This study investigates how AGPAT3, under high glucose/high fat (HG/HF) conditions, triggers osteoblast ferroptosis and impairs bone formation via the ACSL4/ALOX15 pathway. We will first identify AGPAT3 using cellular proteomics in HG/HF-treated osteoblasts. Its role in promoting ferroptosis and inhibiting differentiation will be validated through functional assays. The mechanism of AGPAT3-mediated ACSL4/ALOX15 activation will be explored. Finally, the AGPAT3-ferroptosis-osteogenesis axis will be confirmed in a diabetic mouse model and clinical bone samples from osteoporotic patients.

This study aims to elucidate how AGPAT3 mediates the ACSL4/ALOX15 pathway to induce ferroptosis in osteoblasts and inhibit bone formation under high glucose/high fat (HG/HF) conditions. Using the MC3T3-E1 cell line and human mesenchymal stem cells (hMSCs), we will first identify AGPAT3 as a key protein via quantitative proteomics under HG/HF intervention. The role of AGPAT3 in promoting ferroptosis and inhibiting osteogenic differentiation will then be verified using RNAi, with assessments of ferroptosis markers and mineralization. Furthermore, non-targeted lipidomics and pathway inhibitors will be applied to investigate the mechanism by which AGPAT3 regulates the ACSL4/ALOX15 axis. Finally, the correlation between AGPAT3, ferroptosis, and impaired osteogenesis will be validated in a diabetic osteoporosis mouse model and clinical femoral head samples from patients.

Based on proteomic analysis of osteoblasts under high glucose/high fat (HG/HF) conditions, we identified AGPAT3 as a key upregulated protein associated with impaired osteogenic differentiation and ferroptosis. Functional validation showed that AGPAT3 knockdown alleviated HG/HF-induced ferroptosis (evidenced by reduced Fe²⁺, MDA, ROS, and rescued mitochondrial structure) and restored osteogenic differentiation (increased ALP, mineralized nodules, RUNX2, and COL1A1). Mechanistically, AGPAT3 knockdown remodeled lipid metabolism, inhibited lipid peroxidation, and was found to regulate the ACSL4/ALOX15 axis, a key downstream pathway for inducing ferroptosis. These findings were corroborated in a diabetic mouse model and human femoral head samples from diabetic osteoporosis patients, where elevated AGPAT3, ACSL4, and ALOX15 were consistently linked to suppressed osteogenic markers and activated ferroptosis.

In diabetic osteoporosis, glycolipotoxicity upregulates AGPAT3, which activates the ACSL4/ALOX15 axis to drive ferroptosis in osteoblasts. This cell death process impairs osteogenic differentiation, contributing to the development of diabetes-related bone metabolic disorders.