Sex‑SpecificHepatic FGF21 Activation Mediates Acarbose‑Induced Longevity via Microbiota‑Derived Butyrate

Hypothesis

Acarbose extends lifespan in male mice by increasing colonic butyrate production, which activates the hepatic butyrate receptor GPR109A‑PPARα‑FGF21 axis; this cascade is amplified in males due to higher basal expression of hepatic estrogen receptor β (ERβ) that potentiates PPARα signaling, whereas females exhibit weaker ERβ‑PPARα coupling, limiting FGF21 induction and thus longevity benefit.

Predictions

• Male mice treated with acarbose will show a greater increase in hepatic FGF21 mRNA and serum FGF21 protein than females, correlating with individual butyrate levels.
• Pharmacological blockade of GPR109A or hepatic PPARα will abolish the sex‑difference in lifespan extension, reducing male benefit to female‑level.
• Fecal microbiota transplantation (FMT) from acarbose‑treated male donors into germ‑female recipients will confer a male‑like lifespan extension only if recipients also receive ERβ agonist treatment.
• ERβ knockout in hepatocytes will eliminate the male‑specific lifespan gain without affecting microbiota composition or SCFA production.

Experimental Approach

1. Cohort setup – Use ITP‑derived male and female C57BL/6J mice, start acarbose (1000 ppm diet) at 4 months; include parallel groups receiving GPR109A antagonist (GLPG0974) or PPARα antagonist (GW6471).
2. Microbiota & metabolomics – 16S rRNA sequencing and targeted SCFA quantification (butyrate, formate, acetate) from fecal samples at 1, 3, and 6 months post‑treatment.
3. Hepatic signaling – qPCR and Western blot for GPR109A, PPARα, p‑PPARα, FGF21, and ERβ in liver isolates; serum FGF21 ELISA.
4. Lifespan monitoring – Standard ITP survival analysis; calculate median and 90th percentile lifespan.
5. FMT experiments – Germ‑free male and female recipients receive feces from acarbose‑treated male donors; subsets receive ERβ agonist (DPN) or vehicle.
6. Genetic models – Liver‑specific ERβ knockout (ERβ^LKO) mice treated with acarbose to test necessity of hepatic ERβ.

Potential Outcomes and Falsifiability

• Support – Males show ≥2‑fold higher hepatic FGF21 increase than females; antagonizing GPR109A or PPARα reduces male median lifespan gain from ~22 % to ≤5 % (female‑level); FMT from treated males extends female lifespan only when combined with ERβ agonist; ERβ^LKO males lose the longevity advantage despite normal microbiota and butyrate elevation.
• Refute – No sex difference in hepatic FGF21 induction; antagonists fail to diminish male lifespan benefit; FMT transfers longevity irrespective of recipient sex or ERβ status; hepatic ERβ knockout does not alter lifespan outcome.

Each prediction yields a clear, binary outcome that can be statistically tested, rendering the hypothesis falsifiable. If the data align with the support scenario, we would establish a mechanistic link between microbiota‑derived butyrate, sex‑specific hepatic nuclear receptor signaling, and FGF21‑mediated longevity, explaining the observed male‑biased response to acarbose. If the data align with the refute scenario, the hypothesis would be rejected, prompting investigation of alternative sex‑dependent pathways such as differential drug pharmacokinetics or immune modulation.