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Does ghrelin level change after epileptic seizure in rats?
Seizure 20 (2011) 347–349
Contents lists available at ScienceDirect
Seizure journal homepage: www.elsevier.com/locate/yseiz
Short communication
Does ghrelin level change after epileptic seizure in rats? Z. Ataie b, M.G. Golzar b, Sh. Babri a, H. Ebrahimi b, G. Mohaddes a,b,* a b
Neuroscience Research Centre of Tabriz University of Medical Sciences, Iran Drug Applied Research Centre of Tabriz University of Medical Sciences, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 June 2010 Received in revised form 29 December 2010 Accepted 10 January 2011
Aim: Epilepsy is one of the most common neurologic problems worldwide. A relationship between epilepsy and hormones has been demonstrated. This study was designed to investigate the effect of seizure on blood ghrelin level. Methods: Twenty male Wistar rats were divided into two groups. The control group received saline and the pentylenetetrazole (PTZ) group received a single convulsive dose (50 mg/kg) of PTZ. Thirty minutes later blood samples were collected and acylated and unacylated ghrelin levels in the plasma were assayed. Results: Acylated or active form of ghrelin decreased significantly (p < 0.05) after a PTZ-induced seizure, but the reduction of unacylated and total blood ghrelin levels failed to reach statistical significance. Conclusion: These findings may reflect that PTZ-induced epilepsy decreases AG of plasma. ß 2011 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
Keywords: Seizure Ghrelin PTZ
1. Introduction Epilepsy is one of the most common neurologic problems worldwide1 and characterized by recurrent unprovoked seizures.2 The relationship between epilepsy and the endocrine system has been investigated by neurologists, endocrinologists, and basic scientists.3 Some studies have suggested a relationship between epilepsy and GH, sex hormones, the NPY system, nesfatin-1, ghrelin, and chromogranin A.4 It is also reported that mean daily blood concentrations of ACTH, cortisol and beta-endorphin in patients with epilepsy are higher in comparison with those of the healthy volunteers.5 Ghrelin is a 28-amino acid motilin-related peptide. The peptide is characterized by the presence of an n-octanoylation on the hydroxy group of serine in position 3.6 The octanoylated peptide hormone is produced and secreted by A-like cells within the oxyntic glands of the stomach.7 Ghrelin circulates in the bloodstream in two major forms: acylated (AG; or n-octanoylated) and unacylated (UAG; or desoctanoylated or desacylated).8 Acylation is a post-transcriptional modification that is essential for binding to the growth hormone secretagogue receptor (GHS-R) to a point that ghrelin is originally supposed to be biologically active only in acylated form.6 The acyltransferase that catalyzes ghrelin octanoylation has recently been identified as ghrelin O-acyltransferase (GOAT).9
Ghrelin exerts neuroendocrine effects of eliciting growth hormone release, regulating appetite and energy metabolism, and carries many other functions in a variety of tissues and organs such as gastroenteropancreatic, cardiovascular, reproduction, and immune systems.10 A definite relationship between ghrelin and seizure has also been demonstrated in animals and humans, although the results are controversial.4 Some research has suggested that serum ghrelin levels are enhanced in patients with epilepsy11 and in prepubertal epileptic children treated with valproic acid (VPA).12 On the other hand, some studies have shown that ghrelin levels decreased in epileptic adults13 and normalweight prepubertal children14 taking VPA treatment. In addition, Aydin et al. reported that serum and saliva ghrelin levels were lower in epileptic patients before treatment than in controls.2 To clarify these controversies, evaluation of blood ghrelin levels immediately after induced epileptic seizures in animals is beneficiary. Therefore, the purposes of this study were: (1) to determine AG and UAG levels in blood samples from pentylenetetrazole (PTZ)induced epileptic rats using an enzyme-linked immunosorbent assay (ELISA); (2) to determine any association between ghrelin and epilepsy and clarify the controversial results reported for this hormone. 2. Materials and methods
* Corresponding author at: Dept of Physiology, Faculty of Medicine, Tabriz University of Medical Sciences, Golgasht Ave., Tabriz, Iran. Tel.: +98 411 3364664; fax: +98 411 3364664. E-mail address: [email protected] (G. Mohaddes).
2.1. Chemicals and instruments PTZ was purchased from Tocris Bioscience, UK (Bristol). Blood AG and UAG levels were determined by ELISA using an ELISA reader
1059-1311/$ – see front matter ß 2011 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.seizure.2011.01.001
348
[()TD$FIG]
Z. Ataie et al. / Seizure 20 (2011) 347–349
(Stat Fax, Awarness, USA) and AG and UAG kits (Cat. No. RD 394062400R and Cat. No. RD394063400R, Biovendor co, Czech Republic). 2.2. Animals All experimental procedures were approved by the Regional Ethics Committee of Tabriz University of Medical Sciences. The experiments were performed on male Wistar rats (220–260 g) with food and water available ad libitum, except during the 30 min of procedure. Animals were maintained under controlled temperature (21–23 8C) and light (12 h light, 12 h dark) and with a humidity of 5–10%. All the experimental procedures were carried out between 08.00 and 10.00 a.m. 2.3. Experimental design Twenty rats were divided into two groups: control (n = 10) and PTZ (n = 10). The PTZ group received a single convulsive dose (50 mg/kg) of PTZ and the control group received the same volume of saline, intraperitoneally (i.p.). Seizures were elicited in all PTZreceiving rats. After 30 min of the injections, blood samples were taken from the animals’ heart and collected in tubes containing EDTA and p-hydroxymercuribenzoic acid (1 mM in the final sample volume) to prevent the degradation of AG by protease. Samples were immediately centrifuged at 3500 rpm for 10 min at +4 8C and then, the plasma was transferred into separate tubes. Samples were immediately assayed.
Fig. 2. Effect of PTZ (i.p.) induced seizure on unacylated blood ghrelin (pg/mL) level. Data are expressed as mean standard error of the mean (SEM). n = 10 animals per group.
[()TD$FIG]
2.4. Statistical analysis SPSS 13.0 software was used for statistical comparisons of data, and data expressed as the means SEM. Differences in AG, UAG and total ghrelin levels between the groups were analyzed by independent Student’s t-test (one- or two-tailed) and p < 0.05 was accepted as significant.
Fig. 3. Effect of PTZ (i.p.) induced seizure on total blood ghrelin (pg/mL) level. Data are expressed as mean standard error of the mean (SEM). n = 10 animals per group.
3. Results
4. Discussion
As shown in Fig. 1, an independent Student’s t-test indicated that AG levels of blood decreased significantly (p < 0.05) after i.p. injection of PTZ, compared to saline groups. Figs. 2 and 3 illustrate the effect of PTZ-induced seizure on UAG and total ghrelin levels of blood, respectively. Independent Student’s t-tests indicated that UAG and total ghrelin levels of
To our knowledge, this is the first study to examine blood ghrelin levels in PTZ-induced seizures in rats. The results of this study showed that active form or acylated blood ghrelin levels decreased significantly after PTZ-induced seizures. UAG and total blood ghrelin levels also decreased although they failed to reach statistical significance. The results for the blood ghrelin response to the PTZ-induced seizure showed that measuring the concentration of ghrelin is a useful method for studying plasma ghrelin changes over short time. In recent years, many researchers have focused on the relationships between epilepsy and the endocrine system since epileptic seizures are related to the temporary changes in hormone secretion.2 A definite relationship between ghrelin and seizures has been already demonstrated in animals and humans, although the results are controversial. Some authors have demonstrated that ghrelin levels were increased in children treated with VPA in comparison to healthy controls.12,15 Berilgen et al. also reported that serum ghrelin levels were higher in epilepsy patients who received antiepileptic drug therapy than a control group.11 Contradictorily, circulating ghrelin levels were significantly lower in epileptic patients treated with VPA in pubertal13 and prepubertal periods.14,16 Some studies also showed that both acylated and deacylated forms of serum and salivary ghrelin were depressed in epileptic patients before and after specific treatment with respect to normal controls.2,4
[()TD$FIG]
Fig. 1. Effect of PTZ (i.p.) induced seizure on acylated blood ghrelin (pg/mL) level. Data are expressed as mean standard error of the mean (SEM). n = 10 animals per group. *p < 0.05 compared with the control group.
blood did not change significantly with respect to control group. AG/UAG ratio also was not modified by PTZ-induced seizures.
Z. Ataie et al. / Seizure 20 (2011) 347–349
Our results confirm earlier studies that document that the ghrelin level of blood decreases with epilepsy. In addition our results showed that AG decreased significantly but the reduction of UAG and total levels were not significant. Two possibilities may be considered in this regard: (1) ghrelin is reduced by a feed back system or (2) uptake of AG is increased by CNS structures to modulate epileptic discharges. In accordance with the first possibility, some hormones can reduce the production, acylation or secretion of ghrelin such as somatostatin and leptin. Somatostatin, a peptide produced in the brain, gastrointestinal tract, and pancreas, exerts a broad spectrum of biological activities through paracrine, endocrine, and neuroendocrine mechanisms.17 It is preferentially released from neurons under conditions of elevated activity (i.e. bursting or high frequency neuronal activation), e.g. during seizures.18 It is also previously reported that a single intravenous injection of somatostatin reduces the systemic plasma concentration of ghrelin in rats17 and human healthy volunteers.19 In addition, somatostatin decreases GOAT expression,20 enzyme which catalyzes acylation of ghrelin.9 Therefore, it is possible that blood AG reduction is due to somatostatin released from one of its sources during PTZ-induced seizure. Moreover, a faster rebound of UAG level respect to AG (due to inhibitory effect of somatostatin on GOAT expression) may be occurred. Some studies suggest that kindled seizures induce high serum leptin levels in rats.21,22 In this context, kindled female rats had leptin levels approximately twice as high as the controls during diestrus phase.22 On the other hand, ghrelin is negatively regulated by leptin23 and physiological concentrations of leptin and a moderate hyperleptinemia directly inhibit ghrelin secretion from the isolated perfused rat stomach.24 Therefore, ghrelin reduction might be due to release of leptin during PTZ-induced seizure. In a discussion of the second possibility, human ghrelin exhibits saturable binding and endocytosis in the RBE4 rat cerebral microvessel endothelial cell line.25 On the other hand, blood brain barrier mechanisms regulating ghrelin accumulation by brain may be influenced by pathophysiological events.26 It has also been shown that ghrelin or its agonists can be considered as an antiepileptic agent in rodents.27,28 Therefore, reduction of plasma AG after PTZ-induced seizure could be attributed to its uptake by brain to represent an anti-epileptic effect. In this mater, evaluation of brain ghrelin levels immediately after induced epileptic seizures in animals is beneficiary. Finally, proteolysis of AG29 and degradation into non-UAG metabolites30 could be occurred during seizures. In conclusion, the results obtained from our study showed that AG decreased significantly in blood after PTZ-induced seizures. In the future, a better understanding of the mechanisms responsible for seizure-induced alterations in hormone levels of plasma may help to prevent these changes. Acknowledgment This study was financially supported by the Neuroscience Research Centre of Tabriz University of Medical Sciences. The article is derived from the MSc dissertation of Ms Zohre Ataie entitled ‘‘Evaluation of the antiepileptic effect of intrahippocampal injection of ghrelin through GABAB receptors in adult male rats’’. References 1. Chang BS, Lowenstein DH. Mechanisms of disease: epilepsy. N Engl J Med 2003;349:1257–66.
349
2. Aydin S, Dag E, Ozkan Y, Erman F, Dagli AF, Kilic N, et al. Nesfatin-1 and ghrelin levels in serum and saliva of epileptic patients: hormonal changes can have a major effect on seizure disorders. Mol Cell Biochem 2009;328:49–56. 3. Cramer JA, Gordon J, Schachter S, Devinsky O. Women with epilepsy: hormonal issues from menarche through menopause. Epilepsy Behav 2007;11:160–78. 4. Dag E, Aydin S, Ozkan Y, Erman F, Dagli AF, Gurger M. Alteration in chromogranin A, obestatin and total ghrelin levels of saliva and serum in epilepsy cases. Peptides 2010;31:932–7. 5. Marek B, Kajdaniuk D, Kos-Kudła B, Kapustecki J, Swietochowska E, Ostrowska Z, et al. Mean daily plasma concentrations of beta-endorphin, leu-enkephalin, ACTH, cortisol, and DHEAS in epileptic patients with complex partial seizures evolving to generalized tonic–clonic seizures. Endokrynol Pol 2010;61:103–10. 6. Ferrini F, Salio C, Lossi L, Merighi A. Ghrelin in central neurons. Curr Neuropharmacol 2009;7:37–49. 7. Cowley MA, Smith RG, Diano S, Tscho¨p M, Pronchuk N, Grove KL, et al. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003;37: 649–61. 8. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402: 656–60. 9. Gualillo O, Lago F, Dieguez C. Introducing GOAT: a target for obesity and antidiabetic drugs? Trends Pharmacol Sci 2008;29(8):398–401. 10. Yin X, Li Y, Xu G, An W, Zhang W. Ghrelin fluctuation, what determines its production? Acta Biochim Biophys Sin 2009;41:188–97. 11. Berilgen MS, Mungen B, Ustundag B, Demir C. Serum ghrelin levels are enhanced in patients with epilepsy. Seizure 2006;15:106–11. 12. Gungor S, Yu¨cel G, Akinci A, Tabel Y, Ozerol IH, Yologlu S. The role of ghrelin in weight gain and growth in epileptic children using valproate. Child Neurol 2007;22:1384–8. 13. Greco R, Latini G, Chiarelli F, Iannetti P, Verrotti A. Leptin, ghrelin, and adiponectin in epileptic patients treated with valproic acid. Neurology 2005;65: 1808–9. 14. Prodam F, Bellone S, Casara G, De Rienzo F, Grassino EC, Bonsignori I, et al. Ghrelin levels are reduced in prepubertal epileptic children under treatment with carbamazepine or valproic acid. Epilepsia 2010;51:312–5. 15. Tomoum HY, El-Hadidi ES. Ghrelin and resistin levels in children with epilepsy on valproic acid. Pediatr Neurol 2009;7:223–9. 16. Guzel A, Karasalihoglu S, Kucukugurluoglu Y, Sayar E, Kunduracilar H. Evaluation of serum ghrelin and neuropeptide Y levels in epileptic children under valproate treatment. Univ Tip Fak Derg 2009;26:18–23. 17. Shimada M, Date Y, Mondal MS, Toshinai K, Shimbara T, Fukunaga K, et al. Somatostatin suppresses ghrelin secretion from the rat stomach. Biochem Biophys Res Commun 2003;302:520–5. 18. Vezzani A, Hoye D. Brain somatostatin: a candidate inhibitory role in seizures and epileptogenesis. Eur J Neurosci 1999;11:3767–76. 19. Silva AP, Bethmann K, Raulf F, Schmid HA. Regulation of ghrelin secretion by somatostatin analogs in rats. Eur J Endocrinol 2005;152:887–94. ˜ o JP, Kineman RD, Luque RM. 20. Gahete MD, Co´rdoba-Chaco´n J, Salvatori R, Castan Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach. Mol Cell Endocrinol 2010;317(1– 2):154–60. 21. Bhatt R, Bhatt S, Rameshwar P, Siegel A. Long-term kindled seizures induce alterations in hematopoietic functions: role of serum leptin. Epilepsy Res 2005;65:169–78. 22. Hum KM, Megna S, Burnham WM. Lack of laterality in the effects of right and left amygdala kindling on weight gain in female rats. Epilepsy Res 2009;87(1):40–6. 23. Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, et al. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 2001;120:337–45. 24. Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Oikawa S. Effects of insulin, leptin, and glucagon on ghrelin secretion from isolated perfused rat stomach. Regul Pept 2004;119:77–81. 25. Pan W, Tu H, Kastin AJ. Differential BBB interactions of three ingestive peptides: obestatin, ghrelin, and adiponectin. Peptides 2006;27(4):911–6. 26. Banks WA, Tscho¨p M, Robinson SM, Heiman ML. Extent and direction of ghrelin transport across the blood–brain barrier is determined by its unique primary structure. J Pharmacol Exp Ther 2002;302(2):822–7. 27. Obay BD, Tasdemir E, Tu¨mer C, Bilgin HM, Sermet A. Antiepileptic effects of ghrelin on pentylenetetrazole-induced seizures in rats. Peptides 2007;28: 1214–9. 28. Portelli J, Aourz N, Ver DL, Michotte Y, Smolders I. Anticonvulsant effects of ghrelin receptor ligands against pilocarpine-induced limbic seizures. Acta Physiol Congr 2009;(Suppl. 674):P-03. 29. De Vriese C, Gregoire F, Lema-Kisoka R, Waelbroeck M, Robberecht P, Delporte C. Ghrelin degradation by serum and tissue homogenates: identification of the cleavage sites. Endocrinology 2004;145(11):4997–5005. 30. Ni H, Walia P, Chanoine JP. Ontogeny of acylated ghrelin degradation in the rat. Peptides 2010;31:301–6.
Contents lists available at ScienceDirect
Seizure journal homepage: www.elsevier.com/locate/yseiz
Short communication
Does ghrelin level change after epileptic seizure in rats? Z. Ataie b, M.G. Golzar b, Sh. Babri a, H. Ebrahimi b, G. Mohaddes a,b,* a b
Neuroscience Research Centre of Tabriz University of Medical Sciences, Iran Drug Applied Research Centre of Tabriz University of Medical Sciences, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 June 2010 Received in revised form 29 December 2010 Accepted 10 January 2011
Aim: Epilepsy is one of the most common neurologic problems worldwide. A relationship between epilepsy and hormones has been demonstrated. This study was designed to investigate the effect of seizure on blood ghrelin level. Methods: Twenty male Wistar rats were divided into two groups. The control group received saline and the pentylenetetrazole (PTZ) group received a single convulsive dose (50 mg/kg) of PTZ. Thirty minutes later blood samples were collected and acylated and unacylated ghrelin levels in the plasma were assayed. Results: Acylated or active form of ghrelin decreased significantly (p < 0.05) after a PTZ-induced seizure, but the reduction of unacylated and total blood ghrelin levels failed to reach statistical significance. Conclusion: These findings may reflect that PTZ-induced epilepsy decreases AG of plasma. ß 2011 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.
Keywords: Seizure Ghrelin PTZ
1. Introduction Epilepsy is one of the most common neurologic problems worldwide1 and characterized by recurrent unprovoked seizures.2 The relationship between epilepsy and the endocrine system has been investigated by neurologists, endocrinologists, and basic scientists.3 Some studies have suggested a relationship between epilepsy and GH, sex hormones, the NPY system, nesfatin-1, ghrelin, and chromogranin A.4 It is also reported that mean daily blood concentrations of ACTH, cortisol and beta-endorphin in patients with epilepsy are higher in comparison with those of the healthy volunteers.5 Ghrelin is a 28-amino acid motilin-related peptide. The peptide is characterized by the presence of an n-octanoylation on the hydroxy group of serine in position 3.6 The octanoylated peptide hormone is produced and secreted by A-like cells within the oxyntic glands of the stomach.7 Ghrelin circulates in the bloodstream in two major forms: acylated (AG; or n-octanoylated) and unacylated (UAG; or desoctanoylated or desacylated).8 Acylation is a post-transcriptional modification that is essential for binding to the growth hormone secretagogue receptor (GHS-R) to a point that ghrelin is originally supposed to be biologically active only in acylated form.6 The acyltransferase that catalyzes ghrelin octanoylation has recently been identified as ghrelin O-acyltransferase (GOAT).9
Ghrelin exerts neuroendocrine effects of eliciting growth hormone release, regulating appetite and energy metabolism, and carries many other functions in a variety of tissues and organs such as gastroenteropancreatic, cardiovascular, reproduction, and immune systems.10 A definite relationship between ghrelin and seizure has also been demonstrated in animals and humans, although the results are controversial.4 Some research has suggested that serum ghrelin levels are enhanced in patients with epilepsy11 and in prepubertal epileptic children treated with valproic acid (VPA).12 On the other hand, some studies have shown that ghrelin levels decreased in epileptic adults13 and normalweight prepubertal children14 taking VPA treatment. In addition, Aydin et al. reported that serum and saliva ghrelin levels were lower in epileptic patients before treatment than in controls.2 To clarify these controversies, evaluation of blood ghrelin levels immediately after induced epileptic seizures in animals is beneficiary. Therefore, the purposes of this study were: (1) to determine AG and UAG levels in blood samples from pentylenetetrazole (PTZ)induced epileptic rats using an enzyme-linked immunosorbent assay (ELISA); (2) to determine any association between ghrelin and epilepsy and clarify the controversial results reported for this hormone. 2. Materials and methods
* Corresponding author at: Dept of Physiology, Faculty of Medicine, Tabriz University of Medical Sciences, Golgasht Ave., Tabriz, Iran. Tel.: +98 411 3364664; fax: +98 411 3364664. E-mail address: [email protected] (G. Mohaddes).
2.1. Chemicals and instruments PTZ was purchased from Tocris Bioscience, UK (Bristol). Blood AG and UAG levels were determined by ELISA using an ELISA reader
1059-1311/$ – see front matter ß 2011 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.seizure.2011.01.001
348
[()TD$FIG]
Z. Ataie et al. / Seizure 20 (2011) 347–349
(Stat Fax, Awarness, USA) and AG and UAG kits (Cat. No. RD 394062400R and Cat. No. RD394063400R, Biovendor co, Czech Republic). 2.2. Animals All experimental procedures were approved by the Regional Ethics Committee of Tabriz University of Medical Sciences. The experiments were performed on male Wistar rats (220–260 g) with food and water available ad libitum, except during the 30 min of procedure. Animals were maintained under controlled temperature (21–23 8C) and light (12 h light, 12 h dark) and with a humidity of 5–10%. All the experimental procedures were carried out between 08.00 and 10.00 a.m. 2.3. Experimental design Twenty rats were divided into two groups: control (n = 10) and PTZ (n = 10). The PTZ group received a single convulsive dose (50 mg/kg) of PTZ and the control group received the same volume of saline, intraperitoneally (i.p.). Seizures were elicited in all PTZreceiving rats. After 30 min of the injections, blood samples were taken from the animals’ heart and collected in tubes containing EDTA and p-hydroxymercuribenzoic acid (1 mM in the final sample volume) to prevent the degradation of AG by protease. Samples were immediately centrifuged at 3500 rpm for 10 min at +4 8C and then, the plasma was transferred into separate tubes. Samples were immediately assayed.
Fig. 2. Effect of PTZ (i.p.) induced seizure on unacylated blood ghrelin (pg/mL) level. Data are expressed as mean standard error of the mean (SEM). n = 10 animals per group.
[()TD$FIG]
2.4. Statistical analysis SPSS 13.0 software was used for statistical comparisons of data, and data expressed as the means SEM. Differences in AG, UAG and total ghrelin levels between the groups were analyzed by independent Student’s t-test (one- or two-tailed) and p < 0.05 was accepted as significant.
Fig. 3. Effect of PTZ (i.p.) induced seizure on total blood ghrelin (pg/mL) level. Data are expressed as mean standard error of the mean (SEM). n = 10 animals per group.
3. Results
4. Discussion
As shown in Fig. 1, an independent Student’s t-test indicated that AG levels of blood decreased significantly (p < 0.05) after i.p. injection of PTZ, compared to saline groups. Figs. 2 and 3 illustrate the effect of PTZ-induced seizure on UAG and total ghrelin levels of blood, respectively. Independent Student’s t-tests indicated that UAG and total ghrelin levels of
To our knowledge, this is the first study to examine blood ghrelin levels in PTZ-induced seizures in rats. The results of this study showed that active form or acylated blood ghrelin levels decreased significantly after PTZ-induced seizures. UAG and total blood ghrelin levels also decreased although they failed to reach statistical significance. The results for the blood ghrelin response to the PTZ-induced seizure showed that measuring the concentration of ghrelin is a useful method for studying plasma ghrelin changes over short time. In recent years, many researchers have focused on the relationships between epilepsy and the endocrine system since epileptic seizures are related to the temporary changes in hormone secretion.2 A definite relationship between ghrelin and seizures has been already demonstrated in animals and humans, although the results are controversial. Some authors have demonstrated that ghrelin levels were increased in children treated with VPA in comparison to healthy controls.12,15 Berilgen et al. also reported that serum ghrelin levels were higher in epilepsy patients who received antiepileptic drug therapy than a control group.11 Contradictorily, circulating ghrelin levels were significantly lower in epileptic patients treated with VPA in pubertal13 and prepubertal periods.14,16 Some studies also showed that both acylated and deacylated forms of serum and salivary ghrelin were depressed in epileptic patients before and after specific treatment with respect to normal controls.2,4
[()TD$FIG]
Fig. 1. Effect of PTZ (i.p.) induced seizure on acylated blood ghrelin (pg/mL) level. Data are expressed as mean standard error of the mean (SEM). n = 10 animals per group. *p < 0.05 compared with the control group.
blood did not change significantly with respect to control group. AG/UAG ratio also was not modified by PTZ-induced seizures.
Z. Ataie et al. / Seizure 20 (2011) 347–349
Our results confirm earlier studies that document that the ghrelin level of blood decreases with epilepsy. In addition our results showed that AG decreased significantly but the reduction of UAG and total levels were not significant. Two possibilities may be considered in this regard: (1) ghrelin is reduced by a feed back system or (2) uptake of AG is increased by CNS structures to modulate epileptic discharges. In accordance with the first possibility, some hormones can reduce the production, acylation or secretion of ghrelin such as somatostatin and leptin. Somatostatin, a peptide produced in the brain, gastrointestinal tract, and pancreas, exerts a broad spectrum of biological activities through paracrine, endocrine, and neuroendocrine mechanisms.17 It is preferentially released from neurons under conditions of elevated activity (i.e. bursting or high frequency neuronal activation), e.g. during seizures.18 It is also previously reported that a single intravenous injection of somatostatin reduces the systemic plasma concentration of ghrelin in rats17 and human healthy volunteers.19 In addition, somatostatin decreases GOAT expression,20 enzyme which catalyzes acylation of ghrelin.9 Therefore, it is possible that blood AG reduction is due to somatostatin released from one of its sources during PTZ-induced seizure. Moreover, a faster rebound of UAG level respect to AG (due to inhibitory effect of somatostatin on GOAT expression) may be occurred. Some studies suggest that kindled seizures induce high serum leptin levels in rats.21,22 In this context, kindled female rats had leptin levels approximately twice as high as the controls during diestrus phase.22 On the other hand, ghrelin is negatively regulated by leptin23 and physiological concentrations of leptin and a moderate hyperleptinemia directly inhibit ghrelin secretion from the isolated perfused rat stomach.24 Therefore, ghrelin reduction might be due to release of leptin during PTZ-induced seizure. In a discussion of the second possibility, human ghrelin exhibits saturable binding and endocytosis in the RBE4 rat cerebral microvessel endothelial cell line.25 On the other hand, blood brain barrier mechanisms regulating ghrelin accumulation by brain may be influenced by pathophysiological events.26 It has also been shown that ghrelin or its agonists can be considered as an antiepileptic agent in rodents.27,28 Therefore, reduction of plasma AG after PTZ-induced seizure could be attributed to its uptake by brain to represent an anti-epileptic effect. In this mater, evaluation of brain ghrelin levels immediately after induced epileptic seizures in animals is beneficiary. Finally, proteolysis of AG29 and degradation into non-UAG metabolites30 could be occurred during seizures. In conclusion, the results obtained from our study showed that AG decreased significantly in blood after PTZ-induced seizures. In the future, a better understanding of the mechanisms responsible for seizure-induced alterations in hormone levels of plasma may help to prevent these changes. Acknowledgment This study was financially supported by the Neuroscience Research Centre of Tabriz University of Medical Sciences. The article is derived from the MSc dissertation of Ms Zohre Ataie entitled ‘‘Evaluation of the antiepileptic effect of intrahippocampal injection of ghrelin through GABAB receptors in adult male rats’’. References 1. Chang BS, Lowenstein DH. Mechanisms of disease: epilepsy. N Engl J Med 2003;349:1257–66.
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2. Aydin S, Dag E, Ozkan Y, Erman F, Dagli AF, Kilic N, et al. Nesfatin-1 and ghrelin levels in serum and saliva of epileptic patients: hormonal changes can have a major effect on seizure disorders. Mol Cell Biochem 2009;328:49–56. 3. Cramer JA, Gordon J, Schachter S, Devinsky O. Women with epilepsy: hormonal issues from menarche through menopause. Epilepsy Behav 2007;11:160–78. 4. Dag E, Aydin S, Ozkan Y, Erman F, Dagli AF, Gurger M. Alteration in chromogranin A, obestatin and total ghrelin levels of saliva and serum in epilepsy cases. Peptides 2010;31:932–7. 5. Marek B, Kajdaniuk D, Kos-Kudła B, Kapustecki J, Swietochowska E, Ostrowska Z, et al. Mean daily plasma concentrations of beta-endorphin, leu-enkephalin, ACTH, cortisol, and DHEAS in epileptic patients with complex partial seizures evolving to generalized tonic–clonic seizures. Endokrynol Pol 2010;61:103–10. 6. Ferrini F, Salio C, Lossi L, Merighi A. Ghrelin in central neurons. Curr Neuropharmacol 2009;7:37–49. 7. 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