Hypothesis: In aged cells, the combined action of mTORC1 hyperactivity, Rubicon accumulation, and p300‑mediated VPS34 acetylation raises the autophagic activation threshold, making short fasts insufficient to initiate flux. Restoring NAD+ levels activates SIRT1, which deacetylates TFEB and VPS34, thereby lowering mTORC1‑dependent inhibition and counteracting p300‑VPS34 interference. This dual action predicts that NAD+ boosting will shorten the fasting period required to achieve autophagic flux comparable to that seen in young animals under 16:8 regimens.

Testable Predictions

1. Biochemical – In liver from 24‑month‑old mice, a 16‑hour fast combined with nicotinamide riboside (NR) will increase nuclear TFEB, decrease VPS34 acetylation (measured by immunoprecipitation‑Western), and elevate LC3‑II turnover (with bafilomycin A1) to levels matching those of young mice after a 16‑hour fast alone.
2. Functional – The same NR‑plus‑16h fast condition will reduce p62/SQSTM1 accumulation and increase lysosomal glucosylceramide hydrolase activity, indicating restored autophagosome‑lysosome coupling.
3. Dose‑response – NR alone will not significantly alter flux; low‑dose rapamycin (mTORC1 inhibitor) plus NR will produce additive effects, whereas rapamycin alone will require a longer fast (≥20 h) to reach comparable flux.
4. Genetic validation – Liver‑specific SIRT1 knockout mice will fail to show the NR‑induced reduction in fasting threshold, confirming SIRT1 dependence.

Experimental Design

• Animals: Young (3 mo) and aged (24 mo) C57BL/6 mice; SIRT1 liver‑KO cohort.
• Treatments (n=6 per group): vehicle, NR (400 mg/kg/day, oral), low‑dose rapamycin (0.5 mg/kg i.p., three times/week), NR + rapamycin, each paired with either ad libitum feeding, 16‑hour fast, or 24‑hour fast.
• Readouts (collected at ZT6 after treatment period):
  • Western blot of LC3‑I/II and p62 with/without bafilomycin A1.
  • Immunofluorescence for TFEB subcellular localization.
  • VPS34 immunoprecipitation followed by acetylation‑specific antibody.
  • Lysosomal glucosylceramide measured by LC‑MS/MS.
  • mTORC1 activity via p‑S6K immunoblot.

Mechanistic Rationale

NAD+ fuels SIRT1 deacetylase activity. SIRT1 directly deacetylates TFEB, promoting its nuclear translocation and lysosomal gene expression independent of mTORC1 phosphorylation status. Simultaneously, SIRT1 deacetylates VPS34 at lysine residues targeted by p300, restoring its ability to bind ULK1 and generate phosphatidylinositol‑3‑phosphate for phagophore nucleation. By removing these two acetyl‑based blocks, NAD+ lowers the effective concentration of AMPK‑activating signals needed to overcome mTORC1‑Rubicon‑p300 suppression. Consequently, a fasting interval that merely elevates AMP/ATP ratio in young tissue becomes sufficient in aged tissue when NAD+ is replenished.

Falsifiability

If NR supplementation does not decrease the required fasting duration for autophagic flux in aged mice, or if SIRT1 ablation does not abolish this effect, the hypothesis is refuted. Conversely, observing that NR alone can drive flux without any fasting would also challenge the premise that NAD+ acts primarily to lower the fasting threshold rather than act as a direct autophagy inducer.