Fig. 3: Lactate inhibits osteoclast differentiation and promotes osteoblast differentiation via Gpr81.

A Measuring blood lactate concentration after blood collection from the tail vein. Bars represent mean ± SD. ***p < 0.001. n = 6 per time point. B Representative micro-CT images of the femurs of 8-week-old WT and Gpr81^−/− mice showing the distal femur (top; scale bars = 1 mm) and trabeculae (bottom; scale bars = 1 mm). C Quantitative micro-CT analysis of the trabecular bone parameters of the femurs shown in (B). *p < 0.05, **p < 0.01, n = 6. D Schematic illustration of grouping of BMM extracts: extracts of WT and Gpr81^−/− mice with BMM induced differentiation towards osteoclasts were stimulated by the lactate or PBS, and relevant assays were performed after 7 days. E Representative trap staining images of osteoclast. Scale bars, 200 μm. F Quantifying osteoclast number and Trap-positive area in (E). n = 6. **p < 0.01, ***p < 0.001. For multiple comparisons, experiments were replicated three times independently. G Analysis of Trap, Nfatc1 and CTSK mRNA levels by RT-qPCR in wild-type, Gpr81^−/−, wild-type and Gpr81^−/− stimulated with lactate (10 mM) for 7 days. All data are expressed as mean ± SD, n = 6. **p < 0.01, ***p < 0.001. Two-way ANOVA followed by Dunnett’s test was used for multiple comparative tests with experiments independently repeated three times. H BMSC were isolated and subjected to ALP staining (day 7), Von Kossa staining (day 14) and Alizarin red staining (day 21) from 1-month old Gpr81^−/− mice and WT littermates. I Representative ALP staining, Alizarin Red S staining and Von Kossa staining of osteoblasts from WT littermates and Gpr81-deficient mice. J RT-qPCR analysis of Runx2, ALP and Osterix mRNA levels in WT and Gpr81^−/− osteoblasts stimulated with lactate (10 mM) for 14 days. All data are expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. Two-way ANOVA followed by Dunnett’s test was used for multiple comparative tests with experiments repeated independently three times.