The pellets and floating cells were collected after centrifugation at 500??for 10?min at room heat

The pellets and floating cells were collected after centrifugation at 500??for 10?min at room heat. macrophages (BMDMs) to engulf the lipid droplets released from adipocytes in the?bone marrow of mice. However, the lactate accumulation induced by osteopontin regulation blocks both lipolysis and osteoclastogenesis in BMDMs by limiting the energy regeneration by ATP6V0d2 in lysosomes. Both surgical removal of eWAT and local injection of either clodronate liposomes (for depleting macrophages) or osteopontin-neutralizing antibody show comparable amelioration of HFD-induced bone loss in mice. These results provide Elaidic acid an avenue for developing therapeutic strategies to mitigate obesity-related bone disorders. (secreted phosphoprotein 1, or and in eWAT (in eWAT ((in eWAT significantly increased with prolonged HFD feeding compared with NFD feeding (Fig.?4g). However, the expression of was comparable between the NFD- and HFD-fed groups in the whole iWAT as well as PAT and MAT, except for the PAT collected at week 16, where was significantly higher in the HFD-fed group (Fig.?4g, Supplementary Fig.?4a, b). Almost all ATMs in eWAT express F4/80 and CD11b and can secrete OPN15,25. We found that OPN was predominantly scattered around the F4/80+ macrophages in the stromal vascular fraction (SVF), and it constantly spread up to week 12 of feeding (Fig.?5a, b, and Supplementary Fig.?5a). We isolated the SVF and adipocyte fraction (AF) from adipose tissues of HFD-fed mice at week 12 and found that the expression of in the SVF of eWAT was significantly higher than that in the AF (Fig.?5c). The isolated SVF was digested, and cell sorting was conducted by binning macrophages into the F4/80+CD11b+ ([FC+]) ATM group and the double-negative ([FC?]) ATM group (Fig.?5d and Supplementary Fig.?5b). Elaidic acid The proportion Rabbit Polyclonal to TALL-2 of FC+?macrophages increased approximately four-fold in the HFD-fed group compared with the NFD-fed group at Elaidic acid week 12 (Fig.?5e). mRNA was significantly upregulated in the FC+?subgroup over the FC? subgroup at week 12 in the HFD-fed group (Fig.?5f). Together, these results suggest that OPN is mainly derived from FC+?ATMs in eWAT, especially under HFD feeding conditions. Open in a separate window Fig. 5 OPN is usually primarily secreted by F4/80?+?and CD11b+?macrophages in eWAT.a Representative images of immunofluorescence staining of Elaidic acid OPN in eWAT from NFD- and HFD-fed mice at weeks 8, 12, and 16. Scale bar: 100?m. b Quantification of the F4/80-positive or OPN-positive stained area percentage in eWAT from a (in the adipocyte fraction (AF) and stromal vascular fraction (SVF) from eWAT or iWAT of HFD-fed mice at week 12 (in FC+?and FC? ATMs from HFD-fed mice at week 12 (mRNA was similarly expressed in both FC+?BMDMs with and without HFD feeding, the proportion of FC+?BMDMs was significantly lower in the HFD-fed Elaidic acid group than in the NFD-fed group at week 12 (Supplementary Fig.?7aCc). We also observed significantly less mRNA expression in the bone marrow of HFD-fed mice than NFD-fed mice (Fig.?6c), implying that circulating OPN might be an endogenous supplement for the scarcity of bone marrow. Although the OPN concentrations in serum between the NFD- and HFD-fed groups were comparable at different time points (Supplementary Fig.?8), we found that the concentration of OPN in serum, as well as in bone marrow supernatant, of the HFD?+?eWATx group was significantly lower than that in the HFD?+?Sham group at week 12 (Fig.?6d, e), while the PATx group had a comparable concentration of OPN in serum to the Sham group with or without HFD treatment (Supplementary Fig.?9a, b). The fall in OPN concentrations in serum and bone marrow supernatant after eWATx indicated that eWAT might be a major source of OPN for the circulation and for bone marrow at this time point. Open in a separate windows Fig. 6 eWAT-secreted OPN circulates to the bone marrow.a Representative images of immunohistochemical staining of OPN (left) and the OPN-positive stained areas (right, in bone marrow over the course of feeding (expression in vitro (Fig.?7cCf). We also found that the activation of MMP9, as well as of integrin v3, in the bone marrow of the HFD-fed group at week 12 was significantly higher than that of the NFD-fed group (Fig.?7g). The positive IHC staining intensity of MMP9 was lower in the bone marrow of HFD-fed mice treated with OPN-neutralizing antibody (Neu Ab) than in that of HFD-fed mice treated with saline (Fig.?7h). Bone loss was attenuated after injection of Neu Ab, accompanied by fewer ATMs infiltrating eWAT and less accumulation of rBMAT (Fig.?7iCl and Supplementary Fig.?11a). The decreased Trap+.