Hypothesis: With advancing age, the ileal bile acid transporter ASBT (SLC10A2) is downregulated, reducing enterohepatic bile acid circulation. This diminishes FXR and TGR5 activation in the gut, kidney, and liver, leading to lower expression of NAMPT and sirtuins, which together accelerate NAD+ depletion and promote aging phenotypes. Restoring ASBT function or bile acid signaling should rescue NAD+ levels and extend healthspan, whereas genetic loss of ASBT will exacerbate NAD+ decline and shorten lifespan.

Key predictions that make the hypothesis testable and falsifiable:

1. Expression correlation – Aged mice (≥24 mo) will show significantly reduced ileal ASBT mRNA and protein levels compared to young mice (3 mo), paralleling a drop in hepatic and renal FXR/TGR5 target genes (Shp, Cyp7a1, Sirt1, Sirt3) and NAMPT. NAD+ decline attributed to reduced NAMPT expression, impairing salvage pathway FXR/TGR5 activation boosts SIRT1/SIRT3, enhancing mitochondrial β-oxidation long-lived Ames dwarf mice upregulate renal FXR and TGR5
2. Bile acid pool shift – Loss of ASBT will decrease the total bile acid pool size and increase fecal bile acid excretion, resulting in lower circulating conjugated bile acids that normally activate FXR/TGR5.
3. NAD+ impact – Reduced FXR/TGR5 signaling will lead to decreased NAMPT transcription, lowering NAD+ salvage flux and causing a measurable decline in hepatic and muscular NAD+ concentrations in aged mice.
4. Intervention rescue – Genetic overexpression of ASBT specifically in intestinal enterocytes of aged mice, or dietary supplementation with a FXR agonist (e.g., obeticholic acid) or TGR5 agonist (e.g., INT-777), will restore FXR/TGR5 target gene expression, increase NAMPT and SIRT activity, elevate NAD+ levels, and improve age‑related phenotypes (glucose tolerance, mitochondrial respiration, grip strength). Conversely, intestinal‑specific ASBT knockout in young mice will prematurely mimic the aged NAD+ deficit and accelerate functional decline.
5. Lifespan/healthspan readout – Mice with restored ASBT or bile acid signaling will exhibit median lifespan extension of at least 10 % and improved frailty indices, whereas ASBT‑deficient mice will show shortened lifespan relative to controls.

Mechanistic rationale: FXR and TGR5 activation are known to upregulate SIRT1 and SIRT3, which deacetylate and activate PGC‑1α and FOXO transcription factors, promoting mitochondrial biogenesis and stress resistance. SIRT activity also consumes NAD+; however, their downstream effects include transcriptional upregulation of NAMPT, the rate‑limiting enzyme of the NAD+ salvage cycle. When bile acid signaling wanes, this positive feedback loop is broken: diminished SIRT activity reduces NAMPT expression, NAD+ synthesis falls, and PARP1‑mediated NAD+ consumption (driven by age‑associated DNA damage) further depletes the pool. Thus, the ileal ASBT decline is not a passive byproduct but an active contributor to the metabolic rewiring that limits cellular repair capacity—a "budget cut" that reduces investment in maintenance pathways. This reframes NAD+ loss as, at least partially, a consequence of disrupted bile acid‑FXR/TGR5 signaling rather than solely intrinsic enzymatic damage.

Experimental approach: Use cohorts of C57BL/6J mice at 3, 12, and 24 months. Quantify ileal ASBT (qPCR, Western blot, immunostaining), bile acid pools (LC‑MS), FXR/TGR5 targets, NAMPT, SIRT1/3 activity, and NAD+ levels (enzymatic assay). Manipulate ASBT via AAV‑mediated intestinal overexpression or CRISPR‑mediated knockout, and treat separate groups with FXR/TGR5 agonists. Assess metabolic health (glucose tolerance, indirect calorimetry), mitochondrial function (Seahorse), and survival. Statistical analysis will employ two‑way ANOVA with post‑hoc tests; a p‑value < 0.05 will be considered significant.

If the data show that ASBT loss correlates with reduced FXR/TGR5 signaling, lowered NAMPT/SIRT activity, and NAD+ depletion, and that restoring ASBT or bile acid receptor signaling reverses these changes and extends healthspan, the hypothesis will be supported. Failure to observe these relationships—or demonstration that ASBT manipulation does not affect NAD+ levels or lifespan—would falsify the claim.