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Butyrate, a metabolite of intestinal bacteria, enhances sleep


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Emerging evidence suggests that the intestinal microbiota is a source of sleep-promoting signals. Bacterial metabolites and components of the bacterial cell wall are likely to provide important links between the intestinal commensal flora and sleep-generating mechanisms in the brain. Butyrate is a short-chain fatty acid produced by the intestinal bacteria by the fermentation of nondigestible polysaccharides. We tested the hypothesis that butyrate may serve as a bacterial-derived sleep-promoting signal. Oral gavage administration of tributyrin, a butyrate pro-drug, elicited an almost 50% increase in non-rapid-eye movement sleep (NREMS) in mice for 4 hours after the treatment. Similarly, intraportal injection of butyrate led to prompt and robust increases in NREMS in rats. In the first 6 hours after the butyrate injection, NREMS increased by 70%. Both the oral and intraportal administration of butyrate led to a significant drop in body temperature. Systemic subcutaneous or intraperitoneal injection of butyrate did not have any significant effect on sleep or body temperature. The results suggest that the sleep-inducing effects of butyrate are mediated by a sensory mechanism located in the liver and/or in the portal vein wall. Hepatoportal butyrate-sensitive mechanisms may play a role in sleep modulation by the intestinal microbiota.
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SCIENTIFIC REPORTS | (2019) 9:7035 |
Butyrate, a metabolite of intestinal
bacteria, enhances sleep
Éva Szentirmai
1,2, Nicklaus S. Millican1, Ashley R. Massie1 & Levente Kapás1,2
Emerging evidence suggests that the intestinal microbiota is a source of sleep-promoting signals.
Bacterial metabolites and components of the bacterial cell wall are likely to provide important links
between the intestinal commensal ora and sleep-generating mechanisms in the brain. Butyrate
is a short-chain fatty acid produced by the intestinal bacteria by the fermentation of nondigestible
polysaccharides. We tested the hypothesis that butyrate may serve as a bacterial-derived sleep-
promoting signal. Oral gavage administration of tributyrin, a butyrate pro-drug, elicited an almost 50%
increase in non-rapid-eye movement sleep (NREMS) in mice for 4 hours after the treatment. Similarly,
intraportal injection of butyrate led to prompt and robust increases in NREMS in rats. In the rst 6 hours
after the butyrate injection, NREMS increased by 70%. Both the oral and intraportal administration
of butyrate led to a signicant drop in body temperature. Systemic subcutaneous or intraperitoneal
injection of butyrate did not have any signicant eect on sleep or body temperature. The results
suggest that the sleep-inducing eects of butyrate are mediated by a sensory mechanism located in the
liver and/or in the portal vein wall. Hepatoportal butyrate-sensitive mechanisms may play a role in sleep
modulation by the intestinal microbiota.
Sleep is greatly aected by peripheral metabolic signals, such as satiety and orexigenic hormones13, increased
lipolysis4,5, systemic pro-inammatory signals6, activation of brown adipose tissue7 or the liver8. Recent evidence
points to the importance of the intestinal microbiota in metabolic signaling (reviewed in9). Microbiota-derived
signals modulate complex brain-related functions and various behaviors (reviewed in10).
The brain sleep mechanisms and the gut flora are linked through a dynamic bidirectional relationship.
Depletion of intestinal microbiota induces signicant reduction in sleep suggesting that the gut ora is a source of
sleep-inducing signals11,12, while circadian disruption and chronic sleep fragmentation promote intestinal dysbi-
osis13,14. Cell wall components of bacteria induce sleep when injected systemically1518 suggesting that fragments
of disintegrating intestinal bacteria, once translocated into the portal circulation, could serve in sleep signaling.
In addition to the cell wall fragments, live intestinal bacteria are also a source of biologically active metabolites,
such as short-chain fatty acids (SCFAs), secondary bile acids, indole-derivatives, succinate or hormones and neu-
rotransmitters (reviewed in19). e role of metabolites produced by live bacteria in sleep regulation is, however,
poorly understood.
Butyrate, a SCFA, is a product of anaerobic bacterial fermentation of non-digestible carbohydrates in the
hindgut, a major metabolic product of the clostridial clusters of intestinal ora20. Mammalian cells do not pro-
duce signicant amounts ofbutyrate, the only signicant sources are the microbiota and ingestion of dairy prod-
ucts21,22. Butyrate binds to the FFAR2, FFAR3 and GPR109A receptors2327, and it also acts as a histone deacetylase
inhibitor and aects gene transcription28. It is readily absorbed into the portal circulation and transported directly
to the liver29. e liver represents a major sink for intestinally-produced butyrate as evidenced by the steep con-
centration gradient between portal and systemic levels of butyrate2931. All three butyrate receptors are expressed
in the liver23,32,33.
We hypothesized that intestinally-producedbutyrate acts on hepatoportal sensory mechanisms to promote
sleep. To test this, we investigated the eects of oral administration, direct intraportal injection, as well as systemic
injection of butyrate and tributyrin, a butyrate-yielding pro-drug, in mice and rats. Our results demonstrate
that oral andintraportal administration of butyrate induces robust increases in non-rapid-eye movement sleep
(NREMS), while systemic butyrate treatment has no eect on sleep. ese ndings indicate the existence of a
butyrate-sensitive hepatoportal sleep-inducing sensory mechanism.
1Elson S. Floyd College of Medicine, Department of Biomedical Sciences, Washington State University, Spokane,
Washington, United States of America. 2Sleep and Performance Research Center, Washington State University,
Spokane, Washington, United States of America. Correspondence and requests for materials should be addressed to
É.S. (email:
Received: 12 February 2019
Accepted: 25 April 2019
Published: xx xx xxxx
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Oral gavage administration of tributyrin. Oral gavage administration of tributyrin at the beginning of
the dark phase elicited robust sleep responses in mice (Fig.1, Table1). In the rst four hours aer the treatment,
time spent in NREMS increased by 47% above baseline at the expense of rapid-eye movement sleep (REMS)
and wakefulness (NREMS baseline: 97.9 ± 3.3 min/4 h, tributyrin: 143.7 ± 10.0 min/4 h, p < 0.01; REMS base-
line: 6.8 ± 1.0 min/4 h, t ributyrin: 0.8 ± 0.3 min/4 h, p < 0.001). e increases in NREMS time were due to sig-
nicantly longer NREMS episodes, while REMS decreases were the consequence of a signicant decrease in
the number of REMS episodes [average episode numbers in the rst 6 h on the baseline day: NREMS 34 ± 1.7,
REMS 9 ± 1.1; aer tributyrin treatment: NREMS 32 ± 3.2, REMS 2 ± 0.7 (vs. baseline p < 0.001); average episode
durations in the rst 6 h on the baseline day: NREMS 273 ± 14.2 s, REMS 89 ± 5.7 s; aer tributyrin treatment:
NREMS 407 ± 40.9 min (vs. baseline p < 0.001), REMS 66 ± 15.5 s]. Sleep latency was not aected (baseline:
18.3 ± 4.0 min, tributyrin: 21.3 ± 3.0 min).
e NREMS increase was accompanied by a 0.8–1.2 °C drop in body temperature and greatly suppressed elec-
troencephalographic slow-wave activity (EEG SWA) and motor activity. Subsequently, there was a short rebound
increase in REMS during the second half of the dark, and a decrease in NREMS in the rst half of the light period.
Body temperature and activity were slightly, but signicantly, elevated in the light period.
Intraportal administration of sodium butyrate. Intraportal injection of butyrate induced prompt and
robust NREMS increases in rats (Fig.2, Table2). Sleep latency decreased from the baseline value of 36.1 ± 6.7 to
2.7 ± 2.0 min aer butyrate treatment (p < 0.01). Overall, NREMS increased by 70% in the rst 6 hours aer the
butyrate injection (baseline: 84.6 ± 6.7 min/6 h, butyrate: 144.0 ± 4.2 min/6 h). e animals showed the behavioral
signs of normal sleep, they were easily arousable, and actively engaged with their environment in response to mild
tactile or auditory stimuli. e NREMS response was due to the increased duration of NREMS episodes, while the
number of sleep episodes was not aected (average NREMS episode duration in the rst 6 h on the baseline day:
186 ± 16.2 s, aer butyrate treatment: 301 ± 17.4 s, p < 0.001; number of NREMS episodes in the rst 6 h on the
baseline day: 28 ± 2.7, aer butyrate treatment: 29 ± 1.9).
Figure 1. e eects of oral administration of tributyrin on wakefulness, non-rapid-eye movement sleep
(NREMS), rapid-eye movement sleep (REMS), electroencephalographic (EEG) slow-wave activity (SWA) motor
activity and body temperature. Data are presented in 2-h time blocks; shaded area represents the dark period.
Time “0”: time of the treatments. Asterisks: signicant dierence from baseline, Tukey’s HSD test; error bar: SE.
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REMS also increased aer butyrate treatment as indicated by signicant treatment and treatment x time inter-
actions in ANOVA; post hoc analysis revealed signicant REMS increase in the 5–6 h time block. REMS increase
was due to the combined eects of slightly elevated episode numbers and episode durations; neither change, by
itself, was signicant (average REMS episode duration in the rst 6 h on the baseline day: 79 ± 9.7 s, aer butyrate
treatment: 95 ± 8.0 s; number of REMS episodes in the rst 6 h on the baseline day: 6 ± 1.3, aer butyrate treat-
ment: 10 ± 1.4).
Motor activity decreased by 39% and body temperature by 0.4–1 °C in the rst 6 h. e eects on EEG SWA
were biphasic. In the rst 6 h aer the butyrate injection, there was a tendency towards increased SWA, which was
followed by a slight, but signicant decline below baseline.
Systemic administration of sodium butyrate. Systemic injection of butyrate did not have any signi-
cant eects on sleep, EEG SWA, body temperature and motor activity. In rats, subcutaneous injection of 1 g/kg,
the same dose that promotes sleep aer intraportal administration, did not aect any of the measured parameters
(Fig.2, Table3). Similarly, in mice, intraperitoneal injection of 0.02, 0.1 and 0.5 g/kg butyrate was void of any
signicant eects (Fig.3, Table4).
Evidence suggests that the gut bacteria are a source of sleep-inducing signals11,12, and we hypothesized that SCFAs
may serve as such signal. Our major nding is that orally or intraportally administered tributyrin and butyrate,
respectively, robustly increases NREMS in rats and mice. ese observations are consistent with prior reports
that intravenous injection of butyrate induces slow, high-amplitude EEG waves and behavioral signs of sleep in
rabbits34 or EEG-dened NREMS in cats35.
Butyrate is a four-carbon SCFA, produced by the microbiota in mice and rats. It is the product of the anaer-
obic fermentation of non-digestible carbohydrates by gut bacteria and also a component of dairy products, such
as butter, milk and cheese36. Tributyrin is an ester, composed of three butyric acid molecules and glycerol. It is
considered a pro-drug to deliver biologically active butyrate as lipases in the host organism hydrolyze it resulting
in the release of butyrate37. SCFAs, including butyrate, are readily absorbed from the intestines into the portal
circulation and directly reach the liver29. Plasma levels of butyric acid in the portal circulation aer oral admin-
istration of tributyrin are higher and more prolonged without detectable toxicity in mice and rats as compared
to administration of butyrate itself38. To mimic the eects of intestinally produced butyrate, we administered
tributyrin orally to mice. is treatment elicited an almost 50% increase in NREMS in the rst 4 h supporting the
notion that butyrate from the intestinal tract may potentially serve as a sleep-inducing signal molecule. NREMS
increased at the expense of both REMS and wakefulness. REMS suppression may be due to the mutual inhibitory
interaction between NREMS- and REMS-promoting mechanisms39 or it could be a NREMS-independent eect
of tributyrin. e treatments were administered at dark onset. Since the latency to increased sleep is very short
and the duration of the sleep increases did not exceed 8 hours, the eects of butyrate on sleep were manifested
predominantly during the dark, active, phase.
Sleep-inducing doses of butyrate also elicited a0.4–1.2 °C drop in body temperature. Since naturally occur-
ring NREMS is associated by decreased energy expenditure and body temperature (reviewed in40), it is possible
that the slight hypothermic response is simply the thermic manifestation of enhanced NREMS aer butyrate
treatment. It has been proposed that a drop in core body temperature prompts sleepiness41, thus an alternative
interpretation is also possible, i.e., an initial drop in body temperature in response to butyrate may invoke the
sleep responses.
Baseline sleep recordings were performed on the day before the butyrate treatment. In thoroughly-habituated
rats and mice, such as our experimental animals, sleep and body temperature are remarkably stable across two
successive days. is is evidenced, for example, by the experiments where mice received ip injection of saline on
day 1 and butyrate on the following day (Fig.3). us, it is highly unlikely that the observed sleep-promoting
eects of intraportally or orally administer butyrate are confounded by order of the treatments.
ere is a steep concentration gradient between the high portal levels of butyrate and very low butyrate
concentration in the systemic circulation29,30,42,43, and orally administered tributyrin increases portal, but not
systemic, levels of butyrate44. ese observations indicate that the liver removes almost all butyrate from the
portal blood31. us, the most likely target for butyrate to induce sleep is the hepatoportal system. To investigate
this possibility, we injected butyrate directly into the portal vein in rats. Intra-portal butyrate treatment greatly
reduced NREMS latency and increased the time spent in NREMS. To investigate, if a potential butyrate overow
from the liver into the systemic circulation could be responsible for the sleep eects, we injected the same amount
of butyrate systemically. Systemic administration of butyrate did not have any eect on sleep in rats. Similarly,
NREMS REMS Temperature Activity SWA
df F p df F p df F p df F p df F p
Treatment 1,7 0.7 n.s. 1,7 1.4 n.s. 1,7 2.4 n.s. 1,7 0.0 n.s. 1,7 14.6 <0.01
Time 11,77 15.12 <0.001 11,77 18.3 <0.001 11,77 7.5 <0.001 11,77 11.7 <0.001 11,77 2.7 <0.01
Treatment x
Time 11,77 6.1 <0.001 11,77 4.0 <0.001 11,77 12.8 <0.001 11,77 3.2 <0.01 11,77 3.4 <0.001
Table 1. Oral administration of tributyrin. Non-rapid eye movement sleep (NREMS), rapid-eye movement
sleep (REMS), body temperature, motor activity and electroencephalographic slow-wave activity (SWA):
statistical results.
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none of the systemically-administered doses of butyrate had any eect on sleep-wake activity in mice. ese
ndings indicate that the sleep eects of orally or intraportally administered butyrate are not due to the actions
of butyrate that possibly escaped the hepatic sink. We conclude that butyrate acts on the liver and/or the portal
vein to promote NREMS. ere is prior evidence that the liver is involved in peripheral sleep signaling, since local
warming of the liver increases NREMS8, and depletion of liver Kuper cells impairs recovery sleep responses aer
sleep loss and sleep in a cold environment45.
Hepatoportal sensors have been described for several gut-derived molecules, e.g., glucose, cholecystokinin,
and amino acids4648. ey are located in the wall of the portal vein and the liver and have been implicated in
Figure 2. e eects of intraportal and subcutaneous administration of butyrate on wakefulness, NREMS,
REMS, EEG SWA, motor activity and body temperature. See legend to Fig.1 for details.
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SCIENTIFIC REPORTS | (2019) 9:7035 |
the regulation of glucose and energy homeostasis (reviewed in49). ere is also evidence for hepatoportal SCFA
sensors. Butyrate receptors are present in the hepatoportal region. Butyrate signals through the receptors FFAR2,
FFAR3 and GPR109A, all of which is expressed in the liver32,33,50, thus may serve as hepatic sensors. FFAR3 is
also expressed by the portal vein wall in close proximity to neuronal markers51. e activation of these recep-
tors aects brain circuits as evidenced by the observation that the eects of dietary SCFAs on the activity of the
nucleus tractus solitarius and parabrachial nucleus are abolished by the selective sensory denervation of the per-
iportal area51. e sensory innervation of the hepatoportal region is provided by the vagus and spinal aerents,
both of which have been implicated in sleep signaling5255. Butyrate directly activates vagal aerents56 and the
eects of butyrate on feeding is suppressed by hepatic vagotomy57.
Several bacterial-derived sleep-inducing molecules, such as lipopolysaccharide and fragments of pep-
tidoglycans, have been described before (reviewed in58). All these molecules are components of the bacterial
cell wall, they are released from disintegrating bacteria or from bacteria during cell division. ey have pro-
nounced inammatory actions via the stimulation of the production of pro-inammatory cytokines (reviewed
in59). Sleep responses to systemic bacterial infection are linked to these pro-inflammatory processes. The
properties of butyrate are, however, fundamentally different. Butyrate is produced by live bacteria in the
intestines, and it has strong anti-inammatory properties. It suppresses colonic and liver inammation and
lipopolysaccharide-induced production of pro-inammatory cytokines and NF-κB activation26,6063. is indi-
cates that not only systemic pro-inammatory signals related to bacterial infections, but also bacterial-derived
anti-inammatory signals from the intestinal tract have the potential to modulate sleep.
Animals. Male Sprague-Dawley rats and breeding pairs of C57BL/6 J mice were purchased from e Jackson
Laboratories, Inc.; the mice were further bred at Washington State University. During the experiments, the ani-
mals were housed in temperature-controlled (mice: 30 ± 1 °C, rats: 23 ± 1 °C), sound-attenuated isolation cham-
bers on a 12:12-hour light-dark cycle (lights on at 3 AM). Food and water were available ad libitum throughout
all experiments. Animals were provided regular lab chow (Harlan Teklad, Product no. 2016), in which fats, pro-
teins, and carbohydrates comprise 12%, 22%, and 66% of calories, respectively. All animal procedures were con-
ducted in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of
the National Institutes of Health. All animal protocols were approved by the Institutional Animal Care and Use
Committees at Washington State University.
Surgery. All surgical procedures were performed using ketamine-xylazine anesthesia (87 and 13 mg/kg,
respectively). For sleep-wake activity recordings, 3-month old mice (25.5 ± 0.9 g) and rats (325–350 g) were
implanted with three cortical EEG electrodes, placed over the frontal and parietal cortices, and two nuchal elec-
tromyographic (EMG) electrodes. e EEG and EMG electrodes were anchored to the skull with dental cement.
Telemetry transmitters were implanted intraperitoneally for body temperature and motor activity recordings. In
addition, the rats were implanted with an intraportal cannula64 three weeks prior to the sleep surgery. Briey, a
biocompatible polyurethane was inserted into the superior mesenteric vein and the tip of the cannula routed to
the main stream of the portal vein. e free end of the cannula was routed subcutaneously to the dorsal surface
of the neck and exteriorized. e cannula was sutured to the portal vein, the abdominal muscles and the neck
skin. e patency was maintained by daily ushing with 0.2 ml isotonic saline followed by 0.08 ml of lock solution
containing 500 IU/ml heparin in 50% glycerol solution. e animals were allowed to recover from surgery for at
least 10 days before any experimental manipulation started and handled daily to adapt them to the experimental
NREMS REMS Temperature Activity SWA
df F p df F p df F p df F p df F p
Treatment 1,7 67.7 <0.001 1,7 7.8 <0.05 1,8 2.0 n.s. 1,7 0.2 n.s. 1,7 0.2 n.s.
Time 11,77 38.6 <0.001 11,77 10.2 <0.001 11,88 54.0 <0.001 11,77 37.2 <0.001 11,77 37.2 <0.001
Treatment x
Time 11,77 4.3 <0.001 11,77 2.5 <0.01 11,88 14.0 <0.001 11,77 3.7 <0.001 11,77 3.7 <0.001
Table 2. Intra-portal administration of butyrate. NREMS, REMS, body temperature, motor activity and EEG
SWA: statistical results.
NREMS REMS Temperature Activity SWA
df F p df F p df F p df F p df F p
Treatment 1,4 0.2 n.s. 1,4 2.1 n.s. 1,4 6.6 0.06 1,4 1.4 n.s. 1,4 0.4 n.s.
Time 11,44 42.9 <0.001 11,44 18.1 <0.001 11,44 53.1 <0.001 11,44 21.1 <0.001 11,44 16.6 <0.001
Treatment x
Time 11,44 1.0 n.s. 11,44 1.2 n.s. 11,44 1.6 n.s. 11,44 1.4 n.s. 11,44 0.5 n.s.
Table 3. Subcutaneous administration of butyrate. NREMS, REMS, body temperature, motor activity and EEG
SWA: statistical results.
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Sleep-wake activity recordings and analyses. e animals were tethered to commutators, which were
further routed to Grass Model 15 Neurodata amplier system (Grass Instrument Division of Astro-Med, Inc.,
West Warwick, RI). e amplied EEG and EMG signals were digitized at 256 Hz and recorded by computer. e
high-pass and low-pass lters for EEG signals were 0.5 and 30.0 Hz, respectively. e EMG signals were ltered
with low and high cut-o frequencies at 100 and 10,000 Hz, respectively. e outputs from the 12A5 ampliers
were fed into an analog-to-digital converter and collected by computer using Sleep Wave soware (Bioso Studio,
Hersey, PA). Sleep-wake states were scored visually o-line in 10-s segments. e vigilance states were dened as
NREMS, REMS and wakefulness according to standard criteria as described previously1. EEG power data from
each artifact free 10-s segment were subjected to o-line spectral analysis by fast Fourier transformation. EEG
power data in the range of 0.5 to 4.0 Hz during NREMS were used to compute EEG SWA. EEG SWA data were
normalized for each animal by using the average EEG SWA across 24 h on the baseline day as 100.
Telemetry recordings. Core body temperature and locomotor activity were recorded by MiniMitter telem-
etry system (Starr Life Sciences Corp.) using VitalView soware. Temperature and activity values were collected
every 1 and 10 min, respectively, throughout the experiment and were averaged over 2-h time blocks.
Experimental procedures. Experiment 1: The effects of oral gavage administration of tributyrin in
mice. Eight mice were habituated to the gavage procedure by administering 0.3 ml water for 7 days 5–15 min
before dark onset. Aer the habituation period, a baseline day was recorded aer the oral gavage of 0.3 ml water.
is treatment controls for the non-specic eects of the gavage administration, such as changes in the gastric
Figure 3. e eects of intraperitoneal administration of butyrate on NREMS, REMS, EEG SWA, motor
activity and body temperature. See legend to Fig.1 for details.
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SCIENTIFIC REPORTS | (2019) 9:7035 |
volume. e following day, 0.3 ml tributyrin was administered (Millipore Sigma). e treatments were performed
5–10 min before dark onset. Sleep and telemetric recordings started at dark onset and continued for 23.5 h.
Experiment 2: e eects of intraportal administration of sodium butyrate in rats. Ten rats were habituated to
the injection procedure by daily ushing of the cannula with isotonic saline 5–20 min before dark onset. On the
baseline day, 2 ml/kg isotonic NaCl (vehicle) was administered through the cannula. On the test day, the animals
received 1 g/kg sodium butyrate, dissolved in isotonic NaCl, in a volume of 2 ml/kg. e pH of the butyrate solu-
tion was set to 7.4 by using NaOH. e treatments were performed 5–20 min before dark onset. Sleep and telem-
etric recordings started at dark onset and continued for 23.5 h. Due to the malfunction of some of the implants,
EEG/EMG was obtained only from 8 animals, and body temperature from 9 rats.
Experiment 3: e eects of subcutaneous administration of sodium butyrate in rats. Ten days aer Experiment
2, ve rats were used again to test the eects of sc administration of 1 g/kg butyrate. e animals were habituated
to the treatment by daily sc administration of isotonic saline. On the baseline day, the animals were injected sc
with 2 ml/kg isotonic NaCl (vehicle). On the test day, the animals received 1 g/kg buered sodium butyrate sub-
cutaneously in a volume of 2 ml/kg. e treatments took place 5–10 min before dark onset. Sleep and telemetric
recordings started at dark onset and continued for 23.5 h.
Experiment 4: e eects of intraperitoneal administration of sodium butyrate in mice. ree doses of butyrate
were tested in the same group of mice (n = 7). Aer the habituation period, the animals received 10 ml/kg isotonic
NaCl (vehicle) ip to obtain baseline values. On the test day, the mice were injected with 20 mg/kg sodium butyrate
intraperitoneally. One week later, a new vehicle baseline day was recorded followed by the test day of 100 mg/kg
butyrate. Finally, aer one additional week of recovery, a third vehicle baseline and test day (500 mg/kg butyrate)
were recorded. e treatments took place 5–10 min before dark onset. Sleep and telemetric recordings started at
dark onset and continued for 23.5 h.
Statistics. Time spent in wakefulness, NREMS and REMS, as well as, EEG SWA, body temperature and
motor activity were calculated in 2-h blocks. Two-way repeated measures ANOVA was performed across 24 h
between test days and the corresponding baselines (factors: treatment and time, both repeated). When appropri-
ate, Tukey’s HSD test was applied post hoc. An α-level of P < 0.05 was considered to be signicant.
Data Availability
All data generated or analyzed during this study are included in this published article.
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NREMS REMS Temperature Activity SWA
df F p df F p df F p df F p df F p
20 mg/kg
Treatment 1,6 4.6 n.s. 1,6 1.8 n.s. 1,6 0.8 n.s. 1,5 0.9 n.s. 1,7 0.5 n.s.
Time 11,66 26.4 <0.001 11,66 47.1 <0.001 11,66 60.7 <0.001 11,55 17.1 <0.001 11,77 14.2 <0.001
Treatment x
Time 11,66 0.8 n.s. 11,66 0.8 n.s. 11,66 1.8 n.s. 11,55 0.4 n.s. 11,77 0.8 n.s.
100 mg/kg
Treatment 1,6 2.1 n.s. 1,6 1.8 n.s. 1,6 0.3 n.s. 1,6 0.6 n.s. 1,5 0.0 n.s.
Time 11,66 19.8 <0.001 11,66 26.4 <0.001 11,66 43.5 <0.001 11,66 33.6 <0.001 11,55 7.7 <0.001
Treatment x
Time 11,66 0.4 n.s. 11,66 1.1 n.s. 11,66 0.5 n.s. 11,66 0.6 n.s. 11,55 0.8 n.s.
500 mg/kg
Treatment 1,6 0.0 n.s. 1,6 1.5 n.s. 1,6 0.2 n.s. 1,6 0.0 n.s. 1,5 3.9 n.s.
Time 11,66 1.1 n.s. 11,66 6.5 <0.001 11,66 36.5 <0.001 11,66 18.1 <0.001 11,55 9.4 <0.001
Treatment x
Time 11,66 0.6 n.s. 11,66 1.8 n.s. 11,66 1.3 n.s. 11,66 0.3 n.s. 11,55 0.8 n.s.
Table 4. Intraperitoneal administration of butyrate. NREMS, REMS, body temperature, motor activity and
EEG SWA: statistical results.
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is work was supported by the National Institute of Health, National Heart, Lung, and Blood Institute, Grant
Number R01HL122390, to É.S.
Author Contributions
É.S. & L.K. conceived and designed the experiments, N.S.M., A.R.M. and É.S. conducted the experiments, É.S.
and L.K. analyzed the data. All authors reviewed the manuscript.
Additional Information
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... This same group of researchers later demonstrated that the microbiota induces the cyclic expression of HDAC3 in the small intestine via the TLR adaptor protein MyD88, resulting in the cyclic acetylation and activation of many genes involved in nutrient transport and lipid metabolism [174]. The microbiota is also thought to regulate host metabolism in the intestine and liver via oscillations in microbially produced and modulated metabolites, like SCFAs and bile acids, which influence host epigenetics and transcription [175][176][177][178][179]. In addition, the microbial metabolite butyrate has a purported sleep-promoting effect which may also help reinforce proper circadian cycling [175]. ...
... This same group of researchers later demonstrated that the microbiota induces the cyclic expression of HDAC3 in the small intestine via the TLR adaptor protein MyD88, resulting in the cyclic acetylation and activation of many genes involved in nutrient transport and lipid metabolism [174]. The microbiota is also thought to regulate host metabolism in the intestine and liver via oscillations in microbially produced and modulated metabolites, like SCFAs and bile acids, which influence host epigenetics and transcription [175][176][177][178][179]. In addition, the microbial metabolite butyrate has a purported sleep-promoting effect which may also help reinforce proper circadian cycling [175]. ...
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... 47 Previous studies found that oral or intraportal butyrate administration robustly increased NREM sleep in rats and induced slow, high-amplitude electroencephalographic wave sleep in rabbits. 19,48 Our results were different from previous studies that imposed sleep restriction. In these previous studies, SCFAs did not change after partial SD. ...
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... There is strong evidence that SCFAs have profound effects on brain function through the BGMA. Recent studies have demonstrated that butyrate can potentially act as signaling molecules to induce sleep (Szentirmai et al., 2019). A study of older adults with insomnia found that those who underwent short sleep duration had higher fecal concentrations of SCFAs than those with normal sleep duration (Magzal et al., 2021). ...
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... In neurobiological diseases such as insomnia, SCFAs have been thought to function as mediators linking gut bacteria to mechanisms in the brain 27,28 . Zhang et al. showed differences in gut microbiota, serum metabolites, and serum immune factors between healthy controls and older adults with insomnia 29 . ...
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Physical activity (PA) can improve functional abilities, well-being, and independence in older adults with insomnia. Studies have shown that PA may be linked to changes in the gut microbiota composition and its metabolites’ concentrations. This association among older adults with insomnia, however, is yet to be determined. We explored the relationships between physical activity (PA) levels, gut microbiota composition, and short-chain fatty acid (SCFA) levels in this population. Forty-nine community-dwelling adults with insomnia symptoms, aged 65 and older, participated in this study. Their average daily step-count and sleep continuity measures over a two-week period were calculated based on Actigraphic recordings. Each participant provided fecal samples for the microbiome and SCFA analyses, anthropometric measures, and information via questionnaires on medical history and food consumption. The gut microbiota composition and SCFA concentrations were determined by next-generation sequencing and Gas chromatography-mass spectrometry, respectively. Participants were divided into two groups (more and less active) according to their median step/day count. We compared the microbiota abundance and SCFA concentrations between groups and performed correlation analysis between gut microbiota abundances and study variables. Different microbiota taxa in each PA group and increased SCFAs in feces of less active individuals were found. Changes in step counts were positively or negatively associated with the relative abundance of 19 ASVs, 3 microorganisms at the family level, and 11 microorganisms at the genus level. Furthermore, significant associations were discovered among physical activity, gut microbiota, SCFAs, and sleep parameters. Our findings provide new insights on the relationship between PA, gut microbiota composition, and primary metabolites in older adults with insomnia.
... Validating butyrate synthesis by different human and mouse microbes Butyrate-producing species are also functionally important members of the human gut microbiota and are associated with clinical outcomes of diseases as diverse as inflammatory bowel disease (Parada Venegas et al., 2019) and depression (Caspani et al., 2019). In addition, butyrate is involved in regulating host metabolism (Donohoe et al., 2011), sleep (Szentirmai et al., 2019), and healthy cognitive functioning (Silva et al., 2020) and is important for induction of peripheral T regulatory cells (Atarashi et al., 2011;Furusawa et al., 2013). Faecalibacterium prausnitzii, a canonical butyrate-producing species, has been associated with improved outcomes for inflammatory bowel disease (Carlsson et al., 2013;Sokol et al., 2008), but it does not colonize well in the mouse gut (Aluthge et al., 2020;Lundberg et al., 2020), limiting the utility of mouse models to study these clinical associations. ...
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Human health and disease have increasingly been shown to be impacted by the gut microbiota, and mouse models are essential for investigating these effects. However, the compositions of human and mouse gut microbiotas are distinct, limiting translation of microbiota research between these hosts. To address this, we constructed the Mouse Gastrointestinal Bacteria Catalogue (MGBC), a repository of 26,640 high-quality mouse microbiota-derived bacterial genomes. This catalog enables species-level analyses for mapping functions of interest and identifying functionally equivalent taxa between the microbiotas of humans and mice. We have complemented this with a publicly deposited collection of 223 bacterial isolates, including 62 previously uncultured species, to facilitate experimental investigation of individual commensal bacteria functions in vitro and in vivo. Together, these resources provide the ability to identify and test functionally equivalent members of the host-specific gut microbiotas of humans and mice and support the informed use of mouse models in human microbiota research.
With the development of genome sequencing, many researchers have investigated the mechanism by which the intestinal microbiota influences sleep across the brain-gut axis. However, the relationship between gut microbiota and sleep disorder remains unclear. Thus, we studied the difference in gut microbiota composition between poor sleep quality- and normal populations, which helps set the ground for future research. The recruited college students provided baseline information and stool samples and completed the Pittsburgh Sleep Quality Index (PSQI). We compared the two groups’ gut microbiota composition and functional differentiation by using the 16S rRNA gene sequencing analysis. The main bacterial difference and the most critical effect were mainly concentrated within Tenericutes and Elusimicrobia. Compared with the healthy control group, some functions of the gut microbiota were impaired in the poor sleep quality group, such as butanoate metabolism and propanoate metabolism. Bacterial taxa with significant differences raised the possibility for future diagnosis and treatment of sleep problems.
Sleep is fundamental for survival, contributing to multiple recuperative processes. When sleep is disturbed, including abbreviated sleep and sleep that is too lengthy (> 9 h), the occurrence of illnesses are elevated, including various types of cancer. Such outcomes may be related to effects on energy regulation, immune, and cytokine variations, and those that are tied to tumor suppression and DNA repair processes. Sleep is tied to circadian rhythms, although they can independently affect wellness and the provocation of illnesses. Thus, activities that disturb circadian cycles, such as jobs that involve night shift work, can favor the development of cancer, operating through hormonal, immune, and inflammatory actions. Circadian cycles linked to several clock genes influence the cycles of virtually every cell within the body, including immune cells and microbiota, which are affected by sleep alterations and circadian variations, thereby influencing disease processes. The effectiveness of cancer therapies may be influenced by administering treatments at phases of circadian cycles when cancer cells are most vulnerable to being destroyed.
Naturally occurring carbohydrate polymers containing non-starch polysaccharides (NPs) are a class of biomacromolecules isolated from plants, marine algae, and edible mushrooms, and their biological activities has shown potential uses in the prevention and treatment of human diseases. Importantly, NPs serve as prebiotics to provide health benefits to the host through stimulating the proliferation of beneficial gut microbiota (GM) and enhancing the production of short-chain fatty acids (SCFAs). The composition and diversity of GM play a critical role in regulating host health and have been extensively studied in recent years. In this review, the extraction, isolation, purification, and structural characterization of NPs derived from plants, marine algae, and edible mushrooms are outlined. Importantly, the degradation and metabolism of these NPs in the intestinal tract, the effects of NPs on the microbial community and SCFAs generation, and the beneficial effects of NPs on host health by modulating GM are systematically highlighted. Overall, we hope that this review can provide some theoretical references and a new perspective for applications of NPs as prebiotics in functional food and drug development.
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We previously identified brown adipose tissue (BAT) as a source of sleep-inducing signals. Pharmacological activation of BAT enhances sleep while sleep loss leads to increased BAT thermogenesis. Recovery sleep after sleep loss is diminished in mice that lack uncoupling protein 1 (UCP-1), and also in wild-type (WT) mice after sensory denervation of the BAT. Systemic inflammation greatly affects metabolism and the function of adipose tissue, and also induces characteristic sleep responses. We hypothesized that sleep responses to acute inflammation are mediated by BAT-derived signals. To test this, we determined the effects of systemic inflammation on sleep and body temperature in UCP-1 knockout (KO) and WT mice. Intraperitoneal injections of lipopolysaccharide, tumor necrosis factor-α, interleukin-1 beta and clodronate containing liposomes were used to induce systemic inflammation. In WT animals, non-rapid-eye movement sleep (NREMS) was elevated in all four inflammatory models. All NREMS responses were completely abolished in UCP-1 KO animals. Systemic inflammation elicited an initial hypothermia followed by fever in WT mice. The hypothermic phase, but not the fever, was abolished in UCP-1 KO mice. The only recognized function of UCP-1 is to promote thermogenesis in brown adipocytes. Present results indicate that the presence of UCP-1 is necessary for increased NREMS but does not contribute to the development of fever in systemic inflammation.
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Inflammatory bowel disease (IBD) is associated with a loss of intestinal barrier function and dysregulated immune responses. It has been shown that short chain fatty acids (SCFAs) are protective in IBD and that GPR43 mediates the protective effects of SCFAs. In this study, we investigated the effects of SCFAs in comparison to highly specific GPR43 agonists on human intestinal epithelial and immune cells. Our results confirm that SCFAs are enhancers of barrier function in intestinal epithelial cells. Additionally, SCFAs also displayed potent immunoregulatory properties based upon the ability to inhibit LPS-induced cytokine production in PBMC, and human T cell proliferation and cytokine production. Unexpectedly, and in contrast to the current belief, specific GPR43 agonists failed to exhibit similar barrier enhancing and anti-inflammatory properties. These findings demonstrate that SCFA possess broad protective functions in IBD and agonizing GPR43 alone is unlikely to be beneficial in patients. © 2017 D’Souza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Brown adipose tissue (BAT) is regulated by the sympathetic nervous system via β3-adrenergic receptors (β3-AR). Here we tested the hypothesis that pharmacological stimulation of β3-ARs leads to increased sleep in mice and if this change is BAT dependent. In wild-type (WT) animals, administration of CL-316,243, a selective β3-AR agonist, induced significant increases in non-rapid-eye movement sleep (NREMS) lasting for 4–10 h. Simultaneously, electroencephalographic slow-wave activity (SWA) was significantly decreased and body temperature was increased with a delay of 5–6 h. In uncoupling protein 1 (UCP-1) knockout mice, the middle and highest doses of the β3-AR agonist increased sleep and suppressed SWA, however, these effects were significantly attenuated and shorter-lasting as compared to WT animals. To determine if somnogenic signals arising from BAT in response to β3-AR stimulation are mediated by the sensory afferents of BAT, we tested the effects of CL-316,243 in mice with the chemical deafferentation of the intra-scapular BAT pads. Sleep responses to CL-316,243 were attenuated by ~50% in intra-BAT capsaicin-treated mice. Present findings indicate that the activation of BAT via β3-AR leads to increased sleep in mice and that this effect is dependent on the presence of UCP-1 protein and sleep responses require the intact sensory innervation of BAT.
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Chronic sleep fragmentation (SF) commonly occurs in human populations, and although it does not involve circadian shifts or sleep deprivation, it markedly alters feeding behaviors ultimately promoting obesity and insulin resistance. These symptoms are known to be related to the host gut microbiota. Mice were exposed to SF for 4 weeks and then allowed to recover for 2 weeks. Taxonomic profiles of fecal microbiota were obtained prospectively, and conventionalization experiments were performed in germ-free mice. Adipose tissue insulin sensitivity and inflammation, as well as circulating measures of inflammation, were assayed. Effect of fecal water on colonic epithelial permeability was also examined. Chronic SF-induced increased food intake and reversible gut microbiota changes characterized by the preferential growth of highly fermentative members of Lachnospiraceae and Ruminococcaceae and a decrease of Lactobacillaceae families. These lead to systemic and visceral white adipose tissue inflammation in addition to altered insulin sensitivity in mice, most likely via enhanced colonic epithelium barrier disruption. Conventionalization of germ-free mice with SF-derived microbiota confirmed these findings. Thus, SF-induced metabolic alterations may be mediated, in part, by concurrent changes in gut microbiota, thereby opening the way for gut microbiome-targeted therapeutics aimed at reducing the major end-organ morbidities of chronic SF.
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Sleep is profoundly altered during the course of infectious diseases. The typical response to infection includes an initial increase in nonrapid eye movement sleep (NREMS) followed by an inhibition in NREMS. REMS is inhibited during infections. Bacterial cell wall components, such as peptidoglycan and lipopolysaccharide, macrophage digests of these components, such as muramyl peptides, and viral products, such as viral double-stranded RNA, trigger sleep responses. They do so via pathogen-associated molecular pattern recognition receptors that, in turn, enhance cytokine production. Altered sleep and associated sleep-facilitated fever responses are likely adaptive responses to infection. Normal sleep in physiological conditions may also be influenced by gut microbes because the microbiota is affected by circadian rhythms, stressors, diet, and exercise. Furthermore, sleep loss enhances translocation of viable bacteria from the intestine, which provides another means by which sleep–microbe interactions impact neurobiology.
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The reciprocal interaction between the immune system and sleep regulation has been widely acknowledged but the cellular mechanisms that underpin this interaction are not completely understood. In the present study, we investigated the role of macrophages in sleep loss- and cold exposure-induced sleep and body temperature responses. Macrophage apoptosis was induced in mice by systemic injection of clodronate-containing liposomes (CCL). We report that CCL treatment induced an immediate and transient increase in non-rapid-eye movement sleep (NREMS) and fever accompanied by decrease in rapid-eye movement sleep, motor activity and NREMS delta power. Chronically macrophage-depleted mice had attenuated NREMS rebound after sleep deprivation compared to normal mice. Cold-induced increase in wakefulness and decrease in NREMS, rapid-eye movement sleep and body temperature were significantly enhanced in macrophage-depleted mice indicating increased cold sensitivity. These findings provide further evidence for the reciprocal interaction among the immune system, sleep and metabolism, and identify macrophages as one of the key cellular elements in this interplay.
Background The gut–brain axis facilitates a critical bidirectional link and communication between the brain and the gut. Recent studies have highlighted the significance of interactions in the gut–brain axis, with a particular focus on intestinal functions, the nervous system and the brain. Furthermore, researchers have examined the effects of the gut microbiome on mental health and psychiatric well-being. The present study reviewed published evidence to explore the concept of the gut–brain axis. Aims This systematic review investigated the relationship between human brain function and the gut–brain axis. Methods To achieve these objectives, peer-reviewed articles on the gut–brain axis were identified in various electronic databases, including PubMed, MEDLINE, CIHAHL, Web of Science and PsycINFO. Results Data obtained from previous studies showed that the gut–brain axis links various peripheral intestinal functions to brain centres through a broad range of processes and pathways, such as endocrine signalling and immune system activation. Researchers have found that the vagus nerve drives bidirectional communication between the various systems in the gut–brain axis. In humans, the signals are transmitted from the liminal environment to the central nervous system. Conclusions The communication that occurs in the gut–brain axis can alter brain function and trigger various psychiatric conditions, such as schizophrenia and depression. Thus, elucidation of the gut–brain axis is critical for the management of certain psychiatric and mental disorders.
Fermentable carbohydrate including dietary fibers and resistant starch produce short chain fatty acids (SCFAs), including acetate, propionate and butyrate, through microbial fermentation in the intestine of rodents and humans. Consumption of fermentable carbohydrate and SCFAs suppress food intake, an effect involving the brain. However, their signaling pathway to the brain remains unclear. Vagal afferents serve to link intestinal information to the brain. In the present study, we explored possible role of vagal afferents in the anorexigenic effect of SCFAs. Intraperitoneal (ip) injection of three SCFA molecules (6 mmol/kg) suppressed food intake in fasted mice with the rank order; butyrate > propionate > acetate. The suppressions of feeding by butyrate, propionate and acetate were attenuated by vagotomy of hepatic branch and blunted by systemic treatment with capsaicin that denervates capsaicin-sensitive sensory nerves including vagal afferents. Ip injection of butyrate induced significant phosphorylation of extracellular signal regulated kinase 1/2, cellular activation markers, in nodose ganglia and their projection site, medial nucleus tractus solitaries. Moreover, butyrate directly interacted with single neurons isolated from nodose ganglia and induced intracellular Ca²⁺ signaling. The present results identify the vagal afferent as the novel pathway through which exogenous SCFAs execute the remote control of feeding behavior and possibly other brain functions. Vagal afferents might participate in suppression of feeding by intestine-born SCFAs.
The gut microbiota has emerged as an environmental factor that modulates the host's energy balance. It increases the host's ability to harvest energy from the digested food, and produces metabolites and microbial products such as short-chain fatty acids, secondary bile acids, and lipopolysaccharides. These metabolites and microbial products act as signaling molecules that modulate appetite, gut motility, energy uptake and storage, and energy expenditure. Several findings suggest that the gut microbiota can affect the development of obesity. Germ-free mice are leaner than conventionally raised mice and they are protected against diet-induced obesity. Furthermore, obese humans and rodents have an altered gut microbiota composition with less phylogeneic diversity compared to lean controls, and transplantation of the gut microbiota from obese subjects to germ-free mice can transfer the obese phenotype. Taken together, these findings indicate a role for the gut microbiota in obesity and suggest that the gut microbiota could be targeted to improve metabolic diseases like obesity. This review focuses on the role of the gut microbiota in energy balance regulation and its potential role in obesity.
Unraveling the role of dietary lipids is beneficial to treat obesity and metabolic dysfunction. Nonetheless, how dietary lipids affect existing obesity remains unknown. Arachidonic acid (AA), a derivative of linoleic acid, is one of the crucial n-6 fatty acids. The aim of this study was to investigate whether AA affects obesity through associating microbiota-driven inflammation with hypothalamus-adipose-liver axis. Four-week old C57BL/6J mice were fed with a high-fat diet (HFD, 45% fat) for 10 weeks to induce obesity, and then fed a HFD enriched with 10 g/kg of AA or a continuous HFD in the following 15 weeks. Systemic adiposity and inflammation, metabolic profiles, gut microbiota composition, short-chain fatty acids production, hypothalamic feeding regulators, browning process of adipocytes, hepatosteatosis, and insulin resistance in adipose were investigated. The results indicated that AA aggravates obesity for both genders whereas sex-dependently affects gut microbiota composition. Also, AA favors pro-inflammatory microbiota and reduces butyrate production and circulating serotonin, which augments global inflammation and triggers hypothalamic leptin resistance via microglia accumulation in male. AA exacerbates non-alcoholic steatohepatitis along with amplified inflammation through TLR4-NF-κB pathway and induces insulin resistance. Reversely, AA alleviates obesity-related disorders via rescuing anti-inflammatory and butyrate-producing microbiota, up-regulating GPR41 and GPR109A and controlling hypothalamic inflammation in female. Nevertheless, AA modifies adipocyte browning and promotes lipid mobilization for both genders. We show that AA affects obesity likely through a gut-hypothalamus-adipose-liver axis. Our findings formulate recommendations of n-6 fatty acids like AA from dietary intake for obese subjects preferably in a sexually dimorphic way.