Nitric oxide required for transition to slower hepatic protein synthesis rates during long-term caloric restriction
Hector H. Palacios, Edward Cao, Adelaide Cahill, Hussein Mohamad, Marc K. Hellerstein
Hector H. Palacios, Edward Cao, Adelaide Cahill, Hussein Mohamad, Marc K. Hellerstein
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Research Article Aging Hepatology Metabolism

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

Abstract

Calorie restriction (CR) extends maximal lifespan and maintains cellular homeostasis in various animal models. We have previously shown that CR induces a global reduction of 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 liquid chromatography/mass spectrometry–based (LC/MS-based) flux proteomics in WT and NO-deficient (NO–) mice. We observed a transition to reduced global protein FSRs that occurred rapidly between days 25 and 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– mice. Furthermore, 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.

Authors

Hector H. Palacios, Edward Cao, Adelaide Cahill, Hussein Mohamad, Marc K. Hellerstein

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Figure 2

Time course to CR threshold event transition to slower hepatic protein FSRs.

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Time course to CR threshold event transition to slower hepatic protein F...
Time course of reduction in proteome-wide hepatic protein FSRs during long-term CR. Data represent experiments in which C57Bl/6J male mice underwent CR for different lengths of time, with heavy water labeling for 4 days prior to each termination time point shown. Livers were collected and proteome flux rates were analyzed by LC-MS/MS. Protein FSRs (turnover rates) were calculated by MIDA, as described in Methods. The data show the FSR as log2 values of the FC between the CR and Con groups. Dots represent individual proteins identified by LC-MS/MS, sorted from highest to lowest value. Above the “0” line are the proteins with a higher FSR under CR, while the dose below the “0” line represents the proteins whose FSR was lower under CR. To establish a cutoff threshold value, the CV was averaged, and this average value was used as the cutoff — i.e., a percentage change greater than that of the CV average was considered to be above the cutoff and is marked in red. Vertical gray lines show the dividing line separating faster (left) and slower (right) FSRs. The tables reflect the graphed data and represent the number and percentage of proteins upregulated (higher FSR) or downregulated (lower FSR) in CR compared with the Con. P values show the binomial distribution significance, determined using a 2-tailed binomial distribution. These results are consistent with a modest reduction before day 25 in hepatic FSRs (50%–60% of proteins with lower FSRs than Con) that rarely passes the CV cutoff until after days 25–30. After this transition, there was a global reduction, with 75%–90% of proteins having lower FSRs that exceeded the cutoff compared with the Con. D0, day 0.