Hypothesis The autophagic flux achieved during intermittent fasting is governed by the hepatic glycogen‑to‑ketone ratio rather than fasting duration alone. When liver glycogen falls below ~30% of baseline and circulating β‑hydroxybutyrate rises above 1.0 mM, lysosomal acidification and TFEB nuclear translocation are sufficiently activated to sustain complete autophagosome‑lysosome fusion. Below this combined threshold, autophagy initiates but stalls at phagophore formation, yielding incomplete flux.

Mechanistic Rationale

1. Glycogen depletion activates AMPK, which inhibits mTORC1 and initiates ULK1‑dependent phagophore nucleation.
2. Rising ketone bodies (β‑hydroxybutyrate) act as endogenous HDAC inhibitors, promoting acetylation of lysosomal V‑ATPase subunits, thereby boosting lysosomal acidity and cathepsin activity.
3. Concurrent AMPK activation and HDAC inhibition synergize to drive TFEB dephosphorylation, nuclear entry, and transcription of lysosomal and autophagic genes (e.g., LAMP1, CATB).
4. Thus, the ratio of low glycogen to high ketones reflects a cellular state where both initiation and degradation arms of autophagy are optimally coupled.

Testable Predictions

• In humans undergoing graded fasts (12, 16, 20, 24 h), autophagic flux (measured by LC3‑II turnover with lysosomal blockade) will not increase linearly with time; instead, it will show a sharp rise once the hepatic glycogen‑ketone ratio crosses the predicted threshold.
• Individuals with higher baseline mitochondrial respiration (e.g., athletes) will reach the threshold earlier (≈16 h) due to faster glycogenolysis, whereas insulin‑resistant participants will require >20 h to achieve comparable flux.
• Pharmacological mimicry of the ketone signal (exogenous β‑hydroxybutyrate ester) during a 16‑h fast should rescue autophagic flux in glycogen‑depleted but ketone‑low conditions, while lysosomal alkalisation (e.g., chloroquine) will abolish the flux increase despite adequate glycogen depletion.

Experimental Design

• Recruit 60 volunteers stratified by HOMA‑IR (insulin‑sensitive vs resistant).
• Implement randomized crossover fasts of 12, 16, 20, and 24 h with fed control.
• At each endpoint, acquire: hepatic glycogen concentration via ^13C‑MRS, serum β‑hydroxybutyrate, PBMC LC3‑II/I ratio with and without bafilomycin A1 (to calculate flux), lysosomal pH using exosomes stained with LysoTracker, and TFEB localization by imaging flow cytometry.
• In a sub‑cohort, administer β‑hydroxybutyrate ester during the 16‑h fast or lysosomal alkaliser to test causality.

Falsifiability If autophagic flux correlates strictly with fasting duration independent of glycogen‑ketone ratio, or if manipulating ketones does not alter flux despite fixed glycogen levels, the hypothesis is falsified. Likewise, a lack of difference in TFEB nuclear translocation across the predicted threshold would refute the mechanistic link.

Implications Establishing a quantitative biomarker (glycogen‑ketone ratio) would enable personalized fasting prescriptions, moving beyond arbitrary hour‑based guidelines toward metabolically targeted autophagy activation.