Hypothesis

Age‑dependent decline in ileal bile acid transporter ASBT reduces hepatic FXR activation and intestinal TGR5 signaling, leading to compensatory upregulation of the autophagy inhibitors RUBCN and p300. This creates a feed‑forward loop that actively suppresses autophagy, promotes senescence, and exacerbates metabolic aging.

Mechanistic Model

1. Reduced ileal bile acid reabsorption – With aging, ASBT/SLC10A2 expression falls, decreasing portal bile acid return.

   • This diminishes hepatic FXR agonism, lowering FGF19 secretion.
   • Concurrently, intestinal TGR5 activation drops, reducing cAMP‑PKA signaling that normally inhibits mTORC1.

2. Shift in nutrient signaling – Lower FGF19 and TGR5 activity raise hepatic gluconeogenesis and increase mTORC1 activity in peripheral tissues.

   • Elevated mTORC1 phosphorylates and inhibits ULK1, dampening autophagy initiation.
   • Increased mTORC1 also stimulates transcription of RUBCN and p300 via SREBP‑1c/STAT3 pathways (hypothesized).

3. Direct transcriptional upregulation – The cellular sensing of bile acid deficit triggers a stress‑responsive program that boosts RUBCN and p300 transcription (observed increase with age across species) RUBCN increases with age, p300 increases during aging]

   • Both proteins bind VPS34, blocking its lipid kinase activity and preventing PI3P production, thus halting autophagosome formation.

4. Oxidative amplification – Age‑related ROS further oxidize ATG3/ATG7, blocking LC3 lipidation, while PKA‑mediated LC3B Ser12 phosphorylation adds another layer of inhibition PKA activity increases with age]

5. Functional outcome – Suppressed autophagy leads to accumulation of damaged macromolecules, driving cardiomyocyte senescence as shown in cardiac‑specific Atg7 KO mice suppression of general autophagy caused accumulation of senescent cardiomyocytes]

Testable Predictions

• Prediction 1: Old mice with ileal‑specific ASBT overexpression will restore portal bile acid levels, decrease hepatic mTORC1 activity, and reduce RUBCN/p300 protein levels compared with aged controls.
• Prediction 2: Pharmacological activation of intestinal TGR5 (e.g., with INT‑777) in aged animals will lower RUBCN/p300 expression and rescue autophagic flux (measured by LC3‑II turnover) in liver and intestine.
• Prediction 3: Cholesterol depletion of ileal enterocytes (using methyl‑β‑cyclodextrin) will increase autophagy‑gene transcription, but only when FXR signaling is intact, linking sterol homeostasis to the autophagy‑suppressive axis cholesterol depletion increases transcription of autophagy-related genes].

Experimental Approach

• Use aged (24‑month) C57BL/6 mice; generate villin‑CreERT2;ASBT^fl/fl for inducible ileal ASBT knockdown and villin‑CreERT2;ASBT^fl/fl;Rosa26^LSL‑ASBT for overexpression.
• Measure portal bile acids by LC‑MS, hepatic p‑S6K (mTORC1 read‑out), RUBCN and p300 by Western blot, autophagic flux via chloroquine‑treated LC3‑II accumulation, and senescence markers (p16^Ink4a, SA‑β‑gal) in liver and intestine.
• Treat cohorts with TGR5 agonist or vehicle; assess same endpoints.
• In parallel, isolate ileal enterocytes, treat with methyl‑β‑cyclodextrin ± FXR antagonist (glycyl‑β‑muricholic acid) and quantify autophagy‑gene expression (LC3, ATG5) by qPCR.

If predictions hold, the data will support a model where age‑related ileal bile acid loss actively drives autophagy suppression via FXR/TGR5‑mTORC1‑RUBCN/p300 signaling, repositioning autophagy inhibition from a passive failure to an adaptive, albeit maladaptive, response to enterohepatic dysregulation.