Gut microbiota and intestinal FXR mediate the clinical benefits of metformin

Abstract

The anti-hyperglycemic effect of metformin is believed to be caused by its direct action on signaling processes in hepatocytes, leading to lower hepatic gluconeogenesis. Recently, metformin was reported to alter the gut microbiota community in humans, suggesting that the hyperglycemia-lowering action of the drug could be the result of modulating the population of gut microbiota. However, the critical microbial signaling metabolites and the host targets associated with the metabolic benefits of metformin remained elusive. Here, we performed metagenomic and metabolomic analysis of samples from individuals with newly diagnosed type 2 diabetes (T2D) naively treated with metformin for 3 d, which revealed that Bacteroides fragilis was decreased and the bile acid glycoursodeoxycholic acid (GUDCA) was increased in the gut. These changes were accompanied by inhibition of intestinal farnesoid X receptor (FXR) signaling. We further found that high-fat-diet (HFD)-fed mice colonized with B. fragilis were predisposed to more severe glucose intolerance, and the metabolic benefits of metformin treatment on glucose intolerance were abrogated. GUDCA was further identified as an intestinal FXR antagonist that improved various metabolic endpoints in mice with established obesity. Thus, we conclude that metformin acts in part through a B. fragilis-GUDCA-intestinal FXR axis to improve metabolic dysfunction, including hyperglycemia.

Fig. 1 |. Oral metformin modulates the composition of gut microbiota and bile acids in individuals with T2D.

a, Partial least-square discriminant analysis (PLS-DA). The BM (before the metformin treatment) group is shown in black, and the AM (after 3-d metformin treatment) group is shown in red. PC1 and PC2 account for 12% and 6%, respectively, of the total variance. PERMANOVA with the Bray–Curtis distance was used to assess the significant difference between the two groups, and the result showed significant separation of the BM and AM groups (P = 0.0001). n = 22 individuals/group. b, VIP scores of PLS-DA. VIP scores were used to rank the ability of different taxa to discriminate between BM and AM groups. A taxon with VIP score >1 was considered important in the discrimination. n = 22 individuals/group. c, Different species abundance (percent reads) of Bacteriodes based on metagenomics data. n = 22 individuals/group. q value (FDR-adjusted P value), P value determined by two-tailed Wilcoxon matched-pairs signed rank test. d, Bile acid levels in the stool. n = 22 individuals/group. P value was determined by two-tailed Wilcoxon matched-pairs signed rank test. e,f, Serum FGF19 (e) and C4 (f) levels. n = 22 individuals/group. P value was determined by two-tailed Wilcoxon matched-pairs signed rank test. All the data are presented as the mean ± s.e.m.

Fig. 2 |. GuDCA and tuDCA are identified FXR antagonists.

a, TR-FRET FXR coactivator recruitment assay to assess whether GUDCA is a FXR antagonist in the presence of CDCA (20 μM). n = 4 replicates/treatment. b, Luciferase activity of control (DMSO), different concentrations of GUDCA or TUDCA without or with FXR agonist CDCA treatment. n = 3 replicates/treatment. P value was determined by one-way ANOVA with Tukey’s correction (F_7,16 = 61.69). c, SHP and FGF19 mRNA expression in differentiated Caco-2 cells after treatment with different concentrations of GUDCA or TUDCA with CDCA. n = 3 replicates/treatment. P value was determined by one-way ANOVA with Tukey’s correction (SHP: F_7,16 = 4.871, FGF19: F_7,16 = 15.41). d, Relative expression of the target gene mRNAs of intestinal FXR in mice treated with vehicle, TCA or TCA and CDCA, GUDCA or TUDCA. n = 5 mice/group. P value was determined by one-way ANOVA with Tukey’s correction (Shp: F_4,20 = 38.47, Fgf15: F_4,20 = 25.02). e, Relative expression of intestinal Fxr mRNA and its target gene mRNAs in mice treated with metformin on a HFD for 1 week. n = 5 mice/group. P value was determined by two-tailed Student’s t-test (Shp: t₈ = 2.779, Fgf15: t₈ = 3.383). f, The relative expression of Cyp7a1, Cyp7b1, Cyp8b1 and Cyp27a1 mRNAs in the livers of metformin-treated mice after 1-week HFD feeding. n = 5 mice/group. P value was determined by two-tailed Student’s t-test (Cyp7a1: t₈ = 2.805). g, Relative abundance of intestinal Fxr mRNA and its target gene mRNAs in metformin-treated Ampka1^fl/fl and Ampka1^ΔIE mice on a HFD for 1 week. n = 5 mice/group. P value was determined by one-way ANOVA with Tukey’s correction (Shp: F_3,16 = 8.046, Fgf15: F_3,16 = 11.83). h, Relative expression of intestinal Fxr mRNA and its target gene mRNAs in antibiotics-treated, microbiota-depleted mice treated with metformin on a HFD for 1 week. n = 5 mice/group. P value was determined by one-way ANOVA with Tukey’s correction (Shp: F_3,16 = 6.859, Fgf15: F_3,16 = 8.782). i, In microbiota-depleted mice, metformin had no effect in inhibiting the activation of intestinal FXR on top of TCA. The relative expression of Fxr and its target genes in the ileum. n = 6 mice/group. All the data are presented as the mean ± s.e.m.

Fig. 3 |. Metformin-induced downregulation of B. fragilis abundance was negatively correlated with the modulation of bile acid profiles.

a, Heat map of the correlation between the species of Bacteroides and the bile acid levels in the stool of individuals with T2D in response to metformin treatment. b, Heat map of the correlation between the species of Bacteroides and biochemical indexes in individuals with T2D in response to metformin treatment. n = 22 individuals/group. Spearman’s rank test. *q < 0.05, **q < 0.01 versus BM, q value (FDR-adjusted P value). c, Growth curve of B. fragilis with or without metformin in a laboratory culture. n = 9 replicates/treatment. d,e, The content of 5-methyl-tetrahydrofolic acid (5-methyl-THF (d) and methionine (e) in B. fragilis treated with and without metformin or in a laboratory culture. n = 10 replicates/treatment. P value was determined by two-tailed Student’s t-test (d, t₁₈ = 43.34; e, t₁₈ = 6.249). f, Reconstitution with methionine restored the growth of B. fragilis in the presence of metformin in culture. n = 5 replicates/treatment. g, The abundance of Bsh gene of B. fragilis in individuals with T2D. n = 22 individuals/group. P value was determined by two-tailed Wilcoxon matched-pairs signed rank test. h, The hydrolysis efficiency of d5-GUDCA by stool samples from individuals with T2D before (BM) and after (AM) metformin treatment. n = 22 individuals/group. P value was determined by two-tailed Wilcoxon matched-pairs signed rank test. i, The hydrolysis efficiency of GUDCA mediated by vehicle- and metformin-treated B. fragilis with or without caffeic acid phenethyl ester (CAPE, BSH activity inhibitor) in culture. n = 5 replicates/treatment. P values were determined by one-way ANOVA with Tukey’s correction (F_3,16 = 13). j, The hydrolysis efficiency of GUDCA mediated by B. fragilis with or without TMP in culture. n = 5 replicates/treatment. P value was determined by two-tailed Student’s t-test (t₈ = 15.2). k,l, Bile acid levels (k) and the relative expression of Fxr and its target genes (l) in the ileum after 1 week of TMP treatment on a HFD. n = 6 mice/group. P values were determined by two-tailed Student’ t-test (TβMCA: t₁₀ = 3, Shp: t₁₀ = 2.703, Fgf15: t₁₀ = 2.288). All data are presented as the mean ± s.e.m.

Fig. 4 |. B. fragilis reverses the metabolic improvements of metformin.

Mice were divided to three groups (control, metformin and metformin plus B. fragilis) under HFD treatment for 4 weeks to explore the reversal effects. Mice in the control and metformin groups were given the same dose of heatkilled B. fragilis. a,b, Glucose tolerance test (GTT, a) and area under the curve (AUC, b). n = 5 mice/group. b, F_2,12 = 26.93. c, Insulin tolerance test (ITT). n = 5 mice/group. d,e, Fasting glucose (d) and insulin (e) levels. n = 7 mice/group. d, F_2,18 = 7.043. f, HOMA-IR. n = 7 mice/group. F_2,18 = 5.415. g, Energy expenditure. n = 5 mice/group. h, Bile acid levels in the ileum. n = 7 mice/group. TβMCA: F_2,18 = 43.27, TUDCA: F_2,18 = 31.76. i, Relative mRNA abundance of intestinal Fxr and its target gene mRNAs. n = 7 mice/group. Shp: F_2,18 = 5.37, Fgf15: F_2,18 = 6.213. j, Relative expression levels of the indicated thermogenic genes in sWAT; mice were placed at 4 °C for 24 h before killing. n = 7 mice/group. Ucp1: F_2,18 = 7.604, Elovl3: F_2,18 = 5.488, Cox8b: F_2,18 = 6.511, Tmem26: F_2,18 = 12.09, Pgc1a: F_2,18 = 7.038. All the P value was determined by one-way ANOVA with Tukey’s correction; a,c,g, *P < 0.05, **P < 0.01 versus control; ^#P < 0.05, ^##P < 0.01 versus metformin. All data are presented as the mean ± s.e.m.

Fig. 5 |. Intestinal FXR signaling is essential for the metformin-induced long-term improvements in metabolic diseases.

HFD-fed Fxr^fl/fl and Fxr^ΔIE mice were treated with metformin for 12 weeks. a, Relative mRNA abundance of intestinal Fxr and its target gene mRNAs. n = 4 or 5 (5, 5, 5, 4) mice/group. Fxr: F_3,15 = 23.16, Shp: F_3,15 = 7.384, Fgf15: F_3,15 = 7.121. b,c, GTT (b) and AUC (c). n = 4 or 5 (5, 5, 5, 4) mice/group. c, F_3,15 = 20.58. d, ITT. n = 4 or 5 (5, 5, 5, 4) mice/group. e,f, Fasting glucose (e) and insulin (f) levels. n = 4 or 5 (5, 5, 5, 4) mice/group. e, F_3,15 = 10.23; f, F_3,15 = 5.074. g, HOMA-IR. n = 4 or 5 (5, 5, 5, 4) mice/group. F_3,15 = 11.49. h, Energy expenditure. n = 4 or 5 (5, 5, 5, 4) mice/group. i, Relative mRNA abundance of the indicated thermogenic genes in sWAT; mice were placed at 4 °C for 24 h before killing. n = 4 or 5 (5, 5, 5, 4)/group. Ucp1: F_3,15 = 4.722, Tmem26: F_3,15 = 8.348, Pgc1a: F_3,15 = 5.669. j, Western blot analysis (cropping of blot images) of UCP1 protein expression in sWAT and the statistical graph, mice were placed at 4 °C for 24 h before killing. n = 3 mice/group. F_3,8 = 13. k, UCP1 immunohistologic staining of sWAT sections; mice were placed at 4 °C for 24 h before killing. n = 3 mice/group, three images per mouse. Scale bars, 50 μm. All P values were determined by one-way ANOVA with Tukey’s correction, *P < 0.05, **P < 0.01 versus Fxr^fl/fl + vehicle. All data are presented as the mean ± s.e.m.

Fig. 6 |. GuDCA supplementation had therapeutic effects in improving glucose tolerance dependent on intestinal FXR.

a, Intestinal Fxr and its target gene mRNAs relative abundance after 1-week GUDCA treatment by gavage (5 mg/kg/d, 10 mg/kg/d and 50 mg/kg/d) on a HFD. n = 3 mice/group. Shp: t₄ = 3.178, Fgf15: t₄ (10 mg/kg) = 3.06, t₄ (50 mg/kg) = 5.171. b, Bile acid profiles in the ileum after 1-week GUDCA treatment by gavage (50 mg/kg/d). n = 5 mice/group. TUDCA: t₈ = 2.475, GUDCA: t₈ = 5.369. c, The schematic diagram of animal model applied in GUDCA therapeutic experiments; after a 12-week HFD treatment, mice were given vehicle or GUDCA (50 mg/kg/d) for 4 weeks. DIO, diet-induced obesity. d,e GTT (d) and AUC (e). n = 5 mice/ group. e: t₈ = 4.891. (f) ITT. n = 5 mice/group. g,h Fasting glucose (g) and insulin (h) levels. n = 5 mice/group. (g): t₈ = 2.839, (h): t₈ = 3.332. i, HOMA-IR. n = 5 mice/group. t₈ = 4.321. j, Energy expenditure. n = 5 mice/group. k, Serum active GLP1 levels. Vehicle or GUDCA-treated (50 mg/kg/d) mice on a HFD for 1 week. n = 5 or 6 mice/group. t₉ = 3.072. All P values were determined by two-tailed Student’s t-test, *P < 0.05, **P < 0.01 versus vehicle. All data are presented as the mean ± s.e.m.