Vitamin B₁₂-dependent taurine synthesis regulates growth and bone mass

Abstract

Both maternal and offspring-derived factors contribute to lifelong growth and bone mass accrual, although the specific role of maternal deficiencies in the growth and bone mass of offspring is poorly understood. In the present study, we have shown that vitamin B12 (B12) deficiency in a murine genetic model results in severe postweaning growth retardation and osteoporosis, and the severity and time of onset of this phenotype in the offspring depends on the maternal genotype. Using integrated physiological and metabolomic analysis, we determined that B12 deficiency in the offspring decreases liver taurine production and associates with abrogation of a growth hormone/insulin-like growth factor 1 (GH/IGF1) axis. Taurine increased GH-dependent IGF1 synthesis in the liver, which subsequently enhanced osteoblast function, and in B12-deficient offspring, oral administration of taurine rescued their growth retardation and osteoporosis phenotypes. These results identify B12 as an essential vitamin that positively regulates postweaning growth and bone formation through taurine synthesis and suggests potential therapies to increase bone mass.

Figure 1. B₁₂ deficiency in mice causes growth retardation and low bone mass.

(A) Real-time PCR analysis of Gif expression in WT and Gif^–/– tissues. (B) Serum B₁₂ levels in WT, Gif^–/–_(F1), and Gif^–/–_(F2) mice. (C) BW analysis of WT, Gif^–/–_(F1) and Gif^–/–_(F2) mice. (D) Morphological analysis of 8-week-old WT, Gif^–/–_(F1), and Gif^–/–_(F2) mice. (E and F) Histological analysis of vertebrae (E) and μCT analysis of long bone (F) of WT, Gif^–/–_(F1), and Gif^–/–_(F2) mice. Mineralized bone matrix (black) was stained by von Kossa reagent. BV/TV, bone volume relative to total volume. Ct.Th., cortical thickness. (G) Toluidine blue staining showing reduced osteoblast number on bone surface, with quantification of Ob.N/T.Ar. (H) Photomicrographs showing near-absence of calcein double labeling on the surface of trabecular bone in Gif^–/–_(F2) mice, with quantification of BFR. (I) Photomicrographs showing TRAP-stained osteoclasts on the bone surface (pink), with quantification of osteoclast surface per bone surface (OcS/BS). ^#P < 0.05; *P < 0.01. Values are mean ± SEM. n = 8 [WT and Gif^–/–_(F2)]; 9 [Gif^–/–_(F2)]. Arrowheads on images indicate the location of cell types or parameters measured. Scale bars: 10 mm (D); 1 mm (E and F); 0.1 mm (G); 50 μm (H); 10 μm (H, insets); 0.05 mm (I). See also Supplemental Figure 1.

Figure 2. Maternal B₁₂ regulates offspring growth and bone mass and B₁₂ deficiency during aging regulates bone mass independently of body growth.

(A) Growth curve analysis of WT/VEH, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/B₁₂ mice. (B and C) Histological analysis of vertebral bone (B) and μCT analysis of long bone (C) in 8-week-old WT/VEH, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/B₁₂ mice, with quantification of BV/TV, Ob.N/T.Ar., BFR, OcS/BS, and Ct.Th. (D and E) BW (D) and nose-to-tail length (E) of 48-week-old WT and Gif^–/–_(F1) mice. (F and G) Histological analysis of vertebral bone (F) and Ct.Th. analysis of long bone (G) in 48-week-old WT and Gif^–/–_(F1) mice, with quantification of BV/TV, Ob.N/T.Ar., OcS/BS, and Ct.Th. ^#P < 0.05; *P < 0.01. Values are mean ± SEM. n = 4–6 [WT]; 5 [Gif^–/–_(F2)/VEH], 7 [Gif^–/–_(F2)/B₁₂]; 6 [Gif^–/–_(F1)]. All mice shown are females. Scale bars: 1 mm (B, C, and F). See also Supplemental Figure 2.

Figure 3. B₁₂ deficiency causes GH resistance.

(A) Serum GH levels in WT (n = 6) and Gif^–/–_(F2) (n = 5) mice. (B and C) Real-time PCR analysis of Ghrh in the hypothalamus (Hyp) (B), and Ghr expression in liver and bone (C), of WT and Gif^–/–_(F2) mice (n = 5 per group). (D) Serum IGF1 levels in WT and Gif^–/–_(F2) mice (n = 7–10). (E and F) Western blot analysis of IGF1R (E) and STAT5 (F) phosphorylation in different WT and Gif^–/–_(F2) tissues; blotting was done on the same blot after stripping the membrane for pIGF1R and pSTAT5, respectively. A representative blot from 3 independent experiments is shown; different tissues were run noncontiguously. Relative quantification of pIGF1R and pSTAT5 (normalized to IGF1R and STAT5, respectively) is shown below. (G and H) Real-time PCR analysis of Socs2 (G) and STAT5 target gene (H) expression in WT and Gif^–/–_(F2) liver (n = 5 per group). (I) Enzymatic reactions of MTR and MUT dependent on the B₁₂-generated cofactors methyl-B₁₂ and adenosyl-B₁₂, respectively. (J) Levels of methionine, succinate, and homocysteine (nmol/g liver tissue) in WT and Gif^–/–_(F2) mice (n = 5 per group). (K) ChIP analysis of methylated histones, shown relative to control (assigned as 1), in different regions of Igf1 in Gif^–/–_(F2) liver. P-, promoter; E-, exon; n.d., not detectable. (L) Survival of WT/VEH, WT/IGF1, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/IGF1 mice (n = 5 per group). ^#P < 0.05; *P < 0.01. Values are mean ± SEM. See also Supplemental Figure 3.

Figure 4. Metabolomics analysis identifies taurine as a critical metabolite that connects B₁₂ deficiency with GH signaling.

(A) Supervised hierarchical clustering plot of up- or downregulated metabolites in Gif^–/–_(F2) liver. Metabolites regulated by GH in hepatocytes are shown in red font. (B) Summary plot for quantitative enrichment analysis. Metabolite sets are ranked according to false discovery rate (FDR); dashed lines show FDR value cutoffs. (C) Metabolome view reflects on the x axis increasing metabolic pathway impact according to the betweenness centrality measure, which shows key nodes in metabolic pathways that have been significantly altered upon B₁₂ deficiency. Colored circles correspond to pathways in B. (D) PLSDA-VIP plot. Metabolites are ranked according to their increasing importance to group separation between WT and Gif^–/–_(F2) mice. (E) Measurement of taurine and its derivatives in WT/VEH, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/B₁₂ liver (n = 5 per group). ^#P < 0.05; *P < 0.01. Values are mean ± SEM. See also Supplemental Figure 4.

Figure 5. GH regulates taurine synthesis in a STAT5- and B₁₂-dependent manner.

(A) Relationship among taurine, STAT5, and B₁₂ in the liver. (B) Experimental regimen used to test B₁₂ involvement in GH regulation of taurine synthesis. Also shown is RT-PCR analysis to detect Oleosin transcript in the cells after transfection with empty vector or TCOL construct. (C) STAT5 phosphorylation upon GH treatment in empty vector– or TCOL-transfected HepG2 cells. Blots were run noncontiguously. (D) Taurine levels upon GH treatment in empty vector– or TCOL-transfected HepG2 cells. (E) PLSDA-VIP scores plot of metabolomics data from hepatocytes after empty or TCOL transfection. (F) Experimental regimen used to test STAT5 involvement in GH regulation of taurine production. Photomicrographs show immunohistochemistry of STAT5 in HepG2 cells transfected with nontargeting (empty) or STAT5 (shSTAT5) shRNA. (G) Relative expression of IGF1 upon GH treatment in empty or STAT5 shRNA–transfected HepG2 cells. (H) Taurine levels upon GH treatment in empty or STAT5 shRNA–transfected HepG2 cells. (I) Real-time PCR analysis of enzymes in the taurine synthesis pathway in empty, TCOL, or STAT5 shRNA–transfected cells treated with vehicle or GH. (J) GH regulation of the taurine synthesis pathway. Red metabolites and genes, upregulated (only those upregulated by GH and that do not respond to GH upon STAT5 shRNA or TCOL transfection); green metabolites and genes, downregulated; black metabolites and genes, not altered; gray metabolites and genes, not measured. *P < 0.05; ^#P < 0.01. Values are mean ± SEM. Scale bars: 20 μm (F). See also Supplemental Figure 5.

Figure 6. Oral taurine administration prevents growth retardation and osteoporosis in Gif^–/–_(F2) mice.

(A) Growth curve analysis of WT/VEH, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/TAU mice. (B and C) Serum GH (B) and IGF1 (C) levels in WT/VEH, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/TAU mice. (D) Histological analysis of vertebra in WT, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/TAU mice, with quantification of BV/TV, Ob.N/T.Ar., BFR, and OcS/BS. (E) Pearson correlation scatter plot between BV/TV and serum IGF1 levels in taurine-treated mice. *P < 0.05; ^#P < 0.01. n = 5 per group. Values are mean ± SEM. Scale bar: 500 μm. See also Supplemental Figure 6.

Figure 7. Taurine increases IGF1 synthesis from liver and its action in osteoblasts to regulate bone mass.

(A–D) Liver samples. (A) Levels of methionine and homocysteine in WT, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/TAU liver. (B) B₁₂-dependent (MTR) and -independent (BHMT) methionine synthesis pathways. (C) Western blot analysis of BHMT levels in WT, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/TAU liver. Lanes were run contiguously. U.D., undetectable. (D) Levels of betaine and dimethyl-glycine in liver of WT, Gif^–/–_(F2)/VEH, and Gif^–/–_(F2)/TAU mice. (E–G) MC3T3-E1 osteoblast cells. Changes in BrdU incorporation (E), IGF1R and ERK phosphorylation (F), and Ccnd1 expression (G) in cells treated for 24 hours with vehicle, OSI906, taurine, or taurine plus OSI906. Lanes in F were run contiguously, and blots were stripped and reprobed with IGF1R or ERK. Relative quantification of pIGF1R and pERK (normalized to IGF1R and ERK, respectively) is shown below. A representative blot from 3 different experiments is shown. (H) Growth curve analysis of WT and Gif^–/–_(F2)/TAU+OSI906 mice (n = 5 each). (I) Bone mass analysis (BV/TV) in the vertebra of WT and Gif^–/–_(F2)/TAU+OSI906 mice (n = 5 per group). (J) Gut/liver/bone endocrine axis, illustrating GH/STAT5/B₁₂-dependent changes in serum IGF1 and taurine that regulate osteoblast proliferation and bone mass. *P < 0.05; ^#P < 0.01. Values are mean ± SEM. Scale bar: 500 μm. See also Supplemental Figure 7.

Figure 8. B₁₂ status correlates with taurine and the bone formation marker osteocalcin during early postnatal life and aging in humans.

(A–F) Pearson correlation scatter plots between (A and D) serum B₁₂ and taurine, (B and E) B₁₂ and osteocalcin, and (C and F) osteocalcin and taurine in (A–C) children of B₁₂-deficient mothers (red symbols; n = 5) and of healthy mothers (white symbols; n = 7) and in (D–F) aged B₁₂-deficient subjects (red symbols; n = 8) and healthy controls (white symbols; n = 10). Pearson R as well as P values are shown. See also Supplemental Figure 8.