Abstract Osteoclast is critical in skeletal development and fracture healing, yet the impact and underlying mechanisms of their metabolic state on these processes remain unclear. Here, by using osteoclast-specific small GTPase Rheb1-knockout mice, we reveal that mitochondrial respiration, rather than glycolysis, is essential for cathepsin K (CTSK) production in osteoclasts and is regulated by Rheb1 in a mechanistic target of rapamycin complex 1 (mTORC1)-independent manner. Mechanistically, we find that Rheb1 coordinates with mitochondrial acetyl-CoA generation to fuel CTSK, and acetyl-CoA availability in osteoclasts is the central to elevating CTSK. Importantly, our findings demonstrate that the regulation of CTSK by acetyl-CoA availability is critical and may confer a risk for abnormal endochondral ossification, which may be the main cause of poor fracture healing on alcohol consumption, targeting Rheb1 could successfully against the process. These findings uncover a pivotal role of mitochondria in osteoclasts and provide a potent therapeutic opportunity in bone disorders.
