Hypothesis

Intermittent fasting–induced β‑hydroxybutyrate (BHB) selectively inhibits HDAC3 in human monocytes, leading to histone β‑hydroxybutyrylation (Kbhb) at Nrf2‑target promoters and an enhanced antioxidant stress response.

Rationale

• In C. elegans, 20 mM D‑BHB extends lifespan via HDAC2/3 inhibition, requiring DAF‑16/FOXO and SKN‑1/Nrf2 [D-beta-Hydroxybutyrate Extends Lifespan in C. elegans].
• Biochemical studies show BHB is a direct, reversible inhibitor of class I HDACs (HDAC1, HDAC3, HDAC4) with IC₅₀ ≈ 2.4–5.3 mM, concentrations reached during prolonged fasting or ketosis [Suppression of Oxidative Stress by β-Hydroxybutyrate [β-Hydroxybutyrate May Restore Younger Cells' Responsiveness to Senescent SASP Signals]].
• Human monocytes express high HDAC3 levels and are pivotal sensors of metabolic state; HDAC3 deacetylates Nrf2, limiting its transcriptional activity.
• We propose that, beyond simple inhibition, BHB occupies the HDAC3 catalytic pocket and is transferred onto lysine residues of histones (Kbhb), creating a dual signal: loss of deacetylase activity plus a novel acyl‑mark that recruits bromodomain co‑activators, thereby potentiating Nrf2‑driven transcription.

Testable Predictions

1. Biochemical – In isolated CD14⁺ monocytes from fasted individuals (BHB ≈ 2–4 mM), HDAC3 activity will be reduced by ≥30 % compared with fed controls, and histone H3K9/K14 acetylation plus Kbhb will increase at Nrf2‑target promoters (e.g., NQO1, HMOX1).
2. Transcriptomic – RNA‑seq of the same monocytes will show a ≥1.5‑fold up‑regulation of Nrf2‑dependent genes without a corresponding increase in ROS.
3. Functional – Ex vivo LPS‑stimulated cytokine production (IL‑6, TNF‑α) will be attenuated by ≥20 % in the fasted condition, correlating with monocyte Nrf2 activation.
4. Epigenetic‑clock – After a 4‑week intermittent fasting regimen (2 × 24 h fasts/week), the Horvath DNA‑methylation age of peripheral blood mononuclear cells will decrease by ≤1 year relative to baseline, an effect abolished by concomitant HDAC3 over‑expression via lentiviral transduction in a subset of participants.

Experimental Design

• Study: Randomized, crossover trial, n = 60 healthy adults (age 30‑50).
• Arms: (A) 36‑hour fast (water only) → BHB ≈ 2–4 mM measured by bedside β‑hydroxybutyrate meter; (B) isocaloric fed control.
• Sampling: Peripheral blood drawn at baseline, end of fast, and 2 h after re‑feeding. Isolate CD14⁺ monocytes.
• Assays:
  • HDAC3 fluorometric activity assay.
  • Western blot / mass spectrometry for H3K9ac, H3K14ac, and Kbhb.
  • ChIP‑seq for H3K27ac and Kbhb at Nrf2 loci.
  • RNA‑seq for Nrf2 target expression.
  • LPS (100 ng/mL) stimulation for 4 h, cytokine ELISA.
  • DNA‑methylation array (Epic) for epigenetic age.
• Statistical plan: Paired t‑tests for within‑subject comparisons; Bonferroni correction for multiple endpoints. Power analysis (α=0.05, 80 % power) predicts n = 52 to detect a 25 % change in HDAC3 activity; we recruit 60 to account for drop‑outs.

Falsifiability

If fasting does not reduce monocyte HDAC3 activity, increase histone acetylation/Kbhb, or up‑regulate Nrf2 targets, and if cytokine response and epigenetic age remain unchanged, the hypothesis is falsified. Conversely, a positive result would support the model that BHB acts as a selective HDAC3 inhibitor/modulator that links ketotic metabolism to monocyte‑mediated stress resistance, providing a mechanistic bridge from worm longevity data to human healthspan.

Broader Impact

Confirming this pathway would justify targeted intermittent fasting or BHB‑based regimens as feasible interventions to bolster innate immune resilience and delay inflammaging, offering a testable, mechanism‑driven alternative to broad‑spectrum HDAC inhibitors.