The Gut Microbiota Regulates Endocrine Vitamin D Metabolism through Fibroblast Growth Factor 23

Abstract

To determine the effect of the microbiota on vitamin D metabolism, serum 25-hydroxyvitamin D(25D), 24,25-dihydroxyvitamin D (24,25D), and 1,25-dihydroxyvitamin D (1,25D) were measured in germ-free (GF) mice before and after conventionalization (CN). GF mice had low levels of 25D, 24,25D, and 1,25D and were hypocalcemic. CN of the GF mice with microbiota, for 2 weeks recovered 25D, 24,25D, and 1,25D levels. Females had more 25D and 24,25D than males both as GF mice and after CN. Introducing a limited number of commensals (eight commensals) increased 25D and 24,25D to the same extent as CN. Monocolonization with the enteric pathogen Citrobacter rodentium increased 25D and 24,25D, but the values only increased after 4 weeks of C. rodentium colonization when inflammation resolved. Fibroblast growth factor (FGF) 23 was extremely high in GF mice. CN resulted in an increase in TNF-α expression in the colon 2 days after CN that coincided with a reduction in FGF23 by 3 days that eventually normalized 25D, 24,25D, 1,25D at 1-week post-CN and reinstated calcium homeostasis. Neutralization of FGF23 in GF mice raised 1,25D, without CN, demonstrating that the high FGF23 levels were responsible for the low calcium and 1,25D in GF mice. The microbiota induce inflammation in the GF mice that inhibits FGF23 to eventually reinstate homeostasis that includes increased 25D, 24,25D, and 1,25D levels. The microbiota through FGF23 regulates vitamin D metabolism.

Keywords: fibroblast growth factor 23; inflammation; microbiota; tumor necrosis factor-α; vitamin D.

Figure 1

25D and 24,25D increases following CN of germ-free (GF) mice. (A) Vitamin D, (B) 25D, and (C) 24,25D at 2 and 4 weeks of +D supplementation. Values are ± SEM of n = 10 mice per group, five males and five females. Student’s t-test was used to test significance. (D) Experimental design of conventionalization (CN) and vehicle or +D supplementation. (E) Vitamin D, (F) 25D, and (G) 24,25D levels from plasma of GF before and after 2-week CN. Values are the mean ± SEM of n = 9 mice per group, four males and five females, and two independent experiments. Two-way ANOVA with Bonferroni post hoc tests was used to test significance (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 2

Females produce more 25D and 24,25D than males. (A,B) Vitamin D, (C,D) 25D, and (E,F) 24,25D in vehicle and +D, male and female mice, before and after 2 weeks conventionalization. Values are the mean ± SEM of n = 9 mice per group, four males and five females, and two independent experiments. Two-way ANOVA with Bonferroni post hoc tests was used to test significance (*P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 3

The kinetics of increased 24,25D following conventionalization (CN) of germ-free mice. (A) Vitamin D, (B) 25D, and (C) 24,25D from 0 to 48 h after CN. Values are the mean ± SEM of n = 3–4 males per group. (D) Vitamin D, (E) 25D, and (F) 24,25D from 0 to 2 weeks after CN. Values are the mean ± SEM of n = 5–9 female mice per group. One-way ANOVA with Tukey’s post hoc tests was used to test significance. Bars without a letter in common indicate a significant difference.

Figure 4

Colonization with commensals increased 24,25D after 2 weeks. (A) Vitamin D, (B) 25D, and (C) 24,25D levels following colonization of germ-free (GF) mice with altered Schaedler’s flora (ASF). Values are the mean ± SEM of n = 10–15 mice per group, 5 males and 10 females, and 2 independent experiments. Student’s t-test was used to test significance.

Figure 5

Monocolonization with Citrobacter rodentium increased 24,25D after 4 weeks. (A) Vitamin D, (B) 25D, and (C) 24,25D at 0, 2, and 4 weeks after colonization with 1 × 10⁹ CFU C. rodentium. Values are the mean ± SEM of n = 7–16 mice per group, 6 males and 10 females, and 2 independent experiments. One-way ANOVA with Tukey’s post hoc tests was used to test significance. Bars without a letter in common indicate a significant difference.

Figure 6

Conventionalization (CN) decreases fibroblast growth factor (FGF) 23 and increases calcium and 1,25D levels. (A) Serum Ca following CN of mice. Values are the mean ± SEM of n = 3–21 mice per group, and one to three independent experiments. (B) FGF23 levels following CN of mice. Values are the mean ± SEM of n = 3–6 female mice per group, and one to two experiments. One-way ANOVA with Tukey’s post hoc tests was used to test significance. Bars without a letter in common indicate a significant difference. (C) 1,25D levels following CN of previously germ-free (GF) mice. Values are the mean ± SEM of n = 7 female mice per group, from three independent experiments. (D) Vitamin D, (E) 25D, (F) 1,25D, and (G) 24,25D in GF mice receiving isotype or FGF23-neutralizing antibody. Values are the mean ± SEM of n = 6 mice per group, three males and three females. Student’s t-test was used to test significance.

Figure 7

Conventionalization (CN) induces colonic shortening and TNF-α. (A) Colon length and (B) histological score of inflammation in distal colons from mice following CN. Values are the mean ± SEM of n = 3 females per group. (C) Expression of (C) tnf-α and (D) ifn-γ in the colon. Values are the mean ± SEM of n = 3–6 mice per group, 15 males and 6 females, and 1–2 independent experiments. One-way ANOVA with Tukey’s post hoc tests was used to test significance. Bars without a letter in common indicate a significant difference.

Figure 8

Sterile inflammation reduces 24,25D levels. (A) Vitamin D, (B) 25D, and (C) 24,25D from +D germ-free, 6 and 12 h after LPS injection. Values are the mean ± SEM of n = 4–5 males per group. One-way ANOVA with Tukey’s post hoc tests was used to test significance. Bars without a letter in common indicate a significant difference.