Beta-hydroxybutyrate as a senomorphic modulator of the senescence-associated secretory phenotype via GPR109A–β-arrestin–Nrf2 axis

Max Koch (MD/PhD)· Mar 26, 2026

Hypothesis

Exogenous beta-hydroxybutyrate (BHB) reprograms the secretome of senescent fibroblasts toward a pro‑repair profile by activating the GPR109A–β-arrestin biased signaling pathway, which suppresses NF‑κB–driven inflammatory SASP components (IL‑6, IL‑8) while enhancing Nrf2‑dependent expression of PDGFA and CCN1.

Mechanistic Rationale

BHB is known to inhibit class I HDACs and to activate the G‑protein‑coupled receptor GPR109A (also called HCAR2) in immune and epithelial cells [4], 5].
In senescent cells, GPR109A engagement can recruit β‑arrestin2, which scaffolds a phosphatase complex that dephosphorylates IKKβ, thereby attenuating NF‑κB nuclear translocation [3].
Concurrently, HDAC inhibition by BHB increases histone acetylation at Nrf2 target promoters and disrupts KEAP1‑mediated Nrf2 ubiquitination, favoring Nrf2 nuclear accumulation [1], 2].
Nrf2 drives transcription of antioxidant and matrix‑remodeling genes, including PDGFA and CCN1, which have been shown to accelerate wound healing when secreted by transiently senescent fibroblasts [2], 3]
The combined bias toward β‑arrestin signaling and HDAC inhibition creates a molecular switch that shifts the SASP from a chronic, inflammatory state to a transient, reparative state without eliminating the senescent cell itself.

Experimental Design

In vitro

Induce senescence in human dermal fibroblasts (HDFs) using irradiation (10 Gy) or oncogenic RAS expression; validate by p16^INK4a^ and SA‑β‑gal staining.
Treat cells with 5 mM β‑hydroxybutyrate (BHB) or the ketone ester ester‑BHB for 48 h; include controls: vehicle, GPR109A antagonist (AZD1722), β‑arrestin2 siRNA, and the HDAC inhibitor TSA as a positive control.
Collect conditioned medium and perform multiplex cytokine/chemokine profiling (Luminex) and targeted proteomics for PDGFA, CCN1, IL‑6, IL‑8, MMPs, and TIMPs.
Assess Nrf2 nuclear translocation by immunofluorescence and NF‑κB p65 phosphorylation by Western blot.
Functionally, apply conditioned media to keratinocyte monolayers and measure migration (scratch assay) and proliferation (EdU incorporation).

In vivo

Use aged (18‑month) male and female C57BL/6 mice with experimentally induced diabetic full‑thickness dorsal wounds (db/db model).
Administer BHB via intraperitoneal injection (300 mg/kg) or ketone‑ester‑supplemented drinking water (12 % w/v) daily; control groups receive saline or plain water.
Include a cohort treated with GPR109A antagonist to test receptor dependence.
Harvest wound tissue at days 3, 7, and 14; quantify senescent cells (p16^INK4a^ immunohistochemistry), SASP markers (ELISA for IL‑6, IL‑8, PDGFA, CCN1), and histology (re‑epithelialization length, collagen deposition via Sirius Red).
Measure wound closure rate via planimetry.

Predicted Outcomes

In vitro: BHB treatment will reduce IL‑6 and IL‑8 secretion by ≥40 % while increasing PDGFA and CCN1 levels by ≥30 % relative to vehicle; these changes will be abolished by GPR109A blockade or β‑arrestin2 knock‑down.
Nrf2 nuclear signal will rise ≥2‑fold, and NF‑κB p65 phosphorylation will drop ≥50 % under BHB treatment.
Conditioned media from BHB‑treated senescent fibroblasts will accelerate keratinocyte migration and proliferation compared with media from untreated senescent cells.
In vivo: BHB‑treated diabetic wounds will show a 25‑30 % increase in re‑epithelialization rate at day 7 and a 20 % rise in collagen density at day 14, accompanied by a shift in SASP composition toward PDGFA/CCN1 dominance and reduced IL‑6/IL‑8 levels.
The pro‑repair effect will be attenuated in mice receiving GPR109A antagonist, confirming receptor mediation.

Potential Pitfalls and Alternatives

BHB may also affect non‑senescent cells (e.g., macrophages), confounding wound‑healing readouts; we will isolate senescent cells via p16^INK4a^‑GFP reporter mice to verify cell‑autonomous effects.
Chronic BHB exposure could induce ketosis‑related metabolic shifts; we will monitor blood β‑hydroxybutyrate levels and keep them within physiological fasting range (1‑3 mM).
If GPR109A proves dispensable, we will test whether direct HDAC inhibition (using selective HDAC3 inhibitors) recapitulates the SASP shift, thereby distinguishing receptor‑mediated from epigenetic mechanisms.

This hypothesis is directly falsifiable: if BHB fails to modify the SASP in a GPR109A‑β‑arrestin‑dependent manner, or if the predicted functional improvements in wound healing do not materialize, the proposed senomorphic mechanism will be refuted.

Hypothesis

Mechanistic Rationale

BHB is known to inhibit class I HDACs and to activate the G‑protein‑coupled receptor GPR109A (also called HCAR2) in immune and epithelial cells [4], 5].

In senescent cells, GPR109A engagement can recruit β‑arrestin2, which scaffolds a phosphatase complex that dephosphorylates IKKβ, thereby attenuating NF‑κB nuclear translocation [3].

Concurrently, HDAC inhibition by BHB increases histone acetylation at Nrf2 target promoters and disrupts KEAP1‑mediated Nrf2 ubiquitination, favoring Nrf2 nuclear accumulation [1], 2].

Nrf2 drives transcription of antioxidant and matrix‑remodeling genes, including PDGFA and CCN1, which have been shown to accelerate wound healing when secreted by transiently senescent fibroblasts [2], 3]

The combined bias toward β‑arrestin signaling and HDAC inhibition creates a molecular switch that shifts the SASP from a chronic, inflammatory state to a transient, reparative state without eliminating the senescent cell itself.

Experimental Design

In vitro

Induce senescence in human dermal fibroblasts (HDFs) using irradiation (10 Gy) or oncogenic RAS expression; validate by p16^INK4a^ and SA‑β‑gal staining.

Treat cells with 5 mM β‑hydroxybutyrate (BHB) or the ketone ester ester‑BHB for 48 h; include controls: vehicle, GPR109A antagonist (AZD1722), β‑arrestin2 siRNA, and the HDAC inhibitor TSA as a positive control.

Collect conditioned medium and perform multiplex cytokine/chemokine profiling (Luminex) and targeted proteomics for PDGFA, CCN1, IL‑6, IL‑8, MMPs, and TIMPs.

Assess Nrf2 nuclear translocation by immunofluorescence and NF‑κB p65 phosphorylation by Western blot.

Functionally, apply conditioned media to keratinocyte monolayers and measure migration (scratch assay) and proliferation (EdU incorporation).

In vivo

Use aged (18‑month) male and female C57BL/6 mice with experimentally induced diabetic full‑thickness dorsal wounds (db/db model).

Administer BHB via intraperitoneal injection (300 mg/kg) or ketone‑ester‑supplemented drinking water (12 % w/v) daily; control groups receive saline or plain water.

Include a cohort treated with GPR109A antagonist to test receptor dependence.

Harvest wound tissue at days 3, 7, and 14; quantify senescent cells (p16^INK4a^ immunohistochemistry), SASP markers (ELISA for IL‑6, IL‑8, PDGFA, CCN1), and histology (re‑epithelialization length, collagen deposition via Sirius Red).

Measure wound closure rate via planimetry.

Predicted Outcomes

In vitro: BHB treatment will reduce IL‑6 and IL‑8 secretion by ≥40 % while increasing PDGFA and CCN1 levels by ≥30 % relative to vehicle; these changes will be abolished by GPR109A blockade or β‑arrestin2 knock‑down.

Nrf2 nuclear signal will rise ≥2‑fold, and NF‑κB p65 phosphorylation will drop ≥50 % under BHB treatment.

Conditioned media from BHB‑treated senescent fibroblasts will accelerate keratinocyte migration and proliferation compared with media from untreated senescent cells.

In vivo: BHB‑treated diabetic wounds will show a 25‑30 % increase in re‑epithelialization rate at day 7 and a 20 % rise in collagen density at day 14, accompanied by a shift in SASP composition toward PDGFA/CCN1 dominance and reduced IL‑6/IL‑8 levels.

The pro‑repair effect will be attenuated in mice receiving GPR109A antagonist, confirming receptor mediation.

Potential Pitfalls and Alternatives

BHB may also affect non‑senescent cells (e.g., macrophages), confounding wound‑healing readouts; we will isolate senescent cells via p16^INK4a^‑GFP reporter mice to verify cell‑autonomous effects.

Chronic BHB exposure could induce ketosis‑related metabolic shifts; we will monitor blood β‑hydroxybutyrate levels and keep them within physiological fasting range (1‑3 mM).

If GPR109A proves dispensable, we will test whether direct HDAC inhibition (using selective HDAC3 inhibitors) recapitulates the SASP shift, thereby distinguishing receptor‑mediated from epigenetic mechanisms.