Hypothesis

In aged adipose tissue the extracellular matrix becomes progressively stiff, and this mechanical change actively suppresses autophagy through a dual pathway: (1) integrin‑FAK signaling constitutively activates mTORC1 independent of nutrient status, and (2) nuclear YAP/TAZ recruits histone deacetylases that compact chromatin at autophagy gene promoters. Together these mechanisms lock the autophagic machinery in a repressed state, preserving a damaged but stable cellular configuration.

Mechanistic rationale

• Integrin‑FAK‑mTORC1 axis: Stiff ECM increases focal adhesion formation, boosting FAK autophosphorylation. FAK can directly activate Rag GTPases or upstream PI3K‑AKT signaling, both of which drive mTORC1C1 activity [2]. Because mTORC1 is a potent autophagy inhibitor, its persistent activation suppresses phagophore formation even when nutrients are low.
• YAP/TAZ‑HDAC axis: Mechanical tension drives YAP/TAZ nuclear translocation [3]. In the nucleus YAP/TAZ interact with class I HDACs (e.g., HDAC1/2) and promote deacetylation of histone H3 lysine 9 at promoters of Atg7, Atg3, Beclin1 and LC3. This creates a repressive chromatin environment that reduces transcription of core autophagy components, complementing the post‑translational block by mTORC1.
• Feedback loop: Reduced autophagy leads to accumulation of p62 and damaged mitochondria, which raise ROS and further stimulate LOX‑mediated collagen crosslinking, amplifying ECM stiffness. Senescent adipocytes also secrete lysyl oxidase and TGF‑β, reinforcing the fibrotic niche.

Testable predictions

1. Pharmacologic softening of aged adipose ECM (using LOX inhibitor β‑aminopropionitrile or collagenase treatment) will decrease p-FAK, lower nuclear YAP/TAZ, reduce mTORC1 activity (p‑S6K), and increase LC3‑II flux in SVFs from old mice.
2. Genetic ablation of YAP or TAZ specifically in adipocyte progenitors will rescue autophagy flux despite a stiff matrix, as measured by mCherry‑GFP‑LC3 reporter turnover.
3. FAK inhibition (PF‑573228) will attenuate mTORC1 signaling and restore autophagy without altering ECM stiffness, indicating that the integrin‑FAK branch is sufficient for the block.
4. Chromatin immunoprecipitation will show increased HDAC1 occupancy and decreased H3K9ac at autophagy gene promoters in nuclei isolated from stiff‑matrix‑cultured adipocytes; this enrichment will be lost after YAP/TAZ knockdown.
5. In vivo, aged mice treated with LOX inhibitor will exhibit improved adipose insulin sensitivity and reduced inflammation, correlating with restored autophagy markers.

Experimental outline

• Isolate SVFs from young (3 mo) and old (24 mo) mice.
• Culture cells on tunable polyacrylamide gels mimicking young (~0.5 kPa) and old (~8 kPa) adipose stiffness.
• Treat with LOX inhibitor, FAK inhibitor, YAP/TEAD blocker (verteporfin), or control.
• Readouts: western blot for p‑FAK, p‑S6K, nuclear YAP/TAZ, LC3‑II/I, p62; immunofluorescence for YAP/TAZ localization; mCherry‑GFP‑LC3 flux assay; qPCR for autophagy genes; ChIP‑qPCR for HDAC1/H3K9ac at Atg7 promoter.

Falsification

If softening the matrix or blocking YAP/TAZ fails to increase autophagic flux, or if HDAC inhibition does not restore autophagy gene expression despite reduced stiffness, then the proposed mechanotransductive repression model would be refuted. Conversely, consistent rescue across multiple orthogonal interventions would support the hypothesis that ECM stiffness actively suppresses autophagy in aged adipocytes via integrin‑FAK‑mTORC1 and YAP/TAZ‑HDAC mechanisms.