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Pharmacology Research Unit, Valme University Hospital and Department of Pharmacology, Paediatrics and Radiology, Faculty of Medicine, University of Seville, Seville 41014, Spain
Address all correspondence and requests for reprints to: Prof. Francisco J. Miñano, Pharmacology Research Unit, Valme University Hospital, Avda Bellavista s/n, Seville 41014, Spain. E-mail: jminano{at}us.es.
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Introduction |
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CTR cDNAs have been isolated from multiple species including human, rodent, mouse, guinea pig, and rabbit. Amino acid homology analysis reveals approximately 78% identity between the human and rodent CTR (8). Because peripheral CT cannot enter the CNS through the blood-brain barrier (9) and CT mRNA is either present in only very low concentrations or entirely absent from the normal rat brain (10, 11), it has been supposed that these centrally mediated effects of CT are exerted by an endogenous CT-like peptide via specific binding to CTR (12). Thus, accumulating evidence supports the existence of CT-like peptides, which may act as endogenous regulators of CTR in brain and other nonthyroidal tissues.
According to the molecular structure, tissue distribution, and ligand-binding affinity, two different isoforms (currently termed CT(a) and CT(b)) of the CTR have been cloned and are abundantly and differentially expressed in brain regions of primary importance in the regulation of food intake and energy homeostasis (13, 14). These findings suggest that the two isoforms have different physiological ligands and thus different functional roles. Furthermore, it has recently been shown that the molecular pharmacology of ADM, AMY, and CGRP is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with CRLR and that RAMPs also regulate the interaction of CTR with CT and these peptides (4). Thus, besides pharmacological discrimination (15), the true ligand for CTR in the CNS has not yet been identified. Recently, two research groups discovered other new endogenous peptides named calcitonin receptor-stimulating peptide (16) and intermedin (17); however, it is still not clear whether these peptides act via CTR or via CRLR for ADM, AMY, and/or CGRP.
It has been shown that although the C cells in the thyroid gland are the dominant source of circulating mature CT, nonthyroidal tissue as well as some types of brain cells secrete CT and CT precursors, including procalcitonin (PCT) and its free bioactive peptide N-PCT. PCT is a 116-amino-acid polypeptide that contains three component peptides: 1) the bioactive fragment of PCT, a 7-kDa 57-amino-acid peptide at the amino terminus, named amino-PCT (N-PCT); 2) a centrally positioned 33-amino-acid immature CT; 3) and a 21-amino-acid CT carboxyl-terminal peptide-I (CCP-I or katacalcin) (18, 19). PCT-encoding cDNA has been isolated from several species including human, baboon, rat, mouse, hamster, sheep, pig, dog, chicken, and salmon (20, 21, 22).
N-PCT is present in equimolar amounts and coordinately regulated with CT in vivo and in vitro, and small amounts of free PCT and N-PCT are found in the peripheral circulation of normal subjects and during systemic inflammation (23, 24). Furthermore, studies using a radioreceptor assay revealed the presence of receptors to this prohormone in rat calvarial cells (25), and we recently demonstrated in rats under normal metabolic conditions as well as during endotoxemia that immunoreactive PCT can be detected in serum (26) and hypothalamic areas implicated in appetite control and autonomic regulation (21, 27). Furthermore, the CT sequence shows considerable divergence across species, but all sequences contain 32 amino acids. In each species, the amino acid sequence of PCT is very similar, and the sequence of N-PCT is completely conserved in all mammalian species studied (19), implying that N-PCT plays a critical, and as yet undefined, physiological function. The coelaboration of N-PCT, mature CT, and N-PCT‘s sequence conservation during evolution prompted us to investigate the potential bioactivity of N-PCT. The combined presence of CTR, CTR mRNA, PCT-like immunoreactivity, and receptors to PCT in selective brain areas is strongly suggestive of a physiological role for this prohormone in regulating energy balance.
We here investigated the effect of N-PCT, the bioactive fragment of PCT, on feeding and energy homeostasis. We demonstrated that N-PCT, when given intracerebroventricularly (icv), induced anorexia and body weight loss possibly through activation of neurons localized in hypothalamic feeding regulatory nuclei that ultimately control appetite. In addition, we demonstrate the influence of feeding status on endogenous expression of PCT in the rat hypothalamus and provide an anatomical basis for the possibility that specialized astrocytes immunoreactive for PCT, the physiological precursor of CT, are involved in the function of feeding-regulating neurons.
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Materials and Methods |
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Animals
Adult male Wistar rats (Centro de Producción y Experimentación Animal, Universidad de Sevilla, Spain) weighing 200–225 g were maintained in individual cages under controlled temperature (26–28 C) and light (lights on at 0700 h; lights off at 1900 h) with ad libitum access to tap water and a nutritionally balanced rodent diet (2014 S; Harlan Iberica, Barcelona, Spain), except during the experiments when food availability was manipulated. For fasting, food was withdrawn from cages at the onset of the dark cycle for 24 h, but ad libitum access to water was allowed. All animal experiments were done in accordance with guidelines on animal care and use established by the Seville University Institutional Animal Care and Use Committee.
The icv cannulation
Chronic icv infusion cannulas and injections were performed essentially after standard surgical procedures (28, 29). Animals were anesthetized ip with a mixture of ketamine (100 mg/kg)-xylazine (4 mg/kg), and a 20-gauge stainless steel guide cannula and was implanted just above the lateral cerebral ventricle (coordinates relative to bregma: 1.0 mm lateral, 1.5 mm posterior, and 3.5 mm below the other surface of the skull), according to the stereotaxic coordinates of the rat brain atlas of Paxinos and Watson (30). In some experiments, core body temperature and locomotor activity were monitored in undisturbed animals by remote biotelemetry using radio transmitters implanted into the peritoneum at the same time as cannulation, as described elsewhere (26, 31). After surgery, each animal was housed singly throughout the experiments. Postoperatively, a recovery period of 7 d elapsed before the beginning of the experiments. Rats were sham injected before the study and were weighed and handled daily until completion of the studies. On the test day, each rat received an icv injection of 5.0 µl N-PCT or an equivalent volume of aCSF at pH 7.4 by gravity flow through an injector needle lowered into the ventricular lumen, and the injection cannulas were left in place for an additional 1 min after the injection. Only animals acclimatized to experimental procedures that showed a progressive weight gain and a stable baseline body temperature were used in subsequent experiments. Proper placement of the cannula was verified at the end of the experiments by dye administration. Data from animals whose cannula were found to be incorrectly placed were excluded from analyses (>95% correct cannula placement).
General experimental design
All experiments were performed on adult male Wistar rats that were housed individually under controlled temperature (26–28 C), an ambient temperature within the thermoneutral zone for rats, rather than at 21–23 C, an ambient temperature below the zone of thermoneutrality for rats that could induce abnormal thermoregulatory responses and quantitatively accounts for most of the metabolic efficiency (32). Animals were acclimatized to the facility and handled daily for 1 wk with unrestricted access to water and food before the onset of the experiment. Before experiments, body temperature, food, and animal weights were monitored daily for a minimum of 2 d before injection. Rats were randomly assigned to experimental conditions, and each rat was used for only a single experiment. All drugs were administered at the onset of the dark phase, the normal feeding time for rats. Any inhibition of food intake at this time could be considered to be more physiological than alterations to refeeding after a fast. Animals were monitored for evidence of overt illness and activity after the treatments.
Experiment 1: feeding experiment
To examine the dose effect of icv injection of N-PCT on food intake, 72 rats received icv implants as described above and were divided into six groups (n = 12 per group), and each group received aCSF or 0.02, 0.1, 0.2, 1.0, or 2.0 nmol N-PCT at the beginning of the dark phase (1900 h). Because the physiological ranges of N-PCT in the brain are not known, the doses used in this study (0.02–2.0 nmol) are equivalent to those used in previous studies on food intake with CT or CT-like peptides in the rat (7). A preweighed quantity of food was placed on the cage floors, and water was available from a calibrated bottle. The food was removed, weighed, and returned to the cages immediately after the infusion of N-PCT or aCSF (pH 7.4). Food intake was measured 1, 2, 4, 8, 24, and 48 h after the central injection. The remaining food and spillage was measured to the nearest 0.01 g, and the weight of eaten food was corrected for spillage. To evaluate water intake, each animal was allowed to drink water from a glass bottle calibrated to 0.1 ml. Water intake was measured at 1, 2, 4, 8, 24, and 48 h post injection. Body weight was measured at the beginning of the experiment and after 24 and 48 h at 0800 h. All experiments were performed twice.
To examine the peripheral effects of N-PCT, 5.0 nmol/kg N-PCT (equivalent to 1.0 nmol per animal) or an equivalent volume of PFS (1 ml/kg) was administered by ip injection at 1900 h to rats (n = 10 per group). These experiments were conducted using the crossover methods in which all animals received a central injection of N-PCT (1.0 nmol) or PFS on separate days, and the same paradigm was used for this peripheral dose. Food and water intake were measured 1, 2, 4, 8, and 24 h post injection.
Experiment 2: conditioned taste aversion (CTA) test
To determine whether the anorectic effect of icv administration of N-PCT produces aversive consequences (i.e. nausea) that secondarily reduce food intake independently of the normal regulation of energy balance, a CTA paradigm was used (33). Briefly, 40 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 the 2-h period instead of water, and saccharin consumption was measured. Immediately afterward, five groups of rats (n = 8 rats per group) were administered N-PCT (1.0 and 0.2 nmol, icv), aCSF (icv), LiCl (Sigma 0.15 M, 1 ml/kg, ip), or PFS (1 ml/kg, ip). These doses of N-PCT were chosen because they represented the threshold doses that reliably produced reductions in food intake when administered into the lateral ventricle. 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 2-h fluid consumption percentage was measured.
Experiment 3: body temperature and locomotor activity
Because exercise and thermogenesis are primary mechanisms of energy expenditure, we also investigated the effect of N-PCT on core body temperature and locomotor activity. Twenty-four rats that had received aimed placements of an icv cannula were divided into three subgroups (n = 8 per group) that received either icv injection of aCSF or N-PCT peptide (1.0 and 0.1 nmol; doses used were based on the dose-response study). Furthermore, to examine the peripheral effects of N-PCT, 1.0 nmol N-PCT or an equivalent volume of PFS (1 ml/kg) was administered ip at 1900 h to rats (n = 10 per group). Body temperature and locomotor activity were measured by remote biotelemetry using precalibrated sensitive transmitters (model PDT-4000 E-Mitters; Mini Mitter, Sunriver, OR) implanted into the peritoneal cavities of rats, under general anesthesia induced by a mixture of ketamine and xylazine (same dose as above). Changes in body temperature signals and motor activity were received by an antenna below the rat’s cage and relayed to a signal processor. Core body temperatures of animals were recorded at 5-min intervals beginning at least 24 h before drug injection and continued for at least 2 d after injections. For analysis and clarity of graphical presentation, temperature data were averaged over each 15-min interval and are presented as the change in body temperature from the average baseline over the hour preceding the icv injection. Locomotor activities of animals were recorded at 15-min intervals beginning at least 24 h before drug administration and continued for 8 h after injections. Changes in locomotor activity were detected by changes in the position of the implanted transmitter over the receiver board. This resulted in a change of the signal strength, which was detected by the receiver and recorded as a pulse activity. Cumulative locomotor activity counts were made 3 and 8 h post injection. A data acquisition system (VitalView, Mini Mitter) was used for automatic control of data collection and analysis.
Experiment 4: immunohistochemistry
Tissue preparation.
Four 24-h fasted and 12 ad libitum-fed rats were deeply anesthetized using a mixture of ketamine and xylazine (same dose as above) and transcardially perfused with 300 ml of 0.1 M PBS (pH 7.4), followed by 300 ml of 4% paraformaldehyde in PBS. The brains were removed and postfixed overnight in the fixative and then stored in 30% sucrose in PBS at 4 C. Coronal sections of 30 µm were cut using a freezing microtome, collected in PBS, and stored at –20 C until immunohistochemical processing.
Fos immunohistochemistry.
To determine the metabolic mapping at the cellular level, the expression of c-fos protein in brain was determined after icv administration of N-PCT (1.0 and 2.0 nmol) or aCSF to free-feeding rats (n = 4 per group) 2 h before transcardial perfusion. Floating brain sections were prepared as described previously (34), with modifications (35). Free-floating brain sections were pretreated with 0.1% hydrogen peroxide (H2O2) in PBS for 20 min and incubated with 2% normal goat serum in PBS containing 0.3% Triton X-100 overnight at 4 C. Sections were then incubated with polyclonal Fos antibody (1:10,000 dilution) in PBS with Triton X-100 containing 0.5% normal goat serum for 2 days at 4 C. This anti-c-Fos antibody does not cross-react with other Fos-related gene products (Fos-b, Fra-1, or Fra-2). The sections were rinsed and incubated for 2 h with biotinylated goat antirabbit IgG (1:200; Vector) and successively with avidin-biotin-horseradish peroxidase (diluted 1:300 in PBS, for 2 h; Vectastain Elite ABC kit, Vector). Visualization of the antibody was performed with 0.02% 3,3'-diaminobenzidine and 0.01% H2O2 in 0.05 M Tris/HCl buffer (pH 7.4). Some immunostained sections for Fos protein from each animal were counterstained with cresyl violet to determine whether only neurons or also glial cells were involved in the immunoreaction. Observation of Fos-labeled sections was performed with an Olympus BX41 microscope connected to an Olympus C3030 zoom digital camera. Fos-positive nuclei were identified when structures of the appropriate size and shape demonstrated a clear increase in immunoreactivity as compared with the background level. The anatomical localization of the immunostained cells was identified according to the rat brain atlas by Paxinos and Watson (30). Adobe Photoshop was used to combine photomicrographs into plates. Only the sharpness, contrast, and brightness have been adjusted.
PCT immunohistochemistry.
To investigate the effect of food availability on the expression of PCT, brain sections from 24-h fasted (n = 4) and ad libitum-fed (n = 4) rats were processed for the localization of PCT. For fasting, food was withdrawn from cages at the onset of the dark cycle for 24 h, but ad libitum access to water was allowed. Immunocytochemistry was performed as previously described for PCT in the rat brain using an immunoperoxidase detection system in free-floating sections (21). A mouse monoclonal antibody recognizing the PCT precursor (BioVendor) was used to identify PCT. The sequence of this peptide differs from that of the rat sequence by only one amino acid (19). Briefly, the sections were briefly washed in PBS and then incubated for 15 min in 6% H2O2. After washing in PBS, the sections were incubated with blocking solution (5% goat serum and 0.1% saponin in PBS) for 30 min at room temperature and then incubated for 24 h at 4 C with PCT antibody (1:200). Sections were then incubated with biotinylated goat antimouse IgG (1:200; Biogenex) and with avidin-horseradish peroxidase complex (Vectastain Elite ABC kit, Vector). Finally, the sections were incubated with diaminobenzidine reagent (Dako, Carpinteria, CA). As soon as they turned brown, they were successively washed, counterstained with cresyl violet, dehydrated, and coverslipped. Some sections were also analyzed without counterstaining. Brain sections of fed and fasted rats were postfixed on glass slides under the same conditions to avoid the possibility of varied PCT staining. All antibodies were diluted in 0.1% saponin in PBS. Control experiments included coincubation of the PCT primary antibody with the PCT antigen peptide overnight (BRAHMS, Berlin, Germany; 200 µg/ml); negative controls omitted the primary antibody. In both cases, no positive immunostaining was detected. Digital images of the immunostained sections were captured using a digital camera attached to an Olympus microscope. Images were imported into Adobe Photoshop to compose the figures. Images were not altered, except for small adjustments in brightness and contrast.
Statistical analysis
The data are presented as the mean ± SEM. ANOVA followed by Newman-Keuls post hoc test was used to test the significance of differences among the groups. Additional analysis for dose effect at some time points were evaluated by paired t test (Student’s t test). P < 0.05 was considered to be a significant difference.
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) in a dose-dependent manner with a lowest effective dose of 0.02 nmol. In the groups injected with 0.2 and 1.0 nmol, the responses were virtually identical. The dose of 1.0 nmol N-PCT appeared to be maximal on the anorectic effect. This anorectic effect of N-PCT was rapid (as early as 1 h post injection) and remained up to 48 h post injection (Fig. 2, A and B) but no longer differed from the vehicle-treated group by the 72-h time point (data not shown). However, in contrast to the anorectic effect of N-PCT, the dose dependence of water intake response was more complex. Thus, whereas the lower doses (0.02 and 0.1 nmol) of icv N-PCT decrease cumulative water intake, higher doses (0.2 and 1.0 nmol) have a dipsogenic effect (Fig. 1B). Furthermore, N-PCT produced a significant long-lasting increase in cumulative water intake (Fig. 2, C and D). In addition, the N-PCT-injected rats gained significantly less weight than aCSF-treated rats at 24 and 48 h (Fig. 3, E and F). However, increasing the dose to 2.0 nmol did not result in any significant differences in either food/water intake or in the body weight gain at 24 and 48 h (P > 0.05 vs. aCSF) (Fig. 2, A–F).
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, D and E). By 24 h after ip injection of N-PCT, cumulative food intake, water intake, and body weight gain were not significantly different from vehicle-treated animals (data not shown). Thus, despite substantially increasing circulating N-PCT and demonstrating that the N-PCT is biologically active in parallel icv-injected rats, peripheral administration of N-PCT did not influence feeding and energy balance regulation.
CTA
Another conceivable mechanism underlying the reduction of food intake after icv N-PCT treatment is that central administration of N-PCT is aversive to rats. Nonspecific toxic or aversive effects of N-PCT were ruled out by the CTA test. Saccharin intake was measured after the administration of N-PCT or LiCl, a toxin that causes rats to avoid saccharin. LiCl caused taste aversion, whereas N-PCT (0.2 and 1.0 nmol, icv) did not reduce saccharin intake (Fig. 1C).
Effects of N-PCT on body temperature and locomotor activity
Before drawing any conclusions about whether icv N-PCT administration induces anorexia in rats, we considered the possibility that the reduction in food intake observed after central N-PCT treatment might be secondary to other behavioral effects. Behaviorally, N-PCT-treated animals rested more, groomed less, and ate less. Thus, in a second experiment, rats with radio-telemetric transmitters were monitored for core body temperature and locomotor activity, along with food intake, water intake, and body weight after N-PCT administration (0.1 and 1.0 nmol, icv). As before, overnight food intake was decreased significantly. However, whereas 0.1 nmol N-PCT had no significant effect on body temperature, the icv administration of 1.0 nmol N-PCT caused a significant increase in core body temperature that began to rise after 2 h, remained elevated over control values until approximately 8 h after injection (mean for 0–8 h, vehicle 0.2 ± 0.05 C vs. N-PCT 1.3 ± 0.2 C; P < 0.01), but returned to normal for the remainder of the study (Fig. 3A).
According to these data, it seemed possible that the acute suppression of food intake after N-PCT treatment might be a consequence of impaired locomotor function, thus rendering the rats physically incapable of eating as much as the vehicle-treated animals. Thus, to further investigate this possibility, we performed an experiment to assess whether locomotor activity is altered after acute N-PCT treatment. The ip injection of 5.0 nmol/kg N-PCT (1.0 nmol per animal; n = 8) did not induce any significant change in core body temperature or locomotor activity over the next 8 h compared with PFS-treated animals (Fig. 3B). However, the level of locomotor activity, compared with the pretreatment level, was decreased (P < 0.05) during the first 3 h after icv injection with 0.02, 0.1, 0.2, 1.0, or 2.0 nmol N-PCT (Fig. 3C) but then became normal for the remainder of the observation period (Fig. 3D). Furthermore, there was no evidence of tremors or shivering associated with this dose of N-PCT. These abnormal behaviors were not observed in the PFS-injected rats. In addition, animals that received 1.0 nmol heat-inactivated icv N-PCT (90 C for 2 h) showed no differences in food and water intake, body temperature, locomotor activity and body weight gain relative to the vehicle-treated group (data not shown).
N-PCT-induced Fos expression
To determine the neuronal populations activated by central N-PCT in rat brain, we mapped Fos expression after an icv injection of aCSF or N-PCT. Only positive results of the densities of c-Fos-labeled cells are included. The injection of N-PCT (1.0 nmol) significantly decreased food intake over the 120-min test period compared with vehicle injection (aCSF, 4.6 ± 0.2 g; N-PCT, 0.4 ± 0.1 g; P < 0.01). Fos-immunoreactive cells were observed primarily in regions implicated in the regulation of feeding behavior such as the paraventricular nucleus (PVN) (Fig. 4B), dorsomedial hypothalamic nucleus (Fig. 4D), arcuate nucleus (ARC) (Fig. 4F), supraoptic nucleus (SON) (Fig. 4H), or other regions surrounding the majority of the ventricular system (e.g. locus ceruleus, subfornical organ, and medial preoptic nucleus, data not shown). Fos distributions were similar in the 1.0- and 2.0-nmol N-PCT-injected rats. No significant Fos expression was found in vehicle-treated rats (Fig. 4, A, C, E, and G). No significant staining of cells expressing c-Fos protein was observed in the area postrema or in the nucleus of the solitary tract of animals treated with N-PCT (data not shown).
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, A–H). In addition, the SON, area postrema, subfornical organ, medial preoptic nucleus, tuber cinereum, suprachiasmatic nucleus, dorsal hypothalamic area, and periventricular hypothalamic nucleus also contained a few cells and/or fibers with PCT-like immunoreactivity (data not shown). No strongly labeled cells were found in the PVN. Wherever it was found, PCT was clearly intracellular and indicates, according to previous findings, that PCT-positive cells are astrocytes (20). In free-feeding rats, PCT-positive cells presented elongated cell processes that were sharply delineated in the brain parenchyma (ARC), in contact with Vgl and vascular tissues (Fig. 5, A, C, and E). PCT expression was significantly reduced in fasted rats (Fig. 5, B, D, and F). These results suggest that PCT may be a signaling molecule released from astrocytes.
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An icv administration of N-PCT dose-dependently suppressed dark-phase (feeding-phase) feeding. This effect was extremely potent, with inhibition observed even after administration of only 0.02 nmol N-PCT, although the anorectic effect of the highest dose (2.0 nmol) was comparatively less significant. Initially, these results suggest the existence of putative receptors for N-PCT and that the central receptors mediating N-PCT’s anorectic effect were already fully activated at the lower doses. Indeed, the short latency of the effect (<1 h) suggests a direct action of N-PCT on feeding-regulatory centers. The N-PCT-induced anorexia was accompanied by significant weight loss not clearly dose related. These effects of N-PCT were not accompanied by effects on general activity. Moreover, animals that received heat-inactivated N-PCT showed no significant differences in food intake and body weight gain relative to the aCSF injected. These findings together with the lack of an N-PCT-induced taste aversion at doses that reduce food intake suggest that N-PCT may be a homeostatic regulator of food intake.
Reduction of food intake and body weight gain after the central administration of N-PCT was similar or stronger compared with CT at the time points examined. N-PCT is considerably more potent than CT on a molar basis in this regard, with a threshold dose approximately 10-fold lower than that of CT (7). Often the species of CT used in these experiments were other than human (e.g. human, salmon, porcine, and eel), the amino acid sequences of which differ. It is also known that there are species differences associated with the pharmacology of this peptide family and its receptors, so interactions of CTR with RAMPs may be specific to species homologs (4). Furthermore, pharmacological, not physiological, doses were often employed, and as a result, many actions have been incorrectly imputed to this peptide (36). Taking these aspects into consideration, the resistance of N-PCT to enzymatic degradation by endogenous peptidases, responsible for the short half-life of CT (24) and/or an apparently irreversible binding of N-PCT to AMY receptors (37), could in part explain the potent and longer action of N-PCT on feeding behavior.
CTRs exhibit high affinity for CT peptides, with the general comparative potency of salmon, porcine, rat, and human CT, and a relatively lower affinity for the related peptides AMY, CGRP, and ADM (8). As described before (see introductory section), the CTR gene product can interact with individual RAMPs to generate different high-affinity AMY receptor phenotypes (4). Furthermore, CTRs exist as alternative splice variants (CT(a) and CT(b)). The most abundantly expressed rodent and human CTR isoforms (CT(a) and CT(b), respectively) have similar relative potencies in response to CT peptides; however, differences in absolute potencies of peptides do occur (4). It has been shown that CT(b) receptor was less affinitive and more discriminative toward CT peptides than CT(a), that the coexpression of the CTR with RAMPs form the AMY-specific receptor, and that the CT(a) and that the CT(b) receptor subtypes are codistributed with distinct RAMPs within some hypothalamic regions of primary importance in the regulation of feeding behavior, such as the area postrema and the subfornical organ (13, 14, 37). These findings suggest that the combined activation of CTR subtypes and CRLR in these brain areas is, at least in part, responsible for the short-term central effects of N-PCT on food intake. However, additional studies are necessary to establish the long-lasting actions of N-PCT on food intake and body weight.
Occurring concomitant with a reduction of food intake, icv administration of N-PCT caused a significant rise in body temperature and a decreased locomotor activity, implying that N-PCT increases energy expenditure. However, although N-PCT-treated animals rested more and ate less, by 8 h and for the remainder of the observational period, there was no significant difference between changes in body temperature and locomotor activity compared with the control groups. Thus, like CT or other CT-like peptides, central administration of N-PCT produces weight loss via potent effects on food intake that are not secondary to locomotor impairment or illness. Metabolic heat production can be increased by either shivering or sympathetic excitation in brown adipose tissue and skeletal muscle. The latter mechanism, also called chemical thermogenesis, appears to be the primary mechanism of N-PCT-induced thermogenesis during the first 8 h because shivering was not observed in rats administered N-PCT. Based on our data, N-PCT possibly meets the criteria for a catabolic signaling molecule.
In contrast to the anorectic effect of N-PCT, the dose dependence of the water-intake response was more complex. Thus, whereas the lower doses (0.02 and 0.1 nmol) of icv N-PCT decreased cumulative water intake, higher doses (0.2 and 1.0 nmol) had a dipsogenic effect. These effects of N-PCT are in contrast to the observations of other authors who found that salmon CT given icv dose-dependently decreased water intake possibly due to an activation of neurons in the subfornical organ, a brain region stimulated by CT and CT-related peptides and believed to be involved in control of drinking via excitatory effects on angiotensin II-sensitive neurons or as a consequence of the food intake suppression, i.e. reduced prandial drinking (38). The anti-dipsogenic effect of lower doses N-PCT in rats could therefore be explained by a reduction in prandial drinking. However, the dipsogenic effect of higher doses of N-PCT indicates that there are different threshold requirements for the effects observed on water intake, suggesting that different CTRs and/ or CRLRs may be involved.
Similar to other CT-like peptides such as intermedin (17), the effects of N-PCT on food intake appear to be independent of the effects on water intake. The potent effects of intermedin on food intake have been attributed to central activation of CGRP receptors and are independent of the effects on water intake, which are likely through the ADM receptor in association with RAMPs (39). As described above, the CTR/CRLR gene product can also interact with individual RAMPs to generate different high-affinity ADM and CGRP receptors expressed in brain. Furthermore, the CRLR and RAMPs are expressed throughout the CNS (40), and both ADM and CGRP exert effects within the brain (41). PCT immunoreactivity was found throughout the CNS of normal rats, with the highest peptide levels in the hypothalamus and circumventricular organs (i.e. the subfornical organ), confirming the initial immunohistochemical staining (20). Furthermore, as mentioned above, specific binding sites for CT (CT(a) and CT(b) receptor subtypes) codistributed with distinct RAMPs have been localized in brain areas involved in feeding and fluid and electrolyte homeostasis (13, 14, 37, 42, 43, 44) where there is no diffusional (blood-brain) barrier, so they are directly accessible to thyroidally derived, blood-borne N-PCT. Thus, although these findings could explain, at least in part, the central effects of N-PCT on water intake, additional work including investigating cAMP accumulation at the cellular level is necessary to establish the role of specific brain areas expressing CRLR/CTR subtypes, RAMPs, and N-PCT on drinking behavior. It remains to be determined whether the high levels of PCT detected in the brain reflect uptake of circulating PCT of peripheral origin or mirror peptides synthesized locally within these structures.
We found that very high doses of N-PCT delivered peripherally did not produce any modifications in food and water intake, body temperature, and locomotor activity during at least the first 8 h. These discrepancies between the expression of CTR and PCT and the lack of effect of ip N-PCT suggest that there are different threshold requirements for the activation of CTR and/or CRLR when N-PCT is delivered peripherally. Initially, these observations suggest that cells of the CNS are more likely the critical source of the N-PCT. However, this does not rule out a peripheral mechanism for N-PCT. Thus, one possibility is that PCT and its free bioactive fragment N-PCT may be made in specific regions of the CNS by neurons or other CNS cell types involved in the regulation of energy intake. Another possibility is that peripherally derived N-PCT enters the CNS via an active receptor-mediated uptake system, but there is not an active receptor-mediated uptake system or any evidence indicating that peripherally derived N-PCT enters the CNS. Furthermore, human adipose tissue depots have recently been identified as major sepsis-related nonneuroendocrine CT mRNA expression sites leading to increased serum PCT, and they have a lipolytic effect (glycerol release) (45). Overall, these findings, in contrast with the present results, indicate a potential role of peripheral PCT in situations where the metabolic balance is altered, such as obesity, and suggest that this peptide may act as a circulating factor for integration of adiposity- and satiety-related inputs (46). It will be important in future studies to examine whether N-PCT levels in plasma and tissue fluctuate in concert with various physiological and pathophysiological stimuli.
To study which areas of the brain might be involved in the actions of N-PCT, the distribution and extent of c-fos expression was assessed after a single icv injection of N-PCT. We show that N-PCT modulates feeding behavior in association with neural changes in specific hypothalamic nuclei such as the ARC-ME, DMH, PVN, SON, and other areas surrounding the ventricles (including the peri-third-ventricular hypothalamic region), all regions involved in the integration of hormonal signals for energy homeostasis. These findings are consistent with extensive brain mapping of CT-induced anorexia (47) and the distribution of the CTR/RAMP receptors and CT(a) and CT(b) subtypes in the rat brain (13, 48, 49), especially CT(a)/RAMP3 in the ARC-ME area, which is considered as one of the circumventricular organs where the blood-brain barrier is specially modified to allow entry of peripheral peptides and proteins including insulin and leptin, both of which are considered to be signals of fat (50). So, it can be speculated that PCT plays a role in the integration of hormonal signals for energy homeostasis. However, additional experiments are necessary to study the role of CTR/RAMP and CRLR/RAMP in the central effects of N-PCT on energy homeostasis.
In contrast, c-fos expression was not induced in the brainstem, particularly in the area postrema and the nucleus of the tractus solitarius (data not shown), regions that show abundant expression of CTR, and was substantially induced in the SON, a region involved in water balance, where CTR is not expressed. Thus, these findings are consistent with recent data showing that c-fos expression is present in the SON after icv administration of calcitonin receptor-stimulating peptide-1, a putative endogenous ligand for CTR that has potent effects on energy homeostasis in rats (51). Fos protein is usually expressed only in cells that are activated by a stimulus, and any cells that are inhibited by N-PCT are unlikely to express this immediate early gene. However, it cannot be assumed that c-fos expression in SON and other areas after icv infusions is necessarily due to the direct effects of N-PCT on these cells. These regions are highly interconnected, so that activation of cells locally could induce c-fos at more remote sites by transsynaptic mechanisms. It is also possible that the activity noted in different brain regions may partially represent an indirect response by other cells that are upstream targets of N-PCT. Thus, although the results of the present study cannot discriminate whether the neuronal activation by N-PCT binding within regions involved in feeding behavior and energy homeostasis is mediated by CTR, our results clearly emphasize the important role of these structures as primary targets for N-PCT.
To investigate the anatomic basis for these effects and to demonstrate that either the message or the peptide is endogenously produced under certain physiological changes, we measured PCT expression in rat brain in fed and fasting conditions by immunohistochemistry. As reported previously (21), astrocytes intensely immunoreactive for PCT were abundant in the hypothalamus and circumventricular organs of rats fed ad libitum. The present study reveals an abundance of somata and fibers with PCT-like immunoreactivity in some hypothalamic regions of primary importance in the regulation of energy homeostasis, such as the ARC and the ME. Moreover, similar to some well-characterized anorexia-eliciting paradigms such as systemic inflammation induced by peripheral administration of bacterial endotoxin (27), PCT expression was significantly reduced in these areas during fasting, perhaps as an adaptive response of the brain circuits that regulate appetite against certain specific physiological stressors such as endotoxemia and fasting. Because PCT expression is widespread throughout hypothalamic areas, particularly the ARC, a nucleus linked to the regulation of energy balance and regulated by feeding-regulating molecules, the present findings suggest the possibility of a functional interaction between PCT-positive astrocytes and other feeding-regulating neurons within the ARC that express both orexigenic molecules such as neuropeptide Y (NPY) and agouti-peptide (AgRP) and anorexigenic peptides such as proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) (52). In addition, the present findings are also consistent with the regional distribution of CTR and RAMPs in the rat brain and suggest that different CTR a/b subtypes may be involved in the catabolic effects induced by central administration of N-PCT. However, additional studies are necessary to demonstrate the anatomical basis and the physiological relevance of these possible anatomical relationships.
In summary, although CT has been considered as an enigmatic hormone (36) and there is not a true ligand for CTR, N-PCT, the bioactive fragment of the precursor of CT, has the potential to be an endogenous ligand for central CTR and an important physiological regulator of energy homeostasis. However, caution is needed when interpreting data for any new molecule in the complex pathways involved in energy homeostasis. Thus, additional research including chronic administration of N-PCT will be necessary to determine whether the effects of N-PCT on feeding are merely pharmacological or whether they mimic an effect of endogenous secreted PCT. It will also be necessary to assess N-PCT induction in some well-characterized anorexia-eliciting paradigms such as chronic leptin administration. Another priority is to elucidate how N-PCT can elicit its effects on different brain areas and to determine possible synergies with other endogenous neurotransmitters and neuropeptides in hypothalamic circuits involved in the control of feeding behavior and energy balance and to determine whether the central effects of N-PCT are due to a possible interaction with CT(a)/CT(b) subtypes expressed alone or are in association with RAMPs that have also been shown to govern the pharmacology of the CTR.
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First Published Online December 28, 2006
Abbreviations: aCSF, Artificial cerebrospinal fluid; ADM, adrenomedullin; AMY, amylin; ARC, arcuate nucleus; CGRP, calcitonin gene-related peptide; CNS, central nervous system; CRLR, calcitonin receptor-like receptor; CT, calcitonin; CTA, conditioned taste aversion; CTR, calcitonin receptor; icv, intracerebroventricular(ly); ME, median eminence; N-PCT, amino-procalcitonin; PFS, pyrogen-free saline; PVN, paraventricular hypothalamic nucleus; RAMP, receptor activity-modifying protein; SON, supraoptic nucleus; Vgl, ventral glia limitans.
Received June 13, 2006.
Accepted for publication December 15, 2006.
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