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Endocrinology, doi:10.1210/en.2005-1226
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Endocrinology Vol. 147, No. 4 2043-2050
Copyright © 2006 by The Endocrine Society

Central Effects of Calcitonin Receptor-Stimulating Peptide-1 on Energy Homeostasis in Rats

Hirotake Sawada, Hideki Yamaguchi, Takuya Shimbara, Koji Toshinai, Muhtashan S. Mondal, Yukari Date, Noboru Murakami, Takeshi Katafuchi, Naoto Minamino, Hiroyuki Nunoi and Masamitsu Nakazato

Department of Pediatrics (H.S., H.N.) and Third Department of Internal Medicine (H.Y., T.S., K.T., M.M., Y.D., M.N.), Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan; Department of Veterinary Physiology (N.M.), Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan; and Department of Pharmacology, National Cardiovascular Center Research Institute (T.K., N.M.), Osaka 565-8565, Japan

Address all correspondence and requests for reprints to: Masamitsu Nakazato, M.D., Ph.D., Third Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan. E-mail: nakazato{at}med.miyazaki-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The CT-R [calcitonin (CT) receptor] is expressed in the central nervous system and is involved in the regulation of food intake, thermogenesis, and behaviors. CT-R-stimulating peptide-1 (CRSP-1), a potent ligand for the CT-R, was recently isolated from the porcine brain. In this study, we first confirmed that porcine CRSP-1 (pCRSP-1) enhanced the cAMP production in COS-7 cells expressing recombinant rat CT-R, and then we examined the central effects of pCRSP-1 on feeding and energy homeostasis in rats. Intracerebroventricular (icv) administration of pCRSP-1 to free-feeding rats suppressed food intake in a dose-dependent manner. Chronic icv infusion of pCRSP-1 suppressed body weight gain over the infusion period. Furthermore, icv administration of pCRSP-1 increased body temperature and decreased locomotor activity. The central effects of pCRSP-1 were more potent than those of porcine CT in rats. In contrast, ip administration of pCRSP-1 did not elicit any anorectic or catabolic effects. Administration icv of pCRSP-1 also induced mild dyskinesia of the lower extremities and decreased gastric acid output. Fos expression induced by icv administration of pCRSP-1 was detected in the neurons of the paraventricular nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, locus coeruleus, and nucleus of solitary tract, areas that are known to regulate feeding and energy homeostasis. Administration icv of pCRSP-1 increased plasma concentrations of ACTH and corticosterone, implying that the hypothalamic-pituitary-adrenocortical axis might be involved in catabolic effects of pCRSP-1. These results suggest that CRSP-1 can function as a ligand for the CT-R and may act as a catabolic signaling molecule in the central nervous system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCITONIN RECEPTOR (CT-R), a member of the G protein-coupled receptor family, is abundantly expressed in the central nervous system (CNS), especially in the paraventricular nucleus of the hypothalamus (PVN) and the circumventricular areas (1, 2, 3, 4). Calcitonin (CT), a ligand for CT-R, is a potent hypocalcemic peptide in the peripheral system and can serve as a neurotransmitter or a neuromodulator in the CNS. In fact, central administration of CT induces anorexia (5, 6, 7) and hyperthermia (8, 9), produces analgesia (9, 10, 11), and reduces gastric-acid output (12) and locomotor activity (13, 14). Presently, CT mRNA is not detectable in the CNS. CT in the systemic circulation, secreted from the C-cells of the thyroid gland is not thought to stimulate central CT-R because systemic CT cannot enter the CNS through the blood-brain barrier. Fischer et al. (15) reported that human CT- and C-terminal adjacent peptide-like immunoreactivities were found in extracts of the human periventricular mesencephalic region. Sexton and Hilton (16) detected CT-like immunoreactivity in the rat brain using an antisalmon CT antibody, and this immunoreactive material was reported to stimulate cAMP production in cultured cells and to bind to membrane-bound CT-R in the brain. In 1998, a CT-like peptide containing a unique six-amino acid sequence (EKSQSP) was purified from rat brain and pituitary (17). However, the true ligand for CT-R in the CNS has not yet been identified.

We have recently isolated a novel biologically active peptide, termed CT-R-stimulating peptide (CRSP)-1, as a specific and potent ligand for CT-R. CRSP-1 was originally isolated from acid extracts of porcine brain by monitoring cAMP production in LLC-PK1 cells, which endogenously express CT-R (18). Porcine CRSP-1 (pCRSP-1) was detected in the porcine pituitary, thyroid, and brain, especially in the hypothalamic and midbrain region. Comparison of pCRSP-1 amino acid sequence with other biologically active peptides showed that it has the highest homology with the CT gene-related peptide (CGRP). Furthermore, pCRSP-1 binds to and stimulates both endogenous and recombinant porcine CT-R in vitro, and the potency of pCRSP-1 is 350 times more potent than that of porcine CT (pCT) in the cAMP production assay using recombinantly expressed porcine CT-R. Intravenous administration of pCRSP-1 to rats reduces the plasma calcium concentration in the systemic circulation (18).

To investigate the role of pCRSP-1 in energy homeostasis, we examined the central effect of pCRSP-1 on feeding behavior by intracerebroventricular (icv) injection. We first examined the biological activity of pCRSP-1 on rat CT-R by monitoring the cAMP production to confirm pCRSP-1 can stimulate rat CT-R. We next examined the central effects of pCRSP-1 on food intake, body temperature, locomotor activity, and gastric-acid secretion in rats. We examined whether icv administration of pCRSP-1 induces neuronal activation in the hypothalamus and brainstem, which are pivotal regions in the central regulation of feeding and energy homeostasis by monitoring pCRSP-1-induced Fos expression. In addition, we measured the plasma levels of ACTH and corticosterone after icv administration of pCRSP-1 to examine the downstream signaling of pCRSP-1. Here we report that central administration of pCRSP-1 suppresses feeding, increases body temperature, and possibly plays a role in the central regulation of feeding behavior and energy balance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides
pCRSP-1, -2, and -3 were synthesized by the solid phase method using the Fmoc [N-(9-fluorenyl) methoxycarbonyl] strategy (Peptide Institute, Osaka, Japan). Purity and identity were confirmed by reverse-phase and ion-exchange HPLC, and by amino acid analysis and sequencing, respectively. pCT was purchased from Bachem (Bubendorf, Switzerland).

cAMP-producing activity
COS-7 cells were plated at 100,000 cells/well on 48-well plates and cultured for 24 h. Rat CT-R ligated into pcDNA 3.1 expression vector (Promega Co., Madison, WI) was transfected into the cells with Lipofectamine Plus (Invitrogen Co., Carlsbad, CA) according to the manufacturer’s protocol, and further incubated for 24 h. Mock-transfected COS-7 cells were used as a negative control. Then, the cells were washed twice with DMEM/HEPES (20 mM, pH 7.4) containing 0.5 mM 3-isobutyl-1-methyl xanthine (Sigma, St. Louis, MO) and 0.05% BSA, and incubated in the same medium for 30 min at 37 C. The incubation medium was then replaced with 150 µl of medium in which pCRSP-1 was dissolved and further incubated at 37 C for another 30 min. Aliquots (100 µl) of the incubation media were succinylated, evaporated, and then submitted to RIA for cAMP as reported previously (18).

Animals
Male Wistar rats (Charles River Japan Inc., Shiga, Japan) weighing 300–350 g were maintained in individual cages under controlled temperature (21–23 C) and light (light on 0800 h-2000 h) with ad libitum access to food and water. Cannulation and icv administration were performed as previously described (19). Rats were anesthetized by ip injections of sodium pentobarbital (Abbott Laboratories, Chicago, IL), and proper placement of the cannula was verified at the end of the experiments by dye administration. Rats were sham injected before the study and weighed and handled daily. Only animals that showed progressive weight gain after the surgery were used in subsequent experiments. All experiments were repeated two or three times. pCRSP-1, -2, 3, and pCT were dissolved in 0.9% saline, and 10 µl of solutions were administered icv to rats. All procedures were performed in accordance with the Japanese Physiological Society’s guidelines for animal care.

Feeding experiments
To measure food intake in the dark phase, pCRSP-1 (0.3, 0.5, and 1.0 nmol), pCT (1.0 nmol), or saline was administrated before the onset of the dark phase (1945 h) by icv injection to rats (n = 12 per group). Cumulative food intake for 24 h was measured by weighing the remaining food. The same experiments were carried out with pCRSP-2 (1.0 nmol; n = 9), and pCRSP-3 (1.0 nmol; n = 8). To measure fasting-induced food intake in the light phase, rats were fasted overnight for 14 h, and then pCRSP-1 (0.3, 0.5, and 1.0 nmol), pCT (1.0 nmol), or saline was administrated by icv injection at 1000 h to rats (n = 8 per group), and food intake was measured for 3 h. To examine the peripheral effects of pCRSP-1, 10 nmol of pCRSP-1 or saline was administered by ip injection at 1945 h to rats (n = 6 per group), and food intake was measured for 4 h. These experiments were conducted using the cross-over methods in which all animals received a central injection of pCRSP-1 or control saline on separate days (20). To determine the chronic effects of pCRSP-1 on feeding and body weight gain, 1 nmol of pCRSP-1 or saline was chronically administered to rats (n = 7 per group) by icv injection twice a day for 7 d. The amount of food given to pair-fed rats was determined by measuring the food intake of the pCRSP-1-administered group on the previous day. Daily food intake and body weight were both measured at 0900 h.

Conditioned taste aversion (CTA) test
CTA assessment was performed as described previously (20, 21). Rats were conditioned to 2-h daily access to water from two bottles for 3 d. On the fourth day, rats were given 0.15% saccharin for 2 h instead of water, and saccharin consumption was measured. Immediately afterward, rats (n = 10 per group) were administered with pCRSP-1 (0.5 nmol and 1 nmol, icv), saline (icv), lithium chloride (LiCl; Nacalai Tesque, Kyoto, Japan; 0.15 M, 2 ml/kg, ip), or saline (2 ml/kg, ip). LiCl was used as a positive control for the assessment of CTA. On the fifth day, rats were simultaneously presented saccharin and water for 2 h, and the ratio of fluid consumption was measured.

Body temperature and locomotor activity
The body temperature of the rats was monitored from –30 min to 240 min after icv administration of pCRSP-1 (0.1 and 1.0 nmol), pCT (0.1 nmol), or saline (n = 6 per group) at 1400 h. The same experiments were carried out with pCRSP-2 (5.0 nmol, n = 12), pCRSP-3 (5.0 nmol, n = 12). A sensor tip (measurable range: 25–50 C and measuring error, ± 0.02 C) was inserted into the rectum and the digital signal was transferred to a thermometer (MT-1; Senko Co. Ltd., Tokyo, Japan). Wistar rats were housed in individual cages (36 x 30 x 17 cm) under controlled light (light on 0700–1900 h for locomotor activity study) with ad libitum access to food and water. Before the onset of the dark phase (1845 h), pCRSP-1 (0.3, 0.5, or 1.0 nmol), pCT (1.0 nmol), or saline was administered to rats by icv injection. Locomotor activity was measured for 12 h with a rat locomotor activity recording system (Muromachi Co., Tokyo, Japan) comprising infrared sensors, an interface, and a computer. The infrared sensors were placed above the rat cage and detected all movements. These experiments were conducted using the cross-over methods in which all animals received a central injection of pCRSP-1 or control saline on separate days (20).

Measurement of gastric-acid output
Rats were fasted for 24 h with free access to water and then anesthetized by ip injection of urethane (1 g/kg body weight; Sigma). The pylorus of the rat stomach was ligated through a midline abdominal incision with a 4.0 silk ligature, and the abdominal incision was sutured. One hour after anesthetic administration, pCRSP-1 (1 nmol) or saline was given by icv injection at 0900 h. Two hours after the administration of pCRSP-1, gastric juices were collected from the extirpated stomach and centrifuged at 3000 rpm for 15 min at 4 C. The volume of the supernatant was measured, and its acidity was titrated with an autotitrater (COM-500; Hiranuma Inc., Ibaragi, Japan). Total acid output was calculated from the supernatant volume and acidity.

Immunostaining of Fos
To determine the induced pattern of Fos expression, pCRSP-1 or saline was administered to rats (n = 3 per group) 90 min before transcardial perfusion with fixative containing 4% paraformaldehyde. The rat brain was removed and cut into 40-µm-thick sections. Sections were incubated for 2 d with rabbit anti-Fos antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution 1:2000) and stained with a Vectastain avidin-biotin complex kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s protocol.

Blood sample analysis
Rats (n = 5 per group) were killed by decapitation 15 and 30 min after the icv administration of pCRSP-1 (1.0 nmol) or saline control. Blood samples were collected and were immediately chilled on ice in tubes containing a 10 µl solution of 2.5% EDTA and centrifuged at 4 C. Plasma was then stored at –80 C until analysis. Plasma samples were used for measurement of ACTH and corticosterone with RIA specific for each substance (SRL Co., Tokyo, Japan).

Statistical analysis
All data are expressed as means ± SEM. Groups of data (means ± SEM) were compared using ANOVA and post hoc Fisher’s test. P values less than 0.05 are considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of pCRSP-1 on recombinant rat CT-R
Rat CT-R was transiently expressed in COS-7 cells, and we evaluated the effect of pCRSP-1 on the rat CT-R by measuring the cAMP producing activity. pCRSP-1 stimulated cAMP production in a dose-dependent manner (Fig. 1Go). pCRSP-1 did not stimulate the cAMP production in mock-transfected COS-7 cells (Fig. 1Go). The cAMP production in COS-7 cells expressing rat CT-R showed that pCRSP-1 can stimulate rat CT-R.


Figure 1
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  		FIG. 1. Effects of pCRSP-1 on cAMP production in COS-7 cells expressing rat CT-R. COS-7 cells expressing rat CT-R was stimulated with the indicated concentrations (conc.) of pCRSP-1 (closed circle). pCRSP-1 did not stimulate the cAMP production in mock-transfected COS-7 cells (open circle). The assay media were then succinylated, evaporated, and submitted to RIA for cAMP.

 
Effects of pCRSP-1 on feeding and body weight gain
Administration icv of pCRSP-1 significantly reduced the food intake of free-feeding rats in the dark phase in a dose-dependent manner with a lowest effective dose of 0.3 nmol (Fig. 2AGo). The anorectic effect of pCRSP-1 was stronger than that of pCT. In rats that had fasted for 14 h before icv injection, pCRSP-1 also dose-dependently suppressed food intake for 3 h in the light phase, and the anorectic effect of pCRSP-1 was stronger than that of pCT (Fig. 2BGo). On the other hand, bolus ip injection of pCRSP-1 did not induce a significant change in rat food intake (Fig. 2CGo).


Figure 2
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  		FIG. 2. Effects of icv administration of pCRSP-1 on food intake. A, Cumulative food intake in free-feeding rats in the dark phase (n = 12 per group) was measured after icv administration of saline, pCRSP-1 (0.3, 0.5, 1.0 nmol) or pCT (1.0 nmol). B, Cumulative food intake in 14 h-fasted rats in the light phase (n = 8 per group) was measured after icv administration of saline, pCRSP-1 (0.3, 0.5, 1.0 nmol) or pCT (1.0 nmol) at the light phase. Control rats were given 0.9% saline. *, P < 0.05; **, P < 0.001 (vs. saline controls). a, P < 0.05; b, P < 0.001 (vs. pCT). C, Effect of ip administration of pCRSP-1 (10 nmol) in the dark phase to rats (n = 6 per group). Control rats were given 0.9% saline by ip injection. D, Conditioned taste aversion test. Conditioned rats (n = 10 per group) received icv administration of pCRSP-1 (0.5, 1.0 nmol) or saline and ip administration of LiCl or saline. *, P < 0.05 (vs. ip saline controls). E, Cumulative food intake in free-feeding rats in the dark phase (n = 9 per group) was measured after icv administration of saline or pCRSP-2 (1.0 nmol). F, Cumulative food intake in free-feeding rats in the dark phase (n = 8 per group) was measured after icv administration of saline or pCRSP-3 (1.0 nmol).

 
Saccharin intake was measured after the administration of pCRSP-1 or LiCl, a toxin that causes rats to avoid saccharin. LiCl caused taste aversion, whereas pCRSP-1 (0.5 and 1.0 nmol) did not reduce saccharin intake (Fig. 2DGo).

In contrast with pCRSP-1, neither pCRSP-2 nor pCRSP-3 altered food intake at all by icv injection of 1.0 nmol of the peptide to rats (Fig. 2Go, E and F).

Chronic icv administration of pCRSP-1 (2 nmol/d) significantly suppressed food intake from the third to fifth day of treatment (Fig. 3AGo). However, no anorectic effect of pCRSP-1 was observed on the sixth day. Cumulative body weight gain of pCRSP-1-administrated rats was significantly decreased over the infusion period (Fig. 3BGo). Furthermore, the pCRSP-1-administrated rats gained significantly less weight than pair-fed rats.


Figure 3
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  		FIG. 3. Effects of chronic icv administration of pCRSP-1 on rats. One-day food intake (A) and cumulative body weight gain (B) were measured during icv injection (2.0 nmol/d) for 7 d (n = 7 per group). Each pair-fed rat was given the same amount of food as the paired pCRSP-1-administered rat consumed on the previous day. Control rats were given 0.9% saline. *, P < 0.05; **, P < 0.001 (vs. saline controls). a, P < 0.05 (vs. pair fed).

 
Effects of pCRSP-1 on body temperature and locomotor activity
Administration icv of pCRSP-1 to free-feeding rats dose-dependently increased body temperature during the light phase, with a lowest effective dose of 0.1 nmol (Fig. 4AGo). The thermogenic effect of pCRSP-1 was stronger than that of pCT when 0.1 nmol of each peptide was administered. Neither pCRSP-2 nor pCRSP-3 raised body temperature upon their icv injection of 5.0 nmol of the peptide to rats (Fig. 4BGo).


Figure 4
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  		FIG. 4. Effects of icv administration of pCRSP-1 on body temperature and locomotor activity. A, Body temperature was measured from –30 min to 240 min after administration of pCRSP-1 (0.1, 1.0 nmol) or pCT (0.1 nmol) (n = 6 per group). Control rats were given 0.9% saline. *, P < 0.05; **, P < 0.001 (vs. saline controls). a, P < 0.05 (vs. pCT). B, Body temperature was measured up to 240 min after administration of pCRSP-1 (1.0 nmol), pCRSP-2 (5.0 nmol), or pCRSP-3 (5.0 nmol). Control rats were given 0.9% saline. **, P < 0.001 (vs. saline controls). C, Locomotor activity was measured for 12 h after administration of pCRSP-1 (0.3, 0.5, 1.0 nmol) or pCT (1.0 nmol) (n = 5 per group). Control rats were given 0.9% saline. *, P < 0.05; **, P < 0.001 (vs. saline controls). a, P < 0.05 (vs. pCT).

 
Rats receiving icv injections of pCRSP-1 decreased their locomotor activity in the dark phase at a minimum dose of 0.5 nmol of pCRSP-1 (Fig. 4CGo). The decrease in locomotor activity elicited by pCRSP-1 was greater than that observed with an equal amount (1.0 nmol) of pCT. Moreover, icv administration of pCRSP-1 (1.0 nmol) to freely moving rats elicited mild dyskinesia of the lower extremities and strenuous breathing (n = 8). Dyskinesia consisted of decrease of muscle tone only in the lower extremities but was not associated with loss of consciousness or trunk choreiform movements. The abdomen of pCRSP-1-administrated rats touched the floor, and the body was supported with the upper extremities in the resting position during the dyskinesia. The dyskinesia occurred about 10 min (9.4 ± 2.7 min) after the icv administration and lasted about 40 min (40.3 ± 7.7 min) in all of pCRSP-1-administrated rats. The abnormal behaviors were not observed in the saline-administrated rats.

Effects of pCRSP-1 on gastric acid output
Administration icv of pCRSP-1 to rats that had fasted for 24 h reduced the volume of gastric acid and total acid output (Fig. 5Go, A and B). The volume and total gastric acid content of gastric juice after treatment with pCRSP-1 were reduced to 50% and to 48% of the values obtained in saline-treated controls, respectively.


Figure 5
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  		FIG. 5. Effect of icv administration of pCRSP-1 on gastric acid output. The volumes of gastric acid (A) and total acid output (B) were measured after icv administration of pCRSP-1 (1.0 nmol) or saline. *, P < 0.05 (vs. saline controls).

 
pCRSP-1-induced Fos expression
To determine the target neurons of pCRSP-1 in rat brain, we examined Fos expression induced by icv injection of pCRSP-1. Fos-immunoreactive neurons were observed in the supraoptic nucleus (Fig. 6AGo), locus coeruleus (Fig. 6CGo), preoptic area (Fig. 6EGo), PVN (Fig. 6FGo), dorsomedial hypothalamic nucleus (Fig. 6GGo), arcuate nucleus (ARC) (Fig. 6HGo), periaqueductal gray matter (Fig. 6IGo), and nucleus of solitary tract (Fig. 6JGo). Administration icv of saline did not cause a specific increase of Fos expression in these nuclei (Fig. 6Go, B and D).


Figure 6
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  		FIG. 6. Fos expression in the rat brain after icv administration of pCRSP-1 (1.0 nmol). Photomicrographs demonstrating Fos-immunoreactive nuclei in supraoptic nucleus (A and B), locus coeruleus (C and D), preoptic area (E), PVN (F), dorsomedial hypothalamic nucleus (G), ARC (H), periaqueductal gray matter (I), and nucleus of solitary tract (J). B and D, Negative controls of Fos expression in the rat brain after icv administration of saline. 4V, Fourth ventricle; 3V, third ventricle; Aq, cerebral aqueduct. Scale bars, 500 µm.

 
Effects of pCRSP-1 on plasma ACTH and corticosterone levels
Plasma concentration of ACTH in pCRSP-1-administrated rats was 545 ± 106.3 pg/ml and was significantly higher than that of in saline-administrated rats (97.2 ± 27.9 pg/ml) at 15 min after icv administration (Fig. 7AGo). Plasma concentration of corticosterone in pCRSP-1-administrated rats was 521.8 ± 46.9 ng/ml and was significantly higher than that of in saline-administrated rats (201.4 ± 51.8 ng/ml) at 30 min after the icv administration (Fig. 7BGo).


Figure 7
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  		FIG. 7. A, Plasma levels of ACTH 15 min after the administration of pCRSP-1 (1.0 nmol) or saline (n = 5 per group). B, Plasma levels of corticosterone 30 min after the administration of pCRSP-1 (1.0 nmol) or saline (n = 5 per group). *, P < 0.05 (vs. saline controls).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CRSP-1 is a member of the CGRP family (18) and has been isolated in various mammal including pig, cow, dog, and horse (22). CRSP-1 elicits its biological activity via CT receptor in vitro (22). In in vivo experiments, bolus administration of pCRSP-1 to rats reduced the plasma calcium concentration, indicating that it acts as a CT-R agonist in the peripheral circulation. Although an elaborate search for the counterparts of pCRSP-1 in human and rodents has been undertaken, no cDNA or gene has yet been identified as having significant sequence identity with the pCRSP-1 cDNA. The CT-R in the CNS is abundantly expressed in PVN of the hypothalamus (1, 4), and an icv administration of CT has been reported to reduce food intake (5, 6, 7) and gastric acid secretion (12). To explore the biological effects of CRSP-1 in the CNS of mammals, we first investigated whether pCRSP-1 can stimulate rat CT-R by measuring the cAMP production. pCRSP-1 significantly stimulated rat CT-R, indicating that pCRSP can act on rat CT-R. In this study, we examined the central roles of CRSP-1 by icv administration of pCRSP-1 to rats.

Administration icv of pCRSP-1 dose-dependently reduced food intake both in the dark phase and in the light phase, whereas ip injection of pCRSP-1 did not reduce food intake. Reduction of food intake after the icv administration of 1.0 nmol of pCRSP-1 was 75–95% at the time points examined, being stronger compared with pCT or other potent anorectic peptides. Administration icv of toxic compounds often reduces food intake. However, toxic or aversive effects of pCRSP-1 can be ruled out by a conditioned taste aversion test. Chronic icv administration of pCRSP-1 suppressed daily food intake and cumulative body weight gain over the infusion period. However anorectic effects of pCRSP-1 did not persist on the sixth day in continuous icv infusion. Similar adaptation or tolerance to an anorectic agent has been reported in CT-treated rats (7). Single icv administration of pCRSP-1 to free-feeding rats dose-dependently increased body temperature. Body temperature of chronically pCRSP-1-administrated rats was also higher than that of saline-administrated or pair-fed rats (n = 5 per group) 12 h after the daily icv injection (pCRSP-1; 38.6 ± 1.1 C, saline; 37.3 ± 0.3 C, pair-fed; 37.2 ± 0.2 C, pCRSP-1 vs. saline P < 0.005). The cumulative body weight gain of pCRSP-1-administered rats was significantly lower than that of pair-fed rats, suggesting that the suppression of body weight gain induced by pCRSP-1 is not only due to pCRSP-1-induced anorexia but also to pCRSP-1-induced thermogenesis. Decreased locomotor activity in pCRSP-1-administered rats indicated that elevated body temperature is not related to locomotor activity. Gastric acid secretion is regulated by both central and peripheral pathways, and is activated by various stimuli, including neuropeptides. CT is also reported to decrease its secretion (12). We measured the gastric acid output in the pCRSP-1-administrated rats and demonstrated that pCRSP-1 inhibited gastric acid secretion. These findings, together with the lack of a pCRSP-1-induced taste aversion at doses that reduce food intake, suggest that pCRSP-1 meets the criteria for an anorectic and catabolic signaling molecule in the CNS.

pCRSP-1-induced dyskinetic behaviors were distinct from those induced by other neuropeptides that were involved in locomotor activity, such as TSH-releasing hormone (wet dog shakes) (23), ß-endorphin (generalized rigidity) (24), and ACTH-(1–24) (a contorted posture and an uncoordinated or wobbly gait) (25). The pCRSP-1-induced dyskinesia was not compatible with the rapid rotations along the longitudinal body axis (barrel rotations), which was reported to be produced by somatostatin (26) and sulfated C-terminal fragments of cholecystokinin (27). In addition, dyskinetic movements of pCRSP-1-induced rats were different from those of salmon CT-induced rats (limb and trunk choreiform movements, head shaking) (13). These results implied that pCRSP-1 might be involved in the regulation of motor functions.

The PVN, ARC, and nucleus of solitary tract, where FOS expression was induced by icv administration of pCRSP-1, are known to play important roles in feeding and energy homeostasis. Fos expression was also detected in the locus coeruleus, which is involved in locomotion (28). These results corroborate the behavioral findings that pCRSP-1 elicits anorexia and reduction of locomotion. CT-R has been reported to express in the nucleus accumbens, suprachiasmatic nucleus, preoptic area, PVN, ARC, locus coeruleus, raphe nucleus, and periaqueductal gray matter (1, 2, 3, 4). However, Fos expression was not induced in the nucleus accumbens, suprachiasmatic nucleus, or raphe nucleus. In contrast, Fos expression was induced in the supraoptic nucleus, where CT-R is not expressed. Further study is required to elucidate how pCRSP-1 can elicit its effects on different brain sites lacking the CT-R.

Several lines of evidence that icv administration of pCRSP increased Fos expression in the PVN and decreased food intake and gastric acid secretion along with abundant expression of CT-R in the PVN, lead us to explore whether the anorectic hypothalamic neuropeptide, corticotropin-releasing factor, is involved in pCRSP-1-biological effects. Corticotropin-releasing factor is a neuropeptide that has been demonstrated to decrease food intake (29, 30, 31, 32), gastric acid secretion (33, 34) and increase plasma levels of ACTH and corticosterone. We measured the plasma concentrations of ACTH and corticosterone after icv administration of pCRSP-1 to nonanesthetized conscious rats. pCRSP-1 significantly increased the plasma concentrations of ACTH and corticosterone. These results suggest that the biological effects of pCRSP-1 might be involved in hypothalamic-pituitary-adrenocortical axis.

Recently, pCRSP-2 and canine CRSP-2, as well as pCRSP-3, have been identified (35). These peptides are expressed mainly in the CNS, pituitary, and thyroid in a manner similar to that of CRSP-1. Both pCRSP-2 and pCRSP-3 consist of 37 amino acids and show more than 50% sequence identity with pCRSP-1 and CGRP. pCRSP-2 and pCRSP-3 have very weak effects on cAMP production in LLC-PK1 cells (35). So far as we examined, however, neither pCRSP-2 nor pCRSP-3 elicited significant central effects on food intake and body temperature in rat, indicating that these peptides have properties distinct from those of CRSP-1.

In summary, we have demonstrated that pCRSP-1 can function as a ligand for CT-R and as an anorectic and catabolic signaling molecule in the CNS. Future identification of endogenous CRSP-family peptides in rodents and human would provide valuable insights into the physiological roles of central CRSP/CT-R system.


   Footnotes
 
This study was supported in part by grants-in-aid from the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology, and by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), Japan.

All listed authors have nothing to declare.

First Published Online January 12, 2006

Abbreviations: ARC, Arcuate nucleus; CNS, central nervous system; CRSP, calcitonin receptor-stimulating peptide; CT, calcitonin; CGRP, calcitonin gene-related peptide; CTA, conditioned taste aversion; CT-R, calcitonin receptor; icv, intracerebroventricular; pCRSP-1, porcine CRSP-1; pCRSP-2, porcine CRSP-2; pCRSP-3, porcine CRSP-3; pCT, porcine calcitonin; PVN, paraventricular nucleus of hypothalamus.

Received September 26, 2005.

Accepted for publication December 29, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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