Hypothesis

Acarbose extends lifespan more strongly in male mice because estrogen‑dependent downregulation of the colonic short‑chain fatty acid (SCFA) receptor FFAR2 (GPR43) in females limits SCFA‑driven AMPK activation in the gut‑brain axis, reducing systemic metabolic benefits.

Rationale

• Acarbose increases luminal SCFAs, especially butyrate, which activate AMPK via FFAR2/FFAR3 on enteroendocrine cells and vagal afferents (1).
• AMPK activation improves hepatic insulin sensitivity, reduces inflammation, and promotes autophagy—processes linked to longevity.
• Estradiol suppresses FFAR2 transcription in intestinal epithelium through estrogen receptor‑α (ERα) binding to an upstream enhancer element, a mechanism demonstrated in human colon carcinoma cells (4).
• Consequently, female mice experience a blunted SCFA‑FFAR2 signal despite comparable microbiome shifts, leading to weaker AMPK activation and smaller lifespan gains.

Testable Predictions

1. Receptor Expression: Colonic FFAR2 mRNA and protein levels will be significantly lower in female versus male mice fed acarbose, and this difference will be abolished by ovariectomy or ERα antagonism.
2. Pharmacological Rescue: Treating female acarbose‑treated mice with a selective FFAR2 agonist (e.g., MK‑8666) will restore AMPK phosphorylation in the liver and hypothalamus to male‑like levels and increase median lifespan by ≥15%.
3. Neural Activation: Females will show reduced c‑Fos activation in the nucleus tractus solitarius (NSC) after an oral glucose challenge compared with males; FFAR2 agonism will normalize this response.
4. Microbiota Independence: Germ‑free females colonized with acarbose‑altered microbiota from males will still exhibit a muted longevity response unless FFAR2 signaling is pharmacologically enhanced.

Experimental Design

• Use male and female C57BL/6J mice (n=30 per sex per group) started on acarbose at 4 months of age.
• Groups: vehicle, acarbose, acarbose + FFAR2 agonist, acarbose + ERα antagonist (fulvestrant).
• Measure: colonic FFAR2 expression (qPCR, Western blot), hepatic p‑AMPK, plasma butyrate, glucose tolerance, vagal NSC c‑Fos, and survival.
• Include ovariectomized females to test hormone dependence.
• Perform fecal microbiota transplantation (FMT) from acarbose‑treated male donors into germ‑free female recipients with/without FFAR2 agonist.

Potential Outcomes and Interpretation

• If FFAR2 expression is lower in females and its pharmacological activation rescues lifespan extension, the hypothesis is supported, implicating sex‑hormone‑modulated SCFA signaling as a causal node.
• If FFAR2 levels are equivalent between sexes or agonist treatment fails to improve longevity, the hypothesis is falsified, suggesting other mechanisms (e.g., sex‑specific bile acid metabolism or hepatic drug clearance) underlie the dimorphism.

Broader Implications

Confirming a gut‑brain SCFAFFAR2 axis modulated by estrogen would explain why many interventions that remodel the microbiome show male‑biased benefits in the ITP and would suggest combinatorial strategies (e.g., acarbose + FFAR2 agonists) to achieve equitable longevity outcomes across sexes.