Hypothesis

Extended fasting enhances autophagic flux not merely by time‑dependent glycogen depletion but through a ketone‑driven activation of the transcription factor TFEB, which primes lysosomal biogenesis and sustains LC3‑II turnover. In metabolically healthy individuals, this mechanism yields a threshold‑independent increase in autophagy once β‑hydroxybutyrate exceeds a critical concentration, whereas in insulin‑resistant states the pathway is blunted, making metabolic health a stronger predictor of autophagic response than fasting length.

Mechanistic Basis

• β‑hydroxybutyrate (BHB) acts as a signaling molecule that inhibits histone deacetylases and promotes acetylation of TFEB, favoring its nuclear translocation Mechanism link.
• Nuclear TFEB drives expression of lysosomal genes (LAMP1, CATHEPSINS) and autophagy regulators (LC3, p62) TFEB link.
• Elevated NAD⁺ during fasting activates SIRT1, which deacetylates TFEB and further stabilizes its active state SIRT1 link.
• In obesity/T2D, chronic inflammation and impaired ketone production blunt BHB‑TFEB signaling, reducing lysosomal capacity despite prolonged fasting Inflammation link.

Predictions and Experimental Design

1. Primary prediction: In healthy volunteers, a 20‑hour fast will raise plasma BHB to ≥1.5 mM, correlate with increased TFEB nuclear fraction in peripheral blood mononuclear cells, and show higher LC3‑II/LC3‑I ratio compared with a 16‑hour fast; pharmacological inhibition of ketogenesis (e.g., with etomoxir) will abolish these changes despite identical fasting duration.
2. Falsification: If TFEB nuclear translocation or lysosomal gene expression does not rise with BHB levels, or if blocking TFEB (using siRNA) fails to reduce LC3‑II flux during extended fasting, the hypothesis is falsified.
3. Secondary prediction: In overweight/insulin‑resistant participants, the same fasting protocols will produce blunted BHB rise, minimal TFEB activation, and no significant increase in autophagic markers, indicating that metabolic phenotype overrides duration.
4. Experimental approach: Randomized crossover study with three conditions—16h fast, 20h fast, and 20h fast + etomoxir—collecting blood at 0, 4, 8, 12, 16, 20 h for BHB, TFEB immunofluorescence, LC3‑II via western blot, and cytokine panel. Use mixed‑effects modeling to test interaction between fasting length, ketone level, and TFEB activity on autophagic flux.

Potential Confounders

• Exercise or caffeine intake can alter ketone levels; control with standardized activity logs.
• Genetic variants in HMGCS2 or TFEB may affect responsiveness; genotype participants for major polymorphisms.
• Acute stress hormones (cortisol) can influence autophagy; measure cortisol to adjust models.

By linking ketone signaling to lysosomal priming, this hypothesis shifts the focus from arbitrary hour cutoffs to a measurable metabolic state, offering a falsifiable framework that integrates duration, fuel availability, and health status into a unified model of fasting‑induced autophagy.