IF a triple pharmacologic combination of cell-permeable calcium alpha-ketoglutarate (CaAKG, 2 g/kg/day in chow), rapamycin (14 mg/kg/day enteric-coated chow), and metformin (0.1% w/v in drinking water) — hereafter "RAM-triple" — is administered for 12 continuous weeks to aged (22–24-month), male C57BL/6J mice with already-accumulated hepatic H3K27me3 at aberrantly silenced metabolic and inflammatory loci,

THEN the following will be observed in aged mouse liver:

1. A ≥30% reduction in inter-individual coefficient of variation (CV) of genome-wide H3K27me3 ChIP-seq signal compared to aged vehicle controls (primary endpoint: epigenetic noise reduction, measured by H3K27me3 CUT&Tag on n≥8 livers per group, with per-locus CV computed across animals at aging-gain H3K27me3 sites identified from young vs. aged reference cohorts);
2. A net shift of H3K27me3 enrichment back toward young reference profiles at aberrantly silenced loci, quantified as genome-wide Pearson correlation of per-locus signal to young reference increasing by ≥0.15 units;
3. Partial restoration of H3K27ac at genomic regions showing age-associated H3K27ac loss, particularly at enhancers of metabolic genes;
4. Transcriptional re-activation of genes aberrantly silenced by excess H3K27me3 (hepatic metabolic regulators, Hnf4a targets), measured by RNA-seq;

BECAUSE the following step-by-step causal chain operates:

1. Aging liver accumulates excess H3K27me3 at metabolically critical and developmentally regulated loci due to a progressive deficiency of KDM6A/B (UTX/JMJD3) Jumonji-domain demethylase activity, driven by declining intracellular α-ketoglutarate (α-KG) availability arising from age-associated loss of the SLC1A5 glutamine transporter, which limits glutamine-to-glutamate-to-α-KG flux. (Age-associated KDM6 insufficiency via α-KG depletion produces elevated H3K27me3 and cardiac dysfunction reversible by glutamine supplementation)[https://doi.org/10.1101/2025.05.16.654624]. This mechanism, demonstrated in aged heart, is likely conserved in liver given the shared metabolic dependency of KDM6 enzymes on α-KG as an obligate co-substrate.

2. The accumulated H3K27me3 drift is stochastic across individual hepatocytes and animals, producing inter-individual epigenomic heterogeneity — the "epigenetic noise" phenotype targeted here. (Genome-wide remodeling of H3K27ac and the epigenomic landscape with aging in mice, including mouse liver, produces widespread transcriptional dysregulation)[https://doi.org/10.1101/gr.240093.118]. (Normal liver aging in male mice is characterized by progressive histone modification dynamics that diverge from youthful patterns)[https://doi.org/10.1101/2025.04.21.649824]. Stochastic drift across cells accumulates as measurable inter-individual variance at the bulk-tissue level — the EpiSENS "noise" signal.

3. CaAKG supplementation directly replenishes the rate-limiting obligate co-substrate for KDM6A/B, driving catalytic oxidative demethylation of the...

SENS category: RepleniSENS

Key references: • doi.org/10.1101/2025.05.16.654624]. • doi.org/10.1101/gr.240093.118]. • doi.org/10.1101/2025.04.21.649824]. • doi.org/10.1101/gr.240093.118] • doi.org/10.1101/2025.04.21.649824]