Fig. 5

AP503 increases bone mass in male mice via GPR133/ADGRD1. a Experimental scheme for in vivo treatment (referring to data in b-h). Male wild-type (WT), heterozygous (Het), or homozygous (Homo) Gpr133/Adgrd1 knockout (KO) mice were examined. 5-weeks-old WT, Het or Homo mice were intraperitoneally injected with vehicle or AP503 (2 mg/kg) every day for 4 weeks. Figure created with BioRender. b–h Femora from 9-weeks-old male mice were examined. b Representative µCT images of distal femurs (cross sections and longitudinal sections). c Bone volume/total volume (BV/TV) and (d) trabecular number (Tb.N.) were assessed in the distal femur using µCT. e Number of osteoblasts per bone perimeter (N.Ob./B.pm), (f) number of osteocytes per bone perimeter (N.Ocy./B.pm), and (g) number of osteoclasts per bone perimeter (N.Oc./B.pm) were determined by Tartrate-resistant acid phosphatase (TRAP) staining of femoral bone slides. h Bone formation rate per bone surface (BFR/BS) was assessed by calcein double labeling of tibial bone slides and compared between groups. i Experimental scheme for mechanical load model (referring to data in j-o). 4-weeks-old WT male mice were divided into 4 groups: sedentary control (Sed), Sed+AP503, exercise (Exe) and Exe+AP503. Mice in the Exe and Exe+AP503 group underwent treadmill running following a 5-day-per-week acclimatization protocol at 9 a.m., beginning with a 1-week pre-exercise phase, followed by a 4-week formal exercise period. Mice in the Sed and Exe groups were intraperitoneally injected daily with an equal vehicle, while those in the Sed+AP503 and Exe+AP503 groups received AP503 (2 mg/kg) daily. Figure created with BioRender. j–o Femora from 9-weeks-old male mice were examined. j Representative µCT images of distal femurs (cross sections and longitudinal sections). k Fold change of BV/TV, Tb.N, Tb.Th, and Tb.Sp compared to the Sed group. l N.Ob./B.pm, (m), N.Ocy./B.pm (n), and N.Oc./B.pm were determined by TRAP staining of femoral bone slides. o BFR/BS was assessed by calcein double labeling of tibial bone slides and compared between groups. p Serum calcium and (q) phosphate concentrations from control and AP503 treated male WT, Het or Homo Gpr133/Adgrd1 KO mice. r Serum calcium and (s) phosphate concentrations from male WT mice following exercise and/or AP503 treatment. Data information: The data are presented as the mean ± SEM values, n = 5-14 mice per group. Each dot represents an individual mouse. The data were analyzed via one-way ANOVA with Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001, ^#p < 0.05, ^###p < 0.001, ^$p < 0.05