Nitric oxide required for transition to slower hepatic protein synthesis rates during long-term caloric restriction

Caloric restriction (CR) extends maximal lifespan and maintains cellular homeostasis in various animal models. We have previously shown that CR induces a global reduction in protein fractional synthesis rates (FSRs) across the hepatic proteome in mice, but the timing and regulatory mechanisms remain unclear. Nitric oxide (NO), a bioactive molecule upregulated during CR, is a potential regulator of protein synthesis. To explore the role of NO in hepatic proteome fluxes during CR, we used in vivo deuterium labeling from heavy water and LC/MS-based flux proteomics in wild-type and NO-deficient (NO-) mice. We found a transition to reduced global protein FSRs that occurred rapidly between days 25 to 30 of CR. NO deficiency, whether genetic or pharmacological, disrupted the slowing of proteome-wide fluxes and the beneficial effects on body composition and physiology. Administering the NO donor molsidomine restored the reduction in hepatic FSRs in NO-deficient mice. Further, inhibiting NO pharmacologically, whether starting on day 1, day 14, or day 24 of CR, mitigated the reduction in hepatic protein FSRs at day 32, highlighting NO's critical role during the transition period. These results underscore the importance of NO in CR-induced changes in proteostasis and suggest NO as a potential CR-mimetic target, while offering a specific time window for identifying other signals and testing therapeutic interventions.