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Background: Blood
levels of many hormones oscillate during a 24 hour period and sleep disruption
effects on this circadian rhythm. The ghrelin hormone is
secreted from gastric cells and stimulates hunger.

There are few studies regarding the
24-hour ghrelin secretion pattern in normal subjects
but no studies have been done on the effects of sleep deprivation on this
pattern.

Objective: Assessment
of the 24-hour ghrelin secretion pattern and the effect of sleep deprivation on
this pattern.

Material
and methods: This work was conducted on 16 young military men (19-23
years old) who served in AJA university of Medical sciences. Eight of them had
regular sleep-wake cycle. The second group had not usual arousal cycle. Six
blood samples were taken from each subject within 24 hours. Blood was centrifuged 
and serum was frozen to ?20°C until the various assays were performed. Serum cortisol and ghrelin levels were measured by ELISA
method.

Results

Cortisol
showed a diurnal rhythm with peak was at 0600h in normal and disrupted sleep
subjects. But ghrelin did not show a significant diurnal  rhythm in both of the groups. Sleep
deprivation had no significant effect on the time pattern of the cortisol and
ghrelin secretion.

Conclusion

 It seems,
in real life, ghrelin does not show a diurnal rhythm and sleep
deprivation does not effect on this rhythm.

Key Words: sleep
deprivation; ghrelin; circadian rhythm

 

 

Introduction

Blood levels of many
hormones oscillates during 24 hour period (1-4). This circadian rhythm in hormone secretion is not only affected by
Sleep / wake cycle (such as GH) but also an endogenous biological timing system
(such as cortisol). Appropriate interaction of sleep and biological system is
necessary for regulating  blood hormone
concentration. Under normal situations the sleep/ wake cycle and
endogenous timing system are harmonized and properly regulate levels of
hormones. However, circadian misalignment may have negative adverse health
consequences on metabolic and hormonal factors (5).

Ghrelin, a 28-amino-acid peptide was
purified in 1999 from rat stomach. Ghrelin
is produced by the oxyntic gland in the stomach (6). Peripheral or central injection of ghrelin increases intake
of food and body weight in rodents(7,8). Plasma  human ghrelin levels increase prior the usual meal
times and fall thereafter (9,10).

Therefore, it seems ghrelin secretion regulate by caloric intake.  Ghrelin also  plays a role in neuroendocrine and behavioral responses to stress (11).

Previous reports have
shown the release of hormones such as ghrelin and leptin, which play a central
role in regulating glucose and appetite is dependent on sleep duration and
quality. Therefore, sleep loss may have adverse effects on endocrine function
and metabolism (12).

Few studies have been conducted on the 24-hour pattern of ghrelin
secretion in subjects who have a normal sleep duration (13,14). but with our knowledge, no studies has been done
on the effects of sleep deprivation on this pattern.Therefore, our study
was done to establish whether :

 1) Does the serum ghrelin levels follow a
circadian rhythm in soliders with normal sleep.

 2) Does the shift work affect the pattern of
24 hours of ghrelin secretion?

Material
and methods

Participants

The
study was conducted on healthy young soldiers who served in AJA university of Medical
sciences. They had 19-23 years old. After the
necessary explanations in relation to this research they were invited to
participate in this study. 16 soldiers accepted our
invitation. First group had regular sleep-wake cycle. The soldiers were
sleeping from 22 pm until 6 am and woke the rest of the day and were doing
their daily tasks. The second group consisted of individuals who had not normal
arousal cycle. Their plan within 24 hours was: Two hours
awake for guard, standby two hours and sleep two hours. The program was repeated during 24 hours.

Participants in the
study were given meals three per day (breakfast, lunch, dinner) at specified
times.

Blood sampling and hormone assay

Blood samples were taken taken at four hour intervals during
both wakefulness and sleep for a total of 24 hours (from 10 A.M on day 1 until 6 A.M on day 2). Therefore, six blood
samples were taken from each subject within 24 hours.

Blood
was collected in tubes, which were stored on ice and contained Na-EDTA (1 mg/ml
blood) and aprotinin (300 kallikrein inhibitor units/ml blood). Immediately
after the withdrawal, blood was centrifuged at 2,600 g for 7 min at 4°C, and serum was aliquoted and frozen
to ?20°C until the various assays were performed

Serum cortisol
(DRG Instruments GmbH, Germany) and ghrelin (CRYSTAL DAY CHRISTIAN DAY kit – China) Concentrations
were measured by ELISA method.

The calculations

The average hormone concentrations were
calculated as the area under the curve divided by 24 h [15].

Statistical analysis

All values are expressed as means ± SEM. A normal distribution of
data was evaluated by sample Kolmogorov- Sminov test. To determine the existence of the daily changes in the secretion of the
hormones, we used repeated measures
analysis of variance (ANOVA) with time as within
subject factor and condition (normal sleep vs. sleep deprived) as between
subject factor.

The Independent t-test was used to compare the average
concentration of hormones over 24 hours in the two groups. SPSS software (version
18.0, IBM) was used for all statistical analyses. The
p- value less than 0.05 were considered significant

Results

 

Statistical analysis of the
data using repeated measures of variance showed the main effect of time
on cortisol concentration [F (5,15) =18.9, p=0.006)]. Therefore,there was a
circadian rhythm of cortisol in both groups with peaks were
at 0600h (Fig 1). But there was no significant effect of group on
cortisol secretion pattern [F(1,3)=5.1, p=0.1)]. Concerning
ghrelin, there were not a main effect of group [F(1,7) =0.59, p= 0.46)] and
time [F(5,35) =0.55, p=0.57] on ghrelin concentration. In the other words, In
both groups, control and deprived of sleep, ghrelin did not show obvious
diurnal rhythms and sleep disruption has no significant
effect on the temporal pattern of the serum 
ghrelin level though a non-significant variation was detected (Fig 2). The
average concentration of ghrelin and cortisol throughout the
24 h have been shown in the table 1. The
sleep disruption has no a significant effect on
mean concentrations of cortisol and ghrelin during 24
hours.      

Discussion

Earlier studies have been shown an alteration in activity times, changes
happen in sleep period and feeding behavior together influence circadian
control of the endocrine system (16,17).

In our study, secretion pattern of cortisol is similar to
the 24 hours rythm of cortisol seen in previous reports (3,12) with peak
levels occurring at early morning (06.00 p.m.) and sleep deprivation had no significant effect on this
pattern. The results of Scheer et.al (2009) study showed, the cortisol secretion pattern is more influenced by the internal
daily rhythms rather than behavior (fasting-feeding and sleep/wake) cycles (18).

The study Fumihisa et al. (2013) showed that night
shift (from 0:00 to 8:00) had not effect on circadian rhythm in male nurses. In
addition,  the concentration of
cortisol  in night shift did not differ
from that of the control group (19). 
Also in our study, the average concentration
of cortisol throughout 24 h  was not
significantly higher in the deprived sleep group than the control group.

In agreement with present
study, an earlier study, did not show a significant circadian
rhythm for acylated ghrelin in  the normal
subjects that take 3 meals in a day (14). It seems
that the nutritional state of the person is effective on ghrelin secretion
pulses. Fasting augmented all parameters of ghrelin pulsatile secretion (20). In contrast to our study, some previous studies have
reported circadian rhythm for ghrelin (13). This can be due to
methodological differences that prevented comparison  this study with other works.

1- The number of blood sampling: In our
study, The number of blood sampling was 6 times during 24 hours. Whereas in other
works were between 24-72  during 24 hours
(14,13,21).

2- Age and sex. Most preceding studies
that reported circadian rhythm for ghrelin have been done in female subjects and
a very large age range (13,21).

3-sleep and sleep
deprivation condition. In most previous studies, effects of sleep on a diurnal
or nocturnal pattern of ghrelin secretion investigated in the laboratory
conditions not imitating “real life.

In conclusion, It seems, ghrelin does not show a circadian rhythm and
sleep deprivation has no significant effect
on the 24 h secretion pattern of cortisol and ghrelin.

 

 

 

 

 

Limitations:

The small number of subjects and low
number sampling over 24 h are limitations.