Differential effects of Akkermansia-enriched fecal microbiota transplant on energy balance in female mice on high-fat diet

Abstract

Estrogens protect against weight gain and metabolic disruption in women and female rodents. Aberrations in the gut microbiota composition are linked to obesity and metabolic disorders. Furthermore, estrogen-mediated protection against diet-induced metabolic disruption is associated with modifications in gut microbiota. In this study, we tested if estradiol (E2)-mediated protection against obesity and metabolic disorders in female mice is dependent on gut microbiota. Specifically, we tested if fecal microbiota transplantation (FMT) from E2-treated lean female mice, supplemented with or without Akkermansia muciniphila, prevented high fat diet (HFD)-induced body weight gain, fat mass gain, and hyperglycemia in female recipients. FMT from, and cohousing with, E2-treated lean donors was not sufficient to transfer the metabolic benefits to the E2-deficient female recipients. Moreover, FMT from lean donors supplemented with A. muciniphila exacerbated HFD-induced hyperglycemia in E2-deficient recipients, suggesting its detrimental effect on the metabolic health of E2-deficient female rodents fed a HFD. Given that A. muciniphila attenuates HFD-induced metabolic insults in males, the present findings suggest a sex difference in the impact of this microbe on metabolic health.

Keywords: diabetes; estradiol; estrogens; gut microbiome; metabolism; obesity.

Figure 1

Estradiol treatment but not fecal microbiota transplant (FMT), attenuated obesity and hyperglycemia in female mice on HFD. (A): FMT and cohousing study design and timeline. Mice were fed phytoestrogen-free chow till D11, then switched to HFD. Mice treated with E2 (E donor, n = 7) or Veh (V donor, n = 7) were used as FMT donors. All recipients received Veh implants. V-V and V-E recipients got FMT from V donors or E donors, respectively. Recipient mice were administered antibiotic cocktail for 9 days and starting on D10, were orally gavaged with 9 doses of FMT, on alternate days. G denotes FMT gavage days. (B) Body weight; (C) Fat mass. For (B, C), repeated measures ANOVA, separately on recipients and donors, followed by a t-test. *denotes p < 0.05 between E2 donors and Veh donors). (D) 5h-fasting blood glucose on D19, after one week on HFD. Two-way ANOVA followed by Tukey post-hoc (*p < 0.05). V donor, mice with Veh implants used as FMT donors; E donor, E2-treated mice used as FMT donors; V-V, Veh mice receiving FMT from V donor; V-E, Veh mice receiving FMT from E donor.

Figure 2

Estradiol treatment, but not fecal microbiota transplant (FMT) from lean E2-treated mice supplemented with A. muciniphila, protected ovariectomized mice from HFD-induced obesity. (A) A. muciniphila-enriched FMT study design and timeline. Recipient mice were administered antibiotic cocktail for the first 14 days. The three recipient groups, 1) Veh mice receiving FMT from Veh mice (V-V, n = 4); 2) Veh mice receiving FMT from E2 mice following enrichment with Akkermansia cells (V-E_A, n = 4), and 3) E2-implanted mice receiving FMT from E2 mice (E-E, n = 4) were orally gavaged with a total of 6 gavages on alternate days, starting on D15 and excluding D17. G denotes FMT gavage days; ^F denotes fresh fecal sample collection days. (B) Body weight and (C) Fat mass. *denotes days when E-E groups differ from V-V and V-E_A; # denotes days when E-E differ from V-E_A only (p < 0.05, RM ANOVA, Tukey post-hoc). V-V: mice with Veh implants receiving FMT from Veh mice; E-E: E2-treated mice receiving FMT from E2 mice; V-E_A: mice with Veh implants receiving FMT from E2-treated mice with A. muciniphila supplementation.

Figure 3

Estradiol treatment protected, while A. muciniphila-supplemented fecal microbiota transplant (FMT) from E2-treated lean mice exacerbated, HFD-induced hyperglycemia in female mice. (A) Blood glucose levels during HFD feeding, (B) Glucose tolerance test (GTT) measured on D45. *denotes differences across E-E, V-E_A, and V-V. # denotes differences between E-E and V-E_A (*, # p < 0.05, t-test). E-E, E2-treated mice receiving FMT from E2 mice (n = 4); V-V, mice with Veh implants receiving FMT from Veh mice (n = 4); V-E_A, mice with Veh implants receiving FMT from E2-treated mice supplemented with A. muciniphila (n = 4).

Figure 4

Estradiol, but not fecal microbiota transplant (FMT) from E2-treated mice supplemented with A. muciniphila, protected against changes in energy intake and expenditure in HFD-fed female mice. (A) Food intake, (B) Water intake, (C) Physical activity, (D) Energy expenditure, (E) VO2 consumption, and (F) VCO2 production were measured in metabolic cages on D18–21 (A–D): *p < 0.05, ANOVA, Tukey post-hoc; (E): *p < 0.05, ANCOVA, Tukey post-hoc). E-E, E2-treated mice receiving FMT from E2 mice (n = 4); V-V, mice with Veh implants receiving FMT from Veh mice (n = 4); V-E_A, mice with Veh implants receiving FMT from E2-treated mice supplemented with A. muciniphila (n = 4).

Figure 5

Antibiotics altered gut microbiota α-diversity and β-diversity in adult female mice. Mice received antibiotics in drinking water for two weeks. Starting on D15, mice received a total of 6 doses of FMT gavage with (V-E_A) or without A. muciniphila (E-E and V-V) supplementation. (A) Faith phylogenetic diversity (PD) (± SEM) over time. Gut microbiota data from D23 and D27 (n = 8) were aggregated to isolate the effect during FMT, and D32 and D39 (n = 8), after the FMT was discontinued. (B) Principal component plot showing weighted Unifrac distance between microbiota communities during antibiotic treatment. E-E, E2-treated mice receiving FMT from E2 mice; V-V, mice with Veh implants receiving FMT from Veh mice; V-E_A, mice with Veh implants receiving FMT from E2-treated mice, supplemented with A. muciniphila.

Figure 6

Estradiol, antibiotics, and fecal microbiota transplant (FMT) from E2-treated lean mice supplemented with A. muciniphila altered gut microbiota α-diversity and β-diversity in adult female mice. Mice were administered antibiotics for two weeks. From D15, mice received 6 total doses of FMT gavage with (V-E_A) or without (E-E and V-V) A. muciniphila supplementation. (A) Principal component plot showing weighted Unifrac distance for microbial communities during [●: D17; o: D23 and D27 aggregate (n=8)] after FMT [▽: D32 and D39 aggregate (n=8)]. (B) Microbiota taxa relative abundances at the genus level, across treatment days. E-E, E2-treated mice receiving FMT from E2 mice; V-V, mice with Veh implants receiving FMT from Veh mice; V-E_A, mice with Veh implants receiving FMT from E2-treated mice, supplemented with A muciniphila.