**4. The sleep, circadian rhythm, and the intestine**

The sleep-wake cycle is a good example of the circadian rhythms found in living organisms that are regulated by many biological and environmental factors.

#### *Circadian Clock, Sleep, and Diet DOI: http://dx.doi.org/10.5772/intechopen.100421*

In humans, sleep regulation is governed by homeostatic mechanisms and circadian rhythms, that is, a two-process model [32, 33]. In this model, sleep is regulated by sleep homeostasis (process S) and circadian rhythm (process C; circadian rhythm) (**Figure 2**). Sleep controlled through homeostasis means that sleep debt increases during wakefulness and decreases with sleep; thus, when the sleep debt reaches the sleep threshold, humans fall asleep and when it reaches the lower limit, humans awaken. It is believed that this threshold is dominated by the circadian rhythm, and diurnal variation is observed (process C). This idea is that daytime awakening and nighttime sleep are determined by the sleep debt that accumulates by continuing to stay awake and drowsiness that is induced by the biological clock. It is understood to be a system that compensates for sleep time in response to changes in the environment while physiologically promoting sleep *via* circadian rhythm.

When mammals sleep, rapid eye movement (REM) sleep and non-rapid eye movement sleep (non-REM sleep) occur in a cycle of about 90 min. When you fall asleep, non-REM sleep appears first. Subsequently, light REM sleep appears. REM sleep is accompanied by rapid eye movements, and the body is in a resting state with relaxed skeletal muscles, but the brain is active and awake. The cerebral cortex is more active than during wakefulness, and electroencephalography (EEG) shows mainly theta waves from 4 to 7 Hz and exhibits an amplitude close to that during awakening. Sleep without REM is called non-REM sleep, and the brain is in the so-called state of deep sleep. Low-frequency, high-amplitude brain waves called delta waves ranging from 1-4 Hz are observed in brain waves, and non-REM sleep is characterized as slow-wave sleep based on EEG findings. However, the molecular mechanism(s) underlying the cyclical changes that occur in non-REM sleep and REM sleep are not yet clear.

Sleep disturbances deteriorate the circadian rhythms across various organs. For example, when mice were subjected to sleep disturbances in which the light and dark phases were changed weekly, the circadian rhythm of *Per2* expression in the large intestine disappeared, and the intestinal microbiota was altered, with increased Firmicutes and decreased Bacteroidetes at the phylum level [34]. Other sleep disorders have been reported to increase intestinal permeability and blood LPS levels [35]. In addition, the effects of interventions that cause sleep disorders using tactile stimulation without changing the cycle of light and dark phases have also been investigated, in which the cycle of light stimulation is

#### **Figure 2.**

*A two-process model of sleep. Sleep debt accumulates during awakening, and sleep is encouraged when the sleep threshold is reached. Sleep reduces sleep debt and awakens when the wake threshold is reached. Sleep debt increases during awakening and decreases during sleep (process S). The sleep-wake threshold fluctuates diurnal (process C).*

periodic. An increase in Lachnospiraceae and Ruminococcaceae and a decrease in Lactobacillaceae and Bifidobacteriaceae were observed after a 4-week sleep disturbance [36]. Furthermore, the intestinal contents, such as propionate and citrate, changed after the intervention, indicating functional changes in the intestinal microbiota. When intestinal bacteria from sleep-disturbed mice were transplanted into germ-free mice, the blood levels of inflammatory cytokines, such as IL-6, increased and insulin resistance was exacerbated in the recipient mice. The blood concentration of lipopolysaccharide-binding protein (LBP), which transports LPS to the CD14-TLR4-MD2 complex on the cell membrane of macrophages, was also increased in sleep-disordered mice. The effects of sleep disturbance without alteration of meals and motions in life were also examined in humans. A randomized crossover study was conducted in which nine healthy subjects slept for about 4 h for two days and about 8 h for two days with equal daily activities other than sleep. A short sleep for two days increased Firmicutes, and decreased Bacteroidetes in the intestine as well as worsened insulin resistance and glucose tolerance [37]. These findings demonstrate that sleep disturbances deteriorate the central and peripheral circadian rhythms, leading to metabolic disorders.

## **5. The stimuli from the intestine, circadian rhythm, and sleep**

From the abovementioned examination of sleep disturbances in animals and humans, it was shown that sleep disturbances distort circadian rhythms in the central nervous system and peripheral organs. As mentioned above, the circadian rhythm in the intestine is regulated by the central and peripheral clocks as well as nutritional stimuli, that is, dietary intake. Therefore, the possibility of recovery of the host's circadian rhythm and the control of sleep *via* the intestine is being investigated. In fact, even in Per1/2-deficient mice, diurnal oscillation in the intestinal microbiota was observed by feeding them in a timely fashion.

Recently, Leone et al. compared the expression of clock genes Bmal1 and Clock in the medial basal hypothalamus and liver of germ-free mice with that of control mice and found that circadian rhythms diminished in the germ-free mice [19]. The mechanism by which intestinal bacteria regulate the circadian rhythm of the liver and hypothalamus has also been investigated, and butyric acid, a metabolite of intestinal bacteria, was found to be a key molecule in tuning the circadian rhythm in the CNS and peripheral organs. In fact, when butyric acid was administered to hepatic organoids *in vitro*, an increase in the circadian rhythm of Per2 and Bmal1 expression was observed. Moreover, when butyric acid was injected into germ-free mice every 12 h, the circadian rhythm of the clock gene in the liver reappeared, and the amplitude of the clock gene tended to be enhanced in the medial basal hypothalamus. Consistent with this report, the circadian rhythm of Bmal1 and Cry1 in the intestinal epithelium disappears in mice in which intestinal bacteria are reduced by the administration of a set of antibiotics [38]. Bile acids deconjugated by intestinal bacteria are an important signal for tuning the clock genes in the intestine by dietary stimuli [12, 39]. Therefore, it is considered that changing the circadian rhythm of the intestine by nutritional stimuli could change the circadian rhythm in other organs, including the CNS, and may also affect sleep.

In terms of the stimuli from the intestine to modulate sleep, various modulations have been examined. The muramyl peptide derived from the cell wall of bacteria, LPS, and inflammatory cytokines such as IL-1b, TNF-a, and IL-18 have been reported to promote sleep [40, 41]. These microbial products prolonged and increased non-REM sleep and reduced REM sleep in model animals. In humans without infectious diseases, the levels of serum IL-1b and TNF-a showed

#### *Circadian Clock, Sleep, and Diet DOI: http://dx.doi.org/10.5772/intechopen.100421*

a circadian rhythm, which peaked at night and troughed at dawn, implying that these molecules may be a trigger for falling asleep [42]. Studies on sleep have also been conducted using antibiotic agents to modulate stimuli from the intestine. For example, one study administered a single dose of 200 mg of minocycline or 500 mg of ampicillin to 19 healthy men and found that administration of minocycline significantly reduced the proportion of non-REM sleep, an effect that lasted for two days. No effect was observed on REM sleep, and ampicillin did not affect either non-REM sleep or REM sleep [43]. These findings imply that changes in the gut microbiota may lead to improved sleep quantity and quality. Considering that long-term administration of antibiotics is not realistic in clinics, prebiotics and probiotics have been intensively investigated. It was reported that administration of *Lactobacillus brevis* to mice increased physical activity, prolonged waking hours, and reduced non-REM sleep [44]. Similarly, the administration of prebiotics containing lactoferrin to rats prevented the expected decrease in non-REM sleep due to electric shock [45]. In humans, many clinical trials have been conducted to evaluate the effects of prebiotics and probiotics on sleep. For example, the daily administration of *Lactobacillus gasseri* CP2305 for five weeks was reported to improve sleep quality in healthy volunteers, with a reduction in the amount of intestinal Enterobacteriaceae [46, 47]. Additionally, administration of a probiotic mixture of *Lactobacillus fermentum*, *L. rhamnosus*, *L. plantarum*, and *Bifidobacterium longum* for six weeks was reported to improve sleep quality [48]. Nevertheless, more studies are needed to conclude that modulation of nutritional stimuli from the intestine changes circadian rhythm and sleep quality.
