Sex‑Specific Mitochondrial Stress Signaling Determines Whether MOTS-c Extends Lifespan or Acts as a Metabolic Modulator

Hypothesis

MOTS-c extends mammalian lifespan only when it activates the ATF4‑CHOP mitochondrial stress response in a sex‑specific manner, and this effect is abolished when mTORC1 signaling is concurrently active.

Mechanistic Rationale

Recent surveys of MDP literature highlight a lack of rigorous data, relying on surrogate metabolic readouts without longevity controls 1. In contrast, rapamycin consistently extends lifespan even when started in mid‑life, providing individual survival curves and proper controls 2. The ATF4‑CHOP arm of the integrated stress response (ISR) is known to be triggered by mitochondrial stress and can promote longevity when modulated transiently, while chronic activation is deleterious. We propose that MOTS‑c, upon binding to an as‑yet‑uncharacterized mitochondrial receptor, transiently elevates mitochondrial ROS, activating ATF4‑CHOP in a sex‑dependent fashion due to differences in estrogen‑regulated expression of CHOP and ATF4. This transient ISR upregulates mitochondrial chaperones and improves proteostasis, extending lifespan. However, if mTORC1 is active (e.g., high nutrient signaling), ATF4‑CHOP drives maladaptive responses such as increased autophagy flux that leads to muscle wasting, negating any benefit. Sex stratification is essential because blood concentration differences of drugs like canagliflozin reveal divergent pharmacokinetics between aged females and young males 3.

Testable Predictions

1. In male mice, chronic MOTS‑c treatment will increase ATF4 and CHOP protein levels in liver and muscle after 2 weeks, whereas females will show a delayed, lower‑amplitude peak.
2. Co‑administration of low‑dose rapamycin (which partially inhibits mTORC1) will amplify the ATF4‑CHOP response and extend median lifespan in both sexes, while MOTS‑c alone will extend lifespan only in females.
3. Genetic attenuation of ATF4 (liver‑specific knockout) will abolish MOTS‑c‑induced lifespan extension without affecting its acute glucose‑uptake effect.
4. LC‑MS/MS quantification will reveal higher mitochondrial MOTS‑c concentrations in female skeletal muscle compared with males after matched dosing.

Experimental Design

• Use C57BL/6J mice, n=60 per sex per group to achieve 80 % power to detect a 10 % median lifespan difference 4.
• Groups: vehicle, MOTS‑c (dose based on pilot PK), rapamycin (positive control), MOTS‑c + rapamycin, ATF4 liver‑KO + MOTS‑c, and appropriate sex‑matched controls.
• Primary endpoint: individual survival curves; secondary endpoints: glucose tolerance test, Western blot for ATF4/CHOP in liver/muscle at 1 mo, 3 mo, 6 mo, mitochondrial MOTS‑c levels by LC‑MS/MS, and frailty index.
• Include vehicle and knockout controls, and randomize littermates.

Potential Outcomes and Falsification

If MOTS‑c extends lifespan only in females and this effect is lost in ATF4 liver‑KO mice, the hypothesis is supported. If MOTS‑c extends lifespan equally in both sexes independent of ATF4 status, or if lifespan extension occurs without any detectable ATF4/CHOP activation, the hypothesis is falsified. Likewise, if rapamycin co‑treatment does not further enhance lifespan beyond MOTS‑c alone in males, the proposed mTORC1‑ISR interaction is incorrect.

This framework moves the field beyond anecdotal metabolic assays toward a mechanistically anchored, sex‑stratified longevity test that can definitively place MOTS‑c alongside validated interventions such as rapamycin.