Hypothesis

Combining timed low-dose melatonin supplementation with morning blue-light exposure and early-time-restricted feeding (eTRF) will amplify the melatonin-NAD+-SIRT1 axis, thereby restoring circadian amplitude in middle-aged adults and improving healthspan biomarkers.

Mechanistic Rationale

Melatonin binds MT1/MT2 receptors, inhibiting adenylyl cyclase and lowering cAMP, which reduces protein kinase A (PKA) activity. Lower PKA diminishes phosphorylation of the NAD+-consuming enzyme CD38, leading to decreased NAD+ hydrolysis. Simultaneously, melatonin upregulates nicotinamide phosphoribosyltransferase (NAMPT), boosting the NAD+ salvage pathway. Elevated NAD+ activates SIRT1, which deacetylates core clock components BMAL1 and PER2, enhancing their transcriptional activity and reinforcing circadian oscillations. Morning blue-light advances the phase, increasing daytime alertness and boosting NAD+ production via circadian-driven NAMPT expression. Early TRF aligns nutrient intake with the active phase, further elevating NAD+ during SIRT1’s peak activity. Individuals carrying loss-of-function variants in MT1 (rs10830963) or MT2 (rs1387153) show blunted melatonin-mediated CD38 inhibition, predicting a genotype-dependent response.

Testable Predictions

1. Participants receiving the combined intervention will show a >=20% increase in nocturnal melatonin amplitude compared with baseline, exceeding the increase seen with either melatonin alone or blue-light alone.
2. NAD+/NADH ratio in peripheral blood mononuclear cells will rise by >=15% after 12 weeks, correlating with SIRT1 activity (measured by deacetylation of p53).
3. Circadian phase assessed by dim-light melatonin onset (DLMO) will advance by >=30 minutes relative to control, and interdaily stability will improve.
4. Cognitive performance (executive function) and metabolic markers (fasting insulin, HOMA-IR) will improve significantly only in the combined group.
5. The magnitude of effects will be stratified by MT1/MT2 genotype, with carriers of the protective alleles exhibiting the greatest benefit.

Experimental Design

A 12-month, parallel-group, double-blind RCT enrolling 240 men and women aged 45-60 with suboptimal melatonin amplitude (<10 pg/mL). Four arms: (1) placebo + sham light + ad libitum feeding; (2) melatonin (0.3 mg) 2 h before bedtime + placebo light + ad libitum; (3) morning blue-light (460-480 nm, 30 min) upon waking + placebo melatonin + ad libitum; (4) combined melatonin + morning blue-light + early TRF (10-hour eating window ending 3 h before bedtime). All participants receive blue-blocking glasses after sunset to standardize evening light exposure. Primary outcomes: melatonin amplitude, NAD+/NADH ratio, SIRT1 activity, DLMO, cognitive composite score. Secondary: inflammatory cytokines (IL-6, TNF-alpha), metabolic health, adverse events. Genotyping for MT1/MT2 performed at baseline.

Potential Outcomes

If the combined arm meets or exceeds the predictions, the hypothesis is supported, indicating that synergistic engagement of the melatonin-NAD+-SIRT1 axis can rescue circadian decline and delay age-related dysfunction. Failure to observe additive benefits, or absence of genotype-stratified effects, would falsify the hypothesis and suggest that alternative pathways dominate circadian-aging crosstalk.