Both enantiomers of β-aminoisobutyric acid BAIBA regulate Fgf23 via MRGPRD receptor by activating distinct signaling pathways in osteocytes

Abstract

With exercise, muscle and bone produce factors with beneficial effects on brain, fat, and other organs. Exercise in mice increased fibroblast growth factor 23 (FGF23), urine phosphate, and the muscle metabolite L-β-aminoisobutyric acid (L-BAIBA), suggesting that L-BAIBA may play a role in phosphate metabolism. Here, we show that L-BAIBA increases in serum with exercise and elevates Fgf23 in osteocytes. The D enantiomer, described to be elevated with exercise in humans, can also induce Fgf23 but through a delayed, indirect process via sclerostin. The two enantiomers both signal through the same receptor, Mas-related G-protein-coupled receptor type D, but activate distinct signaling pathways; L-BAIBA increases Fgf23 through Gαs/cAMP/PKA/CBP/β-catenin and Gαq/PKC/CREB, whereas D-BAIBA increases Fgf23 indirectly through sclerostin via Gαi/NF-κB. In vivo, both enantiomers increased Fgf23 in bone in parallel with elevated urinary phosphate excretion. Thus, exercise-induced increases in BAIBA and FGF23 work together to maintain phosphate homeostasis.

Keywords: BAIBA; CP: Metabolism; CP: Molecular biology; FGF23; Gα subunit; MRGPRD; Mas-related G-protein-coupled receptor type D; enantiomer; exercise; osteocyte; phosphate metabolism; β-aminoisobutyric acid.

Figure 1.. Treadmill exercise elevates L-BAIBA, intact FGF23, and urine phosphate levels

(A and B) From left to right, the data display liquid chromatography-tandem mass spectrometry quantitation of plasma L-BAIBA and D-BAIBA, ELISA quantitation of plasma intact FGF23, and colorimetric quantitation of urine phosphate and creatinine obtained from males (A) and females (B). Treadmill exercise significantly increased the levels of L- but not D-BAIBA and elevated intact FGF23, urine phosphate, and creatinine. (C and D) Pearson correlation analysis of L-BAIBA with plasma intact FGF23, as well as intact FGF23 with urine phosphate and creatinine are shown for males (C) and females (D). Positive correlations were observed between L-BAIBA and intact FGF23, while intact FGF23 correlated with urine phosphate and creatinine in males. A positive correlation was observed between intact FGF23 and creatinine in females. The experiments were performed twice each for males and females and the resulting data combined (n = 9–12 male or female animals per group). Data are presented as mean ± SD, and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using unpaired t test (A and B). Correlation coefficient (r) and p values obtained by Pearson correlation analysis are indicated (C and D).

Figure 2.. BAIBA enantiomers differentially regulate Fgf23 and Sost mRNA expression in mature osteocyte culture

(A and B) RT-qPCR analysis of Fgf23 (A) and Sost mRNA (B) in day-28 IDG-SW3 treated with L-BAIBA for 24 h and 72 h. High doses of L-BAIBA increased Fgf23 at 24 h, which was returned to baseline at 72 h. In contrast, 20 μM L-BAIBA decreased Sost mRNA at 72 h but had no effect at 24 h. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (**p < 0.01) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (C and D) RT-qPCR analysis of Fgf23 (C) and Sost mRNA (D) in day-28 IDG-SW3 treated with D-BAIBA for 24 h and 72 h. High doses of D-BAIBA upregulated Fgf23 mRNA at 72 h but had no effect at 24 h. Contrary to Fgf23, Sost mRNA was upregulated at 24 h by 5 and 10 μM D-BAIBA, which was returned to baseline at 72 h. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (E and F) RT-qPCR analysis of Fgf23 (E) and Sost mRNA (F) in osteocyte-enriched murine bone fragments treated with L- and D-BAIBA for 24 h (100 μM) and 72 h (200 μM). Effects of BAIBA in IDG-SW3 cells were replicated in primary osteocytes. There were no sex differences. Data are presented as mean ± SD (n = 4–7 biological replicates per condition), and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. See also Figures S2–S5.

Figure 3.. Both BAIBA enantiomers utilize the same receptor, MRGPRD

(A) Immunoblotting analysis of MRGPRD in day-28 IDG-SW3 treated with 0–20 μM L-BAIBA and D-BAIBA for 24 h. (B) Quantitative data for (A). Both BAIBA enantiomers significantly increased MRGPRD with a 4- to 6-fold greater amount compared to controls. MRGPRD expression levels were normalized by β-tubulin. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (C) 50 μM MU6840, an antagonist against MRGPRD, blocked Fgf23 mRNA expression induced by 10 μM L-BAIBA (left), and 25 and 50 μM MU6840 blocked Sost mRNA expression induced by D-BAIBA in day-28 IDG-SW3. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (D) Both BAIBA enantiomers elevated Fgf23 and Sost mRNA in wild-type (WT) murine osteocyte-enriched bone fragments but not in Mrgprd-KO osteocytes ex vivo. There were no sex differences. Data are presented as mean ± SD (n = 3–6 biological replicates per condition), and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (E) L-BAIBA elevated Fgf23 mRNA in WT bones but not in Mrgprd-KO bones in vivo. There were no sex differences. Data are presented as mean ± SD (n = 3–8 male or female animals per group), and p values (*p < 0.05, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (F) RT-qPCR analysis of Fgf23 and Sost mRNA in day-28 IDG-SW3 pre-treated with 100 and 1,000 μM of one enantiomer before treatment with 10 μM of the opposite enantiomer. Neither enantiomer was blocked by the opposite enantiomer, even if 100-fold excess was used. Data are presented as mean ± SD (n =4 biological replicates per condition), and p values are calculated using one-way ANOVA with Tukey’s multiple comparisons. (G) BAIBA signaling pathway. L- and D-BAIBA utilize MRGPRD to regulate the transcription of Fgf23 and Sost, respectively. MU6840 is an antagonist against MRGPRD. Mrgprd-KO mice were also utilized in this study. See also Figure S6.

Figure 4.. L-BAIBA activates Gαs and Gαq proteins, while D-BAIBA activates the Gαi protein

(A) RT-qPCR analysis of Fgf23 mRNA in day-28 IDG-SW3 treated with 10 μM L-BAIBA in the presence of melittin, a Gαs inhibitor (left), ebselen, a Gαq inhibitor (middle), or pertussis toxin, a Gαi inhibitor (right). 100 nM melittin and ebselen blocked L-BAIBA-induced Fgf23 mRNA expression but not pertussis toxin. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (B) RT-qPCR analysis of Sost mRNA in day-28 IDG-SW3 treated with 10 μM D-BAIBA in the presence of melittin (left), ebselen (middle), or pertussis toxin (right). 10 ng/mL pertussis toxin blocked D-BAIBA-induced Sost mRNA expression, but neither melittin nor ebselen did. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (C) cAMP production in day-28 IDG-SW3 treated with BAIBA enantiomers. Upon 10 μM L-BAIBA treatment cAMP increased after 3 h, peaked at 6 h, and maintained that level for up to 24 h. In contrast, 10 μM D-BAIBA decreased cAMP at 6 h but not at other time points. Data are presented as mean ± SD (n =3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (D) BAIBA signaling pathway. L-BAIBA utilizes Gαs and Gαq coupled to MRGPRD while Gαi is utilized by D-BAIBA. cAMP was upregulated by L-BAIBA whereas it was inhibited by D-BAIBA with different response timing. Melittin is an inhibitor for Gαs, ebselen for Gαq, and pertussis toxin for Gαi, used in this study.

Figure 5.. L-BAIBA induces Fgf23 mRNA via Gαs/cAMP/PKA/CBP/β-catenin and Gαq/PKC/CREB signaling pathways downstream of MRGPRD

(A) RT-qPCR analysis of Fgf23 mRNA in day-28 IDG-SW3 treated with 10 μM L-BAIBA in the presence of PKI5–24, a PKA inhibitor. PKA inhibitor blocked L-BAIBA-induced Fgf23 mRNA expression. Data are presented as mean ± SD (n = 4 biological replicates per condition), and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (B and C) Immunoblotting of phosphorylated β-catenin (Ser675, activated form) and total β-catenin (B) in a whole-cell lysate prepared from IDG-SW3 treated with 10 μM L-BAIBA. (C) Quantification of the immunoblots. Phosphorylation levels of β-catenin at Ser675 were increased 4-fold after 6 h, which was normalized by total β-catenin expression levels. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (D and E) Immunoblotting of phosphorylated β-catenin (Ser675) and total β-catenin (D) in a whole-cell lysate prepared from IDG-SW3 treated with 10 μM L-BAIBA in the presence of 100 nM melittin and 50 nM PKI5–24, Gαs, and PKA inhibitors, respectively. (E) Quantification of the immunoblots. Both Gαs and PKA inhibitors blocked L-BAIBA-induced phosphorylation of β-catenin at Ser675. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (F) RT-qPCR analysis of Fgf23 mRNA in day-28 IDG-SW3 treated with 10 μM L-BAIBA in the presence of ICG-001, a β-catenin inhibitor. High doses of ICG-001 blocked L-BAIBA-induced Fgf23 mRNA expression. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (G) RT-qPCR analysis of Fgf23 mRNA in day-28 IDG-SW3 treated with 10 μM L-BAIBA in the presence of Go6983, a PKC inhibitor. Go6983 blocked L-BAIBA-induced Fgf23 mRNA expression. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (**p < 0.01, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (H and I) Immunoblotting of phosphorylated CREB (Ser133) and total CREB (H) in a whole-cell lysate prepared from IDG-SW3 treated with 10 μM L-BAIBA for 3, 6, and 24 h. (I) Quantification of the immunoblots. L-BAIBA enhanced phosphorylation of CREB at Ser133 at 24 h. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (J and K) Immunoblotting of phosphorylated CREB (Ser133) and total CREB (J) in a whole-cell lysate prepared from IDG-SW3 treated with 10 μM L-BAIBA in the presence of 100 nM ebselen, a Gαq inhibitor. (K) Quantification of the immunoblots. Ebselen inhibited L-BAIBA-induced phosphorylation of CREB. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (L and M) Immunoblotting of phosphorylated CREB (Ser133) and total CREB (L) in a whole-cell lysate prepared from IDG-SW3 treated with 10 μM L-BAIBA in the presence of 100 nM Go6983, a PKC inhibitor. (M) Quantification of the immunoblots. Go6983 blocked L-BAIBA-induced phosphorylation of CREB. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (N) BAIBA signaling pathway. L-BAIBA utilizes Gαs/cAMP/PKA/CBP/β-catenin and Gαq/PKC/CREB signaling pathways downstream of MRGPRD to regulate Fgf23 mRNA. Melittin is an inhibitor for Gαs, ebselen for Gαq, PKI5–24 for PKA, Go6983 for PKC, and ICG-001 for CBP/β-catenin, used in this study.

Figure 6.. D-BAIBA-early-induced sclerostin is responsible for the later induction of Fgf23 mRNA

(A) RT-qPCR analysis of Sost mRNA in IDG-SW3 treated with 10 μM D-BAIBA over a 72-h time course. Sost mRNA expression was increased at 24 h and 48 h but returned to basal levels by 72 h. Data are presented as mean ± SD (n = 4 biological replicates per condition), and p values (*p < 0.05, **p < 0.01 vs. 0 h) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (B) Quantification of sclerostin protein in conditioned media from L- and D-BAIBA-treated IDG-SW3 at 24 h. 10 μM D-BAIBA elevated the secretion of sclerostin protein more than 2-fold compared to controls. Data are presented as mean ± SD (n = 4 biological replicates per condition), and p values (*p < 0.05) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (C) RT-qPCR analysis of Fgf23 mRNA in IDG-SW3 treated with 10 μM D-BAIBA in the presence of AbD09097, a sclerostin-neutralizing antibody, for 72 h. 1,000 pg/mL AbD09097 blocked D-BAIBA-induced Fgf23 mRNA expression. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (D and E) Immunoblotting of phospho NF-κB p65 and total NF-κB p65 (D) in whole-cell lysate from 10 μM D-BAIBA-treated IDG-SW3. (E) Quantification of the immunoblots. D-BAIBA enhanced a 4- to 6-fold of NF-κB p65 phosphorylation after 24–72 h. Data are presented as mean ± SD (n = 3 biological replicates per condition), and p values (*p < 0.05, **p < 0.01) are calculated using one-way ANOVA with Dunnett’s multiple comparisons. (F–I) RT-qPCR analysis of Sost and Fgf23 mRNA expression in IDG-SW3 treated with 10 μM D-BAIBA in the presence of BMS345541, an NF-κB inhibitor for 0–24 h (F), 24–48 h (G), 24–72 h (H), and 48–72 h (I). 500 nM BMS345541 completely blocked D-BAIBA-induced Sost mRNA expression when treated between 0 and 24 h, but Fgf23 mRNA showed no changes. Both Sost and Fgf23 mRNA expressions were elevated by D-BAIBA at 48 h, which were reduced by the treatment of BMS345541 between 24 and 48 h. The treatment of BMS345541 for 24–72 h blocked a late induction of Fgf23 by D-BAIBA, and 48–72 h of treatment of BMS345541 partially reduced D-BAIBA-elevated Fgf23 mRNA, but Sost mRNA showed no changes except a reduction by D-BAIBA in the presence of 500 nM BMS345541 at 72 h. All data are presented as mean ± SD (n = 3 or 4 biological replicates per condition), and p values (*p < 0.05, **p < 0.01, ***p < 0.001) are calculated using two-way ANOVA with Tukey’s multiple comparisons. (J) BAIBA signaling pathway. D-BAIBA utilizes Gαi/NF-κB/sclerostin/NF-κB signaling pathway downstream of MRGPRD to regulate Fgf23 mRNA. BMS34551 is an inhibitor of NF-κB, and AbO09097 is an antibody against sclerostin.