Hypothesis

We propose that the progressive loss of autophagic flux in aged enteroendocrine L‑cells transforms a normal nutrient‑siege response into a maladaptive state where secretory machinery is degraded without adequate recycling, leading to chronically blunted GLP‑1 release. Restoring autophagic rationing—not merely inducing bulk autophagy—should rescue incretin capacity.

Mechanistic Basis

Enteroendocrine L‑cells experience repeated fasting‑fed cycles that create transient nutrient scarcity. During fasting, autophagy acts as a rationing system, selectively degrading damaged organelles and long‑lived proteins to liberate amino acids and lipids that sustain vesicle biosynthesis and hormone secretion [3,4]. With age, autophagic flux declines [2], causing accumulation of defective secretory granules and impaired mitochondrial function. Simultaneously, duodenal nutrient‑sensing deteriorates, reducing glucose‑evoked Ca²⁺ signals that normally trigger GLP‑1 exocytosis [1]. The combined effect is a siege where the cell cannibalizes essential secretory components without sufficient replenishment, producing a net loss of GLP‑1 output.

This differs from the classic view of autophagy as mere housekeeping; instead, it reflects a triage decision where the cell sacrifices secretory capacity to maintain basal survival.

Testable Predictions

1. Flux‑specific rescue: Pharmacological agents that enhance autophagic flux without increasing bulk degradation (e.g., spermidine, low‑dose rapamycin) will improve GLP‑1 secretion in isolated aged L‑cells, whereas agents that induce non‑selective autophagy (high‑dose rapamycin) will not.
2. Selective cargo degradation: Aged L‑cells will show increased ubiquitination of secretory granule proteins (e.g., PCSK1, CHGA) and decreased levels of free amino acids during fasting, measurable by targeted proteomics and metabolomics.
3. mTOR‑autophagy uncoupling: In aged L‑cells, mTORC1 activity will remain elevated despite nutrient restriction, reflecting a failure to shift from growth to rationing mode.
4. In vivo phenotype: Mice with L‑cell‑specific knockout of essential autophagy genes (Atg7) will exhibit an accelerated age‑related decline in plasma GLP‑1 and glucose tolerance, while L‑cell‑specific overexpression of a flux‑promoting factor (e.g., TFEB) will preserve GLP‑1 dynamics.

Experimental Design

• Isolation & culture: Purify L‑cells from young (3 mo) and aged (24 mo) mice using fluorescence‑activated cell sorting for Glu‑GFP reporters.
• Flux assay: Treat cells with bafilomycin A1 and measure LC3‑II turnover via immunoblot; correlate with GLP‑1 release in Krebs‑Ringer buffer under fasting (0.5 mM glucose) and fed (20 mM glucose) conditions.
• Cargo analysis: Perform immunoprecipitation of PCSK1 followed by ubiquitin immunoblot; quantify intracellular leucine and glutamate by LC‑MS/MS.
• mTOR read‑out: Assess p‑S6K and p‑4EBP1 levels under fed/fasted states.
• In vivo validation: Generate Villin‑CreERT2;Atg7^fl/fl mice for inducible L‑cell autophagy deletion; monitor GLP‑1, insulin tolerance, and body composition over 12 months. Parallel cohort receives AAV‑TFEB transduction.
• Falsification: If enhancing flux fails to improve GLP‑1 secretion despite verified LC3‑II turnover, or if aged L‑cells show no increase in secretory cargo ubiquitination, the hypothesis would be refuted.

By framing autophagy as a rationing triage rather than a cleanup crew, this hypothesis links organellar quality control directly to the endocrine deficit that drives age‑related metabolic disease.