We hypothesize that autophagic flux during fasting is primarily driven by intracellular β‑hydroxybutyrate (BHB) concentration modulating lysosomal V‑ATPase activity, rather than by elapsed fasting time per se.

Mechanism

When hepatic glycogen falls (~12 h), adipose lipolysis releases free fatty acids that are oxidized to ketone bodies, chiefly BHB. Rising BHB binds to the lysosomal‑surface GPR109A receptor, triggering a cAMP‑PKA cascade that phosphorylates the V‑ATPase subunit ATP6V0D1, increasing proton pumping and luminal acidity. Acidic lysosomes enhance cathepsin activity and promote autophagosome‑lysosome fusion, completing autophagic flux. This pathway can be activated irrespective of total fasting duration if BHB reaches a threshold (~0.5 mM) that is achievable earlier in individuals with high basal lipolysis or low insulin sensitivity. It's important to note that this doesn't replace the metabolic switch at ~12 h; it simply positions autophagy as a downstream response to ketone signaling.

Prediction

If lysosomal acidification is the gatekeeper, then:

• Pharmacological inhibition of V‑ATPase (e.g., with bafilomycin A1) will abolish the LC3B‑II increase seen after 16 h fasting even when BHB is high.
• Exogenous BHB infusion (raising plasma BHB to 0.8 mM) will rescue autophagic flux in 12‑hour fasted subjects, restoring LC3B‑II turnover to levels comparable with 24‑hour fasting.
• Individuals with a genetic variant that reduces GPR109A expression will require longer fasting periods to achieve the same autophagic response, independent of glycogen status.

Experimental Design

A crossover study in 30 healthy adults will test three conditions in random order, each separated by a week of washout:

1. 16‑hour fast + placebo saline infusion.
2. 12‑hour fast + BHB infusion targeting 0.8 mM plasma.
3. 16‑hour fast + V‑ATPase inhibitor (low‑dose bafilomycin A1) administered orally before the fast.

Primary outcome: change in autophagic flux measured by the ratio of LC3B‑II to LC3B‑I in peripheral blood mononuclear cells, corrected for lysosomal inhibition using chloroquine as a control. Secondary outcomes: plasma BHB, serum phosphatidylethanolamine (PE) levels, and insulin sensitivity (HOMA‑IR).

We're anticipating that condition 2 will show LC3B‑II/LLC3B‑I ratios not different from condition 1, supporting the BHB‑lysosome axis. Condition 3 should show a significant reduction in autophagic markers despite comparable BHB levels, falsifying the hypothesis that fasting duration alone drives autophagy. A null result (no difference between conditions) would refute the proposed mechanism and imply that other duration‑dependent signals dominate.