Hypothesis

NMN‑induced NAD+ elevation directly activates SIRT6, which in turn downregulates the cystine transporter Slc7a11, raising hepatic hydrogen sulfide (H2S) production and deacetylating the autophagy adaptor p62 to shift cargo selectivity toward damaged mitochondria. This SIRT6‑H2S‑p62 axis accounts for the observed sex‑specific lifespan extension by NMN, as estrogen represses Slc7a11 transcription in females, limiting the downstream H2S surge.

Mechanistic Model

• NAD+ boost from NMN increases SIRT6 deacetylase activity (SIRT6 uses NAD+ as a co‑substrate) [3].
• Active SIRT6 deacetylates NAMPT, enhancing NAD+ salvage and creating a feed‑forward loop that sustains SIRT6 activity [7].
• SIRT6 directly deacetylates the cystine transporter Slc7a11 at lysine‑XX, reducing its membrane stability and promoting lysosomal degradation, thereby lowering cystine influx and increasing the intracellular cysteine pool for H2S synthesis via cystathionine beta‑synthase (CBS) [3, 4]
• Elevated H2S modifies p62 through persulfidation, facilitating its deacetylation by SIRT6 and altering its affinity for ubiquitinated mitochondria versus protein aggregates, thereby favoring mitophagy [3]
• In males, lower basal estrogen levels permit stronger Slc7a11 suppression, leading to a larger H2S rise and greater mitophagic flux; in females, estrogen‑driven Slc7a11 expression buffers the effect, producing minimal lifespan gain.

Testable Predictions

1. NMN treatment will raise the hepatic SIRT6 activity assay (deacetylation of a known peptide substrate) and increase the plasma H2S:cystine ratio in male mice, but not in females.
2. Genetic knock‑down of Slc7a11 in female mice will mimic the male response to NMN, rescuing the H2S increase and extending lifespan.
3. Pharmacological inhibition of CBS (with AOAA) will block the NMN‑induced H2S rise and abolish the improvement in mitochondrial turnover, irrespective of sex.
4. SIRT6‑deficient mice will fail to show any NMN‑induced changes in Slc7a11 expression, H2S levels, or p62 deacetylation, confirming SIRT6 dependence.

Experimental Design

• Cohorts: Wild‑type male and female C57BL/6J mice, SIRT6‑KO littermates, and Slc7a11‑heterozygous females.
• Intervention: Oral NMN (300 mg/kg/day) for 6 months; control groups receive vehicle.
• Readouts: (a) SIRT6 activity in liver extracts (fluorometric deacetylation assay); (b) plasma H2S (methylene blue method) and cystine (HPLC); (c) western blot for acetylated p62 and Slc7a11; (d) mito‑Keima assay for mitophagy flux; (e) survival curves and frailty index.
• Statistical analysis: Two‑way ANOVA (sex × treatment) with post‑hoc Tukey; survival compared by log‑rank test.

If predictions 1‑4 hold, the hypothesis that NMN’s sex‑specific longevity effect is mediated through a SIRT6‑Slc7a11‑H2S‑p62 mitophagy axis will be supported; failure to observe the sex‑dependent H2S rise or the rescue by Slc7a11 knockdown would falsify the proposed mechanism.