The beneficial effects of fasting have been recognized and appreciated for a long time across cultures. Some of the health benefits of prolonged fasting (defined as lasting >24 hours and practiced over a variety of intervals, from alternate day, to once weekly, to quarterly) have been described in the literature. Prolonged fasting has been shown to beneficially modulate the immune system, and counteract the process of “inflammaging,” the process by which immune system function is diminished with age, accompanied by increased inflammation. Perhaps most striking is the finding that fasting extends the life of every model organism that has ever been studied, from fruit flies to mice. There is something fundamentally beneficial and innate about the program that gets turned on in all living organisms during an extended fasting period beyond just the typical overnight fast. In the Zivkovic Lab, we wanted to know: what are the acute effects of a single 36h water-only fast on the plasma metabolome, providing a full picture of the metabolic changes that occur in response to fasting, and on the function of innate immune cells, specifically macrophages.

In this study our team recruited 20 healthy, young individuals (10 males, 10 females), who participated in a 3-day intervention including 4 time points:  Day 1, after a standard overnight fast (8AM, Baseline), Day 1, 2 hours after their evening meal (8PM, Fed), Day 3, after a 36h water-only fast (8AM, Fasted), and Day 3, again 2 hours after the evening meal (8PM, Refed). With this study design we were able to compare the effects of the 36h fast to a standard overnight fast, as well as to the postprandial state, and we were also able to assess whether the 36h fasting period had effects that carried over into the next feeding period.

We were not surprised to find that the effects of fasting were universal and profound: the absence of nutrition for this length of time triggered an expected metabolic shift toward the utilization of stored fat and away from carbohydrate metabolism, which was clearly illustrated by the increase in free fatty acids in blood. Also expected was the increase in ketone bodies, which also confirmed that our study participants all adhered to the 36h fast and did not “cheat” by eating foods during the entire 36h period. What was somewhat surprising was the very strong anti-inflammatory effect that 36h fasted plasma had on macrophages, which are part of our innate immune protection arsenal. Compared to not just the postprandial state, which is known to be pro-inflammatory, but also to the overnight fasted state, 36h of fasting profoundly decreased the synthesis of reactive oxygen species, the production of pro-inflammatory signaling molecules, and shifted the phenotype of macrophages from a pro-inflammatory state to what is thought of as a pro-resolving state. We next wanted to find out which specific molecules in plasma contributed to these immunomodulatory effects in macrophages. We performed metabolomic analysis of the plasma and found that >300 metabolites were significantly changed by 36h of fasting, including as mentioned previously, changes in expected molecules such as fatty acids, but also molecules that had not been previously been reported to be altered in this state. Specifically, we were interested in the effects of 4 metabolites that were significantly increased by fasting: spermidine, 1-methylnicotinamide (NMA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA).

We were surprised to find that not only did each of these 4 metabolites individually have a stronger anti-inflammatory effect in our cell model than the well-known ketone body beta-hydroxy butyrate, but the combination of all 4 metabolites had a synergistic effect that reduced the production of pro-inflammatory cytokines even more, such that the effect of the combination was indistinguishable from the negative control in the assay.

We thought this might be too good to be true so we reached out to Dr. JoAnne Engebrecht, a professor of Molecular and Cellular Biology whose research program using C. elegans as a model organism  focuses on fundamental aspects of cellular biology and genetics. When we first reached out to her with the idea that we wanted to study the effects of our metabolites on longevity she was unimpressed and frankly, skeptical that it could work. But she was kind enough to give it a shot. A few weeks later we received the results and saw that each of the individual metabolites prolonged the life of C. elegans, but the really striking finding was the synergistic effect of the combination of the 4 metabolites, which made the worms live nearly twice as long as the unsupplemented worms.

The findings from our study highlight that this study design may be a very powerful discovery tool for finding molecules naturally induced during fasting that may be beneficial to human health. Our study also paves the way for a more thorough mechanistic understanding of all the metabolic programs that are tuned on and off in the prolonged fasted state. Further work is now under way to better understand the effects of fasting on a number of additional endpoints, including the structure and function of HDL, and the plasma lipidome. Findings from this study were also translated to a supplement,  which is now available through a biotech startup from this lab, headed by lead author and former graduate student Chris Rhodes. Check out Chris’s company and supplement at Mimio.