Figure 4. Skeletal interoception promotes osteoblastic bone formation by downregulation of hypothalamic NPY.

(A) Representative images of calcein double labeling of femoral trabecular bone and (B) quantification of mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS) in 3-month-old male Ptger4^fl/fl and Avil^Cre:Ptger4^fl/fl mice injected with vehicle or 10 mg/kg/d SW033291 for 1 month. Scale bars = 20 μm. (C) Representative co-immunofluorescence staining and (D) quantitative analysis of pCREB (green) and osteocalcin (red) from femurs of 3-month-old male Ptger4^fl/fl and Avil^Cre:Ptger4^fl/fl mice injected with vehicle or 10 mg/kg/day SW033291 for 1 month. Scale bars = 50 µm. (E) Quantitative analysis of cAMP concentration in the osteoblasts isolated from 3-month-old male Ptger4^fl/fl and Avil^Cre:Ptger4^fl/fl mice injected with vehicle or 10 mg/kg/day SW033291 for 1 month. (F) RT-PCR quantitative analysis of Runx2 expression in femurs from 3-month-old male Ptger4^fl/fl and Avil^Cre:Ptger4^fl/fl mice injected with vehicle or 10 mg/kg/day SW033291 for 1 month. (G) Representative co-immunofluorescence staining and (H) quantitative analysis (adipocyte number per section and number of osteoblast) of osteocalcin (green) and perilipin (red) from femurs of 3-month-old male Ptger4^fl/fl and Avil^Cre:Ptger4^fl/fl mice injected with vehicle or 10 mg/kg/day SW033291 for 1 month. Scale bars = 50 µm. (I) RT-PCR quantitative analysis of Fatp1 and Cpt1b expression in femurs from 3-month-old male Ptger4^fl/fl and Avil^Cre:Ptger4^fl/fl mice injected with vehicle or 10 mg/kg/day SW033291 for 1 month. N ≥ six per group. *p < 0.05, **p<0.01, and N.S. indicates not significant. Statistical significance was determined by two-way analysis of variance.