Sleep Loss Results in Accumulation of Reactive Oxygen Species in the Gut

Sleep is essential for survival, seen from its ubiquity among all known animals. Clinical evidences have long linked insufficient sleep with defected immunity, defected cognition, increased risks of chronic diseases and weight gain. A research team from Harvard University medical school investigated the association of drosophila food intake and sleep, and found that sleep-deprived flies tended to eat more [1]. Severe sleep deprivation even led to premature death [2,3]. Sleep is regulated by neurons so that the brain is assumed the organ that need sleep most, and that the premature death results from impaired brain function. Sleep is therefore believed helpful to restore brain function. Two leading experimental evidences are that sleep downscales synapses formed during wakefulness and that sleep helps clear harmful substances from interstitial brain areas [4,5]. Because it is not feasible to induce these processes during wakefulness, their contribution to the lethality associated with sleep loss is unknown.

Another proposal is that sleep helps prevent oxidative stress in the brain. Experimental evidences showing antioxidative responses in the brain when drosophila is deprived of sleep supported this proposal. The brain activity has been affected though, there is no significant structural damage [6,7]. So instead, scientists have been searching for oxidative stress in other organs. The Harvard team, using drosophila as model animal, investigated how sleep loss results in premature death [1]. Drosophila requires sleep for a normal and quick lifespan, and shares core attributes of sleep with mammals [8]. They employed three independent methods of sleep deprivation: thermogenetic stimulation, mechanical agitation and sleep regulator inactivation. Results show that regardless of methods, sleep loss results in accumulation of reactive oxygen species (ROS) in the gut. ROS is a group of highly unstable, reactive and oxidative substances that may damage various cellular structures.

Thermogenetic stimulation: engineered drosophila neurons may express a heat-activated cation channel TrpA1, whose conformation is sensitive to temperature. At 21℃ it is closed and does not stimulate the neuron. When temperature rises to 29℃, its conformational change happens and opens channel. Neurons get persistently stimulated and as a consequence, drosophila loses 90% of sleep. Sleep deprived drosophila showed premature death since day 6. The mortality increased around day 10 and reached 100% on day 20. Under same conditions, the lifespan of drosophila that had normal sleep was around 40 days. If the thermogenetic sleep deprivation was stopped on day 10, drosophila restored normal sleep-wake cycles and normal or near normal lifespans. These results indicated at least some major negative consequences of sleep deprivation are reversible.


Figure 1. Thermogenetic stimulation via TrpA1 causes sleep deprivation and ROS accumulation in the gut of drosophila. ROS can be cleared when sleep restores.[Figure source: Vaccaro et al.]

The team then measured multiple biomarkers of cell damage to identify the cause of premature death. Most tissues appeared indistinguishable between sleep-deprived and non-deprived flies, except for one major difference: the guts of deprived flies had increased levels of ROS. What’s more, the ROS levels were highly related to the observed mortality, peaking on day 10.

When sleep deprivation stopped, ROS levels decreased gradually and eventually down to the baseline. To corroborate this conclusion, the team employed another two methods to deprive sleep.

Mechanical agitation: flies were kept in a consistently shaking container to disrupt their sleep. Similarly, accumulation of ROS in the gut was observed after a few days’ sleep deprivation. This method however introduced two undesired variables. The identical strength might impact flies differently: most flies underwent significant sleep loss on the first day and then some got used to it and therefore slept more. Besides, wings and legs were often damaged by the shaking, made it difficult to correlate the observed shortening of lifespan with sleep loss.

Loss-of-function mutation of sleep regulators: the team used RNA interference (RNAi) to induce degradation of mRNAs that encode the sleep regulators, e.g. CycA, Inc, Rye, and SSS. These regulators are essential for sleep signal transduction. As a consequence, accumulation of ROS in the gut was once again observed. All three methods of sleep deprivation arrived at the same conclusion: accumulated ROS in the gut was the direct cause of premature death.

They also found cellular evidence of oxidative stress upon sleep deprivation, which was the widespread DNA damage, increased stress granules and lysosomes throughout the gut. These were the markers of massive apoptosis and necrosis. To be more convincing, the team repeated the sleep-deprivation experiment on mouse, a mammalian animal. Unsurprisingly, sleep-deprived mice showed accumulated ROS in the gut, too. Now it is certain that sleep loss causes premature death through accumulation of ROS in the gut. Luckily, these accumulated ROS can be neutralized by reductants. They tried many reductants and found three effective ones: melatonin, lipoic acid, and NADH. These compounds were able to rescue survival by clearing ROS, without increasing sleep.


Figure 2. Reductants of melatonin, lipoic acid and NAD can clear accumulated ROS in the gut without sleep restoration. [Figure source: Vaccaro et al.] 

“It is unclear why sleep loss would result in ROS accumulation, why in the gut and why so lethal”, according to Prof. Dragana Rogulja, a member of this team. Human trial of sleep deprivation will cause severe health issues to human body and is against the medical ethics, so animal simulation is the only possible way we study it. It is noticeable that sleep loss does not necessarily indicates staying up late.


[1] Vaccaro, A., Dor, Y. K., Nambara, K., Pollina, E. A., Lin, C., Greenberg, M. E., & Rogulja, D. (2020). Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut. Cell, 181(6). doi:10.1016/j.cell.2020.04.049

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