Fig 2: GLP-2 downregulates FGF23 expression by activating GLP-2R/PI3K/AKT signalling in osteoblasts. A Western blot analysis of AKT and p-AKT levels in osteoblasts with or without shRNA-GLP-2R interference. B Relative mRNA expression of FGF23 in osteoblasts treated with or without shRNA-GLP-2R. C Western blot analysis of AKT and p-AKT levels in cells treated with or without LY294002. D Relative mRNA expression of FGF23 following LY294002 intervention. E Western blot analysis of AKT and p-AKT levels in cells treated with or without MK-2206. F Relative mRNA expression of FGF23 after MK-2206 treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 3: Effects of GLP-2 and the PI3K inhibitor LY294002 on FGF23 expression in OVX mice. Forty-four female C57BL/6J mice were randomly divided into five groups: sham-operated (sham, n = 6), OVX treated with vehicle (OVX, n = 8), OVX treated with GLP-2 (OVX + GLP-2, n = 10), OVX treated with LY294002 (OVX + LY294002, n = 10), and OVX treated with GLP-2 plus LY294002 (OVX + GLP-2 + LY294002, n = 10). A IHC staining of FGF23 in the distal femur and corresponding MOD analysis. B Relative mRNA expression of FGF23 in femurs. C Serum FGF23 levels measured by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 4: Transcriptomic analysis of femoral tissues from sham, OVX, and OVX + GLP-2 groups. A Scatter plot of DEGs between the OVX and sham groups (1654 upregulated genes and 2038 downregulated genes). B Scatter plot of DEGs between the OVX + GLP-2 and OVX groups (318 upregulated genes and 396 downregulated genes). C Heatmap of 24 representative DEGs among the three groups. D GO enrichmentresults showing DEGs involved in cellular component, biological process, and molecular function categories. E KEGG signalling pathway diagram. F KEGG enrichment bar chart. G KEGG enrichment scatter plot. H Relative mRNA expression of FGF23, FGF6, and FGF18 in the tibiae of the OVX group

Fig 5: GLP-2 regulates FGF23 expression via the inactivation of FOXO1. A Western blot analysis of FOXO1 and p-FOXO1 levels in osteoblasts treated with or without GLP-2. B Immunofluorescence staining of FOXO1 in osteoblasts (green), counterstained with DAPI (blue). C Relative mRNA expression of FGF23 in osteoblasts treated with the FOXO1 inhibitor AS1842856. D Verification of FOXO1 overexpression by Western blot after PCDH-FOXO1 transfection. E Effect of FOXO1 overexpression on FGF23 mRNA levels in osteoblasts with or without GLP-2 treatment. F Effect of FOXO1 overexpression on the FGF23 concentration in the cell culture supernatant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001