Ketone-Dependent Lysosomal Priming Determines Autophagic Efficacy Beyond Fasting Duration

Hypothesis

Sustained elevation of β-hydroxybutyrate during fasting, not the sheer length of the fasting window, drives maximal autophagic flux by enhancing lysosomal acidification and V‑ATPase assembly, thereby coupling ketosis to efficient autophagosome‑lysosome fusion.

Mechanistic Rationale

• Ketone bodies act as endogenous histone deacetylase inhibitors and can directly bind to lysosomal membrane proteins, modulating the mTORC1‑TFEB axis.[1] This promotes lysosomal biogenesis and increases cathepsin activity.
• Elevated β‑hydroxybutyrate stabilizes the V‑ATPase subunit ATP6V0D2, boosting proton pumping and lysosomal lumen pH to ~4.5, a condition optimal for hydrolase activity.[2]
• When lysosomes are primed, autophagosome‑lysosome tethering via HOPS complex is accelerated, increasing the rate of cargo degradation without necessarily increasing autophagosome formation.[3]
• Thus, autophagy flux becomes a function of ketone concentration rather than fasting duration per se; beyond the point where ketogenesis plateaus, additional fasting time yields diminishing returns on flux.

Predictions

1. In humans undergoing isocaloric fasting regimens, autophagic flux markers (LC3‑II turnover, p62 degradation) will correlate positively with plasma β‑hydroxybutyrate levels across 12‑, 16‑, and 20‑hour fasts, but will not show a significant increase when ketone concentrations are held constant.
2. Pharmacologically raising β‑hydroxybutyrate (via ketone ester ingestion) during a 12‑hour fast will produce autophagic flux comparable to a 20‑hour fast, whereas blocking ketosis with acetoacetate synthase inhibition will blunt flux despite extended fasting.
3. Selective autophagy pathways (mitophagy vs aggrephagy) will exhibit differential sensitivity to lysosomal priming, with mitophagy showing a stronger dependence on V‑ATPase activity due to its reliance on rapid mitochondrial turnover.

Experimental Design

• Recruit 60 healthy adults, randomized to three groups: (A) 12‑hour fast, (B) 16‑hour fast, (C) 20‑hour fast, all with identical caloric intake before fasting.
• Measure plasma β‑hydroxybutyrate every 2 h, and collect blood and peripheral mononuclear cells at 0, 4, 8, 12, 16, and 20 h for LC3‑II/p62 immunoblotting after lysosomal inhibition with bafilomycin A1 to assess flux.
• Include two mechanistic sub‑studies: (i) ketone ester supplementation during the 12‑hour fast to match β‑hydroxybutyrate levels of the 20‑hour group; (ii) siRNA knock‑down of ATP6V0D2 in isolated monocytes to test lysosomal dependence.
• Apply mixed‑effects modeling to test the interaction between fasting duration, ketone concentration, and autophagic flux.

Potential Outcomes and Falsification

• If autophagic flux scales with β‑hydroxybutyrate concentration and plateaus once ketogenesis reaches a steady state, the hypothesis is supported.
• If flux continues to rise with fasting duration independent of ketone levels, or if ketone manipulation fails to alter flux, the hypothesis is falsified.
• A lack of differential effect on mitophagy versus aggrephagy would challenge the predicted selectivity of lysosomal priming.

This framework directly addresses the gap noted in the literature—namely, the absence of comparative flux data across fasting windows—and offers a concrete, falsifiable mechanism linking metabolic state to autophagy efficacy.