European Journal of Pharmacology 764 (2015) 264–270
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Endocrine pharmacology
Central injection of CDP-choline suppresses serum ghrelin levels while increasing serum leptin levels in rats Sinem Kiyici n, Nesrin Filiz Basaran 1, Sinan Cavun, Vahide Savci Uludag University Medical Faculty, Department of Pharmacology, Bursa, Turkey
art ic l e i nf o
a b s t r a c t
Article history: Received 5 June 2015 Received in revised form 3 July 2015 Accepted 6 July 2015 Available online 7 July 2015
In this study we aimed to test central administration of CDP-choline on serum ghrelin, leptin, glucose and corticosterone levels in rats. Intracerebroventricular (i.c.v.) 0.5, 1.0 and 2.0 mmol CDP-choline and saline were administered to male Wistar-Albino rats. For the measurement of serum leptin and ghrelin levels, blood samples were obtained baseline and at 5, 15, 30, 60 and 120 min following i.c.v. CDP-choline injection. Equimolar doses of i.c.v. choline (1.0 mmol) and cytidine (1.0 mmol) were administered and measurements were repeated throughout the second round of the experiment. Atropine (10 mg) and mecamylamine (50 mg) were injected intracerebroventricularly prior to CDP-choline and measurements repeated in the third round of the experiment. After 1 mmol CDP-choline injection, serum ghrelin levels were suppressed significantly at 60 min (P¼ 0.025), whereas serum leptin levels were increased at 60 and 120 min (P ¼0.012 and P¼ 0.017 respectively). CDP-choline injections also induced a dose- and time-dependent increase in serum glucose and corticosterone levels. The effect of choline on serum leptin and ghrelin levels was similar with CDPcholine while no effect was seen with cytidine. Suppression of serum ghrelin levels was eliminated through mecamylamine pretreatment while a rise in leptin was prevented by both atropine and mecamylamine pretreatments. In conclusion; centrally injected CDP-choline suppressed serum ghrelin levels while increasing serum leptin levels. The observed effects following receptor antagonist treatment suggest that nicotinic receptors play a role in suppression of serum ghrelin levels,whereas nicotinic and muscarinic receptors both play a part in the increase of serum leptin levels. & 2015 Elsevier B.V. All rights reserved.
Keywords: CDP-choline Ghrelin Leptin
1. Introduction Appetite is regulated by a complex system of central and peripheral signals. Satiety signals from the gastrointestinal tract act through the arcuate nucleus of the hypothalamus and solitary tract nucleus of the brain system. Ghrelin, a potent gut–brain orexigenic peptide, has a role in the stimulation of food intake and long term regulation of body weight. Ghrelin regulates, in an antagonistic manner to leptin, the synthesis and secretion of several neuropeptides in the hypothalamus that regulate feeding and energy balance (Näslund and Hellström, 2007). CDP-choline is an endogenously-synthesized nucleotide that n Correspondence to: Sevket Yilmaz Education and Research Hospital, Department of Internal Medicine, Mimar Sinan Mah. Emniyet Cad. Yıldırım, Bursa, Turkey. E-mail addresses: [email protected] (S. Kiyici), nesfi[email protected] (N.F. Basaran), [email protected] (S. Cavun), [email protected] (V. Savci). 1 Present Address: Sitki Kocman University Medical Faculty, Department of Pharmacology, Mugla, Turkey.
http://dx.doi.org/10.1016/j.ejphar.2015.07.014 0014-2999/& 2015 Elsevier B.V. All rights reserved.
exerts numerous cellular actions in different experimental models. Exogenous administration of CDP-choline has been shown to affect brain metabolism and exhibit cardiovascular, respiratory, neuroendocrine and neuroprotective actions and has beneficial effects in the treatment of some neurodegenerative and neurovascular disease (Weiss, 1995, Dávalos and Secades, 2011, Topuz et al., 2014, Hurtado et al., 2011). Administered orally, intravenously or intracerebroventricularly (i.c.v.), CDP-choline is rapidly metabolized to choline and cytidine/uridine which results in the elevation of plasma and tissue levels of these metabolites (Lopez et al., 1987, Wurtman et al., 2000). Treatments that raise plasma and tissue choline levels increase the synthesis of acetylcholine and enhance cholinergic transmission (Cohen and Wurtman, 1976, Ulus et al., 1989). Results of studies which investigate the role of the cholinergic system and vagus on serum ghrelin levels were controversial. There is also little data about the role of cholinergic system on serum leptin levels. In a previous study, it has been shown that appetite ratings decline significantly after CDP-choline treatment (Killgore et al., 2010). But there is no data on whether
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there are any alterations in serum ghrelin and leptin levels, or if these alterations are involved in the effect of CDP-choline on appetite. The dense cholinergic innervation of the hypothalamopituitary system and the presence of cholinergic receptors in this area are very well known.When the dense cholinergic innervation of hypothalamus, which has an important role in feeding control (Mason, 1985, Michels et al., 1986), and the effect of vagal stimulus on feeding are taken into consideration, CDP-choline treatment could have an effect on serum ghrelin and leptin levels. Therefore, the present study was designed to determine the effect of centrally injected CDP-choline on serum ghrelin and leptin levels and the involvement of central cholinergic receptors that mediate the effect of CDP-choline. We also aimed to measure serum glucose and corticosterone levels that can interact with serum concentrations of these peptides after central CDP-choline injection.
2. Materials and methods Male Wistar-Albino rats (300–350 g; Experimental Animal Breeding and Research Center, Uludag University Medical Faculty, Bursa, Turkey) were housed under a 12 h light/dark cycle with free access to food and water. The surgical and experimental protocols were approved by the Animal Care and Use Committee of Uludag University and are in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
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i.c.v. In the third set of experiments, the effects of the blockade of central muscarinic or nicotinic acetylcholine receptors on serum ghrelin, leptin and glucose responses were investigated. Rats were pretreated i.c.v. with atropine sulfate (10 μg), mecamylamine HCL (50 μg) or saline (5 μl) 15 min before i.c.v. injection of CDP-choline (1.0 mmol). In the second and third sets of experiments, surgical procedures were performed as described in the first set of experiments. Blood samples (0.5 ml) were taken just before the i.c.v. injections and at 60 and 120 min following the treatments. Time intervals were assessed according to the maximum effect of CDP-choline on serum ghrelin and leptin levels were observed in the first set of experiments. Each blood sample was replaced with equal volume of saline. Blood glucose was detected immediately from samples by a commercially available glucometer using test strips. Blood samples were centrifuged at 3000 g for 15 min at 4 °C and serum samples were separated and stored at 20 °C until analysis. Rats were killed with high dose ether anesthesia after the completion of experiments. 2.3. Drugs The following drugs were used: CDP-choline, choline, cytidine, atropine sulfate, mecamylamine HCL (Sigma, St Louis, MO, USA). Drugs were dissolved in saline (0.9% NaCl). 2.4. Measurements
2.1. Surgical procedures Animals were allowed to acclimate to the animal care facility for 7 days before the experiment. Following the acclimatization period, rats were anesthetized with ether. The left carotid artery was cannulated with PE-50 tubing filled with heparinized saline (250 U/ml) and the cannulas were exteriorized at the nape of the neck and sealed until use. For i.c.v. drug administration, a 21-gauge stainless steel guide cannula was implanted in the right lateral ventricle through a burr hole drilled into the skull. The tip of the guide cannula was positioned 1.5 mm lateral to the midline, 1.0 mm posterior to bregma and 4.5 mm below the skull surface. The cannula was fixed to the skull with acrylic cement. At the end of the surgical procedure, rats were housed in individual cages and allowed to recover from anesthesia for 4–5 h. During this period, rats did not exhibit any sign of pain.
Serum ghrelin, leptin and corticosterone levels were measured by using commercially available RIA kits (Millipore's Ghrelin [Total] Radioimmunoassay Kit, MO, ABD, sensitivity 93 pg/ml), leptin (Linco's Rat Leptin Radioimmunoassay Kit, MO, ABD, sensitivity 0.5 ng/ml) and corticosterone (ImmuChem Double Antibody Corticosterone RIA Kit, Orangeburg, NY, sensitivity 7.7 ng/ml). 2.5. Data and statistical analysis Comparisons between groups were performed using Student's t-test for normally distributed variables, and Mann–Whitney Utest for non-normal variables. Paired t test and Wilcoxon signedrank test were used for within-group comparisons. The data were analyzed using SPSS for Windows, Version 11.5 and shown as mean 7standard deviation. A P value of less than 0.05 was considered statistically significant.
2.2. Experimental procedures In the first set of experiments, dose and time courses of serum ghrelin, leptin, corticosterone, and glucose responses to i.c.v. CDPcholine were studied. Rats were randomized to four groups and various doses of CDP-choline 0.5 (n ¼14), 1.0 (n ¼16), 2.0 (n ¼16) μmol and 5 μl (n¼ 16) saline (0.9% NaCL) were administered. Blood samples (0.5 ml) were taken just before the i.c.v. injection (0 min) of CDP-choline or saline and at 5, 15, 30, 60 and 120 min following administration. Each set of animals was used for the three time intervals and each blood sample replaced with equal volume of saline. Blood glucose was detected immediately from samples by a commercially available glucometer using test strips. Blood samples were centrifuged at 3000 g for 15 min at 4 °C and serum samples were separated and stored at 20 °C until analysis. Rats were killed with high dose ether anesthesia after the first set of experiments. In the second set of experiments, in order to determine the effects of hydrolysis product of CDP-choline, choline, and cytidine, on serum ghrelin, leptin and glucose responses, equimolar dose of choline (1.0 mmol), cytidine (1.0 mmol) or saline (5 ml) was injected
3. Results 3.1. Effects of intracerebroventriculary injected CDP-choline on serum ghrelin, leptin, glucose and corticosterone levels There was no difference between basal serum ghrelin and leptin levels in the groups. Serum ghrelin levels were suppressed significantly at 60 min following 1.0 mmol CDP-choline i.c.v. injection (P ¼0.025) (Fig. 1). When area under the curve (AUC) was calculated, serum ghrelin levels were found significantly suppressed after 0.5 mmol (AUC ¼160711.4 pg dk/ml) and 1.0 mmol (AUC ¼147.57 11.1 pg dk/ml) of i.c.v. CDP-choline injection compared with the saline group (AUC ¼1907 18 pg dk/ml) (P ¼0.035 and P¼0.013, respectively). Administration of 0.5, 1.0 and 2.0 mmol dose of i.c.v. CDP-choline significantly increased serum leptin levels at 60 min (P ¼0.036, P ¼0.012 and P ¼0.043; respectively) compared with basal values and a significant increase was also found at 120 min following 1.0 and 2.0 mmol dose of i.c.v. CDP-choline injection
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Time (min)
0 0
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Fig. 1. Effect of i.c.v. CDP-choline on serum ghrelin (pg/ml) levels. Data are given as mean 7S.D. α ¼statistically significant compared with basal values. Pα ¼ 0.025.
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Fig. 3. Effect of i.c.v. CDP-choline on blood glucose (mg/dl) levels. Data are given as mean7 S.D. α, β, χ, δ, ε, ϕ, γ, η, ι, φ, κ ¼statistically significant compared with basal values. Pα ¼ 0.024, Pβ ¼ 0.018, Pχ ¼0.012, Pδ ¼ 0.012, Pε ¼ 0.012, Pϕ ¼ 0.012, Pγ ¼ 0.012, Pη ¼ 0.025, Pι ¼0.017, Pφ ¼ 0.012, Pκ ¼0.017.
8 Saline
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Corticosterone (ng/ml)
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Fig. 2. Effect of i.c.v. CDP-choline on serum leptin (ng/ml) levels. Data are given as mean 7S.D. α, β, χ, δ, ε ¼statistically significant compared with basal values. Pα ¼ 0.036, Pβ ¼ 0.012, Pχ ¼ 0.043, Pδ ¼ 0.017, Pε ¼ 0.046.
(P ¼0.017 and P¼ 0.046, respectively) (Fig. 2). AUC for serum leptin concentration was 632.37116.0 ng dk/ml after 1.0 mmol i.c.v.CDPcholine and 445.57 80.9 ng dk/ml after i.c.v. saline injection. When the two groups were compared, the difference was statistically significant (P¼ 0.005). CDP-choline injection also caused a dose- and time-related increase in blood glucose and serum corticosterone levels. Blood glucose levels were significantly increased at 15 and 60 min after 0.5 mmol CDP-choline (P ¼0.012 and P ¼0.025, respectively), 5, 15, 30, and 60 min after 1.0 mmol CDP-choline (P¼ 0.024, P ¼0.012, P ¼0.012 and P ¼0.017, respectively) and 5, 15, 30, 60 and 120 min after 2.0 mmol CDP-choline injection (P ¼0.018, P ¼0.012, P ¼0.012, P ¼0.012 and P¼ 0.017, respectively) compared with basal levels (Fig. 3). When the three groups were compared, serum glucose levels were found significantly elevated at 5, 15 and 30 min (P ¼0.020, P ¼0.006 and P ¼0.002, respectively). Serum corticosterone was also found elevated at 15 min following 0.5 mmol CDPcholine (P ¼0.018), at 15 and 30 min following 1.0 mmol CDP-choline (P ¼0.028 and P¼ 0.035) and at 5, 15 and 60 min after 2.0 mmol CDP-choline i.c.v. injection (P¼ 0.027, P ¼0.034 and P ¼0.028, respectively) compared with basal values (Fig. 4).
0 0
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15 30 Time (min)
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Fig. 4. Effect of i.c.v. CDP-choline on serum corticosterone (ng/ml) levels. Data are given as mean 7 S.D. α, β, χ, δ, ε, ϕ¼ statistically significant compared with basal values. Pα ¼ 0.027, Pβ ¼0.018, Pχ ¼0.028, Pδ ¼ 0.034, Pε ¼ 0.035, Pϕ ¼ 0.028.
3.2. Effects of intracerebroventriculary injected choline and cytidine on serum ghrelin, leptin and glucose levels In order to investigate whether i.c.v. administration of equimolar doses of choline or cytidine, which are metabolic products of CDP-choline, can effect serum ghrelin, leptin and glucose levels, rats were injected with choline (1.0 mmol), cytidine (1.0 mmol) or saline (5 ml) i.c.v. There were no statistically significant changes in serum ghrelin levels after cytidine or saline injection, whereas levels were significantly suppressed after choline administration at 60 (P ¼0.043) and 120 (P ¼0.043) min compared with basal levels (Fig. 5). When the three groups were compared, percentage change in ghrelin levels was statistically significant at 120 min (P ¼0.003). No statistically significant changes in serum leptin levels were found after intra- and intergroup comparisons in either group. Choline tended to increase leptin levels especially at 60 min but it did not reach significant levels (Fig. 6). Serum glucose levels were found increased at 60 (P ¼0.018) and 120 (P¼ 0.050) min after choline injection but did not change statistically after cytidine or
S. Kiyici et al. / European Journal of Pharmacology 764 (2015) 264–270
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Fig. 5. Effect of i.c.v. injected equimolar doses of choline or cytidine on serum ghrelin (pg/ml) levels. Data are given as mean 7 S.D. α, β ¼ statistically significant compared with basal values. Pα ¼ 0.043, Pβ ¼ 0.043.
0 Saline
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Mecamylamine
Pretreatment Fig. 8. Effect of atropine or mecamylamine pretreatment on CDP-choline induced suppression in serum ghrelin (pg/ml) levels. Data are given as mean 7 S.D. Rats were injected saline (5 ml), atropine sulfate (10 μg; i.c.v.) or mecamylamine HCL (50 μg; i.c.v) 15 min before of CDP-choline injection (1.0 mmol; i.c.v.). α, β, χ, δ, ε¼ statistically significant compared with basal values. Pα ¼ 0. 018, Pβ ¼0.028, Pχ ¼0.025, Pδ ¼ 0.036, Pε ¼ 0.025.
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Leptin (ng/ml)
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3.3. Effects of blockade of central muscarinic or nicotinic cholinergic receptors on serum ghrelin, leptin and blood glucose levels
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Fig. 6. Effect of i.c.v. injected equimolar doses of choline or cytidine on serum leptin (ng/ml) levels. Data are given as mean 7S.D.
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Glucose (mg/dl)
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Fig. 7. Effect of i.c.v. injected equimolar doses of choline or cytidine on blood glucose (mg/dl) levels. Data are given as mean 7 S.D. α, β ¼ statistically significant compared with basal values. Pα ¼0.018, Pβ ¼0.050.
saline injection compared with basal levels (Fig. 7). When the three groups were compared, serum glucose levels were statistically different at 60 min (P ¼0.042).
To determine if central muscarinic and/or nicotinic cholinergic receptors mediate the effects of CDP-choline on serum ghrelin, leptin and glucose levels, rats were pretreated with atropine sulfate (10 μg; i.c.v.) or mecamylamine HCL (50 μg; i.c.v.) or saline (5 ml; i.c.v.) 15 min prior to injection of CDP-choline (1.0 mmol; i.c. v.). No significant changes were found in serum ghrelin levels of rats pretreated with either saline, atropine sulfate or mecamylamine HCl prior to i.c.v. injection of saline. Serum ghrelin levels were significantly suppressed at 60 (P¼ 0.018) and 120 (P ¼0.028) min in the CDP-choline group pretreated with saline compared with basal levels. Suppression of serum ghrelin levels persisted at 60 and 120 min in the CDP-choline group pretreated with atropine (P ¼0.025 and P ¼0.036, respectively). Observed suppression of serum ghrelin levels after CDP-choline was abolished at 60 min but not at 120 min (P¼ 0.025) in the group pretreated with mecamylamine HCL (Fig. 8). No significant change was found in serum leptin levels of rats pretreated with saline, atropine sulfate or mecamylamine HCL prior to i.c.v. injection of saline.Serum leptin levels were significantly increased at 120 (P ¼0.043) min in saline pretreated CDP-choline group compared with basal levels. Observed increment in serum leptin levels at 120 min compared with basal levels was eliminated in groups pretreated with atropine sulfate or mecamylamine HCL prior to CDP-choline injection (Fig. 9). Blood glucose levels were significantly increased at 60 and 120 min in CDP-choline group pretreated with saline compared with basal levels (P ¼0.028 and P ¼0.028, respectively). The increment of blood glucose levels persisted at 60 and 120 min in CDP-choline group pretreated with atropine (P¼ 0.008 and P¼ 0.008, respectively). The observed increment of blood glucose levels after CDP-choline persisted at 60 min (P ¼0.038) but eliminated at 120 min in group pretreated with mecamylamine HCL (Fig. 10).
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35 0 min 60 min 12 min
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Pretreatment Fig. 9. Effect of atropine or mecamylamine pretreatment on CDP-choline induced increase in serum leptin (ng/ml) levels. Data are given as mean 7 S.D. Rats were injected saline (5 ml; i.c.v.), atropine sulfate (10 μg; i.c.v.) or mecamylamine HCL (50 μg; i.c.v.) 15 min before CDP-choline injection (1.0 mmol; i.c.v.). α ¼statistically significant compared with basal values. Pα ¼ 0.043.
0 min 60 min 120 min
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Glucose (mg/dl)
160 140 120 100 80 60 Saline
Atropine
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Pretreatment Fig. 10. Effect of atropine or mecamylamine pretreatment on CDP-choline induced increase in blood glucose (mg/dl) levels. Data are given as mean 7 S.D. Rats were injected saline (5 ml; i.c.v.), atropine sulfate (10 μg; i.c.v.) or mecamylamine HCL (50 μg; i.c.v.) 15 min before CDP-choline injection (1.0 mmol; i.c.v.). α, β, χ, δ, ε¼ statistically significant compared with basal values. Pα ¼ 0.028, Pβ ¼0.028, Pχ ¼ 0.008, Pδ ¼0.008, Pε ¼0.038.
4. Discussion In this study we found that serum ghrelin levels were suppressed significantly at 60 min (P ¼0.025) whereas serum leptin levels were increased at 60 min and 120 min (P ¼0.012 and P ¼0.017) following 1.0 mmol CDP-choline i.c.v. injection compared with basal values. When AUC was calculated, a significant suppression was observed in serum ghrelin levels after 0.5 mmol and 1.0 mmol CDP-choline i.c.v. injection (P ¼0.035 and P ¼0.013) while an increase was found in serum leptin levels after 1.0 mmol CDPcholine i.c.v. injection (P ¼0.005) compared with the saline group. Central CDP-choline injections also induced a dose- and time-dependent increase in serum glucose and corticosterone levels. It has been clearly demonstrated that the choline concentration of cerebrospinal fluid, hypothalamus, third ventricle and corpus striatum increase after i.c.v. administration of CDP-choline (Lopez
et al., 1987, López-Coviella et al., 1995, Savci et al., 2002, Paroni et al., 1985). CDP-choline is rapidly metabolized to form choline and cytidine in vivo. Choline is also a precursor for the synthesis of the cholinergic neurotransmitter, acetylcholine. Treatments that raise plasma and tissue choline levels increase the synthesis of acetylcholine and enhance cholinergic transmission. CDP-choline treatment has been shown to increase tissue and plasma choline and cytidine concentration to produce pharmacological effects (Wurtman et al., 2000, López-Coviella et al., 1995, Savci et al., 2002, Paroni et al., 1985). In this study, the effects of i.c.v. CDPcholine and choline administration on serum ghrelin and leptin levels were similar, whereas we did not find any change following cytidine injection. Concurrent with previous reports, these present findings suggested that the effect of CDP-choline on serum ghrelin and leptin levels were mediated by its choline component. The dense cholinergic innervations of the hypothalamopituitary system and the presence of cholinergic receptors in this area are very well known (Mason, 1985, Michels et al., 1986). It has also been shown that cholinergic mechanisms have a role in the regulation of some hypothalamopituitary hormone secretions (Kelijman and Frohman, 1991, Savci et al., 1996, Gurun et al., 1997, Cavun and Savci, 2004a, Cavun et al., 2004b). Neuronal networks originating in the hypothalamic arcuate nucleus play fundamental roles in the control of energy balance. Immunoreactivities for proteins required for cholinergic transmission were demonstrated in the cell bodies of arcuate nucleus neurons expressing POMC and CART by the immunohistochemical experiments. These results indicate a role for acetylcholine in control of energy balance, mediating the effects of peripheral hormones such as leptin and insulin (Everitt et al., 1986, Meister et al., 2006). The observed effects of centrally-injected CDP-choline on serum ghrelin and leptin levels in this study confirm and extend previous data reporting that cholinergic mechanisms have a role in energy balance and hormonal regulation. As far as we know this is the first study investigating the effect of i.c.v injected CDP-choline on serum ghrelin and leptin levels in rats. Ghrelin is a powerful orexigenic peptide predominantly secreted by the stomach and vagal activity is important in the secretion and the effect of ghrelin (Date et al., 2002, Korbonits et al., 2004, Sakata et al., 2003). Shrestha et al. (2009) found that acetylcholine (1 mmol) increased ghrelin release from the stomach by approximately 37% whereas insulin (10 nM) decreased it by approximately 30% vs. the control in a study of stomach vascular perfusion model. Maier et al. (2004) reported that atropine alone significantly reduced fasting ghrelin levels by 25%, whereas under acetylcholine esterase inhibitor pyridostigmine alone, ghrelin levels were unaltered in a randomized study on healthy male volunteers. In another human study, it was shown that circulating ghrelin levels were increased following the muscarinic antagonist pirenzepine while decreasing following an indirect cholinergic agonist pyridostigmine (Broglio et al., 2004). In contrast to our results, it was observed that ghrelin levels increased following cholinergic agonists and were suppressed after cholinergic antagonist treatments in these studies. But Shresta et al. (2009) study has an in vitro design with the other two studies comprising of human study formats. Treatments were also administered peripherally in these two studies. In our study, CDP-choline which acts as a cholinergic activator was administered i.c.v. and contrary to expectations, serum ghrelin levels were found to be suppressed. Consistent with these results, ghrelin levels were found increased following i.v. cholinergic blockers and suppressed after i.v. cholinergic accelerator administration in a study which was performed on sheep (Sugino et al., 2003). Adipose tissue is innervated predominantly by the sympathetic system and leptin is a peptide which is mainly produced by adipose tissue (Hube et al., 1996, Berthoud et al., 2006). There are few
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studies investigating the cholinergic system relationship with leptin secretion. Ueda et al. (2001) reported that the intravenous injection of neostigmine, a cholinesterase inhibitor, increased plasma leptin and corticosterone levels in a dose-dependent manner and atropine injection prevented neostigmine-induced increase in leptin. Furthermore, bilateral adrenalectomized rats showed no increase in plasma leptin levels. In addition, it was observed that the expression of leptin messenger ribonucleic acid (mRNA) in white adipose tissue was significantly increased after neostigmine injection. We also found that i.c.v. CDP-choline injection, a cholinergic activator, induced an increase in serum leptin and corticosterone levels consistent with results of the previous study. Obesity and hyperinsulinemia were found associated with increased vagal cholinergic activity in many animal models (Bray and York, 1979, Rohner-Jeanrenaud, 1995, Gilon and Henquin, 2001). The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation (Levey et al.,1994). M3 receptor–deficient mice showed decreased food intake, reduced body weight and peripheral fat deposits and had suppressed serum insulin and leptin levels (Yamada et al., 2001). Nicotinic acetylcholine receptors also have been implicated in the control of appetite and body weight, as well as energy and lipid metabolism. Marrero et al. (2010) reported that a novel alpha 7 nicotinic acetylcholine receptor-selective agonist administration reduced weight gain and food intake in a mouse model of diabetes. There are many different examples which shows that activation of muscarinic and nicotinic receptors have opposite physiological effects compared with each other. Existence of different muscarinic and nicotinic receptor subtypes and different anatomical locations might be the reason of the observed opposite physiological effects of these receptors following activation. We found that suppression of serum ghrelin levels was eliminated by mecamylamine pretreatment while leptin increment was blocked by both atropine and mecamylamine pretreatments. Present findings suggest that the activation of central nicotinic receptors mediates the CDP-choline induced decrease in serum ghrelin levels, while the activation of central muscarinic and nicotinic receptors are both involved in the increase of serum leptin levels. CDP-choline and choline treatments have some pharmacological and metabolic effects. Intraperitoneal administration of choline increased serum insulin as well as plasma levels of catecholamines resulted in hyperglycemia by increasing cholinergic neurotransmission (Ilcol et al., 2002). Intracerebroventricular injection of choline also produced a dose-dependent increase in blood glucose levels and plasma catecholamines (Gurun et al., 2002). It was observed that i.p. injection of CDP-choline, like choline, resulted with hyperglycemia and increased serum insulin and catecholamines levels (Ilcol et al., 2007, Cansev et al., 2008a). Acetylcholine acts as a major neurotransmitter in the neuronal control of insulin and glucagon secretion from pancreatic endocrine cells (Ahrén, 2000). It has been observed that i.p. and i.c.v. administration of CDP-choline increase plasma glucagon levels in rats (Cansev et al., 2008b). In accordance with previous studies we found that i.c.v. administration of CDP-choline induced a time- and dose-dependent increase in serum glucose and corticosterone levels. Previous studies indicate that insulin and hyperglycemia suppress circulating ghrelin levels (Saad et al., 2002, Koerner et al., 2005, Flanagan et al., 2003), and also glucagon seems to act centrally to induce a reduction in ghrelin concentration (Arafat et al., 2005, 2006). Moreover insulin, glucose and corticosterone appear to increase leptin secretion (Koerner et al., 2005).When the effects of choline and CDP-choline administration on serum glucose, insulin, corticosterone and glucagon levels are taken into consideration, the observed effect of i.c.v. CDP-choline on serum
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ghrelin suppression and leptin increase in the present study might be secondary to the mentioned hormonal changes induced by CDP-choline. Moreover, eating behavior can be influenced by both metabolic and non-metabolic stress factors (Zappulla 2008, Monteleone and Maj, 2013). However in this study, we mainly focused on exploring whether CDP-choline administration may influence ghrelin and leptin concentration and if central cholinergic receptors mediate these effects. The involvement of other systems in CDP-choline's effect can be the subject of further research. In conclusion; centrally injected CDP-choline suppresses serum ghrelin levels while increasing serum leptin levels. The observed effects following receptor antagonists’ administration suggest that nicotinic receptors play a role in the suppression of serum ghrelin levels, whereas nicotinic and muscarinic receptors both take a part in the increment of serum leptin levels. The clinical relevance of this cholinergic system should be validated by additional detailed studies that investigate serum ghrelin and leptin levels following peripheral administration of CDP-choline and/or using selective cholinergic receptor antagonists.
Acknowledgments This study was supported by Grants from the Research Fund of Uludag University (Bursa/Turkey; UAP (T)-2009/8).
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Endocrine pharmacology
Central injection of CDP-choline suppresses serum ghrelin levels while increasing serum leptin levels in rats Sinem Kiyici n, Nesrin Filiz Basaran 1, Sinan Cavun, Vahide Savci Uludag University Medical Faculty, Department of Pharmacology, Bursa, Turkey
art ic l e i nf o
a b s t r a c t
Article history: Received 5 June 2015 Received in revised form 3 July 2015 Accepted 6 July 2015 Available online 7 July 2015
In this study we aimed to test central administration of CDP-choline on serum ghrelin, leptin, glucose and corticosterone levels in rats. Intracerebroventricular (i.c.v.) 0.5, 1.0 and 2.0 mmol CDP-choline and saline were administered to male Wistar-Albino rats. For the measurement of serum leptin and ghrelin levels, blood samples were obtained baseline and at 5, 15, 30, 60 and 120 min following i.c.v. CDP-choline injection. Equimolar doses of i.c.v. choline (1.0 mmol) and cytidine (1.0 mmol) were administered and measurements were repeated throughout the second round of the experiment. Atropine (10 mg) and mecamylamine (50 mg) were injected intracerebroventricularly prior to CDP-choline and measurements repeated in the third round of the experiment. After 1 mmol CDP-choline injection, serum ghrelin levels were suppressed significantly at 60 min (P¼ 0.025), whereas serum leptin levels were increased at 60 and 120 min (P ¼0.012 and P¼ 0.017 respectively). CDP-choline injections also induced a dose- and time-dependent increase in serum glucose and corticosterone levels. The effect of choline on serum leptin and ghrelin levels was similar with CDPcholine while no effect was seen with cytidine. Suppression of serum ghrelin levels was eliminated through mecamylamine pretreatment while a rise in leptin was prevented by both atropine and mecamylamine pretreatments. In conclusion; centrally injected CDP-choline suppressed serum ghrelin levels while increasing serum leptin levels. The observed effects following receptor antagonist treatment suggest that nicotinic receptors play a role in suppression of serum ghrelin levels,whereas nicotinic and muscarinic receptors both play a part in the increase of serum leptin levels. & 2015 Elsevier B.V. All rights reserved.
Keywords: CDP-choline Ghrelin Leptin
1. Introduction Appetite is regulated by a complex system of central and peripheral signals. Satiety signals from the gastrointestinal tract act through the arcuate nucleus of the hypothalamus and solitary tract nucleus of the brain system. Ghrelin, a potent gut–brain orexigenic peptide, has a role in the stimulation of food intake and long term regulation of body weight. Ghrelin regulates, in an antagonistic manner to leptin, the synthesis and secretion of several neuropeptides in the hypothalamus that regulate feeding and energy balance (Näslund and Hellström, 2007). CDP-choline is an endogenously-synthesized nucleotide that n Correspondence to: Sevket Yilmaz Education and Research Hospital, Department of Internal Medicine, Mimar Sinan Mah. Emniyet Cad. Yıldırım, Bursa, Turkey. E-mail addresses: [email protected] (S. Kiyici), nesfi[email protected] (N.F. Basaran), [email protected] (S. Cavun), [email protected] (V. Savci). 1 Present Address: Sitki Kocman University Medical Faculty, Department of Pharmacology, Mugla, Turkey.
http://dx.doi.org/10.1016/j.ejphar.2015.07.014 0014-2999/& 2015 Elsevier B.V. All rights reserved.
exerts numerous cellular actions in different experimental models. Exogenous administration of CDP-choline has been shown to affect brain metabolism and exhibit cardiovascular, respiratory, neuroendocrine and neuroprotective actions and has beneficial effects in the treatment of some neurodegenerative and neurovascular disease (Weiss, 1995, Dávalos and Secades, 2011, Topuz et al., 2014, Hurtado et al., 2011). Administered orally, intravenously or intracerebroventricularly (i.c.v.), CDP-choline is rapidly metabolized to choline and cytidine/uridine which results in the elevation of plasma and tissue levels of these metabolites (Lopez et al., 1987, Wurtman et al., 2000). Treatments that raise plasma and tissue choline levels increase the synthesis of acetylcholine and enhance cholinergic transmission (Cohen and Wurtman, 1976, Ulus et al., 1989). Results of studies which investigate the role of the cholinergic system and vagus on serum ghrelin levels were controversial. There is also little data about the role of cholinergic system on serum leptin levels. In a previous study, it has been shown that appetite ratings decline significantly after CDP-choline treatment (Killgore et al., 2010). But there is no data on whether
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there are any alterations in serum ghrelin and leptin levels, or if these alterations are involved in the effect of CDP-choline on appetite. The dense cholinergic innervation of the hypothalamopituitary system and the presence of cholinergic receptors in this area are very well known.When the dense cholinergic innervation of hypothalamus, which has an important role in feeding control (Mason, 1985, Michels et al., 1986), and the effect of vagal stimulus on feeding are taken into consideration, CDP-choline treatment could have an effect on serum ghrelin and leptin levels. Therefore, the present study was designed to determine the effect of centrally injected CDP-choline on serum ghrelin and leptin levels and the involvement of central cholinergic receptors that mediate the effect of CDP-choline. We also aimed to measure serum glucose and corticosterone levels that can interact with serum concentrations of these peptides after central CDP-choline injection.
2. Materials and methods Male Wistar-Albino rats (300–350 g; Experimental Animal Breeding and Research Center, Uludag University Medical Faculty, Bursa, Turkey) were housed under a 12 h light/dark cycle with free access to food and water. The surgical and experimental protocols were approved by the Animal Care and Use Committee of Uludag University and are in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
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i.c.v. In the third set of experiments, the effects of the blockade of central muscarinic or nicotinic acetylcholine receptors on serum ghrelin, leptin and glucose responses were investigated. Rats were pretreated i.c.v. with atropine sulfate (10 μg), mecamylamine HCL (50 μg) or saline (5 μl) 15 min before i.c.v. injection of CDP-choline (1.0 mmol). In the second and third sets of experiments, surgical procedures were performed as described in the first set of experiments. Blood samples (0.5 ml) were taken just before the i.c.v. injections and at 60 and 120 min following the treatments. Time intervals were assessed according to the maximum effect of CDP-choline on serum ghrelin and leptin levels were observed in the first set of experiments. Each blood sample was replaced with equal volume of saline. Blood glucose was detected immediately from samples by a commercially available glucometer using test strips. Blood samples were centrifuged at 3000 g for 15 min at 4 °C and serum samples were separated and stored at 20 °C until analysis. Rats were killed with high dose ether anesthesia after the completion of experiments. 2.3. Drugs The following drugs were used: CDP-choline, choline, cytidine, atropine sulfate, mecamylamine HCL (Sigma, St Louis, MO, USA). Drugs were dissolved in saline (0.9% NaCl). 2.4. Measurements
2.1. Surgical procedures Animals were allowed to acclimate to the animal care facility for 7 days before the experiment. Following the acclimatization period, rats were anesthetized with ether. The left carotid artery was cannulated with PE-50 tubing filled with heparinized saline (250 U/ml) and the cannulas were exteriorized at the nape of the neck and sealed until use. For i.c.v. drug administration, a 21-gauge stainless steel guide cannula was implanted in the right lateral ventricle through a burr hole drilled into the skull. The tip of the guide cannula was positioned 1.5 mm lateral to the midline, 1.0 mm posterior to bregma and 4.5 mm below the skull surface. The cannula was fixed to the skull with acrylic cement. At the end of the surgical procedure, rats were housed in individual cages and allowed to recover from anesthesia for 4–5 h. During this period, rats did not exhibit any sign of pain.
Serum ghrelin, leptin and corticosterone levels were measured by using commercially available RIA kits (Millipore's Ghrelin [Total] Radioimmunoassay Kit, MO, ABD, sensitivity 93 pg/ml), leptin (Linco's Rat Leptin Radioimmunoassay Kit, MO, ABD, sensitivity 0.5 ng/ml) and corticosterone (ImmuChem Double Antibody Corticosterone RIA Kit, Orangeburg, NY, sensitivity 7.7 ng/ml). 2.5. Data and statistical analysis Comparisons between groups were performed using Student's t-test for normally distributed variables, and Mann–Whitney Utest for non-normal variables. Paired t test and Wilcoxon signedrank test were used for within-group comparisons. The data were analyzed using SPSS for Windows, Version 11.5 and shown as mean 7standard deviation. A P value of less than 0.05 was considered statistically significant.
2.2. Experimental procedures In the first set of experiments, dose and time courses of serum ghrelin, leptin, corticosterone, and glucose responses to i.c.v. CDPcholine were studied. Rats were randomized to four groups and various doses of CDP-choline 0.5 (n ¼14), 1.0 (n ¼16), 2.0 (n ¼16) μmol and 5 μl (n¼ 16) saline (0.9% NaCL) were administered. Blood samples (0.5 ml) were taken just before the i.c.v. injection (0 min) of CDP-choline or saline and at 5, 15, 30, 60 and 120 min following administration. Each set of animals was used for the three time intervals and each blood sample replaced with equal volume of saline. Blood glucose was detected immediately from samples by a commercially available glucometer using test strips. Blood samples were centrifuged at 3000 g for 15 min at 4 °C and serum samples were separated and stored at 20 °C until analysis. Rats were killed with high dose ether anesthesia after the first set of experiments. In the second set of experiments, in order to determine the effects of hydrolysis product of CDP-choline, choline, and cytidine, on serum ghrelin, leptin and glucose responses, equimolar dose of choline (1.0 mmol), cytidine (1.0 mmol) or saline (5 ml) was injected
3. Results 3.1. Effects of intracerebroventriculary injected CDP-choline on serum ghrelin, leptin, glucose and corticosterone levels There was no difference between basal serum ghrelin and leptin levels in the groups. Serum ghrelin levels were suppressed significantly at 60 min following 1.0 mmol CDP-choline i.c.v. injection (P ¼0.025) (Fig. 1). When area under the curve (AUC) was calculated, serum ghrelin levels were found significantly suppressed after 0.5 mmol (AUC ¼160711.4 pg dk/ml) and 1.0 mmol (AUC ¼147.57 11.1 pg dk/ml) of i.c.v. CDP-choline injection compared with the saline group (AUC ¼1907 18 pg dk/ml) (P ¼0.035 and P¼0.013, respectively). Administration of 0.5, 1.0 and 2.0 mmol dose of i.c.v. CDP-choline significantly increased serum leptin levels at 60 min (P ¼0.036, P ¼0.012 and P ¼0.043; respectively) compared with basal values and a significant increase was also found at 120 min following 1.0 and 2.0 mmol dose of i.c.v. CDP-choline injection
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Fig. 1. Effect of i.c.v. CDP-choline on serum ghrelin (pg/ml) levels. Data are given as mean 7S.D. α ¼statistically significant compared with basal values. Pα ¼ 0.025.
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Fig. 3. Effect of i.c.v. CDP-choline on blood glucose (mg/dl) levels. Data are given as mean7 S.D. α, β, χ, δ, ε, ϕ, γ, η, ι, φ, κ ¼statistically significant compared with basal values. Pα ¼ 0.024, Pβ ¼ 0.018, Pχ ¼0.012, Pδ ¼ 0.012, Pε ¼ 0.012, Pϕ ¼ 0.012, Pγ ¼ 0.012, Pη ¼ 0.025, Pι ¼0.017, Pφ ¼ 0.012, Pκ ¼0.017.
8 Saline
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Fig. 2. Effect of i.c.v. CDP-choline on serum leptin (ng/ml) levels. Data are given as mean 7S.D. α, β, χ, δ, ε ¼statistically significant compared with basal values. Pα ¼ 0.036, Pβ ¼ 0.012, Pχ ¼ 0.043, Pδ ¼ 0.017, Pε ¼ 0.046.
(P ¼0.017 and P¼ 0.046, respectively) (Fig. 2). AUC for serum leptin concentration was 632.37116.0 ng dk/ml after 1.0 mmol i.c.v.CDPcholine and 445.57 80.9 ng dk/ml after i.c.v. saline injection. When the two groups were compared, the difference was statistically significant (P¼ 0.005). CDP-choline injection also caused a dose- and time-related increase in blood glucose and serum corticosterone levels. Blood glucose levels were significantly increased at 15 and 60 min after 0.5 mmol CDP-choline (P ¼0.012 and P ¼0.025, respectively), 5, 15, 30, and 60 min after 1.0 mmol CDP-choline (P¼ 0.024, P ¼0.012, P ¼0.012 and P ¼0.017, respectively) and 5, 15, 30, 60 and 120 min after 2.0 mmol CDP-choline injection (P ¼0.018, P ¼0.012, P ¼0.012, P ¼0.012 and P¼ 0.017, respectively) compared with basal levels (Fig. 3). When the three groups were compared, serum glucose levels were found significantly elevated at 5, 15 and 30 min (P ¼0.020, P ¼0.006 and P ¼0.002, respectively). Serum corticosterone was also found elevated at 15 min following 0.5 mmol CDPcholine (P ¼0.018), at 15 and 30 min following 1.0 mmol CDP-choline (P ¼0.028 and P¼ 0.035) and at 5, 15 and 60 min after 2.0 mmol CDP-choline i.c.v. injection (P¼ 0.027, P ¼0.034 and P ¼0.028, respectively) compared with basal values (Fig. 4).
0 0
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15 30 Time (min)
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Fig. 4. Effect of i.c.v. CDP-choline on serum corticosterone (ng/ml) levels. Data are given as mean 7 S.D. α, β, χ, δ, ε, ϕ¼ statistically significant compared with basal values. Pα ¼ 0.027, Pβ ¼0.018, Pχ ¼0.028, Pδ ¼ 0.034, Pε ¼ 0.035, Pϕ ¼ 0.028.
3.2. Effects of intracerebroventriculary injected choline and cytidine on serum ghrelin, leptin and glucose levels In order to investigate whether i.c.v. administration of equimolar doses of choline or cytidine, which are metabolic products of CDP-choline, can effect serum ghrelin, leptin and glucose levels, rats were injected with choline (1.0 mmol), cytidine (1.0 mmol) or saline (5 ml) i.c.v. There were no statistically significant changes in serum ghrelin levels after cytidine or saline injection, whereas levels were significantly suppressed after choline administration at 60 (P ¼0.043) and 120 (P ¼0.043) min compared with basal levels (Fig. 5). When the three groups were compared, percentage change in ghrelin levels was statistically significant at 120 min (P ¼0.003). No statistically significant changes in serum leptin levels were found after intra- and intergroup comparisons in either group. Choline tended to increase leptin levels especially at 60 min but it did not reach significant levels (Fig. 6). Serum glucose levels were found increased at 60 (P ¼0.018) and 120 (P¼ 0.050) min after choline injection but did not change statistically after cytidine or
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Fig. 5. Effect of i.c.v. injected equimolar doses of choline or cytidine on serum ghrelin (pg/ml) levels. Data are given as mean 7 S.D. α, β ¼ statistically significant compared with basal values. Pα ¼ 0.043, Pβ ¼ 0.043.
0 Saline
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Pretreatment Fig. 8. Effect of atropine or mecamylamine pretreatment on CDP-choline induced suppression in serum ghrelin (pg/ml) levels. Data are given as mean 7 S.D. Rats were injected saline (5 ml), atropine sulfate (10 μg; i.c.v.) or mecamylamine HCL (50 μg; i.c.v) 15 min before of CDP-choline injection (1.0 mmol; i.c.v.). α, β, χ, δ, ε¼ statistically significant compared with basal values. Pα ¼ 0. 018, Pβ ¼0.028, Pχ ¼0.025, Pδ ¼ 0.036, Pε ¼ 0.025.
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3.3. Effects of blockade of central muscarinic or nicotinic cholinergic receptors on serum ghrelin, leptin and blood glucose levels
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Fig. 6. Effect of i.c.v. injected equimolar doses of choline or cytidine on serum leptin (ng/ml) levels. Data are given as mean 7S.D.
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Fig. 7. Effect of i.c.v. injected equimolar doses of choline or cytidine on blood glucose (mg/dl) levels. Data are given as mean 7 S.D. α, β ¼ statistically significant compared with basal values. Pα ¼0.018, Pβ ¼0.050.
saline injection compared with basal levels (Fig. 7). When the three groups were compared, serum glucose levels were statistically different at 60 min (P ¼0.042).
To determine if central muscarinic and/or nicotinic cholinergic receptors mediate the effects of CDP-choline on serum ghrelin, leptin and glucose levels, rats were pretreated with atropine sulfate (10 μg; i.c.v.) or mecamylamine HCL (50 μg; i.c.v.) or saline (5 ml; i.c.v.) 15 min prior to injection of CDP-choline (1.0 mmol; i.c. v.). No significant changes were found in serum ghrelin levels of rats pretreated with either saline, atropine sulfate or mecamylamine HCl prior to i.c.v. injection of saline. Serum ghrelin levels were significantly suppressed at 60 (P¼ 0.018) and 120 (P ¼0.028) min in the CDP-choline group pretreated with saline compared with basal levels. Suppression of serum ghrelin levels persisted at 60 and 120 min in the CDP-choline group pretreated with atropine (P ¼0.025 and P ¼0.036, respectively). Observed suppression of serum ghrelin levels after CDP-choline was abolished at 60 min but not at 120 min (P¼ 0.025) in the group pretreated with mecamylamine HCL (Fig. 8). No significant change was found in serum leptin levels of rats pretreated with saline, atropine sulfate or mecamylamine HCL prior to i.c.v. injection of saline.Serum leptin levels were significantly increased at 120 (P ¼0.043) min in saline pretreated CDP-choline group compared with basal levels. Observed increment in serum leptin levels at 120 min compared with basal levels was eliminated in groups pretreated with atropine sulfate or mecamylamine HCL prior to CDP-choline injection (Fig. 9). Blood glucose levels were significantly increased at 60 and 120 min in CDP-choline group pretreated with saline compared with basal levels (P ¼0.028 and P ¼0.028, respectively). The increment of blood glucose levels persisted at 60 and 120 min in CDP-choline group pretreated with atropine (P¼ 0.008 and P¼ 0.008, respectively). The observed increment of blood glucose levels after CDP-choline persisted at 60 min (P ¼0.038) but eliminated at 120 min in group pretreated with mecamylamine HCL (Fig. 10).
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Pretreatment Fig. 9. Effect of atropine or mecamylamine pretreatment on CDP-choline induced increase in serum leptin (ng/ml) levels. Data are given as mean 7 S.D. Rats were injected saline (5 ml; i.c.v.), atropine sulfate (10 μg; i.c.v.) or mecamylamine HCL (50 μg; i.c.v.) 15 min before CDP-choline injection (1.0 mmol; i.c.v.). α ¼statistically significant compared with basal values. Pα ¼ 0.043.
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Atropine
Mecamylamine
Pretreatment Fig. 10. Effect of atropine or mecamylamine pretreatment on CDP-choline induced increase in blood glucose (mg/dl) levels. Data are given as mean 7 S.D. Rats were injected saline (5 ml; i.c.v.), atropine sulfate (10 μg; i.c.v.) or mecamylamine HCL (50 μg; i.c.v.) 15 min before CDP-choline injection (1.0 mmol; i.c.v.). α, β, χ, δ, ε¼ statistically significant compared with basal values. Pα ¼ 0.028, Pβ ¼0.028, Pχ ¼ 0.008, Pδ ¼0.008, Pε ¼0.038.
4. Discussion In this study we found that serum ghrelin levels were suppressed significantly at 60 min (P ¼0.025) whereas serum leptin levels were increased at 60 min and 120 min (P ¼0.012 and P ¼0.017) following 1.0 mmol CDP-choline i.c.v. injection compared with basal values. When AUC was calculated, a significant suppression was observed in serum ghrelin levels after 0.5 mmol and 1.0 mmol CDP-choline i.c.v. injection (P ¼0.035 and P ¼0.013) while an increase was found in serum leptin levels after 1.0 mmol CDPcholine i.c.v. injection (P ¼0.005) compared with the saline group. Central CDP-choline injections also induced a dose- and time-dependent increase in serum glucose and corticosterone levels. It has been clearly demonstrated that the choline concentration of cerebrospinal fluid, hypothalamus, third ventricle and corpus striatum increase after i.c.v. administration of CDP-choline (Lopez
et al., 1987, López-Coviella et al., 1995, Savci et al., 2002, Paroni et al., 1985). CDP-choline is rapidly metabolized to form choline and cytidine in vivo. Choline is also a precursor for the synthesis of the cholinergic neurotransmitter, acetylcholine. Treatments that raise plasma and tissue choline levels increase the synthesis of acetylcholine and enhance cholinergic transmission. CDP-choline treatment has been shown to increase tissue and plasma choline and cytidine concentration to produce pharmacological effects (Wurtman et al., 2000, López-Coviella et al., 1995, Savci et al., 2002, Paroni et al., 1985). In this study, the effects of i.c.v. CDPcholine and choline administration on serum ghrelin and leptin levels were similar, whereas we did not find any change following cytidine injection. Concurrent with previous reports, these present findings suggested that the effect of CDP-choline on serum ghrelin and leptin levels were mediated by its choline component. The dense cholinergic innervations of the hypothalamopituitary system and the presence of cholinergic receptors in this area are very well known (Mason, 1985, Michels et al., 1986). It has also been shown that cholinergic mechanisms have a role in the regulation of some hypothalamopituitary hormone secretions (Kelijman and Frohman, 1991, Savci et al., 1996, Gurun et al., 1997, Cavun and Savci, 2004a, Cavun et al., 2004b). Neuronal networks originating in the hypothalamic arcuate nucleus play fundamental roles in the control of energy balance. Immunoreactivities for proteins required for cholinergic transmission were demonstrated in the cell bodies of arcuate nucleus neurons expressing POMC and CART by the immunohistochemical experiments. These results indicate a role for acetylcholine in control of energy balance, mediating the effects of peripheral hormones such as leptin and insulin (Everitt et al., 1986, Meister et al., 2006). The observed effects of centrally-injected CDP-choline on serum ghrelin and leptin levels in this study confirm and extend previous data reporting that cholinergic mechanisms have a role in energy balance and hormonal regulation. As far as we know this is the first study investigating the effect of i.c.v injected CDP-choline on serum ghrelin and leptin levels in rats. Ghrelin is a powerful orexigenic peptide predominantly secreted by the stomach and vagal activity is important in the secretion and the effect of ghrelin (Date et al., 2002, Korbonits et al., 2004, Sakata et al., 2003). Shrestha et al. (2009) found that acetylcholine (1 mmol) increased ghrelin release from the stomach by approximately 37% whereas insulin (10 nM) decreased it by approximately 30% vs. the control in a study of stomach vascular perfusion model. Maier et al. (2004) reported that atropine alone significantly reduced fasting ghrelin levels by 25%, whereas under acetylcholine esterase inhibitor pyridostigmine alone, ghrelin levels were unaltered in a randomized study on healthy male volunteers. In another human study, it was shown that circulating ghrelin levels were increased following the muscarinic antagonist pirenzepine while decreasing following an indirect cholinergic agonist pyridostigmine (Broglio et al., 2004). In contrast to our results, it was observed that ghrelin levels increased following cholinergic agonists and were suppressed after cholinergic antagonist treatments in these studies. But Shresta et al. (2009) study has an in vitro design with the other two studies comprising of human study formats. Treatments were also administered peripherally in these two studies. In our study, CDP-choline which acts as a cholinergic activator was administered i.c.v. and contrary to expectations, serum ghrelin levels were found to be suppressed. Consistent with these results, ghrelin levels were found increased following i.v. cholinergic blockers and suppressed after i.v. cholinergic accelerator administration in a study which was performed on sheep (Sugino et al., 2003). Adipose tissue is innervated predominantly by the sympathetic system and leptin is a peptide which is mainly produced by adipose tissue (Hube et al., 1996, Berthoud et al., 2006). There are few
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studies investigating the cholinergic system relationship with leptin secretion. Ueda et al. (2001) reported that the intravenous injection of neostigmine, a cholinesterase inhibitor, increased plasma leptin and corticosterone levels in a dose-dependent manner and atropine injection prevented neostigmine-induced increase in leptin. Furthermore, bilateral adrenalectomized rats showed no increase in plasma leptin levels. In addition, it was observed that the expression of leptin messenger ribonucleic acid (mRNA) in white adipose tissue was significantly increased after neostigmine injection. We also found that i.c.v. CDP-choline injection, a cholinergic activator, induced an increase in serum leptin and corticosterone levels consistent with results of the previous study. Obesity and hyperinsulinemia were found associated with increased vagal cholinergic activity in many animal models (Bray and York, 1979, Rohner-Jeanrenaud, 1995, Gilon and Henquin, 2001). The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation (Levey et al.,1994). M3 receptor–deficient mice showed decreased food intake, reduced body weight and peripheral fat deposits and had suppressed serum insulin and leptin levels (Yamada et al., 2001). Nicotinic acetylcholine receptors also have been implicated in the control of appetite and body weight, as well as energy and lipid metabolism. Marrero et al. (2010) reported that a novel alpha 7 nicotinic acetylcholine receptor-selective agonist administration reduced weight gain and food intake in a mouse model of diabetes. There are many different examples which shows that activation of muscarinic and nicotinic receptors have opposite physiological effects compared with each other. Existence of different muscarinic and nicotinic receptor subtypes and different anatomical locations might be the reason of the observed opposite physiological effects of these receptors following activation. We found that suppression of serum ghrelin levels was eliminated by mecamylamine pretreatment while leptin increment was blocked by both atropine and mecamylamine pretreatments. Present findings suggest that the activation of central nicotinic receptors mediates the CDP-choline induced decrease in serum ghrelin levels, while the activation of central muscarinic and nicotinic receptors are both involved in the increase of serum leptin levels. CDP-choline and choline treatments have some pharmacological and metabolic effects. Intraperitoneal administration of choline increased serum insulin as well as plasma levels of catecholamines resulted in hyperglycemia by increasing cholinergic neurotransmission (Ilcol et al., 2002). Intracerebroventricular injection of choline also produced a dose-dependent increase in blood glucose levels and plasma catecholamines (Gurun et al., 2002). It was observed that i.p. injection of CDP-choline, like choline, resulted with hyperglycemia and increased serum insulin and catecholamines levels (Ilcol et al., 2007, Cansev et al., 2008a). Acetylcholine acts as a major neurotransmitter in the neuronal control of insulin and glucagon secretion from pancreatic endocrine cells (Ahrén, 2000). It has been observed that i.p. and i.c.v. administration of CDP-choline increase plasma glucagon levels in rats (Cansev et al., 2008b). In accordance with previous studies we found that i.c.v. administration of CDP-choline induced a time- and dose-dependent increase in serum glucose and corticosterone levels. Previous studies indicate that insulin and hyperglycemia suppress circulating ghrelin levels (Saad et al., 2002, Koerner et al., 2005, Flanagan et al., 2003), and also glucagon seems to act centrally to induce a reduction in ghrelin concentration (Arafat et al., 2005, 2006). Moreover insulin, glucose and corticosterone appear to increase leptin secretion (Koerner et al., 2005).When the effects of choline and CDP-choline administration on serum glucose, insulin, corticosterone and glucagon levels are taken into consideration, the observed effect of i.c.v. CDP-choline on serum
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ghrelin suppression and leptin increase in the present study might be secondary to the mentioned hormonal changes induced by CDP-choline. Moreover, eating behavior can be influenced by both metabolic and non-metabolic stress factors (Zappulla 2008, Monteleone and Maj, 2013). However in this study, we mainly focused on exploring whether CDP-choline administration may influence ghrelin and leptin concentration and if central cholinergic receptors mediate these effects. The involvement of other systems in CDP-choline's effect can be the subject of further research. In conclusion; centrally injected CDP-choline suppresses serum ghrelin levels while increasing serum leptin levels. The observed effects following receptor antagonists’ administration suggest that nicotinic receptors play a role in the suppression of serum ghrelin levels, whereas nicotinic and muscarinic receptors both take a part in the increment of serum leptin levels. The clinical relevance of this cholinergic system should be validated by additional detailed studies that investigate serum ghrelin and leptin levels following peripheral administration of CDP-choline and/or using selective cholinergic receptor antagonists.
Acknowledgments This study was supported by Grants from the Research Fund of Uludag University (Bursa/Turkey; UAP (T)-2009/8).
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