Fig 2: Ablation of Adipsin inhibits bone marrow (BM) adipogenesis and protects bone.(A–G) 18-week-old male mice on chow diet ad libitum or subjected to calorie restriction (CR) for 4 weeks. Chow WT (n = 7), Adipsin KO (n = 9); CR WT (n = 6), Adipsin KO (n = 10). (A) Representative osmium tetroxide staining and (B) quantification of femoral marrow adipose tissue (MAT); (C) qPCR analyses of gene expression for markers of adipocytes in the BM from the tibia of CR mice; (D, E) femoral bone mineral density (BMD) in the cortical (D) and trabecular (E) regions; (F, G) bone volume (BV) normalized by total volume (TV) in the cortical (F) and trabecular (G) regions of the femurs determined by µCT scans. (H–L) Adult male mice on HFD for 12 weeks followed by 6 weeks of HFD (n = 8, 10) or HFD supplemented with rosiglitazone (Rosi) treatment (n = 6, 4). (H) Hematoxylin and eosin stain (H&E) staining of the femoral MAT in the same region of three different mice (Rosi only); (I, J) femoral BMD in the cortical (I) and trabecular (J) regions; (K, L) BV normalized by TV in the cortical (K) and trabecular (L) regions of the femurs determined by µCT scans. *p<0.05, **p<0.01, ***p<0.001 for WT vs. Adipsin KO. Data represent mean ± SEM. Two-tailed Student’s t-tests were used for statistical analyses.

Fig 3: Generation of Cfd-null mice. (A) Schematic representation of the Cfd locus in the mouse genome. Cfd gene is located on chromosome 10. Two sgRNAs were designed which targeted the upstream and downstream of the full coding sequence of the Cfd gene. PAM sequences are marked in bold. F1, R1, R2, and R3 represent the position of the primers used to genotype wild type (WT) and KO alleles. (B) PCR analysis of the genomic DNA obtained from the mouse tail at 3 weeks of age. The band for WT allele (primers used: F1 and R2, 576 bp) and that for KO allele (primers used: F1 and R3, 577 bp) were determined. Deletion of the Cfd sequence was confirmed by sequencing.

Fig 4: Bone marrow (BM) Adipsin induces bone marrow adiposity expansion during aging.(A) Immunoblot of Adipsin and Adiponectin from plasma of chow-fed male mice at 6, 24, and 76 weeks of age (L.C. = Coomassie staining of the membrane). (B, C) qPCR analyses of gene expression in the BM from tibia (B) and Cfd expression in the epididymal white adipose tissue and subcutaneous white adipose tissue (C) from chow-fed male mice at 26 and 78 weeks of age (n = 5, 5). *p<0.05 for young vs. aging mice. (D–I) Chow-fed 1-year-old male mice. WT (n = 10) and Adipsin KO (n = 9). (D) Representative osmium tetroxide staining and (E) quantification of femoral MAT; (F, G) femoral bone mineral density (BMD) in the cortical (F) and trabecular (G) regions, and (H, I) bone volume normalized by total voume in the cortical (H) and trabecular (I) regions of the femur determined by µCT scans. **p<0.01 for WT vs. Adipsin KO mice. Data represent mean ± SEM. Two-tailed Student’s t-tests were used for statistical analyses.

Fig 5: Adipsin is robustly induced in the bone marrow (BM) during bone marrow adipose tissue (BMAT) expansion.(A) Representative osmium tetroxide staining of MAT assessed by µCT scanning in the femurs of ad libitum and calorie restriction (CR)-fed mice. (B) Scatterplot displaying the gene expression (y-axis) and fold change (x-axis) in whole bone tissue between CR and control, induced secretory genes is highlighted. (C–E) CR-induced bone loss model: 18-week-old male mice subjected to 30% CR for 4 weeks. (C) qPCR analyses of gene expression in the BM isolated from the tibia (n = 6, 6); (D) immunoblot of plasma Adipsin and Adiponectin (APN) – Coomassie staining of the membrane was used as loading control (L.C.) (n = 4, 4); and immunoblot of Adipsin from bone – HSP90 was used as the loading control (n = 4, 4); (E) quantification of plasma Adipsin and APN from western blot. (F–I) Rosiglitazone (Rosi)-induced bone loss model: adult male mice on HFD for 12 weeks followed by 6 weeks of HFD supplemented with Rosi. (F) Representative osmium tetroxide staining of MAT assessed by µCT scanning in the tibia; (G) qPCR analyses of gene expression in the BM isolated from the femurs (n = 6, 6); (H) immunoblot of plasma Adipsin and APN and bone Adipsin; (I) quantification of plasma Adipsin and APN from western blot (n = 4, 4). (J–L) Adult male mice on HFD for 8 weeks followed by daily injections of 10 mg/kg Rosi or dimethyl sulfoxide solution? (DMSO) (10%) for 3 weeks with continuous HFD feeding. (J) Quantification of femoral MAT (n = 6, 8); (K, L) femoral bone mineral density (BMD) in the cortical (K) and trabecular (L) regions determined by µCT scans (n = 8, 8). *p<0.05, **p<0.01, ***p<0.001 for control group vs. treatment group. Data represent mean ± SEM. Two-tailed Student’s t-tests were used for statistical analyses.