Rapamycin Alters Enterochromaffin Cell Serotonin Signaling via mTORC1-Dependent Regulation of TPH1 and Piezo2, Producing a Gut‑Specific Signal That Distinguishes It from Caloric Restriction

Hypothesis

Rapamycin extends lifespan not by merely mimicking caloric restriction but by selectively suppressing enterochromaffin (EC) cell serotonin production and mechanosensitive release, thereby creating a gut‑derived signal that diverges from the systemic fasting mimetic of CR.

Mechanistic Rationale

mTORC1 promotes translation of key enzymes and transporters through S6K‑mediated phosphorylation of ribosomal protein S6. In EC cells, mTORC1 activity could sustain high TPH1 expression and facilitate trafficking of Piezo2 channels to the apical membrane, both essential for basal serotonin synthesis and mechanosensitive release. Rapamycin‑induced mTORC1 inhibition would therefore reduce TPH1 translation and impair Piezo2 surface residency, lowering luminal 5‑HT levels and slowing gut motility. Caloric restriction, while also lowering mTORC1, preserves EC function through circadian‑linked activation of AMPK and SIRT1, which can maintain TPH1 transcription independent of mTORC1 and promote Piezo2 recycling via deacetylation. This predicts that rapamycin and CR generate opposite EC phenotypes despite overlapping mTORC1 suppression.

Testable Predictions

1. Rapamycin treatment will decrease EC cell TPH1 protein and mRNA levels, whereas CR will maintain or increase them.
2. Rapamycin will reduce Piezo2 expression at the EC apical surface and diminish serotonin release in response to mechanical stretch or butyrate.
3. Gut transit time will be prolonged in rapamycin‑treated mice but unchanged or accelerated under CR.
4. Exogenous 5‑HT or a Piezo2 agonist (e.g., Yoda1) will rescue rapamycin‑induced motility defects without affecting immune phenotypes.
5. Germ‑free mice will show blunted effects of both interventions on EC serotonin, indicating microbiome dependence.

Experimental Design

• Use young (3 mo) and old (20 mo) C57BL/6 mice assigned to ad libitum, 30 % caloric restriction, rapamycin (14 ppm diet), or rapamycin + 5‑HT supplementation groups for 8 weeks.
• Isolate colonic EC cells via FACS for TPH1 and Piezo2 qPCR/Western blot; measure surface Piezo2 by biotinylation assay.
• Ex vivo colonic segments mounted in Ussing chambers: apply luminal pressure steps and quantify serotonin release via HPLC.
• In vivo gut motility assessed by non‑absorbable marker transit (carmined red) and colonic manometry.
• Immune readouts (thymic cellularity, T‑cell naïve/memory ratios) performed to replicate prior divergence.
• Include butyrate supplementation in a subset to test TPH1 inducibility.

Potential Outcomes and Interpretation

If rapamycin lowers TPH1, Piezo2 surface expression, serotonin release, and slows motility while CR does not (or shows opposite trends), the hypothesis is supported: mTORC1 inhibition produces a gut‑specific hardship signal distinct from CR’s preservation of EC function. Rescue of motility by 5‑HT or Piezo2 agonism without altering immune outcomes would further demonstrate that longevity effects can be parsed into separable tissue‑specific axes. Conversely, if rapamycin and CR similarly suppress EC serotonin and motility, the hypothesis is falsified, implying that gut serotonin is not a discriminative downstream effector and that longevity mechanisms converge more tightly across tissues.