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Effects of pinealectomy and exogenous melatonin on immunohistochemical ghrelin staining of arcuate nucleus and serum ghrelin leves in the rat
Neuroscience Letters 410 (2006) 132–136
Effects of pinealectomy and exogenous melatonin on immunohistochemical ghrelin staining of arcuate nucleus and serum ghrelin leves in the rat Sinan Canpolat a , Mehmet Aydin a , Abdullah Yasar a , Neriman Colakoglu b , Bayram Yilmaz a , Haluk Kelestimur a,∗ b
a Firat University Medical School, Department of Physiology, Firat University, Elazig, Turkey Firat University Medical School, Department of Histology and Embriyology ,Firat University, Elazig, Turkey
Received 24 May 2006; received in revised form 7 September 2006; accepted 17 September 2006
Abstract Although the main source of circulating ghrelin is the stomach, it is also present in physiologically relevant amounts in the hypothalamus. It is reported that pharmacological doses of melatonin decrease blood levels of ghrelin. Thus, melatonin (MT) may be a candidate for the regulation of ghrelin synthesis in the brain. This study was therefore undertaken to investigate possible effects of pinealectomy and exogenous melatonin on hypothalamic ghrelin amount. Serum ghrelin levels following pinealectomy and administration of melatonin were also sought. Adult male Sprague-Dawley rats were divided into four groups as sham-operated (SHAM), sham-operated with melatonin treatment (SHAM-MT), pinealectomised (PNX) and melatonin-treated PNX (PNX-MT) groups. Ghrelin staining in the hypothalamus was determined by immunohistochemistry. Hypothalamic ghrelin was not observed in PNX rats. Much higher staining was detected in SHAM-MT rats compared to SHAM group. Lack of effect of melatonin on hypothalamic ghrelin in PNX rats implicates that exogenous melatonin requires an intact pineal to exert its effects. Although there were remarkable changes in the immunohistochemical activity of ghrelin in the hypothalamic arcuate nucleus, neither pinealectomy nor exogenous melatonin significantly changed serum levels of ghrelin. We have demonstrated for the first time that the pineal gland may play a role in ghrelin amount in the hypothalamus. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Arcuate nucleus; Ghrelin; Pinealectomy; Melatonin; Immunohistochemistry
Ghrelin is a 28 amino acid peptide that was first purified from the rat stomach and identified as an endogenous ligand for the growth hormone secretogogue (GHS) receptor [14]. Since ghrelin stimulates growth hormone (GH) secretion [7,14], food intake and body weight gain [1,19,25], factors which affect ghrelin synthesis may have modulatory effects in the regulation of GH secretion and appetite. Ghrelin has recently been suggested to operate as negative modifier of puberty onset [10]. Melatonin (MT) has been reported to have a role in leptin secretion [5], which has generally antagonistic effects to ghrelin in terms of the above-mentioned functions. MT is a candidate for the regulation of ghrelin synthesis in the brain because it has been shown to decrease its circulating levels in the rat [18]. Therefore, we have immunohistochemically investigated possible effects of exogenous melatonin and pinealectomy on ghrelin synthesis
∗
Corresponding author. Tel.: +90 424 2370000/4671; fax: +90 424 2333770. E-mail address: [email protected] (H. Kelestimur).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.09.071
in the hypothalamic arcuate nucleus (ARN), which is the main area of neuronal groups responsible for the regulation of energy metabolism and also involved in reproduction. Serum levels of ghrelin following pinealectomy and exogenous melatonin were also sought. The studies were performed on male Sprague-Dawley rats weighing 250–300 g (Firat University Biomedical Unit). They were housed individually in plastic cages at constant room temperature (21 ◦ C) and in a 12 h light/12 h dark cycle (lights on at 07:00 h). Food and water were supplied ad libitum. In all animals, the food and water intake and urine output were recorded. The remaining food in the cage was weighed out each morning between 10:00 and 11:00 a.m. The average daily food intake was worked out by calculation of all measurements recorded throughout the experiment. The protocol was approved by the Ethics Committee of Medical School. Thirtytwo rats were equally divided into four groups. Sham controls (SHAM) received saline only (1 ml/kg). Another group of sham animals (SHAM-MT) was subcutaneously injected with daily
S. Canpolat et al. / Neuroscience Letters 410 (2006) 132–136
melatonin (0.5 mg/kg/day) for 12 days. Sixteen rats were surgically pinealectomised (PNX) under general anesthesia with ketamine 60 mg/kg plus xylazine 5 mg/kg. Half of these PNX animals were assigned to the PNX group and the remaining rats received daily injection of melatonin (0.5 mg/kg/day, s.c.) at 17:00 h for a period of 12 days (PNX-MT group). At the end, all animals were decapitated between 09:00 and 10:00 a.m., and trunk blood collected. Serum ghrelin levels were determined by using a commercial RIA kit (Linco Research Inc.) with an assay sensitivity of 1 pg/ml. The data were statistically analysed by one-way ANOVA. Level of significance was set at p < 0.05. Results (pg/ml) were presented as mean ± S.E.M. Ghrelin expression in the hypothalamic ARN was detected in 10% formaldehyde-fixed sections of rat brains. Rabbit antighrelin polyclonal antibody and streptavidin-biotin-peroxidase technique were used for immunohistochemistry. The procedure was performed under identical conditions for all sections. Paraffin sections (5 m) were dewaxed in xylene, treated with 0.1% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity, blocked with background blocker for 10 min and incubated overnight at +4 ◦ C with ghrelin antibody (1:400). The sections were then incubated with biotinylated goat anti-rabbit IgG for 30 min, followed by streptavidin-peroxidase for 30 min and 0.5 mg/ml diaminobenzidine plus 0.1% hydrogen peroxide for 3 min. Finally, they were counterstained with hematoxylin, dehydrated through an ascending ethanol series, cleared in xylene and mounted. The sections were viewed and photographed. Immunohistochemical ghrelin staining intensity in the hypothalamus of all four groups was quantitatively evaluated. To determine the number of ghrelin positive cells in the ARCs of the groups, the cells were counted by light microscopy with a 1 cm × 1 cm ocular micrometer. Values were expressed as mean ± S.D. Differences between the values were compared using the Student’s t-test. Differences were considered to be significant if p < 0.05. The cell counts were 10.2 ± 1.94 and 16.7 ± 1.37 in SHAM and SHAM-MT groups, respectively. According to these results, SHAM rats had lower (p < 0.001) ghrelin staining (Fig. 1) compared to SHAM-MT group (Fig. 2). Hypothalamic ghrelin was not immunohistochemically detected in PNX animals (Fig. 3).
Fig. 1. SHAM group: ghrelin positive immune reactivity (arrows) of neuronal cell bodies in the arcuate nucleus. Bar = 10 m.
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Fig. 2. SHAM-MT group: ghrelin positive immune reactivity (arrows) of neuronal cell bodies in the arcuate nucleus of hypothalamus. Bar = 10 m.
Fig. 3. PNX group: ghrelin negative neuronal cell bodies (arrows) in the arcuate nucleus. Bar = 10 m.
Melatonin treatment did not restore hypothalamic ghrelin in PNX rats (Fig. 4). Serum ghrelin levels are shown in Fig. 5. Neither pinealectomy nor exogenous melatonin significantly altered circulatory ghrelin levels. Melatonin administration reduced body weight in SHAM (p < 0.05) and PNX (p < 0.001) rats on the basis of body weight change (%). Body weight change (%) was
Fig. 4. PNX-MT group: ghrelin negative neuronal cell bodies (arrows) in the arcuate nucleus. Bar = 10 m.
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S. Canpolat et al. / Neuroscience Letters 410 (2006) 132–136
Fig. 5. Serum ghrelin levels in the sham controls (SHAM), sham animals injected with melatonin (SHAM-MT), pinealectomised (PNX) and pinealectomised rats injected with melatonin (PNX-MT).
significantly greater in SHAM-MT and PNX-MT groups compared to the SHAM (p < 0.05) and PNX (p < 0.001) groups. PNX group had significantly higher food intake (g/100 g) compared to the SHAM-MT and SHAM groups (p < 0.01) and PNX-MT (p < 0.05) group. There was no significant difference between the groups in terms of water intake and urine volume (Table 1). Although the main source of circulating ghrelin is the stomach [12], it is present in physiologically relevant amounts in the hypothalamic ARN in rats [16]. The results of the present study have confirmed ghrelin presence in the hypothalamic ARN. Furthermore, our study clearly demonstrates that removal of the pineal gland decreases, and exogenous melatonin increases ghrelin amount in this hypothalamic nucleus. Almost totally abolished ghrelin staining following pinealectomy implicates that modulation of ghrelin by melatonin and/or other pineal factors is also a physiological effect. Exogenous melatonin showed its effect only in SHAM rats, but not in the PNX animals, which indicates that exogenous melatonin requires an intact pineal to exert an effect on ghrelin production amount in the hypothalamus. Although there were remarkable changes in the immunohistochemical activity of ghrelin in the ARN, neither pinealectomy nor exogenous melatonin significantly changed serum levels of ghrelin. Therefore, it is conceivable that ghrelin
synthesis in the stomach does not seem to be affected by the pineal gland. In contrast to our results, pharmacological melatonin treatment was reported to decrease plasma ghrelin levels in rats [18], which means that physiological or pharmacological effects of melatonin may be different. Lack of significant changes in serum ghrelin levels following pinealectomy may result from the extensive melatonin synthesis in the gastrointestinal system. It is known that gastrointestinal system includes melatonin independent of the pineal gland [13], which may stimulate ghrelin synthesis peripherally and compansate a possible decline caused by pinealectomy. The interaction between the pineal gland and ghrelin secretion may be of importance for various central regulations. The ARN has been identified as a site with one of the highest concentrations of the GHS-R [20,24], and it is thus possible that locally produced ghrelin might activate these receptors. The decline in GH secretion with aging may result from lack of a melatonin-stimulating effect on ghrelin synthesis since melatonin also shows remarkable decreases with aging. Exogenous melatonin has been shown to stimulate GH secretion [11]. MT may exert its effect by stimulating hypothalamic ghrelin amount. The interaction between the pineal gland and ghrelin synthesis may also be important in the regulation of appetite. Immunohistochemical studies have indicated that ghrelin-containing neural cells are present in the ARN [16], a hypothalamic region involved in regulation of appetite. These ghrelin-containing cells send efferent fibers to the neurones which contain neuropeptide Y (NPY) and agouti-related peptide (AgRP), and stimulate the release of these orexigenic peptides [6]. The hypothalamic ARN, the main active site of ghrelin is also the target site for leptin [9], an appetite-suppressing hormone released from adipose tissues. Leptin inhibits the release of NPY and AgRP, which are synthesized in the same neurones in the ARN [3,4,17,23], and thus it antagonizes appetite-stimulating effect of ghrelin. In our study, body weight gain and food intake were significantly higher in the PNX group, which showed no immunohistochemical ghrelin activity in the hypothalamus or significant difference in serum ghrelin levels. On the contrary, melatonin administration reduced body weight and food intake in the SHAM and PNX rats. Since ghrelin is known to stimulate food intake, the pineal
Table 1 The effects of pinealectomy and exogenous melatonin on the body weight change, food intake, water intake and urine output SHAM (g)a
Body weight Body weight (g)b Body weight change (%) Food intake (g/100 g) Water intake (ml/100 g) Urine volume (ml/100 g) * p < 0.05, ** p < 0,01 a b c d e
312.5 313.3 0.23 5.86 9.99 3.82
± ± ± ± ± ±
compared with SHAM animals. Body weight at the beginning of the experiment. Body weight at the end of the experiment. p < 0.001 compared with PNX animals. p < 0.01 compared with PNX animals. p < 0.05 compared with PNX animals.
SHAM + MT 5.9 6.2 0.25 0.06 0.19 0.07
316.5 312.6 −1.22 5.76 9.94 3.70
± ± ± ± ± ±
2.0 1.8 0.38*,c 0.05d 0.12 0.07
PNX 310.3 314.5 1.36 6.15 10.01 3.80
PNX + MT ± ± ± ± ± ±
4.0 4.5 0.43 0.05** 0.19 0.07
306.9 303.8 −1.2 5.94 10.06 3.50
± ± ± ± ± ±
4.8 4.2 0.38*,c 0.05e 0.11 0.06
S. Canpolat et al. / Neuroscience Letters 410 (2006) 132–136
gland or melatonin may exert its appetite-decreasing effect by other mechanisms rather than through modulation of ghrelin. One important consequence of decreased hypothalamic ghrelin resulting from lack of melatonin may be related to the onset of puberty, since ghrelin has been suggested to operate as a negative modifier of puberty onset [10]. MT is generally thought to have an inhibitory effect on the puberty onset [2,21,22] It may exert its puberty-delaying effect by leading to an increase in hypothalamic ghrelin production. MT has been shown to stimulate NPY synthesis in rat hypothalamic ARN [8] which also potentiates this effect of ghrelin. The pineal hormone has also been shown to decrease leptin production in the pituitary gland [15]. Leptin is suggested to have a permissive effect on the onset of puberty in contrast to the action of ghrelin. Thus, melatonin may have an inhibitory effect on the puberty onset by causing not only an increase in ghrelin synthesis but also a decrease in leptin production. We demonstrated for the first time that the pineal gland may play a role in the amount of hypothalamic ghrelin, which provides new evidence regarding the role of MT in energy homeostasis and the onset of puberty. In view of the relevant literature including our recent studies, it can be postulated that a decrease in ghrelin and increase leptin production resulting from melatonin decline near puberty may have a permissive role in the transition into puberty, since ghrelin is a negative and leptin a positive indicator of nutritional status and also pubertal maturation, respectively. It can also be speculated that central and peripheral production of ghrelin may have different functions and be regulated by different mechanisms. Peripheral ghrelin may be mainly responsible for the regulation of energy homeostasis whereas central ghrelin may be involved in especially puberty onset and growth hormone synthesis. Acknowledgement This work was supported by a grant from TUBITAK (Project No. 104S362). References [1] A. Asakawa, A. Inui, T. Kaga, H. Yuzuriha, T. Nagata, N. Ueno, S. Makino, M. Fujimiya, A. Niijima, M.A. Fujino, M. Kasuga, Ghrelin is an appetitestimulatory signal from stomach with structural resemblance to motilin, Gastroenterology 120 (2001) 337–345. [2] A. Attanasio, F. Borrelli, D. Gupta, Circadian rhythms in serum melatonin from infancy to adolescence, J. Clin. Endocrinol. Metab. 61 (1985) 388–390. [3] W.A. Banks, A.J. Kastin, W. Hunag, J.P. Jaspan, L.M. Maness, Leptin enters the brain by a saturable system independent of insulin, Peptides 17 (1997) 305–311. [4] D.G. Baskin, M.W. Schwartz, R.J. Seeley, S.C. Woods, D. Porte, J.F. Breininger, Z. Jonak, J. Schaefer, M. Krouse, C. Burghdat, L.A. Campfield, P. Burn, J.P. Kochan, Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus, J. Histochem. Cytochem. 47 (1999) 353–362. [5] S. Canpolat, S. Sandal, B. Yilmaz, A. Yasar, S. Kutlu, G. Baydas, H. Kelestimur, Effects of pinealectomy and exogenous melatonin on serum leptin levels in male rat, Eur. J. Pharmacol. 428 (2001) 145–148.
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[6] H.Y. Chen, M.E. Trumbauer, A.S. Chen, D.T. Weingarth, J.R. Adams, E.G. Frazier, Z. Shen, D.J. Marsh, S.D. Feighner, X.M. Guan, Z. Ye, R.P. Nargund, R.G. Smith, L.H. Van der Ploeg, A.D. Howard, D.J. MacNeil, S. Qian, Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein, Endocrinology 145 (2004) 2607– 2612. [7] Y. Date, N. Murakami, M. Kojima, T. Kuroiwa, S. Matsukura, K. Kangawa, M. Nakazato, Central effects of a novel acylated peptide, ghrelin, on growth hormone release in rats, Biochem. Biophys. Res. Commun. 275 (2000) 477–480. [8] E. Diaz, L. Debeljuk, A. Arce, A.I. Esquifino, B. Diaz, Prenatal melatonin exposure affects luteinizing hormone and hypothalamic and striatal neuropeptide Y in the male rat offspring, Neurosci. Lett. 292 (2000) 143– 146. [9] J.K. Elmquist, C.F. Elias, C.B. Saper, From lesions to leptin: hypothalamic control of food intake and body weight, Neuron 22 (1999) 221–232. [10] R. Fern´andez-Fern´andez, V.M. Navarro, M.L. Barreiro, E.M. Vigo, S. Tovar, A.V. Sirotkin, F.F. Casanueva, E. Aguilar, C. Dieguez, L. Pinilla, M. Tena-Sempere, Effects of chronic hyperghrelinemia on puberty onset and pregnancy outcome in the rat, Endocrinology 146 (2005) 3018– 3025. [11] M.L. Forsling, M.J. Wheeler, A.J. Williams, The effect of melatonin administration on pituitary hormone secretion in man, Clin. Endocrinol. (Oxf.) 51 (1999) 637–642. [12] H. Hosoda, M. Kojima, T. Mizushima, S. Shimizu, K. Kangawa, Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing, J. Biol. Chem. 278 (2003) 64–70. [13] G. Huether, B. Poeggeler, A. Reimer, A. George, Effect of tryptophan administration on circulating melatonin levels in chicks and rats: evidence for stimulation of melatonin synthesis and release in the gastrointestinal tract, Life Sci. 51 (1992) 945–953. [14] M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K. Kangawa, Ghrelin is a growth-hormone-releasing peptide from stomach, Nature 402 (1999) 656–660. [15] I. Kus, M. Sarsilmaz, N. Colakoglu, A. Kukne, O.A. Ozen, B. Yilmaz, H. Kelestimur, Pinealectomy increases and exogenous melatonin decreases leptin production in rat anterior pituitary cells: an immunohistochemical study, Physiol. Res. 53 (2004) 403–408. [16] S. Lu, J.L. Guan, Q.P. Wang, K. Uehara, S. Yamada, N. Goto, Y. Date, M. Nakazato, M. Kojima, K. Kangawa, S. Shioda, Immunocytochemical observation of ghrelin containing neurons in the rat arcuate nucleus, Neurosci. Lett. 321 (2002) 157–160. [17] J.G. Mercer, N. Hoggard, L.M. Williams, C.B. Lawrence, L.T. Hannah, P.J. Morgan, P. Trayhurn, Coexpression of leptin receptor and preproneuropeptide Y mRNA in ARC of mouse hypothalamus, J. Neuroendocrinol. 8 (1996) 733–735. [18] A.M. Mustonen, P. Nieminen, H. Hyvarinen, Preliminary evidence that pharmacologic melatonin treatment decreases rat ghrelin levels, Endocrine 16 (2001) 43–46. [19] M. Nakazato, N. Murakami, Y. Date, M. Kojima, H. Matsuo, K. Kangawa, S. Matsukura, A role for ghrelin in the central regulation of feeding, Nature 409 (2001) 194–198. [20] G.S. Tannenbaum, M. Lapointe, A. Beaudet, A.D. Howard, Expression of growth hormone secretagogue-receptors by growth hormone-releasing hormone neurons in the mediobasal hypothalamus, Endocrinology 139 (1998) 4420–4423. [21] F. Waldhauser, P.A. Boepple, M. Schemper, M.J. Mansfield, W.F. Crowley Jr., Serum melatonin in central precocious puberty is lower than in age-matched prepubertal children, J. Clin. Endocrinol. Metab. 73 (1991) 793–796. [22] F. Waldhauser, H. Frisch, M. Waldhauser, G. Weiszenbacher, U. Zeitlhuber, R.J. Wurtman, Fall in nocturnal serum melatonin during prepuberty and pubescence, Lancet 18 (1985) 362–365. [23] Q. Wang, C. Bing, K. Al-Barazanji, D.E. Mossakowaska, X.M. Wang, D.L. McBay, W.A. Neville, M. Taddayon, L. Pickavance, S. Dryden, M.E. Thomas, M.T. McHale, I.S. Gloyer, S. Wilson, R. Buckingham, J.R. Arch, P. Trayhurn, G. Williams, Interactions between leptin and hypothalamic
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Effects of pinealectomy and exogenous melatonin on immunohistochemical ghrelin staining of arcuate nucleus and serum ghrelin leves in the rat Sinan Canpolat a , Mehmet Aydin a , Abdullah Yasar a , Neriman Colakoglu b , Bayram Yilmaz a , Haluk Kelestimur a,∗ b
a Firat University Medical School, Department of Physiology, Firat University, Elazig, Turkey Firat University Medical School, Department of Histology and Embriyology ,Firat University, Elazig, Turkey
Received 24 May 2006; received in revised form 7 September 2006; accepted 17 September 2006
Abstract Although the main source of circulating ghrelin is the stomach, it is also present in physiologically relevant amounts in the hypothalamus. It is reported that pharmacological doses of melatonin decrease blood levels of ghrelin. Thus, melatonin (MT) may be a candidate for the regulation of ghrelin synthesis in the brain. This study was therefore undertaken to investigate possible effects of pinealectomy and exogenous melatonin on hypothalamic ghrelin amount. Serum ghrelin levels following pinealectomy and administration of melatonin were also sought. Adult male Sprague-Dawley rats were divided into four groups as sham-operated (SHAM), sham-operated with melatonin treatment (SHAM-MT), pinealectomised (PNX) and melatonin-treated PNX (PNX-MT) groups. Ghrelin staining in the hypothalamus was determined by immunohistochemistry. Hypothalamic ghrelin was not observed in PNX rats. Much higher staining was detected in SHAM-MT rats compared to SHAM group. Lack of effect of melatonin on hypothalamic ghrelin in PNX rats implicates that exogenous melatonin requires an intact pineal to exert its effects. Although there were remarkable changes in the immunohistochemical activity of ghrelin in the hypothalamic arcuate nucleus, neither pinealectomy nor exogenous melatonin significantly changed serum levels of ghrelin. We have demonstrated for the first time that the pineal gland may play a role in ghrelin amount in the hypothalamus. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Arcuate nucleus; Ghrelin; Pinealectomy; Melatonin; Immunohistochemistry
Ghrelin is a 28 amino acid peptide that was first purified from the rat stomach and identified as an endogenous ligand for the growth hormone secretogogue (GHS) receptor [14]. Since ghrelin stimulates growth hormone (GH) secretion [7,14], food intake and body weight gain [1,19,25], factors which affect ghrelin synthesis may have modulatory effects in the regulation of GH secretion and appetite. Ghrelin has recently been suggested to operate as negative modifier of puberty onset [10]. Melatonin (MT) has been reported to have a role in leptin secretion [5], which has generally antagonistic effects to ghrelin in terms of the above-mentioned functions. MT is a candidate for the regulation of ghrelin synthesis in the brain because it has been shown to decrease its circulating levels in the rat [18]. Therefore, we have immunohistochemically investigated possible effects of exogenous melatonin and pinealectomy on ghrelin synthesis
∗
Corresponding author. Tel.: +90 424 2370000/4671; fax: +90 424 2333770. E-mail address: [email protected] (H. Kelestimur).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.09.071
in the hypothalamic arcuate nucleus (ARN), which is the main area of neuronal groups responsible for the regulation of energy metabolism and also involved in reproduction. Serum levels of ghrelin following pinealectomy and exogenous melatonin were also sought. The studies were performed on male Sprague-Dawley rats weighing 250–300 g (Firat University Biomedical Unit). They were housed individually in plastic cages at constant room temperature (21 ◦ C) and in a 12 h light/12 h dark cycle (lights on at 07:00 h). Food and water were supplied ad libitum. In all animals, the food and water intake and urine output were recorded. The remaining food in the cage was weighed out each morning between 10:00 and 11:00 a.m. The average daily food intake was worked out by calculation of all measurements recorded throughout the experiment. The protocol was approved by the Ethics Committee of Medical School. Thirtytwo rats were equally divided into four groups. Sham controls (SHAM) received saline only (1 ml/kg). Another group of sham animals (SHAM-MT) was subcutaneously injected with daily
S. Canpolat et al. / Neuroscience Letters 410 (2006) 132–136
melatonin (0.5 mg/kg/day) for 12 days. Sixteen rats were surgically pinealectomised (PNX) under general anesthesia with ketamine 60 mg/kg plus xylazine 5 mg/kg. Half of these PNX animals were assigned to the PNX group and the remaining rats received daily injection of melatonin (0.5 mg/kg/day, s.c.) at 17:00 h for a period of 12 days (PNX-MT group). At the end, all animals were decapitated between 09:00 and 10:00 a.m., and trunk blood collected. Serum ghrelin levels were determined by using a commercial RIA kit (Linco Research Inc.) with an assay sensitivity of 1 pg/ml. The data were statistically analysed by one-way ANOVA. Level of significance was set at p < 0.05. Results (pg/ml) were presented as mean ± S.E.M. Ghrelin expression in the hypothalamic ARN was detected in 10% formaldehyde-fixed sections of rat brains. Rabbit antighrelin polyclonal antibody and streptavidin-biotin-peroxidase technique were used for immunohistochemistry. The procedure was performed under identical conditions for all sections. Paraffin sections (5 m) were dewaxed in xylene, treated with 0.1% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity, blocked with background blocker for 10 min and incubated overnight at +4 ◦ C with ghrelin antibody (1:400). The sections were then incubated with biotinylated goat anti-rabbit IgG for 30 min, followed by streptavidin-peroxidase for 30 min and 0.5 mg/ml diaminobenzidine plus 0.1% hydrogen peroxide for 3 min. Finally, they were counterstained with hematoxylin, dehydrated through an ascending ethanol series, cleared in xylene and mounted. The sections were viewed and photographed. Immunohistochemical ghrelin staining intensity in the hypothalamus of all four groups was quantitatively evaluated. To determine the number of ghrelin positive cells in the ARCs of the groups, the cells were counted by light microscopy with a 1 cm × 1 cm ocular micrometer. Values were expressed as mean ± S.D. Differences between the values were compared using the Student’s t-test. Differences were considered to be significant if p < 0.05. The cell counts were 10.2 ± 1.94 and 16.7 ± 1.37 in SHAM and SHAM-MT groups, respectively. According to these results, SHAM rats had lower (p < 0.001) ghrelin staining (Fig. 1) compared to SHAM-MT group (Fig. 2). Hypothalamic ghrelin was not immunohistochemically detected in PNX animals (Fig. 3).
Fig. 1. SHAM group: ghrelin positive immune reactivity (arrows) of neuronal cell bodies in the arcuate nucleus. Bar = 10 m.
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Fig. 2. SHAM-MT group: ghrelin positive immune reactivity (arrows) of neuronal cell bodies in the arcuate nucleus of hypothalamus. Bar = 10 m.
Fig. 3. PNX group: ghrelin negative neuronal cell bodies (arrows) in the arcuate nucleus. Bar = 10 m.
Melatonin treatment did not restore hypothalamic ghrelin in PNX rats (Fig. 4). Serum ghrelin levels are shown in Fig. 5. Neither pinealectomy nor exogenous melatonin significantly altered circulatory ghrelin levels. Melatonin administration reduced body weight in SHAM (p < 0.05) and PNX (p < 0.001) rats on the basis of body weight change (%). Body weight change (%) was
Fig. 4. PNX-MT group: ghrelin negative neuronal cell bodies (arrows) in the arcuate nucleus. Bar = 10 m.
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S. Canpolat et al. / Neuroscience Letters 410 (2006) 132–136
Fig. 5. Serum ghrelin levels in the sham controls (SHAM), sham animals injected with melatonin (SHAM-MT), pinealectomised (PNX) and pinealectomised rats injected with melatonin (PNX-MT).
significantly greater in SHAM-MT and PNX-MT groups compared to the SHAM (p < 0.05) and PNX (p < 0.001) groups. PNX group had significantly higher food intake (g/100 g) compared to the SHAM-MT and SHAM groups (p < 0.01) and PNX-MT (p < 0.05) group. There was no significant difference between the groups in terms of water intake and urine volume (Table 1). Although the main source of circulating ghrelin is the stomach [12], it is present in physiologically relevant amounts in the hypothalamic ARN in rats [16]. The results of the present study have confirmed ghrelin presence in the hypothalamic ARN. Furthermore, our study clearly demonstrates that removal of the pineal gland decreases, and exogenous melatonin increases ghrelin amount in this hypothalamic nucleus. Almost totally abolished ghrelin staining following pinealectomy implicates that modulation of ghrelin by melatonin and/or other pineal factors is also a physiological effect. Exogenous melatonin showed its effect only in SHAM rats, but not in the PNX animals, which indicates that exogenous melatonin requires an intact pineal to exert an effect on ghrelin production amount in the hypothalamus. Although there were remarkable changes in the immunohistochemical activity of ghrelin in the ARN, neither pinealectomy nor exogenous melatonin significantly changed serum levels of ghrelin. Therefore, it is conceivable that ghrelin
synthesis in the stomach does not seem to be affected by the pineal gland. In contrast to our results, pharmacological melatonin treatment was reported to decrease plasma ghrelin levels in rats [18], which means that physiological or pharmacological effects of melatonin may be different. Lack of significant changes in serum ghrelin levels following pinealectomy may result from the extensive melatonin synthesis in the gastrointestinal system. It is known that gastrointestinal system includes melatonin independent of the pineal gland [13], which may stimulate ghrelin synthesis peripherally and compansate a possible decline caused by pinealectomy. The interaction between the pineal gland and ghrelin secretion may be of importance for various central regulations. The ARN has been identified as a site with one of the highest concentrations of the GHS-R [20,24], and it is thus possible that locally produced ghrelin might activate these receptors. The decline in GH secretion with aging may result from lack of a melatonin-stimulating effect on ghrelin synthesis since melatonin also shows remarkable decreases with aging. Exogenous melatonin has been shown to stimulate GH secretion [11]. MT may exert its effect by stimulating hypothalamic ghrelin amount. The interaction between the pineal gland and ghrelin synthesis may also be important in the regulation of appetite. Immunohistochemical studies have indicated that ghrelin-containing neural cells are present in the ARN [16], a hypothalamic region involved in regulation of appetite. These ghrelin-containing cells send efferent fibers to the neurones which contain neuropeptide Y (NPY) and agouti-related peptide (AgRP), and stimulate the release of these orexigenic peptides [6]. The hypothalamic ARN, the main active site of ghrelin is also the target site for leptin [9], an appetite-suppressing hormone released from adipose tissues. Leptin inhibits the release of NPY and AgRP, which are synthesized in the same neurones in the ARN [3,4,17,23], and thus it antagonizes appetite-stimulating effect of ghrelin. In our study, body weight gain and food intake were significantly higher in the PNX group, which showed no immunohistochemical ghrelin activity in the hypothalamus or significant difference in serum ghrelin levels. On the contrary, melatonin administration reduced body weight and food intake in the SHAM and PNX rats. Since ghrelin is known to stimulate food intake, the pineal
Table 1 The effects of pinealectomy and exogenous melatonin on the body weight change, food intake, water intake and urine output SHAM (g)a
Body weight Body weight (g)b Body weight change (%) Food intake (g/100 g) Water intake (ml/100 g) Urine volume (ml/100 g) * p < 0.05, ** p < 0,01 a b c d e
312.5 313.3 0.23 5.86 9.99 3.82
± ± ± ± ± ±
compared with SHAM animals. Body weight at the beginning of the experiment. Body weight at the end of the experiment. p < 0.001 compared with PNX animals. p < 0.01 compared with PNX animals. p < 0.05 compared with PNX animals.
SHAM + MT 5.9 6.2 0.25 0.06 0.19 0.07
316.5 312.6 −1.22 5.76 9.94 3.70
± ± ± ± ± ±
2.0 1.8 0.38*,c 0.05d 0.12 0.07
PNX 310.3 314.5 1.36 6.15 10.01 3.80
PNX + MT ± ± ± ± ± ±
4.0 4.5 0.43 0.05** 0.19 0.07
306.9 303.8 −1.2 5.94 10.06 3.50
± ± ± ± ± ±
4.8 4.2 0.38*,c 0.05e 0.11 0.06
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gland or melatonin may exert its appetite-decreasing effect by other mechanisms rather than through modulation of ghrelin. One important consequence of decreased hypothalamic ghrelin resulting from lack of melatonin may be related to the onset of puberty, since ghrelin has been suggested to operate as a negative modifier of puberty onset [10]. MT is generally thought to have an inhibitory effect on the puberty onset [2,21,22] It may exert its puberty-delaying effect by leading to an increase in hypothalamic ghrelin production. MT has been shown to stimulate NPY synthesis in rat hypothalamic ARN [8] which also potentiates this effect of ghrelin. The pineal hormone has also been shown to decrease leptin production in the pituitary gland [15]. Leptin is suggested to have a permissive effect on the onset of puberty in contrast to the action of ghrelin. Thus, melatonin may have an inhibitory effect on the puberty onset by causing not only an increase in ghrelin synthesis but also a decrease in leptin production. We demonstrated for the first time that the pineal gland may play a role in the amount of hypothalamic ghrelin, which provides new evidence regarding the role of MT in energy homeostasis and the onset of puberty. In view of the relevant literature including our recent studies, it can be postulated that a decrease in ghrelin and increase leptin production resulting from melatonin decline near puberty may have a permissive role in the transition into puberty, since ghrelin is a negative and leptin a positive indicator of nutritional status and also pubertal maturation, respectively. It can also be speculated that central and peripheral production of ghrelin may have different functions and be regulated by different mechanisms. Peripheral ghrelin may be mainly responsible for the regulation of energy homeostasis whereas central ghrelin may be involved in especially puberty onset and growth hormone synthesis. Acknowledgement This work was supported by a grant from TUBITAK (Project No. 104S362). References [1] A. Asakawa, A. Inui, T. Kaga, H. Yuzuriha, T. Nagata, N. Ueno, S. Makino, M. Fujimiya, A. Niijima, M.A. Fujino, M. Kasuga, Ghrelin is an appetitestimulatory signal from stomach with structural resemblance to motilin, Gastroenterology 120 (2001) 337–345. [2] A. Attanasio, F. Borrelli, D. Gupta, Circadian rhythms in serum melatonin from infancy to adolescence, J. Clin. Endocrinol. Metab. 61 (1985) 388–390. [3] W.A. Banks, A.J. Kastin, W. Hunag, J.P. Jaspan, L.M. Maness, Leptin enters the brain by a saturable system independent of insulin, Peptides 17 (1997) 305–311. [4] D.G. Baskin, M.W. Schwartz, R.J. Seeley, S.C. Woods, D. Porte, J.F. Breininger, Z. Jonak, J. Schaefer, M. Krouse, C. Burghdat, L.A. Campfield, P. Burn, J.P. Kochan, Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus, J. Histochem. Cytochem. 47 (1999) 353–362. [5] S. Canpolat, S. Sandal, B. Yilmaz, A. Yasar, S. Kutlu, G. Baydas, H. Kelestimur, Effects of pinealectomy and exogenous melatonin on serum leptin levels in male rat, Eur. J. Pharmacol. 428 (2001) 145–148.
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