Background: Bloodlevels of many hormones oscillate during a 24 hour period and sleep disruptioneffects on this circadian rhythm. The ghrelin hormone issecreted from gastric cells and stimulates hunger. There are few studies regarding the24-hour ghrelin secretion pattern in normal subjectsbut no studies have been done on the effects of sleep deprivation on thispattern.Objective: Assessmentof the 24-hour ghrelin secretion pattern and the effect of sleep deprivation onthis pattern.Materialand methods: This work was conducted on 16 young military men (19-23years old) who served in AJA university of Medical sciences. Eight of them hadregular sleep-wake cycle.
The second group had not usual arousal cycle. Sixblood 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 ELISAmethod.Results Cortisolshowed a diurnal rhythm with peak was at 0600h in normal and disrupted sleepsubjects.
But ghrelin did not show a significant diurnal rhythm in both of the groups. Sleepdeprivation had no significant effect on the time pattern of the cortisol andghrelin secretion.Conclusion It seems,in real life, ghrelin does not show a diurnal rhythm and sleepdeprivation does not effect on this rhythm. Key Words: sleepdeprivation; ghrelin; circadian rhythm IntroductionBlood levels of manyhormones oscillates during 24 hour period (1-4). This circadian rhythm in hormone secretion is not only affected bySleep / wake cycle (such as GH) but also an endogenous biological timing system(such as cortisol).
Appropriate interaction of sleep and biological system isnecessary for regulating blood hormoneconcentration. Under normal situations the sleep/ wake cycle andendogenous timing system are harmonized and properly regulate levels ofhormones. However, circadian misalignment may have negative adverse healthconsequences on metabolic and hormonal factors (5).Ghrelin, a 28-amino-acid peptide waspurified in 1999 from rat stomach. Ghrelinis produced by the oxyntic gland in the stomach (6). Peripheral or central injection of ghrelin increases intakeof food and body weight in rodents(7,8).
Plasma human ghrelin levels increase prior the usual mealtimes 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 haveshown the release of hormones such as ghrelin and leptin, which play a centralrole in regulating glucose and appetite is dependent on sleep duration andquality. Therefore, sleep loss may have adverse effects on endocrine functionand metabolism (12). Few studies have been conducted on the 24-hour pattern of ghrelinsecretion in subjects who have a normal sleep duration (13,14). but with our knowledge, no studies has been doneon the effects of sleep deprivation on this pattern.Therefore, our studywas done to establish whether : 1) Does the serum ghrelin levels follow acircadian rhythm in soliders with normal sleep.
2) Does the shift work affect the pattern of24 hours of ghrelin secretion?Materialand methodsParticipantsThestudy was conducted on healthy young soldiers who served in AJA university of Medicalsciences. They had 19-23 years old. After thenecessary explanations in relation to this research they were invited toparticipate in this study. 16 soldiers accepted ourinvitation. First group had regular sleep-wake cycle.
The soldiers weresleeping from 22 pm until 6 am and woke the rest of the day and were doingtheir daily tasks. The second group consisted of individuals who had not normalarousal cycle. Their plan within 24 hours was: Two hoursawake for guard, standby two hours and sleep two hours. The program was repeated during 24 hours.
Participants in thestudy were given meals three per day (breakfast, lunch, dinner) at specifiedtimes.Blood sampling and hormone assayBlood samples were taken in both sleep and wakefulness atintervals of 4 hours for a total of 24 hours (from 10A.M on day 1 until 6 A.M on day 2). Therefore, six blood samples were takenfrom each subject within 24 hours.Bloodwas collected in tubes, which were stored on ice and contained Na-EDTA (1 mg/mlblood) and aprotinin (300 kallikrein inhibitor units/ml blood). Immediatelyafter the withdrawal, blood was centrifuged at 2,600 g for 7 min at 4°C, and serum was aliquoted and frozento ?20°C until the various assays were performedSerum cortisol(DRG Instruments GmbH, Germany) and ghrelin (CRYSTAL DAY CHRISTIAN DAY kit – China) Concentrationswere measured by ELISA method.
The calculationsThe average hormone concentrations werecalculated as the area under the curve divided by 24 h 15.Statistical analysisAll values are expressed as means ± SEM. A normal distribution ofdata was evaluated by sample Kolmogorov- Sminov test. To determine the existence of the daily changes in the secretion of thehormones, we used repeated measuresanalysis of variance (ANOVA) with time as withinsubject factor and condition (normal sleep vs.
sleep deprived) as betweensubject factor.The Independent t-test was used to compare the averageconcentration of hormones over 24 hours in the two groups. SPSS software (version18.0, IBM) was used for all statistical analyses. Thep- value less than 0.05 were considered significantResults Statistical analysis of thedata using repeated measures of variance showed the main effect of timeon cortisol concentration F (5,15) =18.9, p=0.006).
Therefore,there was acircadian rhythm of cortisol in both groups with peaks wereat 0600h (Fig 1). But there was no significant effect of group oncortisol secretion pattern F(1,3)=5.1, p=0.1). Concerningghrelin, there were not a main effect of group F(1,7) =0.59, p= 0.46) andtime F(5,35) =0.
55, p=0.57 on ghrelin concentration. In the other words, Inboth groups, control and deprived of sleep, ghrelin did not show obviousdiurnal rhythms and sleep disruption has no significanteffect on the temporal pattern of the serum ghrelin level though a non-significant variation was detected (Fig 2). Theaverage concentration of ghrelin and cortisol throughout the24 h have been shown in the table 1. Thesleep disruption has no a significant effect onmean concentrations of cortisol and ghrelin during 24hours. DiscussionEarlier studies have been shown an alteration in activity times, changeshappen in sleep period and feeding behavior together influence circadiancontrol of the endocrine system (16,17).In our study, secretion pattern of cortisol is similar tothe 24 hours rythm of cortisol seen in previous reports (3,12) with peaklevels occurring at early morning (06.00 p.
m.) and sleep deprivation had no significant effect on thispattern. The results of Scheer et.al (2009) study showed, the cortisol secretion pattern is more influenced by the internaldaily rhythms rather than behavior (fasting-feeding and sleep/wake) cycles (18).The study Fumihisa et al. (2013) showed that nightshift (from 0:00 to 8:00) had not effect on circadian rhythm in male nurses.
Inaddition, the concentration ofcortisol in night shift did not differfrom that of the control group (19). Also in our study, the average concentrationof cortisol throughout 24 h was notsignificantly higher in the deprived sleep group than the control group.In agreement with presentstudy, an earlier study, did not show a significant circadianrhythm for acylated ghrelin in the normalsubjects that take 3 meals in a day (14).
It seemsthat the nutritional state of the person is effective on ghrelin secretionpulses. Fasting augmented all parameters of ghrelin pulsatile secretion (20). In contrast to our study, some previous studies havereported circadian rhythm for ghrelin (13). This can be due tomethodological differences that prevented comparison this study with other works.
1- The number of blood sampling: In ourstudy, The number of blood sampling was 6 times during 24 hours. Whereas in otherworks were between 24-72 during 24 hours(14,13,21). 2- Age and sex. Most preceding studiesthat reported circadian rhythm for ghrelin have been done in female subjects anda very large age range (13,21).3-sleep and sleepdeprivation condition.
In most previous studies, effects of sleep on a diurnalor nocturnal pattern of ghrelin secretion investigated in the laboratoryconditions not imitating “real life.In conclusion, It seems, ghrelin does not show a circadian rhythm andsleep deprivation has no significant effecton the 24 h secretion pattern of cortisol and ghrelin.