Discussion
DISEASE
In this pilot clinical study, we showed that PF is capable of dramatically altering human plasma functionalities and the plasma metabolome; treatment of nonfasted macrophage with fasted plasma is capable of producing significant anti-inflammatory effects; these effects are mediated, at least in part, by specific bioactive metabolites that are upregulated after 36 h of fasting; and these metabolites are capable of extending lifespan in A key advantage of the current study was the rigorous and controlled design of the human trial. Unlike other studies of fasting, we controlled for dietary intake within individuals using a controlled habitual diet, assessed compliance to fasting through personal glucose monitoring, and collected 4 distinct nutritional states from each individual across the study timecourse. These measures allowed us the ability to sensitively assess differences not only in a prolonged fasted state versus a postprandial state, as most other fasting trials have performed, but also between a 36-h fasted state and an overnight fasted state and postprandial states both before and after 36 h of fasting. This was critical in determining the distinct effects and mediators of PF beyond what is achieved during a typical overnight fast and the carryover effects of PF into the re-fed state without confounding dietary influences.In this study, PF was capable of significantly increasing plasma antioxidant capacity as well as the cholesterol efflux ability of participant plasma over both the baseline and fed states, and these increases carried over to the re-fed state. Treatment of nonfasted macrophage with fasted plasma also induced powerful anti-inflammatory effects significantly reducing TNF-α secretion, intracellular ROS accumulation, M1 polarization responses, and total COX activity of stimulated human macrophage versus the baseline and fed states. To our knowledge, these are the first studies to show that PF is capable of beneficially modifying human plasma functionalities and treatment of human macrophage with fasted plasma can induce significant anti-inflammatory effects beyond what is achieved by an overnight fast. We are similarly the first to report that PF is capable of ameliorating Fc-γ receptor-induced macrophage activation and reducing total COX activity in macrophage, further elucidating the molecular pathways involved in the immunomodulatory effects of PF. Additionally, although other studies have shown that PF is capable of modulating macrophage polarization in mice [The importance of investigating these potential plasma-borne mediators cannot be understated as they represent a direct pathway by which the beneficial effects of PF may be replicated without the need to fast. The discovery of such mediators and their development into fasting mimetics represents great potential as therapeutic or preventative interventions for health and disease. In our investigations of these mediators, we showed that the human plasma metabolome in the fasted state versus the baseline state was remarkably altered with 389 metabolites being significantly different between the 2 states. Further analysis and screening of significantly upregulated metabolites in the fasted state revealed spermidine, 1-MNA, PEA, and OEA to have significant anti-inflammatory effects on stimulated macrophage even beyond the effects of BHB, which is often considered to be the major immunomodulatory metabolite during PF [Finally, we also showed that lifelong treatment of In conclusion, the results of this trial identified metabolites and mechanisms of action needed to develop future interventions designed to mimic the effects of PF. This study revealed new functionalities and pathways affected by PF in humans, identified 4 candidates for use as fasting mimetics, showed their effects to be most potent when used in combination, and discovered a previously unknown role of PEA and OEA in lifespan extension. Moreover, this study serves as a strong proof of concept for a human-based approach for the discovery of clinically relevant longevity and health molecules. As with PEA and OEA, by first identifying bioactive molecules in humans and then assessing their roles in longevity extension in model organisms, it may be possible to greatly increase the pace of discovery of longevity targets that could then be immediately translatable to a human health context. Although spermidine has already been investigated for its potential as a caloric restriction mimetic [The authors’ responsibilities were as follows—CR and AZ: conceived of and planned the clinical study design and all experimental analyses performed; CR: performed the clinical study, blood processing, cell isolation and culturing, and in vitro functional assays with assistance from JA; CZ and XT: performed the statistical analysis of the in vitro functional data and metabolomic data; QL and JE: designed and executed the 