Hypothesis

Leucine‑pulse timing during intermittent prolonged fasting enhances skeletal‑muscle autophagy via SIRT2‑dependent ATG4B activation without suppressing hepatic ketogenesis.

Background

Prolonged fasting robustly induces hepatic autophagy through SIRT2‑mediated ATG4B deacetylation and mTORC1 suppression, yet skeletal muscle fails to show comparable LC3, LAMP1, or BCLN1 increases after 72‑hour weekly fasts[1]. Meanwhile, metabolic flexibility—measured by the speed of glucose‑to‑fat shift—predicts slower cognitive and cardiovascular decline[4]. Nutrient‑dense caloric restriction preserves muscle when protein intake is adequate[5], but the interaction between fasting‑induced SIRT2 activation and amino‑acid signaling in muscle remains unclear.

Mechanistic Insight

We propose that a brief leucine‑rich pulse (≈5 g) administered 2 hours before the end of a 24‑hour fasting window transiently activates mTORC1 in muscle, which paradoxically stimulates SIRT2 activity through NAD⁺ salvage pathways, leading to ATG4B deacetylation and increased autophagosome formation. In the liver, the same pulse does not raise mTORC1 enough to inhibit ketogenesis because hepatic glycogen stores are already depleted and SIRT2 remains active due to high NAD⁺/NADH ratio.

Testable Predictions

1. Human volunteers undergoing a 24‑hour fast will show a higher muscle LC3‑II/I ratio and increased ATG4B deacetylation when a leucine bolus is given at hour 22 compared with fast alone.
2. It's simple to administer and we don't expect hepatic β‑hydroxybutyrate levels to differ between leucine‑pulse and control fasts, indicating preserved ketogenesis.
3. The muscle autophagic response will correlate with improved metabolic flexibility scores (measured by respiratory exchange ratio shift) during the subsequent refeeding phase.

Experimental Design

• Participants: 30 healthy adults (age 30‑50), randomized to leucine‑pulse or placebo control.
• Protocol: 24‑hour fast (water, electrolytes) on day 1; at hour 22 receive either 5 g leucine dissolved in water or isocaloric non‑essential amino acid placebo.
• Measurements: Muscle biopsy (vastus lateralis) at hour 24 for LC3‑II/I, acetylated ATG4B (Western blot); blood β‑hydroxybutyrate at hours 0, 12, 24; indirect calorimetry to assess RER shift before and after refeeding.
• Analysis: Two‑way ANOVA (treatment × time) with post‑hoc tests; significance set at p<0.05.

If the leucine pulse augments muscle autophagy without blunting hepatic ketogenesis, it would refine fasting‑based longevity protocols by adding a muscle‑targeted nutrient timing strategy that leverages the same SIRT2‑ATG4B axis activated in liver.