Heat-treating FM with Clamshell Powder Ameliorate Postmenopausal Osteoporosis by Regulating Bone-Fat Metabolism.

Postmenopausal osteoporosis is characterized by a disruptive bone‑fat imbalance, yet effective therapies remain limited. Here, for the first time, we link the traditional use of fish maw heat‑treated with clam shell powder (CPFM) to the restoration of bone‑fat balance. Using INFOGEST simulated digestion followed by LC‑MS/MS analysis, we found that CPFM peptides were predominantly low‑molecular‑weight species (84.6% below 1 kDa), with heptapeptides as the most abundant fraction (20%). In ovariectomized mice, CPFM peptides reduced bone marrow fat accumulation and improved bone microstructure (increased BV/TV and BMD). In bone marrow stromal cells (BMSCs), they concurrently inhibited adipogenic differentiation (reduced Oil Red O‑positive area) and promoted osteogenesis (enhanced ALP activity and mineralization). From CPFM digests, we systematically screened and identified an absorbable tetrapeptide, FLLL, based on its high relative abundance (37.8%), predicted bioactivity (PeptideRanker >0.5), cell‑penetrating ability (CPPpred >0.33), and favorable ADMET properties. In cultured BMSCs, FLLL recapitulated the dual effects of CPFM, promoting osteogenesis while suppressing adipogenesis. Mechanistically, both CPFM and FLLL downregulated adipogenic factors (PPARγ, FABP4, FASN) and upregulated osteogenic markers (RUNX2, COL1α2, BGLAP). Surface plasmon resonance confirmed direct binding of FLLL to RUNX2 (KD = 32.5 μM), and rescue experiments indicated that FLLL regulates the bone‑fat balance in association with the PPARγ/RUNX2 axis. Collectively, this study establishes a chain of evidence spanning peptide profiling, in vivo efficacy, active component identification, and mechanistic insight. These findings support CPFM as a promising dietary candidate and identify FLLL with well-defined in vitro bioactivity.