Fig 2: 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 3: 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 4: Top genes enriched in CD11b+CD45hi cells reflected peripheral myeloid cell profiles in WD-fed mice. a, b Normalized gene expression plot (reproduced from ImmGen datasets, see the “Methods” section) showing relative gene expression values for 34 CD11b+CD45lo cell-enriched DE genes (a) or 73 CD11b+CD45hi cell-enriched DE genes (b) within macrophage (green), monocytes (pink), and neutrophils (red). Normalized expression means log2(gene expression value/average expression value of all genes). ImmGen immune cell type abbreviations: MF = macrophage, Mo = monocytes, GN = neutrophils, PC = peritoneal cavity, Bl = blood, Sp = spleen, BM = bone marrow, Alu_Lu = alveolar cell in the lung, Fem = female, CNS = central nervous system. (c) Fold change (FC) of top 10 CD11b+CD45hi cell-enriched genes (FC compared to CD11b+CD45lo cells) in WD-fed mice, colored by the significance level using -log10(FDR). d Numbers of genes involved in GO term of Biological Process and cellular component analysis based on 73 CD11b+CD45hi cell-related genes in WD-fed mice, colored by significance level using -log10(BJ). e Gene interaction network generated in IPA containing 37 of the 73 CD11b+CD45hi cell-enriched genes in WD-fed mice. f Normalized expression levels of Itgal (blood-derived immune cell marker), Cxcl2, Spp1, Stfa2l1 (specific neutrophil marker), and Aif1 (brain myeloid cell marker) across macrophage, monocytes, and granulocytes reproduced from ImmGen datasets (same datasets as a and b)

Fig 5: eWAT-secreted OPN circulates to the bone marrow.a Representative images of immunohistochemical staining of OPN (left) and the OPN-positive stained areas (right, n = 5 biologically independent samples) of the proximal tibiae of NFD- and HFD-fed groups. Scale bar: 50 µm. b Schematic representation of the isolation of bone marrow supernatant for ELISA (left). Free OPN levels in the bone marrow supernatant of NFD- and HFD-fed mice (right, n = 6 biologically independent samples). c Relative expression of Spp1 in bone marrow over the course of feeding (n = 4 biologically independent samples). d OPN expression in bone marrow supernatant from mice with or without eWATx at week 12 of the indicated diet (n = 6 biologically independent samples). e OPN expression in the serum of the HFD-fed subgroups with and without eWATx at weeks 8, 12, and 16 (n = 5 biologically independent samples). f OPN concentration in eWAT (from NFD- and HFD-fed mice at weeks 8, 12, and 16)-derived conditioned medium (n = 5 biologically independent samples). g OPN concentration in iWAT (from NFD- and HFD-fed mice at weeks 8, 12, and 16)-derived conditioned medium (n = 4 biologically independent samples). h Ex vivo imaging (IVIS200 system) of the indicated organs and tissues collected 1 h (left part of the right box) or 24 h (right part of the right box) after unilateral eWAT injection of rOPN-FITC derived from HFD-fed mice at week 12. i, j Representative images of immunohistochemical staining of OPN (anti-human OPN antibody) in the spine, femur, tibia (proximal and distal), eWAT (ipsilaterally injected depot and contralateral depot) (i), lung, heart, liver, kidney and iWAT (j) of mice injected with rOPN-FITC into the unilateral eWAT for 1 h (top row) and 24 h (bottom row), respectively. Scale bar: 100 µm. BM: bone marrow. Images are representative of 3 independent experiments. All data are presented as mean ± SD. Two-way ANOVA with Sidak’s post-hoc test (a–c, e–g) and two-way ANOVA with Tukey’s post-hoc test (d) were used. Source data are provided as a Source Data file. See also Supplementary Table 6.