Gereç ve Yöntem: Bu çalışmada 30-40 gr ağırlığında yetişkin erkek fareler kullanılmıştır. Hayvanlar her biri 5 hayvandan oluşacak şekilde 2 ana gruba ayrıldı. Kontrol gruba farelere pineal bezinin çıkarılması işlemine benzer şekilde sham operasyon yapıldı. Deney grubunda farelere pinealektomi yapıldı ve iyileşmeleri için iki hafta süre verildi. Farelere günlük i.p olarak melatonin (5 mg/kg/day, i.p.) ve luzindol (10 mg/kg/day, i.p.) uygulandı. Leptin 0.1 mL 15 mM HCl içerisinde 25 μg/fare olacak şekilde i.p olarak enjekte edildi. Kontrol hayvanlarına ise aynı hacimde çözücü maddeler uygulandı.

Bulgular: Pinealektomi farelerde gıda alımında bir azalmaya neden olmuştur. Melatonin ise pinealektomiden kaynaklanan bu gıda alımındaki azalmayı geri çevirmiştir. Melatonin antagonisti olan luzindol ise melatoninin neden olduğu bu artışı geri döndürememiştir. Leptin hem pinealektomize hemde sham-pinealektomize hayvanlarda anlamlı bir şekilde gıda alımını azaltmıştır.

Sonuç: Sonuç olarak melatonin farelerde gıda alımını hem fizyolojik hem de farmakolojik olarak artırmıştır. Melatoninin farelerde gıda alımı üzerine etkilerini hangi mekanizmalar ile gerçekleştirdiğinin belirlenmesi için daha ileri çalışmalara gerek vardır.

Melatonin may have a role in the regulation of food intake due to its effects on the hormones regulating food intake. Adipose tissue is also an important target for several hormones, including melatonin¹⁴. Melatonin has been reported to affect leptin^15,16 and ghrelin release^11,17, which are the main hormones having a role in food intake.

The present study was designed to determine whether pinealectomy has an effect on food intake in mice. The efficiency of melatonin in restoring the possible effect of pinealectomy on food intake was also investigated. To determine whether melatonin has a modulatory role in the effect of leptin on food intake, the effect of leptin on food intake in pinealectomized animals were also examined.

Melatonin (Sigma, St Louis, MO, USA) and luzindole (Acros Organics, Belgium) were dissolved in ethanol and further diluted in saline to give a final ethanol concentration of 5%. Mice received daily injections of melatonin (5 mg/kg/day, i.p.) and luzindole (10 mg/kg/day, i.p.). Mice were i.p. injected with leptin (Sigma, mouse recombinant) in a dose of 25 μg/mouse in 0.1 mL 15 mM HCL. Control animals were injected with the respective vehicle (0.5 ml 15 mM HCL and 0.3 mL 7.5 mM NaOH). The drugs were i.p. injected between 9:⁰⁰ a.m. and 10:⁰⁰ a.m.

The animals were divided into two main groups, each consisting of 5 animals. The mice of the control group were exposed to sham-pinealectomy. In the experimental group, mice were pinealectomized and allowed to recover for a period of two weeks. These animals intraperitonally (i.p.) received daily injections of melatonin for 5 days. Then, luzindole, a melatonin receptor antagonist, was i.p. given together with melatonin for 5 days and then i.p. injections of leptin were carried out for the last five days. The mice belonging to the control group were injected with only vehicles that were used for melatonin, luzindole or leptin injections. During the experimental period, food intake and body weights were measured.

Mice were anaesthetized by i.p. injection of ketamin (100 mg/kg) plus xylazine (5 mg/kg) and placed in a stereotaxic frame. Pinealectomy was performed as follows: Briefly, a longitudinal midline incision was made above the skull. By means of a dental drill, a rectangular piece of skull bone was removed. The dura was then cut. The superior sagittal sinus was ligated with surgical threads and resected. Pineal gland was exposed beneath the confluence sinuum by retracting the caudal portion of the ligated superior sagittal sinus posteriorly, and removed with a miniature forceps. The procedure was completed by returning the reflected superior sagittal sinus to its original position, re-apposing the dura and closing the wounds. Sham-pinealectomy was conducted almost identically, except pineal gland left intact. Mice were placed in individual cages. Food intake was measured between 9:⁰⁰ a.m. and 10:⁰⁰ a.m. each day. The animals were weighed daily.

The data were statistically analysed by Wilcoxon Signed Ranks Test. Level of significant was set at P<0.05. Results are expressed as g/day and presented as means±S.E.M.

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Figure 1: (A) Effects of sham–operated and vehicle on food intake in mice. (B) Effects of pinealectomy (Px), melatonin (MT) and luzindole (LZ) on food intake in mice. a,b,c P<0.05 as compared to pinealectomized mice.

Five days after melatonin injection, a combination of luzindole and melatonin injections did not have a significant reducing effect on food intake, i.e. food intake level was similar to the melatonin treatment level (Figure 1B).

After the treatment of melatonin combined with luzindole, food intake continued to decrease for another period of 5 days. Then, leptin injection caused a further significant decrease in food intake (Figure 2A-2B). There was no significant difference between leptin–injected pinealectomized and sham–operated animals compared to respective vehicle–injected groups.

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Figure 2: Effects of leptin on food intake in sham–operated (A) and pinealectomized (B) mice. *P<0.05 as compared to sham–operated or pinealectomized mice.

As shown in Figure 3, body weights increased slightly from the experimental period. Only, leptin administration caused a significant decrease in the last 5 days. The mean values of the body weights decreased from 38.12±0.1 and 36.84±0.09 to 37.96±0.27 and 34.9±0.2 g in sham–operated and pinealectomized animals, respectively.

Büyütmek İçin Tıklayın

Figure 3: The mean values of the body weights during the experimental period.

Melatonin may have a role in the regulation of food intake for many reasons. Our previous studies^15,16 and other studies^4,18, showed that melatonin decreases serum leptin levels in rats. The leptindecreasing effect of melatonin is expected to lead to higher food intake because diminution of leptin stimulates food intake. In the present experiment, the stimulatory effect of melatonin on food intake might occur due to the influence of melatonin on leptin release although the levels of serum leptin have not been measured.

The fact that melatonin has different effects on food intake in animal species may be due to its different effects on ghrelin secretion. Ghrelin is known to increase appetite of rodents⁹. In the animal species such as rats in which melatonin decreases plasma ghrelin levels^17, it has an inhibitory effect on food intake whereas melatonin treatment results in a higher food intake in the voles¹³. The stimulatory effect of melatonin on food intake in the voles may have been mediated via elevated ghrelin levels of the melatonin-treated animals.

Melatonin has been suggested to be useful for the treatment of neoplastic cachexia in human because melatonin was found to stimulate appetite by decreasing TNF secretion²⁰. Determination of the effect of melatonin on the appetite - regulating hormone such as leptin and ghrelin in human may help the exact mechanism by which melatonin exerts its effect on appetite to be established.

To determine the mechanism by which melatonin exerts its stimulatory effect on food intake in mice, a melatonin antagonist, luzindole (MT2 receptor antagonist) was used. Luzindole did not reverse the increase in food intake caused by melatonin. It was suggested that Melatonin shows an inhibitory effect on food intake in rat¹⁴ and goldfish via luzindole-sensitive melatonin receptors¹¹. Thus, stimulatory and inhibitory effects of melatonin appear to be mediated by different mechanisms.

Plasma glucose concentration has an important role in the regulation of food intake. As plasma glucose concentration increases, brain satiety mechanisms are stimulated²¹ resulting in a decrease in food intake or vice versa. It has been reported that pinealectomy leads to an increase in plasma glucose concentration^22, which may cause a decrease in food intake. So, the decrease in food intake following pinealectomy may have resulted from an increase in plasma glucose concentration, which stimulates brain satiety mechanisms. In that study, melatonin replacement has been shown to restore 24-h mean plasma glucose concentrations in pinealectomized rats.

In the present experiment, leptin administration resulted in a lower food intake in both shampinealectomized and pinealectomized rat. The efficiency of leptin in reducing food intake were similar in the two groups, which shows pinealectomy did not cause a further enhancement in the effect of leptin on food intake.

The body weights of the animals were daily measured during the experimental study. Until leptin injection, which was performed on 25 day, the body weights of the groups tended to increase. Leptin injection decreased body weights in sham–operated and pinealectomized animals. Percentage decrease in body weight was higher in the pinealectomized group (5.2%) compared with sham-operated group (0.4%). Thus, melatonin seems to prevent leptin-induced decrease in body weight.

In conclusion, melatonin was found to increase food intake physiologically and pharmacologically in mice. Further studies are needed to determine the mechanism by which melatonin exerts its effect on food intake in mice.

Acknowledgements
The authors thank Dr. Bayram Yilmaz of University at Yeditepe, Faculty of Medicine, Department of Physiology for his critical review of the manuscript.

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