Fig 2: Osteopontin regulates bone marrow-derived macrophages through the lactate/ATP6V0d2 axis.a Relative expression of different V-ATPase subunits in BMDMs treated with or without (ctrl) rOPN (0.5 µg/ml) under osteoclastogenic induction for 3 days in vitro (n = 3 biologically independent samples). b Representative western blot images (left) and quantification (right, n = 3 biologically independent samples) of ATP6V0d2 in BMDMs, Oc and rOPN-treated Oc for 4 days in vitro. c Total intracellular ATP concentration in BMDMs (ctrl), rOPN (0.5 µg/ml)-treated BMDMs, and BMDMs treated with rOPN (0.5 µg/ml) plus antagonist 27 (100 nM) under osteoclastogenic induction for 0.5 h (n = 4 biologically independent samples). d Intracellular lactate concentration in BMDMs (ctrl), rOPN (0.5 µg/ml)-treated BMDMs, and BMDMs treated with rOPN (0.5 µg/ml) plus antagonist 27 (100 nM) under osteoclastogenic induction for 10 h (n = 4 biologically independent samples). e Relative expression of the indicated V-ATPase subunits in BMDMs stimulated with a gradient concentration of lactate for 24 h under osteoclastogenic induction. #represents significantly decreased (P < 0.0001) expression of Atp6v0d2 in 50 mM lactate-treated BMDMs compared with untreated BMDMs (n = 3 biologically independent samples). f, g Representative images of TRAP staining (f) and counting of Trap+ osteoclasts (n = 6 biologically independent samples) (g) after osteoclastogenic induction in the presence or absence (ctrl) of rOPN (0.5 µg/ml), lactate (50 mM), and rOPN with siRNA Cd61 for 5 days. Scale bar: 50 µm. Images are representative of 3 independent experiments. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s post-hoc test (a, e) and one-way ANOVA with Tukey’s post-hoc test (b–d, g) were used. Source data are provided as a Source Data file.

Fig 3: OPN induces lysosomal-dependent lipid metabolism in BMDMs.a Representative western blots (left) and quantification (right, n = 3 biologically independent samples) of OPN receptor av and ß3 expression in BMDMs and metabolically activated macrophages (MMes) after culture for 4 days in vitro. b Schematic diagram illustrating the co-culture system as indicated below (c). c, d Representative images (c) and quantification (n = 5 biologically independent samples) (d) of crystal violet staining of BMDMs (ctrl) and MMes treated with or without rOPN for 24 h in a Transwell system. Scale bar: 50 µm. e Immunofluorescence images of buoyant BMDMs (F4/80) containing lipid vesicles (Perilipin 1) isolated from bone marrow supernatant at week 12. Scale bar: 15 µm. f FACS analysis of FC + (F4/80+ and CD11b+ , top) and Perilipin1+ (bottom) BMDMs from NFD- (left) and HFD-fed (right) mice. P9, FC + and Perilipin1+ BMDM population from Q2. g Percentage of FC + and Perilipin1+ BMDMs (n = 5 biologically independent samples). h Lactate concentration in bone marrow supernatant (n = 5 biologically independent samples). i Schematic diagram illustrating the co-culture system as described below (j). j Immunofluorescence staining of the co-cultured BMDMs for 4 days in vitro; the eWAT from mice fed the HFD for 8 weeks was placed in the upper chamber; Neu Ab (2.0 µg/ml) was added into the lower chamber. Scale bar: 50 µm. The right-most image is an enlarged view of the field in the white dashed box. Scale bar: 12.5 µm. k Total intracellular ATP concentration (left, co-culture for 0.5 h) and lactate concentration (right, co-culture for 10 h) in BMDMs with the treatments indicated in j (n = 4 biologically independent samples). l Relative expression of different V-ATPase subunits in ctrl (BMDMs), MMes, and MMes+ rOPN after culture for 24 h (n = 3 biologically independent samples). rOPN, 0.5 µg/ml. Images are representative of 3 independent experiments. All data are presented as mean ± SD. Two-tailed Welch’s t-test (a), one-way ANOVA with Tukey’s post-hoc test (d, k), two-way ANOVA with Sidak’s post-hoc test (g, h), and two-way ANOVA with Tukey’s post-hoc test (l) were used. Source data are provided as a Source Data file.

Fig 4: Schematic diagram showing that macrophages in epididymal adipose tissue secrete OPN to regulate bone homeostasis.eWAT-secreted OPN accumulates in the bone marrow compartment where OPN promotes bone resorption and lipophagocytic mobilization, but the excess accumulation of lactate inhibits pre-osteoclast fusion and neutral lipid hydrolysis via lysosomal-dependent ATP6V0d2 in BMDMs.

Fig 5: (A and B) Gene expression of Atp6v0d2, Atp6v1a in aorta of mice in each group (**p<0.01, ***p<0.001, ****p<0.0001). (C) The proportion of positive area of co-localization of CD68 and Atp6v0d2 by immunofluorescence double staining of aortic sinus (****p<0.0001). (D) Representative immunofluorescence analysis for detecting the colocalization (yellow particles) of CD68 (specific for monocyte macrophages, green particles) and Atp6v0d2 (red particles) in frozen sections from the aortic sinus of ApoE−/− mice; red arrow: the co-localization of CD68 and Atp6v0d2; Objective magnification 4×, 18×.