Disuse osteoporosis is a metabolic bone disease resulting from skeletal unloading (e.g., during extended bed rest, limb immobilization, and spaceflight), and the slow and insufficient bone recovery during reambulation remains an unresolved medical challenge. Here, we demonstrated that loading-induced increase in bone architecture/strength was suppressed in skeletons previously exposed to unloading. This reduction in bone mechanosensitivity was directly associated with attenuated osteocytic Ca²⁺ oscillatory dynamics. The unloading-induced compromised osteocytic Ca²⁺ response to reloading resulted from the HIF-1α/PDK1 axis–mediated increase in glycolysis, and a subsequent reduction in ATP synthesis. HIF-1α also transcriptionally induced substantial glutaminase 2 expression and thereby glutamine addiction in osteocytes. Inhibition of glycolysis by blockade of PDK1 or glutamine supplementation restored the mechanosensitivity in those skeletons with previous unloading by fueling the tricarboxylic acid cycle and rescuing subsequent Ca²⁺ oscillations in osteocytes. Thus, we provide mechanistic insight into disuse-induced deterioration of bone mechanosensitivity and a promising therapeutic approach to accelerate bone recovery after long-duration disuse.

Substantial evidence has indicated that osteoblastic differentiation may be regulated by mechanical loads or bone morphogenetic protein-2 (BMP-2). BMP-2-induced in vivo osteogenesis can be significantly enhanced in the presence of mechanical stimuli, revealing the therapeutic potential of the combined application of BMP-2 and mechanical loads in clinical bone diseases (e.g., bone fractures and osteoporosis); however, the underlying mechanisms remain elusive. In this study, we found that cyclic stretch or BMP-2 alone increased the expression of osteoblastic differentiation markers, including alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), as shown by RT-qPCR, western blot analysis and ALP activity test. Furthermore, our results revealed that cyclic mechanical stretch with 10% elongation at 0.1 Hz significantly enhanced the BMP-2-induced upregulation of ALP and Runx2 expression in osteoblast-like MC3T3-E1 cells. Cyclic stretch also inhibited the BMP-2-induced upregulation of Hes-related family bHLH transcription factor with YRPW motif 1 (Hey1, measured by RT-qPCR and immunofluorescence staining), a potent negative regulator of osteogenesis. Moreover, the transient transfection of a Hey1 expression plasmid (pcDNA3.1-Hey1) significantly reversed the effects of cyclic stretch on the BMP-2-induced upregulation of differentiation markers in the MC3T3-E1 cells. This revealed the importance of Hey1 in modulating BMP-2-induced osteoblastic differentiation in response to cyclic stretch. Taken together, our results demonstrated that cyclic stretch enhanced the BMP-2‑induced osteoblastic differentiation through the inhibition of Hey1. The present study broadens our fundamental knowledge of osteoblastic mechanotransduction and also sheds new insight into the mechanisms through which the combined application of BMP-2 and mechanical load promotes osteogenesis.