Hypothesis

Aging‑associated decline in circulating osteocalcin removes an endogenous brake on the mTORC1‑Rubicon autophagy‑suppressive circuit, leading to active inhibition of autophagy in multiple tissues.

Mechanistic Rationale

Osteocalcin, when carboxylated, binds the GPRC6A receptor on adipocytes, hepatocytes, and immune cells, triggering a cascade that activates AMPK and inhibits mTORC1 signaling (see refs [1][5]). In young organisms, sufficient osteocalcin keeps mTORC1 activity low, limiting Rubicon transcription and permitting basal autophagy. With age, osteocalcin serum levels fall sharply, reducing GPRC6A‑mediated AMPK activation. Consequently, mTORC1 remains hyperactive, phosphorylating ULK1 and TFEB to block autophagy initiation, while simultaneously up‑regulating Rubicon expression, which stalls autophagosome maturation. This creates a feed‑forward loop: suppressed autophagy diminishes clearance of damaged mitochondria, raising ROS, which further stabilizes mTORC1 signaling [7]. The systemic nature of the defect explains why young plasma can rescue autophagy in aged tissues [6]—the replete osteocalcin in young circulation reinstates GPRC6A‑AMPK signaling and overrides the suppressive cues.

Testable Predictions

1. Genetic: Mice lacking osteocalcin ('Ocn‑/‑') will show elevated phospho‑ULK1 (Ser757) and Rubicon protein levels in liver, muscle, and brain at young ages, mimicking the autophagic suppression seen in wild‑type aged mice.
2. Pharmacological: Chronic administration of recombinant osteocalcin to aged wild‑type mice will decrease mTORC1 activity (reduced p‑S6K), lower Rubicon expression, and increase LC3‑II/I ratio and p62 turnover, effects abrogated by GPRC6A antagonism.
3. Cell‑autonomous: Primary hepatocytes treated with osteocalcin will exhibit increased AMPK phosphorylation and reduced mTORC1 signaling, leading to enhanced autophagic flux measured by mCherry‑GFP‑LC3 reporter; this effect will be lost in AMPK‑α1/α2 double‑knockout cells.
4. Physiological: Osteocalcin‑deficient mice will exhibit accelerated accumulation of ubiquitinated protein aggregates and increased senescence markers (p16^Ink4a, SA‑β‑gal) in tissues, which are rescued by concurrent rapamycin treatment, indicating that the phenotypes stem from mTORC1‑driven autophagy suppression.

Experimental Approach

• Cohorts: Young (3 mo) and aged (24 mo) wild‑type, 'Ocn‑/‑', and 'Ocn‑/‑' + osteocalcin‑treated groups (n = 10 per group).
• Readouts: Western blot for p‑ULK1, total ULK1, p‑S6K, Rubicon, LC3, p62; immunofluorescence for autophagosome‑lysosome co‑localization; serum osteocalcin ELISA; metabolic assays (insulin tolerance, glucose tolerance).
• Interventions: Recombinant osteocalcin (30 µg/kg/day, i.p.) for 4 weeks; GPRC6A antagonist (if available) co‑administration; rapamycin (1 mg/kg/day) as positive control for autophagy induction.
• Analysis: Two‑way ANOVA with genotype and age as factors; post‑hoc Tukey test. Flux assays using chloroquine blockade to distinguish increased synthesis from decreased degradation.

Falsifiability

If osteocalcin supplementation fails to reduce mTORC1 activity or Rubicon levels, or if 'Ocn‑/‑' mice do not display premature autophagy suppression despite low circulating osteocalcin, the hypothesis would be refuted. Conversely, observing that GPRC6A blockade abolishes the autophagic rescue by osteocalcin would strengthen the mechanistic link.