Effect of oral melatonin on plasma growth hormone concentration in rhesus monkeys (Macaca mulatta)

This study has been carried out to discover the cause of exogenous melatonin on plasma growth hormone concentration (PGHC) in four adult Macaca mulatta (rhesus monkeys). To inspect the result of solitary dose (5mg) of exogenous melatonin on PGHC four adult individual of Macaca mulatta with mean body weight 8.62 ± 1.05 were utilized. Sharp decrease in PGHC was noticed at 105 minutes 3.13 ± 0.88 mIU/L, and 120 minutes 2.86 ± 0.87 mIU/L. The growth hormone levels recorded after every 15-minutes showed gradual decrease with the advance time. Growth hormone levels regressed negatively with time (b= 0.9202 ± 0.071). Extremely imperative reduction have been noticed through regression analysis in growth hormone levels with the advance in time (F(1,6)=163.5,P<0.0001). Results of this study supports that melatonin sharply increases plasma growth hormone concentrations for short time and then starts decreasing.


Introduction
For some 50 years ago pineal gland was known as rudimentary gland in the body. Basically, pineal gland recognized about 2000 years ago [1]. In 1958, circumstances altered when Aaron Lerner and his colleagues discovered melatonin (pineal active substance) with its chemical structure "N acetyl-5-methoxytryptamine" [2, 3]. MT1, MT2 and MT3 are known as three types of melatonin receptors which were discovered on the basis of their molecular structures [4,5]. Along with some other sides, melatonin also produced inside the retina and pineal gland [6]. Due to strong association of pineal and reproductive system in animals the reproduction in gonadal axis normalized by melatonin [7]. It has been investigated that for the physiological development of the normal puberty melatonin perform a key function [7,8]. Abnormal fluctuations in the level of melatonin linked with delayed and precocious puberty [9,10]. Monkeys and humans are non-seasonal breeders so the pineal played major role to regulate the reproductive physiology and neuroendocrine regulation of puberty [11]. The somatotrope cells of the anterior pituitary produced Growth hormone (GH) which is a protein made up of 191 amino acids [12]. In prepubescent children the significant reduction in GH has been figure out while in adult there is no any effect on the level of GH by melatonin treatment [13,14]. In comparison, additional research suggests that in normal humans there is little increase in the level of basal Growth hormone by large dose of exogenous melatonin [15]. In rodents, clear inhibitory results of melatonin injections have been observed [16]. Contrastingly, some results showed that IGF-I and circulating concentrations of growth hormone were increasing by evening melatonin injections [17]. Number of studies has been carried out to show the effect of melatonin harmone in various organisms with an assortment of aspects [7,8,11,13,14,19,23,25] but no significant study has been carried out to explore the effects of exogenous melatonin on plasma growth hormone concentration correlated with time and treatment. Present study has been conducted to show the effect of exogenous melatonin administration on plasma growth hormone concentration in adult male rhesus monkeys (Macaca mulatta).

Materials and Methods
In this study, experimental study of four adult male rhesus monkeys (8-10 years age) was performed. Each animal was assigned with particular number: 0702, 0703, 0704 and 0705. Dental formula was used to calculate the ages of animals [18]. During experiment, body weight of used animals was weighed as ranged from 6.8-10.5 (kg). In Primate Facility, each individual was housed in specific cage and sustained in standard colony circumstances at Quaid-i-Azam University, Islamabad. Vegetables and fresh fruits were supplied to those animals as standard monkey food. Water availability was facilitated ad-libitum. Before the beginning of experiment (1 month ago) the appetite of the animals was monitored. All individuals utilized in this experiment were capable to eat their food within limited time of 5-10 minutes. The food protocol was maintained under proper time schedule (Fruits, boiled potatoes, eggs and bread at 6:00am, 9:00am, 11:00am and 1:00pm respectively) on daily basis. All animals were provided diet on behalf of their body weight.

Experimental protocol
Bleeding started from -30 minutes and melatonin was given orally at 0 minute ( Fig.  1).

Figure 1. Experimental Protocol showing the bleeding time before and after melatonin treatment
Chair restraining method Before 8 weeks of beginning of the experimental study the animals were adapted for chair restraining. On the chair, each animal was restrained for two hours on daily basis. Animals were also taught to get their foodstuff although chair restrained. Ketamine hydrochloride (5mg/kg BW) was used intramuscularly in order to create effortlessness situation to fix and remove from the chair animals were knocked out. Catheterization Prior to handle, 5 mg/kg of Ketamine was used to anaesthetize animals. Beginning of sampling (before 60 minutes) in the sephanous vein a Teflon cannula was introduced and animals were restrained to the chair. Through a butterfly tubing (20g weight and 300mm length) the terminal part of the cannula was affixed to a syringe. The given amount of ketamine was enough to halt status of the animal but not enough to induce narcosis.

Bleeding
After every 15 minutes, sequential blood samples (2.0 ml of each) were achieved before single dose of melatonin orally (5 mg).
The blood samples were achieved in hepranized syringes following melatonin administration and the same volume (5 IU/ml) of hepranized saline inserted in tubing. Whole bleeding was taken out during 9:00-10:00 am and for 10 minutes at 3000 rpm samples was centrifuged and extracted plasma was preserved at temperature of -15°C.

Immunoradiometric (IRMA) assay for growth hormone
For the proper analysis of data mean and standard error were calculated. For the comparison of mean student's t test was applied. Regression analysis of variance was used to show the plasma Growth hormone concentrations regress on time following treatment with melatonin.

Results
Results of this designed study examined the response of exogenous melatonin in rhesus monkeys (male adult) on plasma growth hormone concentrations. Mean body weight (kg) of all the four monkeys studied shown out in (Table 1).

Effect of exogenous melatonin on mean plasma growth hormone concentration (mIU/L) in adult male rhesus monkeys (Macaca mulatta)
Prior and later to single dose of melatonin (5mg) administration PGHC (mIU/L) mentioned in (Table 2 & Fig. 2). Melatonin treatment was given to four adult male rhesus monkeys. Plasma growth hormone concentration was recorded before treatment with melatonin. Mean concentration at -30 minutes for the four monkeys was 4.95 ± 0.67 mIU/L. The plasma growth hormone concentration increased at -15 minutes 5.27 ± 1.04 mIU/L, but this decreased at 0 minute 4.51 ± 0.87 mIU/L. Melatonin was administered orally with a dose of 5mg to the four monkeys at 0 minute. The rapid increasement (8.83 ± 2.79 mIU/L) has been recorded in mean PGHC after 15 minutes of treatment. This increase in growth hormone concentration (GHC) was not considerably dissimilar from that at 0 minute (t (6) = 1.18; P> 0.1). There was a slight decrease at 30minutes time 8.71 ± 2.81 mIU/L but at 45 minutes time there was sudden decrease in concentration 7.65 ± 3.22 mIU/L. A steady decrease in plasma growth hormone concentration was observed at 60 minutes 6.54 ± 2.97 mIU/L, 75 minutes 5.62 ± 3.11 mIU/L, and 90 minutes 5.42 ± 2.46 mIU/L after treatment. Sharp decrease in plasma growth hormone concentration was observed at 105 minutes 3.13 ± 0.88 mIU/L, and 120 minutes 2.86 ± 0.87 mIU/L.    (Fig. 3).

Effect of exogenous melatonin on individual plasma growth hormone concentration (mIU/L) in adult male rhesus monkeys (Macaca mulatta)
Individual plasma growth hormone concentration (mIU/L) prior and later single dose of melatonin (5mg) administration showed in (Table 2 & Fig. 4a, b). Melatonin treatment was given to four adult male rhesus monkeys individually. Plasma growth hormone concentration was recorded before treatment with melatonin. Individual plasma growth hormone concentration of animals 0702, 0703, 0704, and 0705 at 0 minute timing was 3.09, 2.99, 6.51 and 5.43 (mIU/L) respectively. Melatonin was administered orally with a dose of 5mg to all four monkeys at 0 minute. After 15 minutes of treatment rapid increase in PGHC to 4.87, 5.27, 16.91 and 8.29 (mIU/L) has been recorded respectively, the level of plasma growth hormone concentration (mIU/L) raised upto 30 minutes in all individuals, then it started decreasing slowly in all individuals from 45 to 120 minutes and reached to 1.38, 3.62, 4.97 and 1.45 (mIU/L) respectively.  Meeking examined the oral melatonin (5mg) effect on exercise induced growth hormone secretion in seven healthy male subjects having BMI (body mass index) of 23.9 ± 0.9 kg/m -2 . Subjects studied on two circumstances, receiving either melatonin or placebo in random order. The duration of each study was 180 minutes and began at 1400 h [24]. Zeman demonstrated that a single melatonin injection was able to affect growth hormone secretion in Japanese quail [25] and in man by [15]. This increase in growth hormone concentration was time dependant, it started increasing from 0 to 20 minutes time and it was highest at 40 minutes and then decreased to 60 minutes, where as in present study oral melatonin administration has increased growth hormone concentration for short time and then decreased with the passage of time which shows time dependant effect in adult male monkeys.

Conclusion
Present study supports that melatonin sharply increases plasma growth hormone concentrations for short time and then starts decreasing.
Consequently results in significant decrease in growth hormone with increase in time. Thus, it shows the time dependent effect of melatonin on plasma growth hormone concentrations.