Effects of ghrelin on hypothalamic glucose responding neurons in rats

Brain Research 1055 (2005) 131 – 136 www.elsevier.com/locate/brainres

Research Report

Effects of ghrelin on hypothalamic glucose responding neurons in rats Xi Chen a, Yin-Lin Ge a, Zheng-Yao Jiang a,*, Chang-Qin Liu a, Inge Depoortere b, Theo L. Peeters b a

Department of Physiology, Qingdao University School of Medicine, Qingdao 266021, P.R. China b Centre for Gastroenterological Research, Catholic University of Leuven, Leuven, Belgium Accepted 30 June 2005 Available online 9 August 2005

Abstract Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) with potent stimulatory effects on food intake. The aim of the present study was to investigate the effects of ghrelin on neuronal activity of hypothalamic glucose responding neurons. Single unit discharges in the lateral hypothalamic area (LHA), the ventromedial hypothalamic nucleus (VMH), and the parvocellular part of the paraventricular nucleus(pPVN) were recorded extracellularly by means of four-barrel glass micropipettes in anesthetized rats. The activity of glucose-sensitive neurons (GSNs) in the LHA, pPVN, and of glucoreceptor neurons (GRNs) in the VMH modulated by administration of ghrelin was analyzed. In the LHA, the majority of GSNs (17/25) increased in frequency due to ghrelin. Whereas the majority of VMH-GRNs (27/33) and pPVN-GSNs (9/13) was inhibited. The responses to ghrelin were abolished by pretreatment of [d-Lys-3]-GHRP-6, ghrelin receptor antagonist. These data indicate that the glucose responding neurons in the LHA, VMH, and pPVN are also involved in the orexigenic actions of ghrelin in the hypothalamic circuits, although AgRP/NPY neurons in the arcuate nucleus (ARC) are the primary targets of ghrelin. D 2005 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Central control of ingestion Keywords: Ghrelin; Glucose-sensitive neuron; Glucoreceptor neuron; LHA; VMH; pPVN

1. Introduction Ghrelin is a recently identified endogenous ligand of the growth hormone secretagogue receptor (GHS-R) [15]. It was originally isolated from the stomach [5,6], but has also shown to be present in the rat hypothalamus [4,18]. Recent data have led to the recognition that ghrelin plays an important role in body-weight regulation and energy Abbreviations: AgRP, agouti-related peptide; CART, cocaine- and amphetamine-regulated transcript; POMC, proopiomelanocortin; GSN, glucose-sensitive neuron; GRN, glucoreceptor neuron; ARC, arcuate nucleus; LHA, lateral hypothalamic area; pPVN, parvocellular part of paraventricular nucleus; VMH, ventromedial hypothalamic nucleus; GHSR, growth hormone secretagogue receptor * Corresponding author. Fax: +86 532 83801449. E-mail address: [email protected] (Z.-Y. Jiang). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.06.080

homeostasis because its administration increases food intake and causes fat and weight gain in rodents [23,31]; the orexigenic effect of ghrelin seems to be independent of its GH-releasing activity [14]. It has been found that circulating levels of ghrelin increase following a 48-h fast, and infusion of glucose into the stomach decreases plasma ghrelin concentration [22,31]. Information accumulated over the past decade has revised our views on the hypothalamic control of appetite. Hypothalamic areas including the paraventricular nucleus (PVN), perifornical area (PFA), and the lateral hypothalamic area (LHA) are richly supplied by axons from the arcuate nucleus (ARC) NPY/AgRP and POMC/CART neurons [8,29]. The recent studies have shown that injection of ghrelin into the cerebrospinal fluid (CSF) induces c-fos expression in the PVN, dorsomedial (DMH), VMH, and

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ARC of the hypothalamus, as well as in the nucleus of the solitary tract (NTS) and area postrema (AP) of the brain stem [17]. It has been established that the glucose-sensitive neurons (GSNs) in the LHA, pPVN, and glucoreceptor neurons (GRNs) in the VMH are involved in the control of food intake [25]. Furthermore, it has been shown that the activity of GSNs in the LHA was suppressed by leptin, whereas the activity of GRNs in the VMH was facilitated; in contrast, orexin-A had opposite effects [30]. The present study was undertaken to examine the effects of ghrelin on the glucose responding neurons in the LHA, VMH, and pPVN.

(Gut Hormone Laboratory, Leuven, Belgium). Drugs were ejected on the surface of firing cells with short pulse gas pressure (1500 ms, 5.0 –15.0 psi) [13]. The intrabarrel drug concentrations were chosen on the basis of their efficacy to reliably alter cell firing. Volumes less than 1 nl of ghrelin were applied to the firing cells during extracellular recording. The recorded electrical signals were amplified and displayed on a Memory Oscilloscope (VC-11, Nihon Kohden), the analog signals were fed into a signal analyzer and computer which incorporated a signal discriminator to allow unitary data to be stored on-line. 2.3. Histological verification

2. Materials and methods 2.1. Animals Adult Wistar rats (Qingdao Institute for Drug Control) of either sex, weighing 220– 280 g, were used. They were housed under conditions of controlled illumination (12:12-h light/dark cycle, lights on/off: 8:00 a.m./8:00 p.m.), humidity, and temperature (22 T 2 -C) for at least 7 days prior to the experiments. Standard laboratory chow pellets and tap water were available ad libitum. All animal experiments were carried out in accordance with the ethic guidelines of Qingdao University for animal care. 2.2. Electrophysiological recordings Rats were anesthetized with urethane (1.0 g/kg, i.p.) and a maintenance dose of anesthetics was given whenever necessary. Anesthetized animals were positioned in a stereotaxic apparatus (Narishige SN-3, Tokyo, Japan) with the incisor bar 3.3 mm below the center of ear bars, the dorsal surface of the brain was exposed. Stereotaxic coordinates were as follows: LHA (1.8 –2.3 mm posterior to the bregma, 1.5– 2.5 mm lateral to the sagittal sinus, 7.5– 9.0 mm ventral from the dura); VMH [P: 2.8 –3.3 mm, L(R): 0.2 – 1.0 mm, H: 9.3– 10 mm]; pPVN [P: 1.8 – 2.3 mm, L(R): 0.1– 0.4 mm, H: 7.7– 8.4 mm] [26]. Rectal temperature was maintained at 36– 38 -C. Four-barrel glass microelectrode (total tip diameter 3 – 10 Am, resistance 5 – 20 MV) was used for electrophysiological recording and micro-pressure injection. The recording glass microelectrode was filled with 0.5 M sodium acetate and 2% Pontamine sky blue. The other three barrels connected with 4channel pressure injector (PM2000B, Micro Data Instrument, Inc., USA) were filled with 2 M solution of glucose (pH 7.4), 15 nM solution of ghrelin, and 28 nM solution of [d-Lys-3]GHRP-6 (each was dissolved in 0.9% NaCl) and 0.5 M NaCl, respectively. The barrel filled with 0.5 M NaCl was used to rule out the osmotic effects and any neurons that responded to Na+ or Cl applications were omitted from the results. Rat ghrelin and the ghrelin receptor antagonist ([d-Lys3]-GHRP-6) were generously supplied by Dr. T.L. Peeters

To check the position of the recording electrode, at the end of each experiment a direct current (10 AA, 20 min) was passed through the electrode to form an iron deposit of Pontamine sky blue. The rats were perfused transcardially with 0.9% saline, followed by 10% buffered Formalin solution. The brains were removed, 50-Am frozen coronal sections were cut through the regions of the hypothalamus, stained with Neutral red, cleared with xyline, and coverslipped. 2.4. Data analysis Data were expressed as means T standard error of the mean (SEM). Comparisons of agents induced responses before (pre-) and after (post-) treatment were made by Student’s t test; the differences of the percentages between GSNs and non-GSNs responding to ghrelin or [d-Lys-3]GHRP-6 or not responding on LHA, VMH, and pPVN neurons were tested by means of the v 2 test. Differences were considered to be significant at P < 0.05.

3. Results Results of ghrelin on hypothalamic GSNs and non-GSNs are summarized in Table 1. 25 (35%) GSNs in 72 LHA neurons were identified by their suppression in response to applied glucose. Of 25 LHA-GSNs tested with ghrelin, 17 (68%) GSNs were excited. 33 (40%) GRNs in 81 VMH neurons were identified by their facilitation in response to Table 1 Effects of ghrelin on hypothalamic neurons Decrease LHA 25 GSNs 47 Non-GSNs VMH 33 GRNs 48 Non-GRNs pPVN 13 GSNs 36 Non-GSNs

Increase

No effect

3 8

17 2

5 37

27 5

2 19

4 24

9 5

1 9

3 22

X. Chen et al. / Brain Research 1055 (2005) 131 – 136

applied glucose. Of 33 VMH-GRNs tested with ghrelin, 27 (81.88%) GRNs were inhibited. 13 (26%) pPVN-GSNs were identified by their suppression in response to glucose. Of 13 pPVN-GSNs tested with ghrelin, 9 (70%) pPVNGSNs were suppressed. 3.1. Effects of ghrelin on LHA GSNs The effects of pressure-ejected application of glucose and ghrelin were studied in a total of 72 LHA neurons in 85 rats. Neurons were said to be glucose-sensitive if the activity of the neuron is decreased by application of glucose [25]. Glucose inhibited about 35% (25/72) neurons in LHA, which were identified as GSNs. In LHA, 17 out of 25 (68%) GSNs and 2 out of 47 (4.3%) non-GSNs showed an excitation in response to the administration of ghrelin, whereas 1.2% (3/25) GSNs and 17% (8/47) non-GRNs were inhibited. The changes in the firing rate in response to micro-pressure injection of glucose and ghrelin are illustrated in Fig. 1. Administration of ghrelin increased the firing rate of LHA-GSNs by 62.0 T 12.9%. This increase in neuronal activity was statistically significant compared with control level ( P < 0.05). The ghrelin-induced response lasted for 148.2 T 29.3 s. In contrast, ghrelin had no effect on 20% (5/25) GSNs and 78.7% (37/47) non-GSNs. These data show that ghrelin had an excitatory effect on a large proportion of GSNs in the LHA. In addition, the effectiveness of ghrelin receptor antagonist was tested by the administration of [d-Lys-3]-GHRP-6 on the 10 LHA-GSNs. After treatment with [d-Lys-3]-GHRP-6, the ghrelininduced response was abolished.

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3.2. Effect of ghrelin on VMH-GRNs Out of a total of 81 VMH neurons in 50 rats, 33 (40%) were identified as GRNs by their facilitation in response to applied glucose (Fig. 2). 33 VMH-GRNs were tested for response to ghrelin. Administration of ghrelin decreased the firing rate by 67.2 T 4.3% in 27 GRNs (27/33, 81.8%), this decrease was statistically significant compared to 0.5 M NaCl-injected controls ( P < 0.05). The duration of ghrelininduced response was about 187.37 T 39.34 s. It is important to note that a substantial non-GRNs (19/48, 40%) responded to ghrelin with an increase in activity. The difference of the ghrelin-induced responses between GRNs and non-GRNs is significant ( P < 0.001). In 7 VMHGRNs, after treatment with [d-Lys-3]-GHRP-6, administration of ghrelin failed to cause any changes in the firing rate. 3.3. Effect of ghrelin on pPVN GSNs In 43 rats, 13 pPVN-GSNs, identified by their suppression in response to applied glucose, were tested for response to ghrelin. As shown in Fig. 3, of 13 pPVN-GSNs tested, the activity of 70% (9/13) of the GSNs was suppressed significantly by ghrelin ( 69.3 T 9.0% compared with 0.5 M NaCl, P < 0.05). The duration of inhibitory effect lasted about 173.75 T 33.85 s after ghrelin. 9 out of 36 (25%) nonGSNs were excited by ghrelin. Similar to VMH, ghrelin had opposite effects on pPVN GSNs and non-GSNs ( P < 0.001). [d-Lys-3]-GHRP-6 antagonized the effect of ghrelin on 4 GSNs.

Fig. 1. Effects of ghrelin, [d-Lys-3]-GHRP-6 on firing rate of GSNs in LHA. (A) Changes in firing rate in response to administration of 2 M glucose, 0.5 M NaCl, and ghrelin. Application of 2 M glucose and ghrelin caused a significant decrease and an increase in the neuronal activity, respectively; administration of 0.5 M NaCl had no effect. (B) Similar responses were observed after treatment with 2 M glucose and ghrelin; after [d-Lys-3]-GHRP-6 treatment, ghrelininduced excitatory response was abolished.

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Fig. 2. Effect of ghrelin, [d-Lys-3]-GHRP-6 on the firing rate of GRNs in VMH. (A) Administration of 2 M glucose and ghrelin caused a significant increase and a decrease in the neuronal activity, respectively; application of 0.5 M NaCl had no effect. (B) After [d-Lys-3]-GHRP-6 treatment, ghrelin-induced inhibitory response was abolished.

The changes in firing rate of LHA-GSNs, VMH-GRNs, and pPVN-GSNs in response to administration of ghrelin are summarized in Table 2.

4. Discussion Recent study has demonstrated that i.c.v. administration of ghrelin stimulated feeding and activated several hypo-

thalamic brain regions in rat, including the ARC, PVN, LHA, VMH, and dorsomedial hypothalamic nucleus [17]. There is still debate about the mechanism by which ghrelin modifies feeding. In the present study, results clearly show that ghrelin significantly increases GSNs activity in comparison to the non-GSNs in the LHA. An excitation in the activity of ghrelin on the LHA-GSNs would fit nicely with the notion that the LHA appears to be one of the main targets of ghrelin-derived input [10,19]. In contrast, ghrelin

Fig. 3. Effect of ghrelin, [d-Lys-3]-GHRP-6 on the firing rate of GSNs in pPVN. (A) Administration of 2 M glucose and ghrelin caused a similar significant decrease in the neuronal activity; application of 0.5 M NaCl had no effect. (B) After [d-Lys-3]-GHRP-6 treatment, ghrelin-induced inhibitory response was abolished.

X. Chen et al. / Brain Research 1055 (2005) 131 – 136 Table 2 Changes in firing rate of glucose responding neurons after ghrelin

LHA-GSNs VMH-GRNs pPVN-GSNs

n

Changes in firing rate (%)

17 27 9

+62.0 T 12.9 67.2 T 4.3 69.3 T 9.0

P < 0.05 Compared with 0.5 M NaCl group.

inhibits a large proportion of the GRNs in the VMH. This finding is in agreement with the traditional notion that the VMH mediates the cessation of eating. It has been postulated that the PVN plays a pivotal role in the putative brain network involved in the control of satiety and energy balance [12]. Information of both anorexigenic systems and orexigenic peptidergic systems seems to converge in the PVN [2,11]. We were able to show that about 70% of GSNs in pPVN were inhibited by administration of ghrelin. A suppression in the activity of ghrelin on the pPVN GSNs fits well into the concept that the pPVN GSNs might be the second-order catabolic effectors located downstream of the ARC which play a role in the satiety regulation [29]. It is also interesting to note that leptin has opposite effects. It inhibits GSNs in the LHA and enhances activity of the glucose responsive neurons in the VMH and the pPVN [30]. There is convincing evidence that the notion of specific Fcenter_ of the brain that controls food intake and body weight has been replaced by the distinct hypothalamic neuropeptide-containing pathways [29,32]. There is also new hypothesis that populations of first-order NPY-AgRP neurons and POMC/CART neurons in the ARC are regulated by leptin and project to the pPVN, LHA, VMH, and perifornical area (PFA), which are locations of secondorder hypothalamic neuropeptide neurons involved in the regulation of food intake and energy homeostasis [29]. Moreover, the LHA GSNs containing orexin project directly to the glucose-sensitive NPY neurons in the medial ARC (mARC) where both NPY and AgRP are released [7,27]. Furthermore, it has been shown that leptin suppresses the activity of the GSNs in the mARC [9,28], whereas it enhances the activity of GRNs in the lateral ARC (lARC). The GSNs in mARC contain NPY [21], and the GRNs in lARC are POMC/CART neurons [16,20]. It has been suggested that ghrelin exerts its effect on energy balance primarily by binding to the growth hormone secretagogue(GHS)-receptor located on ARC neurons [1]. In this study, we found that the effects of ghrelin on the hypothalamic glucose responding neurons in the LHA, VMH, and pPVN are completely abolished by pretreatment with [d-Lys-3]-GHRP-6, ghrelin receptor antagonist. It is well known that ghrelin producing neurons are located mainly in the ARC, whereas GHS-Rs are distributed in various regions of the rat brain, including the ARC, VMH, PVN, LHA, infundibular nucleus, periventricular nucleus, lateral mammillary nucleus, and the hippocampus [19]. Olzewski et al. [24] recently demonstrated that intra-LH injection of ghrelin at a dose inducing food intake resulted

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in a significant increase of activation of orexin-A-containing neurons. In a recent paper, Chen et al. [3] demonstrated that the NPY/AgRP neurons in the ARC were the mediator of ghrelin-stimulated feeding. With the new evidence provided by Cowley et al. [4], ghrelin was expressed in a previously uncharacterized group of neurons in the hypothalamus. These neurons lay in the space between the lateral, arcuate, ventromedial, dorsomedial, and paraventricular nuclei, and they sent projections to several of these nuclei as well as outside the hypothalamus. In summary, we conclude that the glucose responding neurons in the LHA, VMH, and pPVN are also involved in the orexigenic actions of ghrelin in the hypothalamic circuits, although the AgRP/NPY neurons in the ARC are the primary targets of ghrelin-stimulated feeding.

Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30370467) and by the grant from the Bilateral Scientific and Technological Cooperation between Flanders and China (Contract BIL01/13).

References [1] M. Bagnasco, G. Tulipano, M.R. Melis, A. Argiolas, D. Cocchi, E.E. Muller, Endogenous ghrelin is an orexigenic peptide acting in the arcuate nucleus in response to fasting, Regul. Pept. 111 (2003) 161 –167. [2] C. Broberger, J. Johansson, M.S. Challing, T. Hokfelt, The neuropeptide Y/agouti-related protein(AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 15043 – 15048. [3] H.Y. Chen, M.E. Trumbauer, A.S. Chen, D.T. Weingarth, J.R. Adams, E.G. Frazier, Z. Shen, D.J. Marsh, S.D. Feighner, X.M. Guan, Z. Ye, R.P. Nargund, R.G. Smith, L.H. Van der Ploeg, A.D. Howard, D.J. MacNeil, S. Qian, Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein, Endocrinology 145 (2004) 2607 – 2612. [4] M.A. Cowley, R.G. Smith, S. Diano, M. Tschop, N. Pronchuk, K.L. Grove, C.J. Strasburger, M. Bidlingmaier, M. Esterman, M.L. Heiman, L.M. Garcia-Segura, E.A. Nillni, P. Mendez, M.J. Low, P. Sotonyi, J.M. Friedman, H. Liu, S. Pinto, W.F. Colmers, R.D. Cone, T.L. Horvath, The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis, Neuron 37 (2003) 649 – 661. [5] Y. Date, M. Kojima, H. Hosoda, A. Sawaguchi, M.S. Mondal, T. Suganuma, S. Matsukura, K. Kangawa, M. Nakazato, Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans, Endocrinology 141 (2000) 4255 – 4261. [6] D. Dornonvile, M. Bjorkqvist, A.K. Sandvik, I. Bakke, C.M. Zhao, D. Chen, R. Hankanson, A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control, Regul. Pept. 99 (2001) 141 – 150. [7] C.F. Elias, C.B. Saper, E. Maratos-Flier, N.A. Tritos, C. Lee, J. Kelly, M. Yanaigawa, J.K. Elmquist, Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area, J. Comp. Neurol. 402 (1998) 442 – 459. [8] J. Elmquist, C. Elias, C. Saper, From lesions to leptin: hypothalamic control of food intake and body weight, Neuron 22 (1999) 221 – 232.

136

X. Chen et al. / Brain Research 1055 (2005) 131 – 136

[9] H. Funahashi, T. Yada, S. Muroya, M. Tagigawa, T. Ryushi, S. Horie, Y. Nakai, S. Shioda, The effect of leptin on feeding-relating neurons in the rat hypothalamus, Neurosci. Lett. 264 (1999) 117 – 120. [10] X.M. Guan, H. Yu, O.C. Palyha, K.K. McKee, S.D. Feighner, D.J. Sirinathsinghiji, R.G. Smith, L.H. Van der Ploeg, A.D. Howard, Distribution of mRNA encoding the growth hormone secretagogue receptor in the brain and peripheral tissue, Brain Res. Mol. Brain Res. 48 (1997) 23 – 29. [11] C. Haskell-Luevano, P. Chen, C. Li, K. Chang, M.S. Smith, J.L. Cameron, R.D. Cone, Characterization of the neuroanatomical distribution of agouti-related protein immunoreactivity in the rhesus monkey and the rat, Endocrinology 140 (1999) 1408 – 1415. [12] T.L. Horvath, S. Diano, P. Sotonyi, M. Heiman, M. Tschop, Minireview: ghrelin and the regulation of energy balance—A hypothalamic perspective, Endocrinology 142 (2001) 4163 – 4169. [13] J.H. Jhamandas, R.W. Lind, L.P. Renau, Angiotensin II may mediate excitatory neurotransmission from the subfornical organ to the hypothalamic supraoptic nucleus: an anatomical and electrophysiological study in the rat, Brain Res. 487 (1989) 52 – 56. [14] J. Kamegai, H. Tamura, T. Shimizu, S. Ishii, H. Sugihara, I. Wakabayashi, Central effect of ghrelin, an endogenous growth hormone secretagogue, on hypothalamic peptide gene expression, Endocrinology 141 (2000) 4797 – 4800. [15] M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K. Kangawa, Ghrelin is a growth-hormone releasing acylated peptide from stomach, Nature 402 (1999) 656 – 660. [16] P. Kristensen, M.E. Judge, L. Thim, U. Rubel, KN. Christjansen, B.S. Wulff, J.T. Clausen, P.B. Jensen, O.D. Madsen, N. Brang, P.J. Larsen, S. Hastrup, Hypothalamic CART is a new anorectic peptide regulated by leptin, Nature 393 (1998) 72 – 76. [17] C.B. Lawrence, A.C. Snape, F.M. Baudoin, S.M. Luckman, Acute central ghrelin and GH secretagogues induce feeding and activate brain appetite centers, Endocrinology 143 (2002) 155 – 162. [18] S. Lu, J.L. Guan, Q.P. Wang, K. Uehara, S. Yamada, N. Goto, Y. Date, M. Nakazato, M. Kojima, K. Kangawa, S. Shioda, Immunocytochemical observation of ghrein-containing neurons in the rat arcuate nucleus, Neurosci. Lett. 321 (2002) 157 – 160. [19] V. Mitchell, S. Bouret, J.C. Beauvillain, A. Schilling, M. Perent, C. Kordon, Comparative distribution of mRNA encoding the growth hormone secretagogue-receptor (GHS-R) in Mirocebus murinus

[20]

[21]

[22]

[23]

[24]

[25]

[26] [27]

[28]

[29] [30]

[31] [32]

(primate, lemurian) and rat forebrain and pituitary, J. Comp. Neurol. 429 (2001) 469 – 489. T.M. Mizuno, H. Bergen, C. Priest, J. Robersts, Gold-thioglucose (GTG) reduces hypothalamic proopiomelanocortin (POMC) mRNA correlated body weight gain and impaired sensitivity, Abstr. - Soc Neurosci. 24 (1998) 705. S. Muroya, T. Yada, S. Shioda, M. Tagigawa, K. Goto, Glucosesensitive neurons in the rat arcuate nucleus contain neuropeptide Y, Neurosci. Lett. 264 (1999) 113 – 116. E. Nakagawa, N. Nagaya, H. Okumura, et al., Hyperglycaemia suppresses the secretion of ghrelin, a novel growth-hormone-releasing peptide: responses to the intravenous and oral administration of glucose, Clin. Sci. (Lond) 103 (2002) 325 – 328. M. Nakazato, N. Murakami, Y. Date, M. Kojima, H. Matsuo, K. Kangawa, S. Matsukura, A role for ghrelin in the central regulation of feeding, Nature 409 (2001) 194 – 198. P.K. Olzewski, D.H. Li, M.K. Billington, C.M. Kotz, A.S. Levine, Neural basis of orexicgenic effects of ghrelin acting within lateral hypothalamus, Peptides 24 (2003) 597 – 602. Y. Oomura, Endogenous chemical sense in control of feeding, in: D. Ottoson, E.R. Perl, R.F. Schmidt, H. Shimazu, W.D. Willis (Eds.), Progress in Sensory Physiology, Springer-Verlag, Heidelberg, 1989, pp. 171 – 191. G.T. Paxinos, C. Waston, The Rat Brain in Stereotaxic Coordinates, 4th edR, Academic Press, New York, 1998. C. Peyron, D.K. Tighe, A.N. van den Pol, L. de Lecea, H.C. Heller, J.G. Sutecliffe, T.S. Kilduff, Neurons containing hypocrtin (orexin) project to multiple neuronal systems, J. Neurosci. 18 (1998) 9996 – 10015. K. Sakai, K. Nagamori, T. Shiraishi, K. Muramoto, Y. Oomura, Effects of leptin and neuropeptide Y (NPY) on rat arcuate neurons, Abstr. Soc. Neurosci. 24 (1998) 449. M. Schwartz, S. Woods, D.J. Porte, R. Seeley, D. Baskin, Central nervous system control of food intake, Nature 404 (2000) 661 – 671. T. Shiraishi, Y. Oomura, K. Sasaki, M.J. Wayner, Effects of leptin and orexin-A on food intake and feeding related hypothalamic neurons, Physiol. Behav. 71 (2000) 251 – 261. M. Tschop, D.L. Smiley, M.L. Heiman, Ghrelin induces adiposity in rodents, Nature 407 (2000) 908 – 913. S. Woods, R. Seeley, D.J. Porte, M. Schwartz, Signals that regulate food intake and energy homeostasis, Science 280 (1998) 1378 – 1385.