass of the mice by NMR miniSpec LF50, n = 4 per group. f PCoA plots of the cecal microbiome based on the Jaccard similarity. g Mean abundance of the phyla among the three groups. h The bar plots show the mean percentage of (non-)12OH and (un)conjugated BAs in the serum. i The BAs composition profiles in the cecal contents among three groups. j The composition of unconjugated non-12OH BAs (LCA and UDCA). p-values were adjusted by the FDR’s method. k Relative mRNA expression of Tgr5 and Gcg in the distal ileum. l Active GLP-1 level in serum. m Relative mRNA expression of Ucp1 and Pgc1α in BAT. n UCP1 protein expression in BAT. o The body weight of UKO mice. p The fasting blood glucose level of UKO mice. n = 5 in the CR + HF + PD, CR + HF + Vehicle, CR + HF + HKPD, and CR + HF + PD (UKO) groups. n = 4 in the CR + HF + Vehicle (UKO) group. All differences were assessed by the two-tailed multiple T-test in the GraphPad software. All box and whiskers plots showed the box (from the 25th to 75th percentiles), the median value (in the transverse line), and the whiskers (go down to the smallest value and up to the largest), data in bar plots were expressed as means $\pm$ SEM. Source data are provided as a Source data file. High-fat diet (HFD); principal coordinate analysis (PCoA); 12α-hydroxylated bile acids (12OH BAs); non-12α-hydroxylated bile acids (non-12OH BAs); T-conjugated (taurine-conjugated); cholic acid (CA); deoxycholic acid (DCA); chenodeoxycholic acid (CDCA); muricholic acid (MCA); lithocholic acid (LCA); ursodeoxycholic acid (UDCA); bile acid (BA); Takeda G protein-coupled receptor 5 (Tgr5); glucagon-like peptide-1 (GLP-1); uncoupling protein 1 (Ucp1); peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc1α); brown adipose tissue (BAT).

Fig. 7. The administration of non-12OH BA attenuated the weight regain after CR diet.

a Body weight (*p < 0.05. Differences between the CR + CD group and the CR + HF + Vehicle group are marked in lilac asterisks, differences between the CR + HF + UDCA group and the CR + HF + Vehicle group are marked in purple asterisks.) and the weight gain during the 4-week HFD. b Average energy intake during the first week of HFD. c Fasting blood glucose level at the end of the experiment. d AUC of the OGTT and ITT. e Fat mass and lean mass of the mice by NMR miniSpec LF50, n = 4 per group. f The bar plots show the mean percentage of (non-)12OH and (un)conjugated BAs in the serum among the three groups. g The BAs composition profiles in the serum among three groups. p-values were adjusted by the FDR’s method. h Relative mRNA expression of BAs synthesis-related liver enzymes. i Relative mRNA expression of Ucp1 and Pgc1α in BAT. j The body weight of UKO mice. k The fasting blood glucose level of UKO mice. n = 6 in the CR + CD, CR + HF + Vehicle and CR + HF + UDCA groups. n = 5 in the CR + HF + UDCA (UKO) group and n = 4 in the CR + HF + Vehicle (UKO) group. All p-values in figures were assessed by the two-tailed unpaired T-test (towards CR + HF + Vehicle group) in the GraphPad software. All box and whiskers plots showed the box (from the 25th to 75th percentiles), the median value (in the transverse line), and the whiskers (go down to the smallest value and up to the largest), other data in bar plots were expressed as means $\pm$ SEM. Source data are provided as a Source data file. High-fat diet (HFD); ursodeoxycholic acid (UDCA); area under the curve (AUC); oral glucose tolerance test (OGTT); insulin tolerance tests (ITT); 12α-hydroxylated bile acids (12OH BAs); non-12α-hydroxylated bile acids (non-12OH BAs); T-conjugated (taurine-conjugated); cholic acid (CA); deoxycholic acid (DCA); chenodeoxycholic acid (CDCA); muricholic acid (MCA); lithocholic acid (LCA); cytochrome P-450 cholesterol 7α-hydroxylase (Cyp7a1); oxysterol 7α-hydroxylase (Cyp7b1); sterol-12α-hydroxylase (Cyp8b1); cytochrome P-450-27A1 (Cyp27a1); brown adipose tissue (BAT); uncoupling protein 1 (Ucp1); peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc1α).