Hypothesis

Physiological β‑hydroxybutyrate (BHB) concentrations achieved during sustained ketosis selectively inhibit HDAC3 in human monocytes, leading to a trained‑immunity phenotype that delays inflammaging and extends healthspan. This effect occurs only when intracellular nuclear BHB exceeds ~2 mM, a threshold attainable through combined dietary ketosis and intermittent high‑intensity exercise, which enhances mitochondrial BHB import.

Rationale

• The reported IC50 for BHB inhibition of class I/IIa HDACs ranges 2.4‑5.3 mM (1), yet histone acetylation rises at 1 mM in vitro, suggesting isoform‑specific sensitivity.
• HDAC3 is a key regulator of monocyte inflammatory gene expression and metabolic reprogramming (2).
• Trained immunity—epigenetically reinforced heightened responsiveness of innate cells—depends on HDAC activity shifts and correlates with lower systemic inflammation in long‑lived individuals.
• No human study has linked BHB‑driven HDAC3 inhibition in accessible immune cells to functional healthspan outcomes.

Novel Mechanistic Insight

We propose that BHB does not act as a broad HDAC inhibitor at physiological levels but instead preferentially occupies the HDAC3 active site due to its unique tunnel architecture, which accommodates the β‑hydroxybutyrate moiety more effectively than class I HDAC1/2. This selective inhibition reduces deacetylation of H3K27 at promoters of glycolytic genes (e.g., HK2, PFKFB3), fostering a Warburg‑like metabolic state in monocytes that supports trained immunity. Concurrently, BHB‑mediated activation of HCAR2 (GPR109A) in neutrophils amplifies IL‑10 release, creating an anti‑inflammatory feedback loop that tempers chronic inflammaging.

The threshold effect arises because nuclear BHB accumulation depends on both cytosolic concentration and the activity of the mitochondrial monocarboxylate transporter MCT1, which is up‑regulated by exercise‑induced AMPK phosphorylation. Thus, ketosis alone may yield subthreshold nuclear BHB (<1.5 mM), whereas ketosis plus repeated sprint intervals pushes nuclear BHB above the HDAC3 inhibitory threshold.

Testable Predictions

1. Dose‑response in humans – Participants undergoing a 4‑week ketogenic diet (target plasma BHB 1‑3 mM) will show no change in monocyte HDAC3 activity or H3K27ac, whereas adding three weekly HIIT sessions (plasma BHB peaks 3‑5 mM) will produce a ≥30 % increase in monocyte H3K27ac and a concomitant decrease in HDAC3 activity, measured via ex vivo enzyme assay and western blot of isolated PBMCs.
2. Functional immune readout – The combined ketosis + HIIT group will exhibit enhanced monocyte responsiveness to a secondary LPS challenge (increased TNF‑α, IL‑6 production) indicative of trained immunity, alongside reduced baseline plasma IL‑6 and CRP.
3. Healthspan correlation – After 12 weeks, the trained‑immunity signature (monocyte H3K27ac ↑, HDAC3 activity ↓) will predict improvements in frailty index and gait speed better than plasma BHB levels alone.
4. Specificity control – Pharmacologic HDAC3 inhibition (using RGFP966) will mimic the immunological and metabolic shifts seen with ketosis + HIIT, whereas pan‑HDAC inhibition (SAHA) will not reproduce the trained‑immunity phenotype, confirming isoform selectivity.

Experimental Design (outline)

• Population: 120 healthy adults aged 50‑70, randomized to four arms (control, ketogenic diet only, HIIT only, ketogenic + HIIT).
• Intervention: 4‑week ketogenic diet (≤50 g carbs/day); HIIT protocol: 4 × 4‑min intervals at 90 % HRmax, 3×/week.
• Readouts: fasting plasma BHB, monocyte isolation, HDAC3 activity fluorometric assay, western blot for H3K27ac, RNA‑seq for glycolytic gene signature, cytokine panel after LPS restimulation, clinical frailty and gait assessments.
• Analysis: Mixed‑effects models testing interaction between diet and exercise on monocyte HDAC3 activity; mediation analysis to link epigenetic changes to healthspan metrics.

Falsifiability

If ketogenic + HIIT fails to raise monocyte H3K27ac above control levels, or if the observed epigenetic changes do not correlate with improved trained‑immunity biomarkers or healthspan outcomes, the hypothesis is refuted. Conversely, a positive result would support a threshold‑dependent, isoform‑specific HDAC3 mechanism as a viable translation route for BHB‑mediated longevity effects in humans.