Hypothesis Timed inhibition of mTORC1 during the fasting phase restores its ability to oscillate between fed and fasted states in aged duodenal L-cells, thereby rescuing nutrient‑responsive GLP-1 secretion and improving systemic glucose homeostasis.

Mechanistic Rationale Aging locks mTORC1 in a constitutively high, nutrient‑insensitive state [1][4], which impairs the cyclic synthesis of nutrient‑sensing GPCRs and transporters needed for L‑cell activation [2]. Simultaneously, the lack of a fasting‑phase dip suppresses autophagy‑mediated quality control of secretory vesicles and mitochondria [3], leading to depleted GLP-1 stores and vesicular trafficking defects. Because mTORC1 activity also drives transcription factors such as TFEB that coordinate lysosomal biogenesis, its loss of dynamism disrupts the coupling between nutrient influx and secretory replenishment. The result is a ‘stuck dial’ where L‑cells cannot up‑receptorial machinery during meals nor clear damaged components during rest, producing a secretory exhaustion phenotype.

We propose that brief, circadian‑aligned rapamycin exposure—delivered during the early fasting window—creates a transient mTORC1 trough that permits autophagy flux and resets the signaling circuit. When nutrients reappear, mTORC1 reactivates, driving de novo synthesis of GLP-1 secretory machinery (e.g., SGLT1, FFAR2, CaSR) and refilling vesicles. This restores the physiological feed‑fast cycle that young L‑cells naturally exhibit.

Predictions

1. Aged mice receiving intermittent rapamycin synchronized with the dark‑phase fasting period will show increased amplitude of mTORC1 activity oscillations in isolated duodenal L‑cells (measured by phospho‑S6K immunohistochemistry) compared with continuously treated or untreated aged controls.
2. These mice will exhibit enhanced GLP-1 secretion ex vivo in response to glucose or fatty acid challenges, matching levels seen in young adult mice.
3. In vivo, they will demonstrate improved oral glucose tolerance and increased insulin secretion, effects that are abrogated by GLP-1 receptor antagonism.
4. Transcriptomic profiling of sorted L‑cells will reveal restored expression of nutrient‑sensor genes (e.g., Gpr119, Slc5a1) and autophagy markers (e.g., LC3B, Lamp1) only in the intermittently treated group.

Experimental Approach

• Animals: Young (3 mo) and aged (20 mo) C57BL/6 mice.
• Treatment groups: (a) Vehicle control, (b) Continuous rapamycin (2 mg/kg/day via chow), (c) Intermittent rapamycin (same total dose administered only during the first 4 h of the dark phase, 5 days/week) for 8 weeks.
• Readouts:
  • In vivo: oral glucose tolerance test (OGTT) with plasma GLP-1 and insulin measurements.
  • Ex vivo: isolated intestinal loops or cultured enteroids subjected to nutrient stimulations; GLP-1 release quantified by ELISA.
  • Molecular: duodenal mucosal sections stained for p‑S6K (mTORC1 activity) and LC3B (autophagy) at zeitgeber times corresponding to fed and fasted states; flow‑sorting of L‑cells (GFP‑GLP-1 reporter) for RNA‑seq.
  • Functional: transmission electron microscopy to assess secretory vesicle density and mitochondrial morphology.

Potential Outcomes and Interpretation If intermittent rapamycin rescues mTORC1 oscillatory dynamics, we expect to see restored feeding‑fast cycling of p‑S6K, increased autophagy markers during fasting peaks, and a concomitant rise in GLP-1 secretory capacity. This would support the idea that longevity‑associated mTOR modulation works not by static suppression but by reinstating the physiological rhythm that governs cellular ‘civilization’ versus ‘survival’ modes. Failure to observe these changes would falsify the hypothesis, indicating that either mTORC1 dysregulation is not the primary limiter of L‑cell function in aging or that additional downstream blocks (e.g., irreversible epigenetic silencing) dominate the phenotype.

Significance Demonstrating that a temporally precise mTORC1 intervention can rejuvenate an enteroendocrine hormone axis would shift the therapeutic paradigm from chronic inhibition to chronotherapeutic restoration of signaling dynamics, offering a strategy to mitigate age‑related metabolic decline without sacrificing the anabolic functions essential for tissue maintenance.