Am J Physiol Regul Integr Comp Physiol 298: R467–R477, 2010.
First published November 25, 2009; doi:10.1152/ajpregu.00557.2009.
Restricted feeding-induced sleep, activity, and body temperature changes in
normal and preproghrelin-deficient mice
Éva Szentirmai,1,2,3 Levente Kapás,1,2,3 Yuxiang Sun,4 Roy G. Smith,5 and James M. Krueger2,3
1
Washington, Wyoming, Alaska, Montana and Idaho (WWAMI) Medical Education Program, Washington State University,
Spokane; 2Sleep and Performance Research Center and 3Department of Veterinary and Comparative Anatomy,
Pharmacology, and Physiology, Washington State University, Pullman, Washington; 4United States Department of
Agriculture, Agricultural Research Service Children’s Nutrition Research Center, Huffington Center on Aging, Departments
of Pediatrics and Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas; and 5Department of
Metabolism and Aging, The Scripps Research Institute, Scripps Florida, Jupiter, Florida
Submitted 2 September 2009; accepted in final form 24 November 2009
food-entrainable oscillator; food anticipatory activity; electroencephalographic slow-wave activity; food deprivation; hypothermia
INTERACTIONS BETWEEN ENERGY homeostasis, feeding, and sleep
are well-documented. For example, meal size correlates with
the subsequent duration of sleep (8). Starvation induces sleep
loss in rats (16). These sleep-loss effects develop more rapidly
if the initial energy levels of the body are low (3, 10). In
contrast, refeeding after food deprivation increases sleep in rats
(3, 33). Rats kept on a calorie-rich “cafeteria diet” tend to
overeat and have an increased amount of sleep (7, 15). Intravenous infusion of a variety of nutrients differently modulates
sleep in rats (9). Chronic sleep deprivation leads to hyperphagia and weight loss in rats (30), whereas chronic short sleep
duration is associated with obesity in humans (34, 41). When
food availability is restricted to the light period, it alters the
Address for reprint requests and other correspondence: É. Szentirmai,
WWAMI Medical Education Program, Washington State Univ., Spokane, P.O.
Box 1495, Spokane, WA 99210-1495 (e-mail: [email protected]).
http://www.ajpregu.org
diurnal distribution of sleep in rats (29, 31). These findings
indicate a strong relationship between sleep and feeding.
The diurnal rhythm of sleep and feeding is driven by
biological clock(s). The biological clock within the suprachiasmatic nucleus (SCN) is entrained to a 24-h rhythm by light. In
addition to light, another potent entraining signal is periodic
feeding. The biological clock that is affected by feeding is called
the food-entrainable oscillator (FEO), which is located outside the
confines of the SCN. One manifestation of the activity of the
FEO is food-anticipatory activity (FAA). FAA is characterized
by increases in behavioral activity, corticosterone secretion,
and body temperature 1– 4 h before scheduled feeding time
when food availability is restricted to a few hours a day.
The site of the FEO remains elusive. Lesions of hypothalamic and extrahypothalamic areas (19, 22, 23) or subdiaphragmatic vagotomy (28) all failed to abolish FAA in rats. Recently, the dorsomedial hypothalamus (DMH) was posited to
be an integrator of light- and food-entrainable circadian
rhythms (12, 13); these findings, however, remain controversial (18, 20, 26). Genetic deletion (17, 24, 46) or postnatal
lesions (e.g., Refs. 1, 21, and 25) of feeding-related signals in
mice or rats have also failed to eliminate FAA. Clock genes are
expressed in various peripheral organs, including the gastrointestinal system, suggesting the possibility that the FEO is
located in the gastrointestinal system. Irrespective whether the
FEO is peripheral or central, gut-to-brain signaling is required
either as an output signal from a peripheral clock to the brain
to elicit behavioral responses or as an afferent signal from the
gut to a centrally located oscillator. Gastrointestinal hormones,
the secretions of which are phase-locked to feeding, are likely
candidates to serve as such a signal.
Ghrelin is an orexigenic peptide, produced mainly by the
stomach. Its secretion is locked to feeding activity; ghrelin
levels are elevated during fasting and suppressed after eating
(2, 6, 43). In an experimental restricted feeding (RF) paradigm,
plasma ghrelin levels increase in parallel to FAA (11). Ghrelin
is also produced by the brain; ghrelin-immunoreactive cell
bodies are present in the hypothalamic area between the arcuate
nucleus (ARC), paraventricular nucleus (PVN), DMH, and lateral
hypothalamus (5). Ghrelin stimulates food intake via the activation of neuropeptide Y (NPY)-ergic neurons in the hypothalamic
ARC. Intracerebroventricular administration or hypothalamic
microinjections of ghrelin promote wakefulness in rats (37,
38). Ghrelin receptors are expressed in the ARC, SCN, DMH,
and PVN of the hypothalamus (14, 27, 47), all of which are
implicated in feeding and sleep regulation.
0363-6119/10 $8.00 Copyright © 2010 the American Physiological Society
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Szentirmai É, Kapás L, Sun Y, Smith RG, Krueger JM. Restricted feeding-induced sleep, activity, and body temperature changes
in normal and preproghrelin-deficient mice. Am J Physiol Regul Integr
Comp Physiol 298: R467–R477, 2010. First published November 25,
2009; doi:10.1152/ajpregu.00557.2009.—Behavioral and physiological rhythms can be entrained by daily restricted feeding (RF), indicating the existence of a food-entrainable oscillator (FEO). One
manifestation of the presence of FEO is anticipatory activity to
regularly scheduled feeding. In the present study, we tested if intact
ghrelin signaling is required for FEO function by studying food
anticipatory activity (FAA) in preproghrelin knockout (KO) and
wild-type (WT) mice. Sleep-wake activity, locomotor activity, body
temperature, food intake, and body weight were measured for 12 days
in mice on a RF paradigm with food available only for 4 h daily
during the light phase. On RF days 1–3, increases in arousal occurred.
This response was significantly attenuated in preproghrelin KO mice.
There were progressive changes in sleep architecture and body temperature during the subsequent nine RF days. Sleep increased at night
and decreased during the light periods while the total daily amount of
sleep remained at baseline levels in both KO and WT mice. Body
temperature fell during the dark but was elevated during and after
feeding in the light. In the premeal hours, anticipatory increases in
body temperature, locomotor activity, and wakefulness were present
from RF day 6 in both groups. Results indicate that the preproghrelin
gene is not required for the manifestation of FAA but suggest a role
for ghrelinergic mechanisms in food deprivation-induced arousal in
mice.
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Based on these observations, we hypothesized that ghrelin
might be an integrator of feeding- and arousal-related signals
directed to the FEO, or ghrelinergic neurons may be part of the
FEO itself. To test this hypothesis, we studied the short (1–3
days)- and longer (8 –12 days)-term effects of RF on sleep,
locomotor activity, and body temperature in preproghrelin
knockout [KO (originally named as ghrelin⫺/⫺; see Ref. 36)]
and wild-type (WT) mice. The wake-promoting effects of
short-term RF were attenuated in preproghrelin KO animals,
but they displayed normal FAA, suggesting that ghrelin is not
required for the normal function of the FEO but it plays a
significant role in arousal responses to fasting.
MATERIALS AND METHODS
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Table 1. The amounts of wakefulness, NREMS, and REMS
during the 12 h of dark and light phases and the 24-h
amounts of each vigilance state on the baseline and RF day
8 in preproghrelin WT and KO mice
WT (n ⫽ 8)
Baseline
Wakefulness, min
Dark
Light
Daily
NREMS, min
Dark
Light
Daily
REMS, min
Dark
Light
Daily
RF Day 8
KO (n ⫽ 8)
Baseline
RF Day 8
434.2⫾16.6 366.2⫾18.4* 416.7⫾17.6
233.8⫾11.0 319.2⫾13.2* 205.6⫾12.5
668.0⫾26.4 685.4⫾23.2 616.1⫾21.8
331.6⫾9.4*
313.0⫾18.2*
643.4⫾15.3
265.0⫾15.8 317.4⫾16.4* 282.2⫾15.9
424.8⫾13.2 352.1⫾12.6* 454.8⫾10.9
689.8⫾27.6 669.5⫾23.0 742.0⫾21.6
340.1⫾7.5*
357.7⫾14.1*
699.1⫾11.1
20.7⫾1.8
61.3⫾3.9
82.0⫾5.3
37.4⫾2.8*
48.3⫾3.6*
85.7⫾3.8
20.8⫾3.2
59.1⫾4.1
81.0⫾4.1
45.3⫾4.2*
49.2⫾4.3*
95.2⫾5.9
Values are means ⫾ SE; n, no. of mice. WT, wild type; KO, knockout; RF,
restricted feeding; NREMS, non-rapid-eye movement sleep; REMS, rapid-eye
movement sleep. There was no significant difference between genotypes in the
time spent in the vigilance states under ad libitum feeding conditions and on
RF day 8. RF led to decreased sleep during the day and increased sleep during
the dark period in both genotypes. The daily, 24-h amounts of NREMS,
REMS, and wakefulness remained at baseline levels in both groups. *Significant difference between baseline and RF day 8 within the same genotype
(paired t-test, P ⬍ 0.05).
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Animals and surgery. Breeding pairs of preproghrelin KO and
WT mice with a C57BL6J/129SvEv genetic background, backcrossed to C57BL6J for 10 generations, were generated at Baylor
College of Medicine (Houston, TX), and further bred at Washington State University. The procedures used to generate the mutant
mice were described previously (36). Each mouse used was genotyped (Transnetyx, Cordova, TN) from tail-tip samples. Male mice,
4 mo of age at the time of surgery, were maintained, under
veterinary supervision, in animal quarters approved by the Association for Assessment and Accreditation of Laboratory Animal
Care. The procedures employed were approved by the Washington
State University Institutional Animal Use and Care Committee.
Mice were anesthetized with an intraperitoneal injection of ketamine-xylazine mixture (87 and 13 mg/kg, respectively). The
animals were implanted with cortical electroencephalographic
(EEG) electrodes, placed over the frontal and parietal cortexes and
stainless steal electromyographic (EMG) electrodes in the dorsal
neck muscles. The EEG and EMG electrodes were connected to a
pedestal that was fixed to the skull with dental cement. Wax-coated
telemetry transmitters (model XM; Mini Mitter, Bend, OR) were
implanted intraperitoneally in each animal. After surgery, mice
were housed individually in standard plastic cages and maintained
at 29 ⫾ 1°C (a thermoneutral ambient temperature for mice) in
sound-attenuated environmental chambers on a 12:12-h light-dark
cycle (light on at 9:00 A.M.). During a 7- to 10-day recovery
period, the animals were handled daily and connected to the
recording cables for habituation to the experimental conditions.
Water was available ad libitum throughout the experiment.
Experimental protocol. Baseline body temperature, food intake,
and sleep-wake activity data were obtained from the animals over a
48-h period (baseline days 1 and 2), when food was available ad
libitum. From the 3rd day, mice were restricted to a 4-h daily meal,
beginning 4 h after lights-on, for 12 consecutive days (RF). Body
weight of mice was assessed daily at the beginning of the feeding
period, and then preweighed amounts of food pellets were placed in
the cages. The leftover was collected and weighed after 4 h. The RF
condition was followed by a 2-day recovery period during which mice
had ad libitum access to food.
Biotelemetry and sleep-wake recordings. Core body temperature,
locomotor activity, and EEG data were collected simultaneously from
each animal with the exception of three preproghrelin KO mice from
which only body temperature and activity data were obtained (sleep:
n ⫽ 8 for both WT and KO; temperature and activity: n ⫽ 8 for WT
and n ⫽ 11 for KO). The transmitter-emitted signals were converted
to temperature and locomotor activity data using VitalView Series
3000 data acquisition software. Body temperature and locomotor
activity values were collected in every 10 s or 10 min, respectively,
and were averaged into 1-h time blocks for each day. Animals were
connected to recording cables 3 days after surgery for habituation to
the experimental conditions. Recording cables were attached to commutators that were connected to amplifiers. The amplified EEG and
EMG signals were digitized and recorded by computer using SleepWave Software (Biosoft Studio). EEG was filtered below 0.1 Hz and
above 40 Hz. EMG activity was used for the vigilance state determination and was not further analyzed. The vigilance states [i.e., wakefulness, non-rapid-eye movement sleep (NREMS), and rapid-eye
movement sleep (REMS)] were visually determined off-line in 10-s
epochs by using the conventional criteria as described previously (37).
Briefly, low-amplitude, mixed-frequency EEG accompanied by high
EMG activity was defined as wakefulness. NREMS was identified by
high-amplitude EEG waves, predominant EEG power in the delta
range (0.5– 4 Hz), and low EMG activity. REMS was identified by
highly regular low-amplitude EEG, the dominance of theta activity,
and minimal EMG activity. Power density values in the delta range
during NREMS [also known as EEG slow-wave activity (SWA) and
used as a measure of NREMS intensity] were averaged across the
entire 48-h baseline recording period to obtain a reference value for
each mouse. EEG SWA values are expressed as a percentage of this
reference value in 2-h time blocks. The total number of wakefulness,
NREMS, and REMS episodes and the average length of these episodes were determined for each day. Time spent in wakefulness,
NREMS and REMS, body temperature, and locomotor activity were
expressed in 1-h time blocks. Data from the first (days 1, 2, and 3),
middle (days 4 and 6), and the last (days 8 and 12) days of RF were
collapsed and are referred to as RF days 1–3, RF days 4 – 6, and RF
days 8 –12, respectively.
Statistical analyses. Two-way ANOVA for repeated measures
(factors: time and day) was performed to compare the durations of
wake, NREMS and REMS, EEG SWA during NREMS, body temperature, and locomotor activity between baseline and RF days within
the same genotype. When significant day effect or day ⫻ time
interaction was found, a paired t-test was performed post hoc. Body
weight and food intake were analyzed by ANOVA (repeated measure:
day, independent factor: genotype) followed by paired t-test for
comparisons between days within a genotype. For the comparison of
the number and duration of wake, NREMS, and REMS episodes
between genotypes, two-tailed Student’s t-test was used. Body temperature during the 4-h period before feeding on RF days and during
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the corresponding hours on the baseline and recovery days were
further analyzed by 1) computing linear regression daily for each
genotype (dependent variable: temperature, independent variable:
time) and 2) testing for parallelism between the regression lines of
WT and preproghrelin KO groups. Increasing body temperature
(hence the slope of the regression lines) in these hours is indicative of
FAA. Significant differences in parallelism mean different slopes and
would indicate differences in temperature-defined food anticipation.
P values ⬍0.05 were considered significant.
RESULTS
Table 2. Results of statistical analyses [2-way ANOVA for repeated measures (factors: time and day)] performed on
wakefulness, NREMS, REMS, SWA body temperature, and locomotor activity data separately for WT and KO mice
Treatment
WT RF 1–3
Wake
NREMS
REMS
SWA
Body temperature
Activity
KO RF 1–3
Wake
NREMS
REMS
SWA
Body temperature
Activity
WT RF 4 and 6
Wake
NREMS
REMS
SWA
Body temperature
Activity
KO RF 4 and 6
Wake
NREMS
REMS
SWA
Body temperature
Activity
WT RF 8 & 12
Wake
NREMS
REMS
SWA
Body temperature
Activity
KO RF 8 & 12
Wake
NREMS
REMS
SWA
Body temperature
Activity
Time
Interaction
F value
P
F value
P
F value
P
⫽
⫽
⫽
⫽
⫽
⫽
⬍0.05
⬍0.05
⬍0.05
NS
⬍0.05
⬍0.05
F(23,161) ⫽ 30.4
F(23,161) ⫽ 26.6
F(23,161) ⫽ 30.8
F(11,77) ⫽ 9.9
F(23,161) ⫽ 30.9
F(23,161) ⫽ 16.5
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(23,161) ⫽ 6.1
F(23,161) ⫽ 6.5
F(23,161) ⫽ 2.6
F(11,77) ⫽ 5.4
F(23,161) ⫽ 12.5
F(23,161) ⫽ 2.6
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(1,7) ⫽ 6.5
F(1,7) ⫽ 6.9
F(1,7) ⫽ 0.4
F(1,7) ⫽ 0.3
F(1,7) ⫽ 16.0
F(1,11) ⫽ 0.01
⬍0.05
⬍0.05
NS
NS
⬍0.05
NS
F(23,161) ⫽ 31.9
F(23,161) ⫽ 28.1
F(23,161) ⫽ 25.9
F(11,77) ⫽ 14.6
F(23,161) ⫽ 32.5
F(23,253) ⫽ 24.1
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(23,161) ⫽ 4.4
F(23,161) ⫽ 4.3
F(23,161) ⫽ 2.4
F(11,77) ⫽ 3.5
F(23,161) ⫽ 16.9
F(23,253) ⫽ 3.2
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⫽
⫽
⫽
⫽
⫽
⫽
NS
NS
NS
NS
⬍0.05
NS
F(23,161) ⫽ 13.2
F(23,161) ⫽ 12
F(23,161) ⫽ 12.3
F(11,77) ⫽ 3.5
F(23,161) ⫽ 16.3
F(23,161) ⫽ 13.3
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(23,161) ⫽ 5.9
F(23,161) ⫽ 5.6
F(23,161) ⫽ 5.3
F(11,77) ⫽ 4.0
F(23,161) ⫽ 21.9
F(23,161) ⫽ 4.0
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(1,7) ⫽ 0.3
F(1,7) ⫽ 0.7
F(1,7) ⫽ 2.7
F(1,7) ⫽ 2.7
F(1,7) ⫽ 75.4
F(1,11) ⫽ 13.1
NS
NS
NS
NS
⬍0.05
⬍0.05
F(23,161) ⫽ 12.7
F(23,161) ⫽ 11.5
F(23,161) ⫽ 12.4
F(11,77) ⫽ 6.2
F(23,161) ⫽ 16.8
F(23,253) ⫽ 14.5
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(23,161) ⫽ 6.3
F(23,161) ⫽ 6.0
F(23,161) ⫽ 4.3
F(11,77) ⫽ 5.2
F(23,161) ⫽ 34.5
F(23,253) ⫽ 3.1
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⫽
⫽
⫽
⫽
⫽
⫽
0.1
0.2
0.7
3.5
40.1
0.03
NS
NS
NS
NS
⬍0.05
NS
F(23,161) ⫽ 17.0
F(23,161) ⫽ 15.4
F(23,161) ⫽ 13.4
F(11,77) ⫽ 4.4
F(23,161) ⫽ 28.9
F(23,161) ⫽ 10.5
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(23,161) ⫽ 13.5
F(23,161) ⫽ 13.3
F(23,161) ⫽ 6.9
F(11,77) ⫽ 7.5
F(23,161) ⫽ 58.1
F(23,161) ⫽ 5.2
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(1,7) ⫽ 0.0
F(1,7) ⫽ 0.1
F(1,7) ⫽ 3.7
F(1,7) ⫽ 3.5
F(1,7) ⫽ 40.3
F(1,11) ⫽ 6.2
NS
NS
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(23,161) ⫽ 14.5
F(23,161) ⫽ 14.1
F(23,161) ⫽ 9.5
F(11,77) ⫽ 5.2
F(23,161) ⫽ 27.7
F(23,253) ⫽ 10.9
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(23,161) ⫽ 11.0
F(23,161) ⫽ 10.1
F(23,161) ⫽ 8.6
F(11,77) ⫽ 8.5
F(23,161) ⫽ 71.3
F(23,253) ⫽ 4.9
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
⬍0.05
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
F(1,7)
12.2
7.4
6.9
0.9
9.8
5.2
1.2
0.4
1.8
0.03
21.7
0.5
Individual tests compare baseline day and one of the three different time blocks of RF within the same genotype (RF 1–3, RF 4 – 6, and RF 8 –12). SWA, slow
wave activity; NS, not significant.
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Wakefulness, NREMS, and REMS. Under baseline conditions, both groups of mice showed characteristic nocturnal
sleep-wake patterns; the amounts of NREMS and REMS were
greater during daylight hours and less during the dark. There
was no significant difference between genotypes in the time
spent in the vigilance states under ad libitum feeding conditions (Figs. 1– 4 and Table 1).
RF elicited progressive changes in the diurnal distribution of
sleep-wake activity in both groups (see Table 2 for statistics).
On RF days 1–3 (Fig. 1), wakefulness during the first 8 h of the
dark period was significantly elevated above baseline in both
genotypes; these increases were significantly higher in WT
mice compared with KO [79.7 ⫾ 15.4 min above baseline in
WT mice compared with 43.2 ⫾ 11.5 min in KO mice
(Student’s t-test, P ⬍ 0.05)]. Simultaneously, NREMS was
significantly suppressed in both groups, whereas REMS was
suppressed only in the WT animals. On the first 3 days of RF,
neither group of mice showed an anticipatory increase in
wakefulness before the scheduled feeding time (Fig. 2B). In the
first hour of the daily meal time, wakefulness was significantly
increased and NREMS and REMS suppressed in both preproghrelin KO and WT mice.
On RF days 4 – 6 (Fig. 3), changes in wakefulness showed a
biphasic pattern during the night in control animals; it remained
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Fig. 1. The diurnal rhythm of wakefulness, nonrapid-eye-movement sleep (NREMS), rapid-eyemovement sleep (REMS), electroencephalographic
(EEG) slow-wave activity (SWA) during NREMS,
body temperature, and locomotor activity of wildtype [WT (left), n ⫽ 8] and preproghrelin knockout
[KO (right); sleep data: n ⫽ 8 ; temperature and
activity data: n ⫽ 11] mice on the baseline days
(open symbols) and on the restricted feeding (RF,
filled symbols) days 1–3. Data are averaged across
RF days 1, 2, and 3 and expressed in 1-h bins for
each parameter except for EEG SWA where data
points represent 2-h averages. Error bars, SE; gray
shaded area, dark phase of the day. *Significant
difference between baseline days and treatment
days (univariate tests of significance for planned
comparison, P ⬍ 0.05).
significantly elevated in the first half but was significantly reduced
below baseline in the second half of the night. In KO animals,
only the second response phase was present. During the feeding
time, wakefulness increased in both genotypes. Changes in
NREMS and REMS mirrored those in wakefulness.
On RF days 8 –12, changes in the sleep-wake pattern led to
the partial reversal of the normal, nocturnal-type sleep-wake
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distribution. Nighttime wakefulness, mainly during the second
half of the night, decreased below, and daytime wakefulness
increased above, baseline in both KO and WT mice (Fig. 4 and
Table 1). The daily, 24-h amounts of sleep and wakefulness
remained at baseline levels. Both groups of mice exhibited
FAA from RF day 8 as indicated by the significantly increased
preprandial wakefulness (25.4 ⫾ 8.5 min increase in WT,
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29.6 ⫾ 8.3 min increase in KO) 1 h before the scheduled
feeding time, i.e., hour 16 (Fig. 2B). There was no significant
difference in the anticipatory increases in wakefulness between
control and KO animals. During the 4 h of the daily mealtime,
wakefulness was significantly increased, and duration of
NREMS and REMS was suppressed in both groups. Sleepwake activity returned to baseline on recovery day 1 (data not
shown), and the anticipatory increase in wakefulness was
absent (Fig. 2B).
Episode number and average duration of episodes. Under ad
libitum feeding conditions, there was a tendency toward an
increased number of wake and NREMS episodes and decreased average duration of these episodes in the KO animals
compared with WT mice, although these differences were not
significant (Figs. 5 and 6).
During RF days 1–3, there was an increase in sleep consolidation as indicated by the reduced number and the increased
average duration of wake and NREMS episodes in both
groups. Duration of REMS episodes remained unaltered, and
there was a slight but significant decrease in REMS episode
number in WT mice. On RF days 4 – 6, the number and
duration of the vigilance state episodes did not show any
significant difference from baseline in either group of mice,
but, when compared with control animals, KO mice had a
significantly higher number of wake, NREMS, and REMS
episodes. The average length of wake episodes was significantly shorter in KO animals. NREMS episode duration
showed a similar trend, but the difference was not significant
between the two genotypes.
On RF days 8 –12, wake, NREMS, and REMS episode
numbers and durations showed no significant difference from
baseline in either group of mice. There was a general tendency
toward increased episode numbers and decreased episode
AJP-Regul Integr Comp Physiol • VOL
lengths in KO animals, but the differences were not significant.
Similar tendencies were present on recovery day 1.
EEG SWA. EEG delta power during NREMS showed a
characteristic diurnal rhythm in both genotypes. SWA gradually increased during the dark, peaking at the beginning of the
light, and then gradually decreased during the remaining daylight hours. RF significantly altered EEG SWA rhythms in both
KO and control mice. During the dark phases of RF days 1–3,
EEG SWA was significantly elevated above baseline in WT
but not in KO animals (Fig. 1). During the following RF days,
the nocturnal rise in SWA was attenuated in both genotypes,
which led to the decreased diurnal amplitude of the EEG SWA
rhythms (Fig. 3). On RF days 8 –12, WT animals exhibited two
peaks in EEG SWA, one at the beginning of the dark and one
during the feeding period, in the middle of the light phase; the
second peak was absent in preproghrelin KO mice (Fig. 4). On
the recovery day, SWA returned to the baseline level (data not
shown) (Figs. 1, 3, and 4).
Body temperature. Baseline body temperature showed normal diurnal rhythms in both genotypes, with higher body
temperatures during the dark period and lower during the
daylight; there was no significant difference between the two
genotypes. RF induced similar changes in body temperature in
control and KO mice with three distinct daily response phases.
The first phase was a hypothermic response during the dark
period, and this was already apparent on the first RF day. The
hypothermia became progressively deeper each day, leveling
off at the end of dark on RF day 8, when body temperature was
2.4 ⫾ 0.2°C and 3.0 ⫾ 0.3°C below baseline in WT and KO
mice, respectively (Figs. 1– 4).
The hypothermia was followed by an increase in body
temperature during the 4 h before feeding time, a characteristic
sign of food anticipation. This response appeared on RF day 6
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Fig. 2. A: hourly body temperature values (⫾SE) with regression lines during the 4-h period before food access on RF days 1, 2, 3, 4, 6, 8, and 12 and during
the same hours on the baseline and first recovery days. Open symbols: WT; filled symbols: KO. A significant positive correlation was apparent between time
and temperature on RF days 8 and 12 in both genotypes, indicating food anticipation. On the recovery day, the correlation was absent. There was no significant
difference between the parallelism (slopes) of the regression lines of WT and KO groups on any of the RF days, indicating that the anticipation was not
statistically different in the two groups. B: amount of wakefulness in the 1-h period preceding food availability on the RF days or in the same hour on the recovery
day. Values are expressed as change from baseline (⫾SE). *Significant change from baseline (P ⬍ 0.05, paired t-test).
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Fig. 3. The diurnal rhythm of wakefulness, NREMS, REMS,
EEG SWA, body temperature, and locomotor activity of WT
(left) and preproghrelin KO (right) mice on the baseline days
(open symbols) and on the RF days 4 – 6. Data are averaged
across RF days 4 and 6. *Significant difference between
baseline days and treatment days (univariate tests of significance for planned comparisons, P ⬍ 0.05). See legend for Fig.
1 for details.
in both groups and became more pronounced during the following days (Fig. 2A). There was no significant difference in
the slopes of the temperature increases between genotypes,
indicating that this facet of FAA was similar in both strains.
A third response phase, hyperthermia during feeding and the
following 4 h, was already present on RF days 1–3 and became
progressively more pronounced over the course of the experiment. There was a consistent tendency toward accentuated
hyperthermic responses in the KO animals compared with
controls throughout the entire experiment; on RF day 12, the
AJP-Regul Integr Comp Physiol • VOL
difference between WT and KO groups reached statistical
significance (1.1 ⫾ 0.1°C above baseline in KO vs. 0.8 ⫾
0.1°C in WT mice, P ⬍ 0.05, t-test). Body temperature
returned to baseline on the recovery day (data not shown), and
the anticipatory increases during the first 4 h of the light were
absent (Fig. 2A).
Activity. In general, changes in activity paralleled changes in
wakefulness in both genotypes. In the 1st h of feeding, activity
was increased on all days in both groups. Locomotor activity
was significantly elevated in WT animals during the first 8 h of
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FOOD ANTICIPATORY ACTIVITY AND GHRELIN
the night on RF day 1–3 and RF day 4 – 6. In KO mice, this
response was absent. Starting from RF days 4 – 6, motor activity became suppressed during the latter part of the dark in both
groups. On RF day 8 –12, anticipatory increases in locomotor
activity were evident in both groups in hour 16. On the
AJP-Regul Integr Comp Physiol • VOL
recovery day, activity returned to baseline (data not shown)
(Figs. 1, 3, and 4).
Body weight and food intake. There was no significant
difference in the baseline body weight (WT mice: 30.8 ⫾ 1.3 g,
KO mice: 32.2 ⫾ 0.5 g) and food intake (Fig. 7 and Table 3)
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Fig. 4. The diurnal rhythm of wakefulness, NREMS,
REMS, EEG SWA, body temperature, and locomotor
activity of WT (left) and preproghrelin KO (right)
mice on the baseline days (open symbols) and on
RF days 8 –12. Data are averaged across RF days
8 and 12. *Significant difference between baseline days and treatment days (univariate tests of
significance for planned comparisons, P ⬍ 0.05).
See legend for Fig. 1 for details.
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FOOD ANTICIPATORY ACTIVITY AND GHRELIN
normal daily food intake; feeding increased during the following days, reaching ⬃90% of baseline on RF day 8. By this day,
both KO and WT mice adapted to the RF regimen and were
able to consume their normal daily food intake during the 4-h
access period.
DISCUSSION
The current findings indicate that RF alters the diurnal
distribution of sleep and wakefulness, EEG SWA, locomotor
activity, and body temperature in preproghrelin KO and WT
mice. Changes during the first 3 days of RF were different
between the two genotypes, but, by RF day 8, both groups of
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Fig. 5. The average episode numbers of wakefulness, NREMS, and REMS
episodes in WT (open bars) and preproghrelin KO (filled bars) mice during the
experiment. Data are expressed as average durations during the 24-h periods.
Bars represent the baseline day, the average of RF days 1–3, the average of RF
days 4 – 6, the average of RF days 8 –12, and recover day 1. Error bars, SE.
*Significant difference between baseline days and treatment days for both
genotypes (univariate tests of significance for planned comparison, P ⬍ 0.05).
#Significant difference between WT and KO (univariate tests of significance
for planned comparisons, P ⬍ 0.05).
between the two genotypes. In response to RF, both groups of
mice showed similar changes in body weight and food intake.
Body weight in both genotypes gradually decreased from RF
day 1– 6 when it reached 92.2% of baseline in WT mice and
90.4% in KO mice. On RF day 1, mice ate ⬃20 –25% of their
AJP-Regul Integr Comp Physiol • VOL
Fig. 6. The average duration of wakefulness, NREMS, and REMS episodes in
WT (open bars) and preproghrelin KO (filled bars) during the experiment.
*Significant difference between baseline days and treatment days for both
genotypes (univariate tests of significance for planned comparisons, P ⬍ 0.05).
#Significant difference between WT and KO (univariate tests of significance
for planned comparisons, P ⬍ 0.05). See legend for Fig. 5 for details.
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mice developed food anticipatory behavior. This suggests that
intact ghrelin signaling is not required for the normal function
of the FEO or for afferent signaling or efferent mechanisms
arising from it, but ghrelin likely plays a role to arousal
responses to fasting.
During the first 3 days of RF, mice of both genotypes were
partially food deprived since they consumed ⬍50% of their
normal daily food intake. Short-term food deprivation induces
wakefulness, and our results with WT mice confirm this
finding. We hypothesized that increases in wakefulness and
feeding at night in nocturnal rodents (“dark onset syndrome”)
are the result of the activation of the hypothalamic ghrelinorexin-NPY neuronal circuit by metabolic and circadian signals (37, 38). The finding that the arousal responses to partial
food deprivation were significantly attenuated in preproghrelin
KO mice is in line with this hypothesis. In a separate pilot
study, we found a similar attenuated wakefulness response to
48 h of fasting in preproghrelin KO mice (n ⫽ 12, unpublished
observation). It is unlikely that the drive for feeding is attenuated in the KO animals, since we found no significant difference in food intake of the two genotypes, which also confirms
previous observations (32, 36). Similar to our findings in
preproghrelin KO mice, orexin neuron-ablated mice show no
appreciable increase in arousal during fasting (44).
During the first 3 days of RF, there was a net sleep loss in
both genotypes that was significantly more robust in the WT
animals. The sleep loss was accompanied by increased EEG
SWA in WT mice. EEG SWA is, in part, a function of prior
AJP-Regul Integr Comp Physiol • VOL
Table 3. Results of statistical analyses (2-way mixed
ANOVA, repeated measure: day, independent measure:
genotype) performed on daily body weight and food intake
data
Body Weight
Food Intake
F value
P
F value
P
Genotype
Day
Genotype ⫻ day
interaction
F(1,18) ⫽ 1.72
F(12,216) ⫽ 25.1
F(12,216) ⫽ 0.99
NS
⬍0.05
NS
F(1,18) ⫽ 0.2
F(12,216) ⫽ 60.4
F(12,216) ⫽ 0.6
NS
⬍0.05
NS
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Fig. 7. Daily body weight and food intake of WT (open symbols) and KO
(filled symbols) mice on the baseline day (B) and during the course of RF.
Error bars, SE. *Significant difference between baseline and RF days for WT
group. #Significant difference between baseline and RF days for KO (paired
t-test, P ⬍ 0.05). There was no significant difference in body weight and food
intake between the control and KO mice on any day.
wakefulness and considered an indicator of NREMS intensity
and sleep pressure (4). It is likely that increased EEG SWA in
the second half of the dark period in WT mice is due to the
prior sleep loss because sleep loss induces elevated EEG SWA
during the rebound NREMS. In KO mice, the moderate sleep
loss did not lead to increases in EEG SWA, although these
mice have a normal capacity to enhance EEG SWA after sleep
loss (39).
Long-term (4 –11 days) food deprivation induces sleep fragmentation in rats, as indicated by the higher number and
decreased duration of vigilance state episodes (3, 16). In the
present experiment, however, during the first 3 days of RF,
wakefulness and NREMS became more consolidated than
under ad libitum feeding conditions in both groups of mice.
Consistent with these observations, we previously found that
normal mice respond to short-term food deprivation in a cold
environment with more consolidated sleep architecture (40). It
is possible that increased sleep consolidation is an adaptive,
energy-saving mechanism when food availability is transiently
decreased. Longer-term food deprivation is accompanied by
more severe metabolic changes, stress responses, and more
desperate food-seeking behavior, which together could lead to
increased sleep fragmentation.
During the RF day 4 – 6, the daily amount of sleep and
wakefulness gradually returned to baseline in both genotypes,
but profound changes in the diurnal distribution of sleep/
wakefulness remained in both groups. When food availability
is restricted to the 12-h light period, the diurnal distribution of
REMS is reversed, and the amount of NREMS becomes
equally distributed between the light and dark phases in rats
(29, 31). We found that 4 h of RF during the light period also
alters the normal diurnal distribution of sleep and wakefulness,
and the amount of wakefulness, NREMS, and REMS became
equally (⬃50%) distributed between the dark and light phases.
This is consistent with a previous finding in which 4 h RF
increased daytime wakefulness from 31 to 52.1% in rats (13).
Another study reported a more robust, complete reversal of
sleep-wake rhythms in normal and orexin⶿ataxin mice kept
under 4 h RF conditions (21). Importantly, the daily amounts of
sleep and wakefulness in preproghrelin KO and WT mice did
not change during the course of RF, confirming findings of
previous studies (13, 21, 31).
Twelve hours of fasting during the light phase do not alter
the diurnal distribution of EEG SWA during NREMS in rats
(31). We found that, in response to 4 h of RF, WT animals
exhibited two peaks in EEG SWA. In contrast, preproghrelin
KO mice had only a single peak in EEG SWA despite the
comparable sleep loss between KO and WT during the light
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AJP-Regul Integr Comp Physiol • VOL
role in the given function or, if it has, its lack can be compensated for.
Perspective and Significance
Our results show that intact ghrelin signaling is not required
for the normal function of the FEO or for its input or output
mechanisms. Ghrelinergic mechanisms, however, play a role in
food deprivation-induced arousal. This supports the hypothesis
that ghrelinergic neurons are part of those hypothalamic circuits that are responsible for triggering behavioral activation at
the beginning of the active phase of the day. In rats and mice,
the first part of the dark period is characterized by increased
arousal and enhanced feeding, a set of concerted responses
named dark-onset syndrome. In humans, similar responses
occur at the beginning of the light period. Night-eating syndrome is possibly related to the inadequate timing of the
hypothalamic ghrelinergic activating mechanisms.
ACKNOWLEDGMENTS
We thank Kenneth Carias, Jacob Pellinen, and Sean Woodward for help
analyzing the data.
GRANTS
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS-27250.
DISCLOSURES
No conflicts of interest are declared by the authors.
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