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Endocrinology Vol. 147, No. 4 1744-1752
Copyright © 2006 by The Endocrine Society

Galanin-Like Peptide Stimulates Food Intake via Activation of Neuropeptide Y Neurons in the Hypothalamic Dorsomedial Nucleus of the Rat

Motoki Kuramochi, Tatsushi Onaka, Daisuke Kohno, Satoshi Kato and Toshihiko Yada

Department of Physiology, Division of Integrative Physiology (M.K., T.O., D.K., T.Y.), and Department of Neuropsychiatry (M.K., S.K.), Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan

Address all correspondence and requests for reprints to: Dr. Toshihiko Yada, Department of Physiology, Division of Integrative Physiology, Jichi Medical School, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. E-mail: tyada{at}jichi.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Galanin-like peptide (GALP), a 29-amino-acid neuropeptide, is located in the hypothalamic arcuate nucleus (ARC), binds to galanin receptor subtype 2, and induces food intake upon intracerebroventricular (icv) injection in rats. However, neural mechanisms underlying its orexigenic action remain unclear. We aimed to identify the nuclei and neuron species that mediate the food intake in response to icv GALP injection. Intracerebroventricular injection of GALP, as powerfully as that of neuropeptide Y (NYP), increased food intake for the initial 2 h. GALP injected focally into the dorsomedial nucleus (DMN), but not the ARC, lateral hypothalamus, or paraventricular nucleus (PVN), stimulated food intake for 2 h after injection. In contrast, galanin injected into the DMN had no effect. DMN-lesion rats that received icv GALP injection showed attenuated feeding compared with control rats. Intracerebroventricular GALP injection increased c-Fos expression in NPY-containing neurons in the DMN, but not the ARC. GALP increased the cytosolic calcium concentration ([Ca2+]i) in NPY-immunoreactive neurons isolated from the DMN, but not the ARC. Furthermore, both anti-NPY IgG and NPY antagonists, when preinjected, counteracted the feeding induced by GALP injection. These data show that icv GALP injection induces a potent short-term stimulation of food intake mainly via activation of NPY-containing neurons in the DMN.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GALANIN IS A 29-amino-acid neuropeptide isolated from the porcine intestine (1). It is widely distributed in the central and peripheral nervous systems and has various activities, including regulation of feeding behavior, cognition, nociception, and secretion of pituitary hormones (2, 3, 4). Three distinct galanin receptor subtypes, GalR1, GalR2, and GalR3, have been identified (5). These receptors show different tissue distributions: GalR1 is expressed mainly in the central nervous system, whereas GalR2 abundantly and GalR3 at low levels are expressed in both the central nervous system and peripheral tissue.

Galanin-like peptide (GALP), a 60-amino-acid peptide whose residues 9–21 are identical with galanin-(1–13), was isolated from the porcine hypothalamus (6). GALP has a higher affinity for GalR2 than GalR1. Therefore, GALP was initially suggested to be the endogenous ligand for GalR2 (6). Later, it was reported that the action of GALP in the hypothalamus was observed in GalR2-deficient mice (7). Thus, the receptor for GALP remains to be clarified. Several actions of GALP have been shown. Intracerebroventricular (icv) injection of GALP stimulates LH secretion, and it is blocked by treatment with antagonists of LHRH (8). In relation to the energy metabolism, GALP-containing neurons express leptin receptors, and deficiency of leptin function is associated with reductions in GALP protein and mRNA expression (9, 10). Gene expression of GALP is positively regulated by leptin, insulin, and thyroid hormone, the hormones implicated in energy metabolism (11, 12).

It has been reported that GALP mRNA is expressed in the arcuate nucleus (ARC) (9, 13), a feeding regulatory center, and that icv administration of GALP increases food intake in rodents (14), although the underlying mechanisms remain largely unknown. The present study aimed to identify the nucleus and neuron species that serve as effectors for the orexigenic action of GALP in rats. First, the target nucleus was studied using the following methods. The effects of focal injections of GALP into several hypothalamus nuclei on food intake were examined. Whether destruction of specific nucleus affects orexigenic effect of icv GALP injection was examined. The expression of c-Fos protein was also examined to specify the area and neuron that were activated by icv GALP injection. Secondly, the target neuron was explored with special attention to the neuropeptide Y (NPY) neurons, in considering their central role in stimulation of feeding. NPY-containing neurons are located mainly in the ARC and dorsomedial nucleus (DMN) in the hypothalamus (15). NPY antisense injected into the ARC decreased NPY levels in this nucleus and suppressed food intake (16). Studies using animals with lesions of the DMN (DMNL) have shown that this nucleus plays a role in sensing feeding-related signals from the periphery, including cholecystokinin (CCK) (17). Therefore, we performed both in vivo and in vitro studies to determine whether GALP activates NPY neurons in the ARC and/or DMN. Furthermore, we examined the effects of icv injections of anti-NPY IgG and NPY receptor antagonists on GALP-induced feeding. We obtained results to support the idea that icv GALP injection stimulates feeding by activating NPY-containing neurons in the DMN.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The icv injections were carried out in 0.9% NaCl solution. The cytosolic calcium concentration ([Ca2+]i) measurements were carried out in a solution composed of 129 mmol/liter NaCl, 5.0 mmol/liter NaHCO3, 4.7 mmol/liter KCl, 1.2 mmol/liter KH2PO4, 1.8 mmol/liter CaCl2, 1.2 mmol/liter MgSO4, and 10 mmol/liter HEPES (pH 7.4; HKRB). Rat GALP was provided by Takeda Co. (Osaka, Japan). Rat galanin was obtained from Phoenix Pharmaceuticals (Belmont, CA). Ghrelin was provided by Dr. Kangawa and later obtained from Peptide Institute (Osaka, Japan). Rat NPY was obtained prom Peptide Institute. Fura-2/acetoxymethylester was purchased from Dojin Chemical (Kumamoto, Japan). All chemicals were obtained from Wako Biochemicals (Osaka, Japan) or Sigma-Aldrich Corp. (St. Louis, MO).

Animals
Adult male Sprague Dawley rats, weighing 280–320 g (7–8 wk old; Japan SLC, Hamamatsu, Japan; Sprague Dawley) at the time of surgery were kept in individual cages under controlled temperature (21–30 C) and light (12-h light, 12-h dark cycle; lights on at 0730 h) with ad libitum access to food (CE-2; Clea, Osaka, Japan) and water. All procedures were reviewed and approved by the institutional animal care and use committee of the Jichi Medical School and were performed in accordance with the Japanese Physiological Society’s guidelines for animal care.

Intracerebroventricular or intranuclear cannulation
Male rats were anesthetized by ip injection of Avertin (tribromoethanol, 200 mg/kg, ip) and placed in a stereotaxic frame. A stainless steel guide cannula (26 gauge) was stereotactically placed into the brain with the tip in the right lateral ventricle, PVN, DMN, ARC/ventromedial hypothalamus (VMH), and lateral hypothalamus (Lh) (coordinates: lateral ventricle, 0.8 mm posterior from the bregma and 1.6 mm lateral from the midline, 3.5 mm from the skull; PVN, 1.8 mm posterior from the bregma and 0.4 mm lateral from the midline, 7.2 mm from the skull; DMN, 3.4 mm posterior from the bregma and 0.6 mm lateral from the midline, 7.5 mm from the skull; ARC/VMH, 3.4 mm posterior from the bregma and 0.5 mm lateral from the midline, 9.8 mm from the skull; and Lh, 2.1 mm posterior from the bregma and 2.0 mm lateral from the midline, 7.4 mm from the skull) (18). Rats were allowed 1 wk to recover from the surgical procedure and were handled daily to minimize nonspecific stress responses. Substances were administered via a stainless steel injector (30 gauge), which extended 1 mm beyond the tip of the guide cannula. Experiments were carried out during the light phase (1400–1600 h). Rats received an injection of vehicle, 0.3 nmol GALP, or 0.3 nmol NPY in a volume of 5 µl into the ventricle and an injection of vehicle, 0.03 nmol galanin, 0.06 nmol galanin, or 0.03 nmol GALP in a volume of 0.5 µl into the DMN, PVN, ARC/VMH, and Lh. Food intake during the 2 and 24 h after icv or intranuclear injection was calculated by weighing the remaining pellets and spillage at the appropriate time. Sections of the hypothalamus were histologically examined at the end of the study, and the placement of the cannulas was verified. The animals whose cannulas were outside the lateral ventricle, PVN, DMN, ARC/VMH, or Lh were excluded from the data analysis.

Assessment of the effects of GALP, anti-NPY IgG, BIBP3226, 1229U91, and vehicle injection into the lateral ventricle on food intake
Rat received an icv injection of 0.3 nmol GALP and 5 µl vehicle, nonpeptidic NPY receptor antagonist BIBP3226 (60 nmol/5 µl; Sigma-Aldrich Corp.) and 5 µl vehicle, a mixture of BIBP3226 and GALP, or vehicle (35% ethanol and 0.45% NaCl) in a volume of 10 µl. After injection, animals were returned to their cages. One series of rats was injected with 0.3 nmol GALP, a peptidic NPY receptor antagonist 1229U91 (30 µg/5 µl; Sigma-Aldrich Corp.), a mixture of 0.3 nmol GALP and 1229U91, or vehicle (0.9% NaCl) in a volume of 10 µl. The other series of rats was injected with 0.3 nmol GALP, mouse IgG (0.5 µg/5 µl; Sigma-Aldrich Corp.), a mixture of 0.3 nmol GALP and mouse IgG, or a mixture of GALP and anti-NPY IgG (0.5 µg/5 µl; Yanaihara Institute, Shizuoka, Japan). Food intake during the 2 h after icv injection was measured.

c-Fos protein immunohistochemistry
GALP was injected icv via a cannula implanted 1 wk in advance. Male rats were anesthetized by ip injection of Avertin, and colchicine (0.05 mg/10 µl, icv) was icv injected 1 d before GALP injection. After icv GALP injection, food was deprived, and 2 h after injection, the rats were anesthetized by i.p. injection of 1.5 g/kg urethane (ethyl carbamate) and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 15 min. The brain was removed, postfixed for 24 h, and immersed in 30% sucrose for 48 h at 4 C. It were rapidly frozen on dry ice and stored at –80 C. Frozen tissue was sectioned at 30 µm with a sledge microtome. Coronal hypothalamic sections were made. The expression of Fos protein was examined as described previously (19). In brief, tissues were washed three times in PB, then incubated for 15 min in 1.5% H2O2 solution to quench endogenous peroxidase. After rinsing, they were incubated with 10% normal goat serum for 1 h, then with a rabbit antiserum against c-Fos protein (Ab-5; Oncogene Research Products, San Diego, CA) at a dilution of 1:10,000 that contained 5% normal goat serum. After 48 h of incubation, the slices were washed three times in PB and incubated with a goat antirabbit IgG-peroxidase complex (Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1:500 for 24 h. After extensively rinsing in PB, these slices were incubated in 0.1 M acetate buffer and processed with a glucose-oxidase-based, nickel-intensified, 3,3'-diaminobenzidine procedure. For dual immunostaining, Fos-immunolabeled sections were then washed and incubated in 1.5% H2O2 solution for 15 min. These tissues were briefly washed, blocked with 10% normal goat serum, and incubated with a rabbit anti-NPY antiserum (DiaSorin, Stillwater, MN) diluted to 1:6,000 at 4 C for 24 h. The slices were washed three times in PB and processed using an avidin-biotin-peroxidase complex method (Vector Laboratories, Inc.). Slices were incubated in biotinylated antirabbit antibody for 1 h, washed, incubated in avidin-biotinylated peroxidase for 1 h, washed, and developed with diaminobenzidine. Slices were then washed, mounted on slides, and coverslipped with Malinol (Muto, Osaka, Japan).

Cell counting for c-Fos- and NPY-containing neurons
Analysis of Fos protein positive neurons was performed under a light microscope. The number of c-Fos-immunoreactive cells per section was counted in 12 sections including the DMN at a 180-µm interval.

Assessment of the effect on food intake of GALP injection into lateral ventricle in DMN-lesioned rats
Male rats were anesthetized by ip injection of Avertin and placed in a stereotaxic frame. A stainless steel cannulas (30 gauge) was stereotactically placed into the brain with the tip in the bilateral DMN (3.4 mm posterior from the bregma and 0.6 mm lateral from the midline, 7.5 mm from the skull). Vehicle or ibotenic acid (IBO; 3 µg; Sigma-Aldrich Corp.) in a volume of 3 µl was injected into the DMN. Substances were administered via stainless steel cannulas, which extended 5 min. These rats were left for 1 wk to recover from the surgical procedure and were handled daily to minimize nonspecific stress responses. Experiments were carried out during the light phase (1400–1600 h). Rats received an injection of vehicle or 0.3 nmol GALP in a volume of 5 µl into the lateral ventricle. Food intake during the 2 h after icv injection was calculated by weighing the remaining pellets and spillage at the appropriate time. Sections of the hypothalamus were histologically examined at the end of the study, and placement of the cannula was verified. The animals whose cannulas were outside the lateral ventricle or DMN were excluded from the data analysis.

Assessment of the effect on food intake of GALP injection into lateral ventricle in ARC-lesioned rats
Neonatal rats received sc injections of monosodium glutamate (MSG) in doses of 4 mg/g body weight on d 1, 3, 5, 7, and 9 after birth. Control rats were treated with saline. At 7 wk of age, the rats were treated by icv cannulation. Rats were allowed 1 wk to recover from the surgical procedure and were handled daily to minimize nonspecific stress responses. Experiments were carried out during the light phase (1400–1600 h). Rats received an injection of vehicle, 0.3 nmol GALP, or 0.3 nmol ghrelin in a volume of 5 µl into the lateral ventricle. Food intake during the 2 h after icv injection was calculated by weighing the remaining pellets and spillage at the appropriate time. Sections of the hypothalamus were histologically examined at the end of the study, and placement of the cannula was verified. The animals whose cannulas were outside the lateral ventricle were excluded from the data analysis.

Preparation of single neurons from the DMN
The DMN was isolated from the rat brain, then single neurons were prepared according to the procedures reported previously (20, 21) with slight modifications. Briefly, rats were deeply anesthetized with an ip injection of carbamic acid ethyl ester (900 mg/kg) and decapitated. The brain was removed, slices containing the DMN were prepared, and the whole DMN was punched out. The tissue was washed with HKRB supplemented with 10 µM glucose. After washing in HKRB, the tissues were incubated in HKRB with 20 U/ml papain (Sigma-Aldrich Corp.), 0.015 mg/ml deoxyribonuclease, and 0.75 mg/ml BSA for 16 min at 36 C in a shaking water bath, followed by gentle mechanical trituration for 5–10 min. The cell suspension was centrifuged at 100 x g for 5 min. The pellet was resuspended in HKRB and distributed onto coverslips. The cells were kept at 18 C in moisture-saturated conditions until use.

Measurements of [Ca2+]i in single DMN neurons
Two to 5 h after cell preparation, [Ca2+]i was measured by ratiometric fura-2 microfluorometry combined with digital imaging, as reported previously (20, 21, 22). Briefly, after incubation with 2 µM fura-2/acetoxymethlylester for 60 min at 18 C, the cells were mounted in a chamber and superfused at 1 ml/min at 34 C with HKRB. Fluorescence images due to excitation at 340 nm and those at 380 nm were alternately detected every 8 sec with an intensified change-coupled device camera, and the ratio images were produced by an Argus-50 or Aquacosmos imaging system (Hamamatsu Photonics, Shizuoka, Japan). Ratio images were converted to [Ca2+]i images according to calibration curves (22).

Immunocytochemistry and identification of NPY neurons
After [Ca2+]i measurements, the cells were fixed with 4% paraformaldehyde overnight. They were incubated with a rabbit antiserum against NPY (1:10,000 dilution; DiaSorin) for 24 h at 4 C, followed by incubation with biotinylated antibodies raised against rabbit IgG for 1 h and then with avidin-peroxidase complex for 1 h. The sections were developed with 3,3'-diaminobenzidine. Control experiments were carried out by omitting the primary antiserum. Correlation of [Ca2+]i and immunocytochemical data was preformed using the procedure reported previously (20, 21, 22). In brief, at the end of [Ca2+]i imaging, we took phase-contrast photographs of all cells in the microscopic field subjected to [Ca2+]i measurements. Based on these photographs, the cells in which [Ca2+]i was recorded were correlated with their corresponding immunocytochemical results.

Data analysis
All data are presented as the mean ± SEM. Statistical analysis was carried out using Student’s t test for two-group experiments and an ANOVA, followed by Duncan’s new multiple range test, for experiments containing three or more groups. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GALP injection into the ventricle
GALP and NPY, when injected into the third ventricle, markedly increased food intake, whereas in control rats, icv vehicle injection had no effect on the feeding behavior (Fig. 1AGo). GALP-induced food intake was first detected at 5–15 min and continued for 1.5 h after injection (Fig. 1AGo). Notably, GALP injection stimulated food intake for the initial 2 h as strongly as NPY, a strong orexigenic substance described previously (23). In contrast, cumulative food intake 24 h after icv GALP injection was not significantly different from the control value after icv vehicle injection. In contrast, icv NPY injection tended to increase cumulative food intake at 24 h (Fig. 1BGo).


Figure 1
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  		FIG. 1. GALP icv injection stimulated food intake in rats. A, Cumulative amount of food intake after GALP, NPY, or vehicle icv injection. B, Food intake 24 h after GALP, NPY, or vehicle icv injection. There was a significant difference in the amount of food intake at 2 h between GALP- and vehicle-treated (P < 0.05), but there is not a significant difference between GALP and NPY. At 24 h, the cumulative amount of food intake between GALP-, NPY-, and vehicle-treated were not significantly different (n = 5 animals/group).

 
GALP injection into DM, Lh, ARC/VMH, and PVN
To determine the effector site for the orexigenic action of GALP, rats were microinjected with 0.03 nmol GALP into several areas of the hypothalamus that are concerned with feeding regulation. GALP injection into the DMN significantly increased food intake 2 h after injection, but injection into the PVN, Lh, or ARC/VMH did not significantly increase food intake (DMN, 1.41 ± 0.25 with GALP vs. 0.32 ± 0.14 in controls; P < 0.05; Fig. 2AGo). GALP injection into the DMN stimulated food intake mainly during the period of 40 min after injection. There was no statistically significant difference in the amounts of food intake during the 24-h period among groups injected with GALP in the DMN, Lh, PVN, and ARC/VMH. In another series of rats, GALP, galanin, or vehicle was injected into the DMN. Injection of GALP (0.03 nmol) into the DMN significantly increased food intake compared with vehicle injection, whereas injection of galanin at the same (0.03 nmol) and higher (0.06 nmol) doses did not significantly increase food intake during a 2-h period after injection [0.24 ± 0.16 g with vehicle; 1.32 ± 0.18 g with GALP (P < 0.05); 0.46 ± 0.20 g with 0.03 nmol galanin; 0.39 ± 0.11 g with 0.06 nmol galanin; Fig. 2BGo].


Figure 2
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  		FIG. 2. GALP injection into the DMN stimulated food intake. A, GALP injection into the DMN stimulated feeding, whereas injection into other nuclei did not significantly altered feeding. B, Food intake after injection of 0.03 nmol GALP, 0.03 nmol galanin, 0.06 nmol galanin, and vehicle into the DMN. GALP injection into the DMN stimulated feeding, whereas galanin at the same and 2-fold higher doses did not significantly increase food intake. *, P < 0.05 vs. control, PVN, Lh, and ARC/VMH; **, P < 0.05 vs. vehicle and galanin (n = 6 animals/group).

 
Food intake after GALP injection in DMN-lesioned rats
To examine the role of the DMN in food intake after icv GALP injection, neurons in the DMN were destroyed by bilateral injections of IBO (Fig. 3Go, A–F). IBO injection destroyed the cells of the DMN. The number of NPY-containing neurons in the DMN was reduced in DMNL rats (Fig. 3GGo). At 1 wk after cannulation and treatment with IBO, food intake per day and body weight were not statistically different between IBO- and vehicle-injected rats (Fig. 3HGo). In rats treated with IBO, food intake after GALP injection was significantly less than that of vehicle-injected rats (Fig. 3IGo).


Figure 3
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  		FIG. 3. GALP icv injection failed to increase food intake in DMN-lesioned rats. Nissl-stained cells in the hypothalamus (A and D) and DMN (B and E) and anti-NPY-immunostained cells in the DMN (C and F) in DMNL (A–C) and sham (D–F) rats. IBO (A–C) and vehicle (D–F) were injected into the bilateral DMN. Squares in A and D indicate the compact zone. G, The number of NPY-containing cells in the DMN is significantly reduced in DMNL rats. H, Food intake for 24 h was not significantly different between vehicle-treated (sham) and IBO-treated (DMNL) rats. I, Food intake for 2 h after icv injection of GALP was depressed in DMNL rats compared with sham rats. Bar, 50 µm (B and E) and 80 µm (C and F). #, P < 0.05 vs. sham; ##, P < 0.05 vs. sham plus vehicle, DMNL plus vehicle, and DMNL plus GALP; ###, P < 0.05 vs. sham plus vehicle, DMNL plus vehicle, and sham plus GALP (n = 5 animals/group).

 
Double staining for c-Fos and NPY
The icv injection of GALP significantly increased c-Fos expression in several brain regions, including the DMN, Lh, and periventricular nucleus. In the DMN, some Fos-positive cells were immunoreactive to NPY (Fig. 4AGo). There were also Fos-positive cells not immunoreactive to NPY in the DMN. Fos-positive cells were also found in the Lh. They contained neither orexin nor melanin-concentrating hormone (data not shown). In the DMN, the percentage of Fos-positive cells among NPY-immunoreactive cells increased after icv GALP injection, which was significantly larger than that in vehicle-injected control rats (39.8 ± 4.2% with GALP vs. 10.0 ± 5.1% with vehicle; P < 0.05; Fig. 4BGo).


Figure 4
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  		FIG. 4. GALP icv injection increased the incidence of c-Fos-expressing cells in NPY-containing cells of the DMN. A, c-Fos- and NPY-immunoreactive cells in the DMN after GALP icv injection. B, GALP icv injection, compared with vehicle injection, increased the incidence of c-Fos immunoreactivity in NPY-containing cells in the DMN. Bar, 20 mm. *, P < 0.05 vs. vehicle (n = 6 animals/group).

 
GALP increased [Ca2+]i in NPY-containing neurons in the DMN
Administration of 10–10 M GALP increased [Ca2+]i in a single neuron isolated from the DMN (Fig. 5Go, left), which was subsequently shown to be immunoreactive to NPY (Fig. 5Go, right). The time lag until the onset of the [Ca2+]i response to GALP was 1–4 min; upon washout of GALP, [Ca2+]i returned to the baseline. Administration of 10–10 M GALP increased [Ca2+]i in five of 36 NPY-containing neurons (13.9%) of the DMN examined. Galanin at 10–10 M did not increase [Ca2+]i in these GALP-responsive neurons in the DMN. The results revealed that GALP directly acts on NPY-containing neurons in the DMN.


Figure 5
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  		FIG. 5. GALP increased [Ca2+]i in NPY-immunoreactive neurons of the DMN. GALP, but not galanin, increased [Ca2+]i in a single neuron isolated from the DMN (left panel), which was subsequently shown to contain NPY by immunocytochemistry with anti-NPY antiserum (right panel). These results were representative of five neurons. Bar, 10 µm.

 
Anti-NPY IgG and NPY-Y1 receptor antagonists inhibited GALP-stimulated food intake
The stimulated food intake during 2 h after icv GALP injection was significantly decreased by icv injection of anti-NPY IgG, whereas control nonimmune rabbit IgG had no effect. The attenuated level of food intake was not significantly different from that in the control vehicle-injected rats (Fig. 6AGo). Similarly, icv injection of NPY-Y1 receptor antagonists, nonpeptidic BIBP 3226 and peptidic 1229U91, significantly counteracted the stimulation of food intake during 2 h after GALP injection (Fig. 6Go, B and C).


Figure 6
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  		FIG. 6. The effects of anti-NPY IgG and NPY-Y1 receptor antagonists on food intake after GALP icv injection. Preinjection of anti-NPY IgG (A) and the Y1 antagonists BIBP3226 (BIBP) (B) and 1229U91 (C) reduced the food intake following GALP icv injection to the levels not significantly different from those in controls without GALP injection. *, P < 0.05 vs. rabbit IgG plus vehicle, anti-NPY IgG plus vehicle, and anti-NPY IgG plus GALP; **, P < 0.05 vs. vehicle plus vehicle, BIBP plus vehicle, and BIBP plus GALP; #, P < 0.05 vs. vehicle plus vehicle, 1229U91 plus vehicle, and 1229U91 plus GALP (n = 6 animals/group).

 
Food intake after GALP injection in ARC-lesioned rats
To examine the role of the ARC in food intake after icv GALP injection, neurons in the ARC were destroyed by MSG injection (Fig. 7Go, A–D). Food intake per day and body weight were impaired in MSG-treated rats at 7 wk of age [food intake per day, 20.2 ± 1.4 g for MSG vs. 26.8 ± 0.8 g for vehicle (P < 0.01); body weight, 249.3 ± 8.6 g for MSG vs. 294.5 ± 5.0 g for vehicle (P < 0.05)]. Vehicle-injected rats showed little feeding behavior during the light period. In contrast, in MSG-treated rats, significant feeding behavior was observed during the light period, and cumulative food intake tended to increase. In rats treated with MSG, GALP was injected into the lateral ventricle, and the increase in food intake after GALP injection was not significantly decreased compared with the vehicle-injected group. In contrast, food intake stimulated by icv ghrelin injection was significantly reduced by MSG treatment (Fig. 7EGo).


Figure 7
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  		FIG. 7. Treatment with MSG decreased NPY neurons in the ARC, but not the DMN. In the ARC, MSG-injected rats (B) showed diminished NPY-containing cells compared with vehicle-injected rats (A). In the DMN, MSG-injected rats (D) showed a similar number of NPY-containing cells as vehicle-treated rats (C). E, In the rats treated with MSG, icv injection of GALP stimulated the cumulative food intake at 2 h, whereas icv injection of ghrelin failed to significantly increase the cumulative food intake. MSG- plus vehicle-treated rats showed feeding behavior during the light period and tended to eat more compared with the vehicle- plus vehicle-treated group. *, P < 0.05 vs. vehicle plus ghrelin; **, P < 0.05 vs. MSG plus GALP. Bar, 10 µm (n = 5 animals/group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It was previously reported that GALP icv injection stimulates food intake in rats (14). However, the effector site and mechanism underlying the orexigenic action of icv GALP injection have remained unknown. In this study, rats injected with GALP into the ventricle showed the stimulated feeding behavior during the initial 2 h. Notably, food intake for 2 h after icv injection of GALP was as large as that after NPY, a strong orexigenic substance. However, food consumption at 24 h after injection was not different between the GALP-injected and control rats, confirming previous reports (24, 25). The results suggest that icv GALP acts as a strong and short-term stimulator of feeding. Moreover, when GALP was injected into the DMN, ARC/VMH, PVN, and Lh of the rats, nuclei known to participate in feeding control (26, 27, 28, 29), only GALP injection into the DMN stimulated feeding. Stimulation of feeding by GALP icv injection was markedly attenuated in the DMNL rats. Furthermore, GALP icv injection activated c-Fos expression in NPY-containing neurons in the DMN. GALP increased [Ca2+]i in NPY-containing neurons isolated from the DMN. Intracerebroventricular injection of anti-NPY IgG and NPY Y1 receptor antagonists inhibited the feeding following icv GALP injection. These results demonstrate that the short-term orexigenic effect of icv GALP injection is mediated at least partly by activation of NPY-containing neurons in the DMN in rats.

Among feeding-related peptides localized in the hypothalamus, NPY is a remarkably strong orexigenic peptide. The NPY antisense injected into the ARC decreased NPY levels in this nucleus and suppressed food intake (4). NPY-containing neurons in the ARC have been implicated in the orexigenic function (30). In addition, NPY-containing neurons are located in the DMN (15). Although much less is known about them compared with ARC NPY neurons, the feeding-promoting function of the DMN, particularly that of NPY-containing neurons located in this nucleus, has been suggested. Animals with DMNL showed hypophagia. Several studies have suggested that this nucleus underlies the neural signaling of satiety factors (31, 32). For instance, 24-h food deprivation increased NPY mRNA specifically in the DMN in Otsuka Long-Evans Tokushima fatty rats, a hyperphagic and obese model, due to lack of CCK-A receptor. Melanocortin-4 receptor-null mice displayed excessive NPY mRNA expression in neurons of the DMN (32), whereas the activity of ARC NPY neurons was not altered. Thus, impairment of anorectic signaling pathways of CCK and proopiomelanocortin appear to be linked with overstimulation of NPY neurons in the DMN, which may be a cause of the hyperphagia and obesity observed in these animals. Moreover, elevation of NPY expression in the DMN has been reported in two physiological hyperphagic models: lactation (31) and diet-induced obese mice (33). NPY neurons in the DMN project to many areas in the hypothalamus, which includes especially intense innervation to the PVN (34, 35). The PVN has been considered the final common pathway through which the brain controls feeding (36). Therefore, it is suggested that GALP icv injection stimulates feeding via activating NPY neurons in the DMN that could innervate the PVN.

In this study, GALP was injected into the ARC/VMH, Lh, PVN, and DMN, nuclei that are concerned with the control of feeding. Only rats given GALP injection into the DMN showed elevation of feeding behavior. As a loss of function approach, DMNL rats were produced by IBO injection into the bilateral DMN, a procedure known to cause cell death by increasing the [Ca2+]i. The destruction of cells was limited in the area of DMN, and the number of NPY-containing neurons decreased significantly compared with that in vehicle-injected rats. DMNL rats showed alterations in neither food intake per a day nor body weight, suggesting that NPY in the DMN is not concerned with the regulation of daily food consumption and energy metabolism. However, DMNL rats showed markedly reduced food consumption for 2 h after GALP icv injection compared with control rats. These data indicate that the DMN is involved in the feeding behavior following GALP icv injection.

The increase in food intake for 2 h after injection of GALP into the DMN was approximately half that after icv injection of GALP. This result is reasonable, because the focal injection of GALP was performed into lateral, not bilateral, DMN, whereas icv GALP injection was expected to affect bilateral DMN.

Furthermore, we found that when GALP was icv injected, c-Fos expression occurred in NPY-containing neurons in the DMN. The c-Fos-positive cells also included neurons that did not contain NPY in the DMN and cells of the Lh. These results are in accord with a previous report that GALP icv injection stimulates c-Fos expression, especially in the DMN and Lh (25). In the present study, the c-Fos-expressing neurons in the Lh contained neither orexin nor melanin-concentrating hormone, feeding-stimulating peptides. It was shown that icv GALP also induces c-Fos expression in the ARC (25, 37), but the c-Fos-expressing cells in the ARC may not be neurons, but, rather, astrocytes (25). The neurochemical identity of Fos-positive, NPY-negative cells in the DMN and that of Fos-positive cells in the Lh remain to be clarified. Thus, among the neurons containing orexigenic peptides, the NPY neuron in the DMN is the only identified target of GALP.

Next, whether the activation is via a direct action of GALP was examined. We found that GALP at 10–10 M increased [Ca2+]i in NPY-containing neurons isolated from the DMN. In contrast, galanin at the same concentration did not affect [Ca2+]i in NPY-containing neurons in the DMN. The [Ca2+]i increase often results from depolarization of the plasma membrane and is the key signal for triggering the release of neurotransmitters/hormones and gene expression (22, 38). Thus, the [Ca2+]i increase is a good indicator of neuronal activation. However, the incidence of Fos-positive NPY-containing neurons in response to icv GALP injection was higher than that of single NPY-containing neurons that responded to GALP with [Ca2+]i increases. This apparent discrepancy may be due to the fact that the [Ca2+]i increase reflects a direct effect of GALP to activate NPY neurons, whereas GALP icv injection is expected to activate NPY neurons both directly and indirectly via possible effects on other neurons that secondarily activate NPY neurons. Furthermore, we found that GALP-induced food intake was inhibited by icv injection of anti-NPY IgG and NPY Y1 receptor antagonists. Taken together, the results indicate that icv GALP injection stimulates feeding at least partly via activation of NPY-containing neurons in the DMN.

The current study showed that GALP, but not galanin, activates NPY-containing neurons in the DMN and stimulates feeding when injected into the DMN. These results could suggest an involvement of GalR2, because GALP is a more potent agonist than galanin for GalR2 (6), and GalR2 mRNA is expressed abundantly in the DMN (39, 40). In this study, however, galanin injected into the DMN, even at a 2-fold higher concentration, failed to stimulate food intake. It is not known whether NPY-containing neurons in the DMN express GalR2. Moreover, Krasnow et al. (7) recently reported that GalR2 knockout mice injected with GALP into the ventricle showed the same response as wild-type mice, suggesting that GALP activates an as yet unidentified GALP-specific receptor. Additional studies are needed to identify the receptor subtype that mediates the effect of GALP in the DMN.

Six NPY receptor subtypes are known. Of these, Y1 and Y5 receptors have been implicated in food intake (26, 27, 28). BIBP3226, a specific nonpeptidic NPY Y1 receptor antagonist, blocked NPY-induced food intake when injected icv (41). The icv injection of 1229U91, a Y1 antagonist and Y4-selective agonist, was shown to block food intake after NPY icv injection. Thus, the Y1 receptor appears to play a central role in the orexigenic action of NPY. The anti-NPY IgG used in the present study was reported to inhibit the feeding following icv injection of melanin-concentrating hormone (42). We found that GALP-induced food intake was inhibited by icv preinjection of anti-NPY IgG, BIBP3226 and 1229U91 in rats. Y1 receptors (43) and Y5 receptors (44) are located in various hypothalamic areas, including the PVN. Taken together, these findings suggest that the feeding response elicited by GALP is mediated mainly by release of NPY and activation of the Y1 receptor in the PVN.

The ARC contains NPY neurons, which are also concerned with orexigenic functions. Injection of MSG into neonatal rodents causes specific lesions in the hypothalamic ARC, resulting in a 70–90% destruction of neuronal cell bodies, including NPY-, GHRH-, and agouti-related protein-containing neurons (45, 46, 47, 48). Thus, MSG-treated rats in this study could be used as a model to study NPY-, GHRH-, and agouti-related protein deficiency. The results of the present study show that in rats neonatally treated with MSG, icv injection of GALP stimulated food intake to a level similar to that in control rats treated with saline. Furthermore, GALP failed to increase [Ca2+]i in NPY-containing neurons isolated from the ARC, confirming our previous report (49). These results indicate that the ARC is not involved in feeding behavior after icv injection of GALP. In contrast, we found that the feeding induced by icv injection of ghrelin in control rats was not significantly observed in MSG-treated rats. Similar results were previously reported by Tamura et al. (50). These results suggest that the food intake induced by icv ghrelin injection is mediated by neurons in the ARC, most likely NPY-containing neurons, because they are the direct target of ghrelin (49, 51). In our study and previous report (50), the basal level of food intake tended to increase with MSG treatment. This could be explained by the fact that MSG-treated rats eat food constantly in both light and dark photoperiods (52).

GALP stimulated food intake for the initial 2-h period in rats, but the cumulative food consumption over 1 d was not changed. Thus, under physiological conditions, GALP could be a rapid and short-term stimulator of feeding, instead of a long-term regulator of feeding and energy balance. In contrast, it was reported that NPY neurons in the DMN are stimulated for a longer period when rats are in particular situations, such as lactation, diet-induced obesity, or a defect of melanocortin and CCK-A receptor signaling (35, 53, 54). Our present finding suggests that GALP is a candidate mediator that links these (patho)physiological conditions to the DMN NPY neurons, although underlying neural mechanisms for the activation of the DMN NPY neurons remain unknown. It was reported that GALP is positively regulated by leptin (9) and that the plasma leptin level is elevated in obese compared with lean animals and humans. Therefore, GALP may be up-regulated in obesity and stimulate feeding, possibly contributing to the obesity-associated hyperphagia. Moreover, we previously reported that foot shock increased the expression of c-Fos in GALP-containing neurons (55). Several types of stresses are known to temporarily stimulate food intake (56). Therefore, GALP may participate in feeding behavior in bulimia nervosa and hyperphagia under stress conditions. However, additional studies are definitely needed to clarify the role of GALP in the regulation of feeding.


   Acknowledgments
 
We thank Dr. Tetsuya Ohtaki (Discovery Research Laboratories I, Takeda Chemical Industries, Ltd.) for providing anti-GALP antiserum and for valuable discussion.


   Footnotes
 
This work was supported by the Research Award to Jichi Medical School (JMS) Graduate Student (to M.K.). Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to T.O. and T.Y.), a Grant-in-Aid for Scientific Research on Priority Areas (15081101) from the Japan Society for the Promotion of Science (to T.Y.), a grant from the 21st Century Center of Excellence (COE) Program (to T.Y.), and a grant from Takeda Pharmaceutical Co. Ltd. (Osaka, Japan; to T.O. and T.Y.).

First Published Online January 12, 2006

Abbreviations: ARC, Arcuate nucleus; [Ca2+]i, cytosolic calcium concentration; CCK, cholecystokinin; DMN, dorsomedial nucleus; DMNL, lesions of the DMN; GALP, galanin-like peptide; GalR, galanin receptor; HKRB, HEPES-containing Krebs-Ringer bicarbonate buffer; IBO, ibotenic acid; icv, intracerebroventricular; Lh, lateral hypothalamus; MSG, monosodium glutamate; NPY, neuropeptide Y; PB, phosphate buffer; PVN, paraventricular nucleus; VMH, ventromedial hypothalamus.

Received July 19, 2005.

Accepted for publication January 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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