Hyperglycaemia impairs mineralisation by disrupting coordination of bone and mTOR pathways during osteocyte differentiation

Diabetic patients exhibit increased bone fragility despite variable bone mineral density, yet hyperglycaemia has shown discordant effects on mineralisation in different studies. Notably, its impact on the differentiation and function of osteocytes, the most abundant bone cell type, remains poorly understood. We examined osteoblast-to-osteocyte differentiation by exposing murine IDG-SW3 cells to normoglycaemic or hyperglycaemic conditions. Hyperglycaemia initially enhanced matrix production but later proved detrimental. Whole-cell proteomics revealed that under normoglycaemia, differentially expressed proteins (DEPS) converged towards bone differentiation and mTOR signalling pathways; this was disrupted under hyperglycaemia, with DEPs dispersed across unrelated processes, indicating loss of programme coordination and specialisation. Both proteomics and functional analyses convergently identified a critical transition at week 3-4, where high glucose drives premature osteogenesis arrest. Hyperglycaemia also induced mitochondrial and lysosomal dysfunction, senescence-associated β-galactosidase activity and proliferation arrest; however, nuclear shrinkage rather than enlargement differentiates this state from classical senescence. mTOR inhibitor studies revealed temporal complexity in mTOR's role. Pan-mTOR inhibition during the first week alone was sufficient to impair mineralisation at week 4, establishing an early mTOR-dependent sensitivity window. While pan-mTOR inhibitors and rapamycin inhibited mineralisation at week 3, only rapamycin rescued hyperglycaemia-impaired mineralisation by week 4, indicating that mTORC1 shifts from supporting to inhibiting mineralisation during this transition. These findings establish pathway coordination as a key determinant of successful osteocyte differentiation and explain contradictory reports of glucose effects on mineralisation through timing-dependent mechanisms. They also highlight the need for caution in choice, dose and timing of mTOR inhibitors as senotherapeutics in diabetic patients.