Hypothesis

Acarbose extends lifespan primarily by reshaping the gut microbiome to increase production of secondary bile acids that activate hepatic fibroblast growth factor 21 (FGF21) signaling. This microbiome‑bile‑acid‑FGF21 axis is more pronounced in males, explaining the sex‑specific longevity benefit.

Rationale

• Acarbose raises undigested starch in the colon, stimulating saccharolytic bacteria that produce short‑chain fatty acids (SCFAs) and concurrently alter bile acid biotransformation (3).
• Certain gut microbes (e.g., Clostridium scindens, Bacteroides spp.) deconjugate and 7‑α‑dehydroxylate primary bile acids, generating secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid (LCA) that act as signaling molecules (2).
• Secondary bile acids activate the nuclear receptor FXR and the membrane receptor TGR5, leading to increased hepatic FGF21 expression, a hormone linked to metabolic health and lifespan extension in mice (4).
• Sex differences in bile acid pool composition and FXR/TGR5 expression have been documented, with males showing higher baseline levels of DCA and greater FGF21 responsiveness (5).

Predictions

1. Male mice treated with acarbose will exhibit a significant increase in fecal and circulating secondary bile acids (DCA, LCA) compared with untreated males and with females receiving acarbose.
2. The rise in secondary bile acids will correlate with heightened hepatic FGF21 mRNA and protein levels, and with improved metabolic markers (lower insulin, higher adiponectin).
3. Depleting the gut microbiome with broad‑spectrum antibiotics or germ‑free conditions will abolish acarbose‑induced secondary bile acid elevation, hepatic FGF21 up‑regulation, and lifespan extension in males.
4. Supplementing secondary bile acids (e.g., DCA) to acarbose‑treated female mice will recapitulate the male‑specific lifespan benefit.
5. FXR or TGR5 knockout mice will fail to show acarbose‑mediated FGF21 induction or lifespan extension, despite similar glucose blunting.

Experimental Design

• Cohorts: Male and female C57BL/6 mice (n=30 per group) receiving control diet, acarbose (400 ppm), acarbose + antibiotics (cocktail in water), or acarbose + secondary bile acid supplement (DCA 0.1 % w/w). Include germ‑free male and female subsets.
• Duration: Treatment begins at 16 months and continues until natural death; interim sacrifices at 3 and 6 months for mechanistic read‑outs.
• Measurements:
  • Fecal and plasma bile acid profiles (LC‑MS/MS).
  • Hepatic Fgf21 mRNA (qPCR) and protein (ELISA).
  • Serum metabolic panel (glucose, insulin, adiponectin, lipids).
  • Inflammatory markers (NF‑κB p65 phosphorylation, TNF‑α).
  • Survival analysis (Kaplan‑Meier, log‑rank test).
• Controls: Pair‑fed groups to isolate caloric intake effects; vehicle‑treated microbiota‑transfer experiments to test causality.

Potential Outcomes

• Supportive: Male acarbose mice show ↑ secondary bile acids, ↑ hepatic FGF21, ↓ inflammation, and median lifespan extension (~18 %). Antibiotic or germ‑free treatment abolishes these changes and the longevity benefit. Female mice given DCA supplement display male‑like FGF21 induction and lifespan gain.
• Refutative: Acarbose alters bile acids equally in both sexes without sex‑specific FGF21 response, or lifespan extension persists despite microbiome depletion, indicating a primary role of glucose blunting.

This framework directly tests whether the microbiome‑bile‑acid‑FGF21 axis causally links acarbose to its sex‑biased longevity effect, providing a clear, falsifiable path forward.