Aging is a complex biological process associated with systemic and cellular dysfunctions. Both fasting (i.e., time-restricted feeding) and dietary protein restriction (PR) are among the most promising interventions to promote healthy aging. Since the two paradigms are hardly compatible, it has remained unclear whether they may exert synergistic effects. However, recent studies have shown that the endogenous polyamine spermidine increases during fasting in both the fruit fly, Drosophila, and humans. In my dissertation, I therefore treated Drosophila with a combination of dietary spermidine supplementation (SPD) and PR to assess whether their effects are additive or synergistic. My observations confirmed that both interventions act through orthogonal mechanisms, which encourages future clinical studies in this direction. The Drosophila study included behavioral analyses (lifespan, locomotion, and fecundity) combined with polyamine quantification and proteome profiling. Both SPD and PR alone promoted healthspan and lifespan in flies, while their combination provided additional benefits — including extended lifespan, improved locomotion, and prolonged fecundity in aging flies. SPD (but not PR) increased putrescine and spermidine levels in PR-fed flies and reprogrammed the proteome toward specific metabolic pathways — including mitochondrial metabolism, autophagy, and hypusination. Hypusination is a posttranslational modification of the eukaryotic initiation factor 5A (eIF5A), in which spermidine donates an aminobutyl group to a specific lysine residue, forming hypusine (Nε-[4-amino-2-hydroxybutyl]-lysine). ____________________________________________________________________________________________________ In the second part of this work (ImmuneAge trial in human participants), we investigated the role of SPD in immune rejuvenation and conducted a comprehensive analysis of polyamine biokinetics in blood fractions (plasma, serum, and cellular components) in collaboration Charité – Universitätsmedizin Berlin, German Institute of Human Nutrition Potsdam-Rehbrücke (DIfE), Leibniz Institute for Analytical Sciences – ISAS – e.V., and The Longevity Labs GmbH (TLL). The ImmuneAge Trial used a multi-parameter molecular profiling approach, including ELISA for inflammatory markers, LC-MS for polyamine quantification, proteomics, western blot analysis for autophagy and hypusination markers, and flow cytometry to evaluate immune responses. We showed that a 20-day SPD significantly enhanced autophagy and hypusination in peripheral blood mononuclear cells (PBMCs), particularly in younger participants (20–40 years), with similar trends observed in older individuals (60–90 years). These effects correlated with SPD’s strong ability to counteract the age-related decline of spermidine levels in PBMCs. Moreover, SPD reduced inflammaging and improved aspects of both innate and adaptive immune responses to the SARS-CoV-2 spike peptide, with stronger effects in younger participants. Plasma proteomics revealed that SPD strengthened the immune system, lowered thrombosis risk, and reduced inflammation in older participants, while in younger participants, it improved lipid metabolism and triggered changes considered cardioprotective.
