Rapamycin‑induced bone phenotype reflects famine mimicry rather than true matrix rejuvenation

Rapamycin extends lifespan by acting as a caloric‑restriction mimetic, but its effects on bone may reflect a stress‑adapted state rather than genuine rejuvenation of the extracellular matrix. We hypothesize that mTORC1 inhibition reduces the flux through the pentose‑phosphate pathway, limiting NADPH‑dependent regeneration of vitamin K hydroquinone, the cofactor required for γ‑glutamyl carboxylase (GGCX) activity. Consequently, osteocalcin is synthesized in greater amounts (as seen with rapamycin‑induced increases in total osteocalcin)[1] but remains undercarboxylated because the carboxylation step is impaired. Undercarboxylated osteocalcin binds hydroxyapatite less efficiently, altering crystal nucleation and growth, which leads to a bone mineral that is more amorphous or poorly crystalline despite a balanced formation/resorption phenotype.

If this mechanism is correct, rapamycin‑treated animals will show: (1) elevated serum total osteocalcin with a decreased carboxylated‑to‑total ratio; (2) unchanged or reduced vitamin K hydroquinone levels in bone tissue; (3) hydroxyapatite that exhibits lower crystallinity index and higher carbonate substitution as measured by X‑ray diffraction and FT‑IR spectroscopy; and (4) nanoindentation measurements showing reduced elastic modulus and hardness compared with young controls, even though trabecular number and cortical thickness appear preserved. Importantly, supplementing rapamycin‑treated mice with vitamin K2 (menaquinone‑7) should restore osteocalcin carboxylation without affecting mTOR signaling, thereby normalizing the carboxylated‑to‑total ratio and improving crystal quality. A rescue of bone mechanical properties under vitamin K2 co‑treatment would support the hypothesis; a lack of improvement would falsify it.

To test this, we propose a 2 × 2 factorial study in aged mice: rapamycin vs vehicle, each with or without vitamin K2 supplementation, alongside a pair‑fed caloric‑restriction group as a positive control for famine signaling. Primary endpoints will be serum osteocalcin isoforms (ELISA), bone vitamin K hydroquinone (HPLC), crystallinity index (XRD), carbonate/phosphate ratio (FT‑IR), and nanoindentation modulus. Secondary endpoints include lifespan and frailty index. Statistical interaction between rapamycin and vitamin K2 on carboxylation and crystal parameters will indicate whether the rapamycin‑induced bone phenotype is mediated through impaired vitamin K recycling. This design directly challenges the view that rapamycin merely suppresses maladaptive mTOR‑driven osteoclast hyperfunction and instead posits that it imposes a molecular famine signal that compromises a key post‑translational modification essential for mature bone matrix.