Hypothesis

Sustained elevation of the ketone body β‑hydroxybutyrate (BHB) to ≥1 mM in circulation directly stimulates lysosomal biogenesis and autophagosome‑lysosome fusion, thereby rescuing measurable autophagy markers (LC3B‑II/I ratio, p62 degradation) even when fasting periods are limited to 16:8 or 18:6 time‑restricted eating (TRE).

Mechanistic Rationale

1. BHB as an epigenetic modulator – BHB inhibits class I histone deacetylases (HDACs), increasing acetylation of autophagy‑related gene promoters (e.g., LC3B, ATG5, TFEB). This transcriptional priming lowers the threshold for autophagy induction independent of AMPK activation [1].
2. BHB‑GPR109A signaling – Binding of BHB to the G‑protein‑coupled receptor GPR109A on immune and metabolic cells raises intracellular Ca²⁺, activating CaMKKβ‑AMPK and inhibiting mTORC1 through Raptor phosphorylation [2]. Concurrent AMPK activation phosphorylates ULK1, initiating autophagosome formation.
3. Lysosomal priming via TFEB – Elevated BHB promotes nuclear translocation of TFEB by inhibiting mTORC1‑dependent phosphorylation, expanding lysosomal capacity and enhancing autophagosome‑lysosome fusion [3]. Increased lysosomal acidification and cathepsin activity accelerate substrate degradation, normalizing LC3B‑II turnover.
4. Metabolic independence from prolonged fasting – While 24‑48 h fasts raise BHB via hepatic ketogenesis, exogenous BHB esters or a ketogenic diet can achieve comparable blood concentrations within 2‑4 h, circumventing the need for extended nutrient deprivation [4].

Testable Predictions

• Prediction 1: In healthy adults undergoing 16:8 TRE, supplementation with β‑hydroxybutyrate ester (maintaining plasma BHB ≥ 1 mM) will produce a statistically significant increase in peripheral blood mononuclear cell (PBMC) LC3B‑II/I ratio and decrease in p62 after 2 weeks compared with placebo‑matched 16:8 TRE (p < 0.05, two‑tailed t‑test).
• Prediction 2: The autophagy enhancement observed in Prediction 1 will be attenuated in participants receiving a GPR109A antagonist (e.g., MK‑683) or HDAC inhibitor control, confirming receptor‑ and epigenetic‑mediated mechanisms.
• Prediction 3: Muscle biopsy samples from the ketone‑supplemented group will show increased LAMP1‑positive lysosome density and colocalization of LC3B with lysosomal markers (measured by immunofluorescence) without a corresponding rise in circulating insulin or IGF‑1 levels, indicating autophagy induction occurs despite unchanged nutrient‑sensing hormones.
• Prediction 4: Participants with baseline insulin resistance (HOMA‑IR > 2.5) will exhibit a greater relative increase in autophagy markers than insulin‑sensitive counterparts, reflecting a larger compensatory role for ketone‑driven lysosomal priming when endogenous fasting‑ketosis is blunted.

Falsifiability

If, under rigorously controlled conditions, ketone supplementation fails to raise LC3B‑II/I or reduce p62 beyond placebo levels, or if the effect is completely abolished by AMPK inhibition (Compound C) but persists despite GPR109A or HDAC blockade, the hypothesis would be falsified. Likewise, absence of lysosomal biogenesis changes in tissue biopsies despite elevated BHB would refute the mechanistic link.

Significance

This hypothesis reframes the ‘fasting duration equals autophagy’ dogma by positioning ketone bodies as proximate signaling agents that uncouple autophagic activation from prolonged caloric restriction. It offers a practical avenue—nutritional ketosis or exogenous ketone delivery—to harness autophagy‑related health benefits (improved proteostasis, inflammation modulation) while preserving adherence to shorter, more sustainable eating windows.