Aminoprocalcitonin-mediated suppression of feeding involves the ...

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Am J Physiol Endocrinol Metab 304: E1251–E1262, 2013. First published April 9, 2013; doi:10.1152/ajpendo.00590.2012.

Aminoprocalcitonin-mediated suppression of feeding involves the hypothalamic melanocortin system Eva Tavares,1 Rosario Maldonado,1 and Francisco J. Miñano1,2 1

Unidad de Farmacología Experimental y Clínica, Hospital Universitario de Valme, Seville, Spain; and 2Departamento de Farmacología, Pediatría y Radiología, Facultad de Medicina, Universidad de Sevilla, Seville, Spain Submitted 26 November 2012; accepted in final form 2 April 2013

Tavares E, Maldonado R, Miñano FJ. Aminoprocalcitonin-mediated suppression of feeding involves the hypothalamic melanocortin system. Am J Physiol Endocrinol Metab 304: E1251–E1262, 2013. First published April 9, 2013; doi:10.1152/ajpendo.00590.2012.—Aminoprocalcitonin (N-PCT), a neuroendocrine peptide encoded by the calcitonin-I (CALC-I) gene, suppresses food intake when administered centrally in rats. However, the neural pathways underlying this effect remain unclear. N-PCT and calcitonin receptors (CT-R) have been identified in hypothalamic regions involved in energy homeostasis, including the arcuate nucleus (ARC). Here, we hypothesized an involvement of the hypothalamic ARC in mediating the anorexic effects of central N-PCT based on its content of peptidergic neurons involved in feeding and its expression of N-PCT and CT-R. Fasting strongly reduced expression of the N-PCT precursor gene CALC-I in the ARC, and central immunoneutralization of endogenous N-PCT increased food intake. Intracerebroventricular administration of NPCT reduced food intake in fed and fasted rats, and its effect was attenuated by a neutralizing anti-N-PCT antibody. Immunohistochemistry for N-PCT showed that it is expressed in astrocytes and neurons in the ARC and is colocalized with anorexigenic proopiomelanocortin (POMC) neurons. Fasting reduced coexpression of N-PCT and POMC, and N-PCT administration activated hypothalamic neurons, including rostral POMC neurons. We also found that N-PCT stimulates POMC mRNA expression in fed and fasted rats, whereas it reduced the expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP) only in fasted rats in which those mRNAs are normally elevated. Finally, we showed that the melanocortin-3/4 receptor antagonist SHU 9119 attenuates the intake-suppressive effect of N-PCT. These data demonstrate that hypothalamic N-PCT is involved in control of energy balance and that its anorexigenic effects are mediated through the melanocortin system. food intake; aminoprocalcitonin; arcuate nucleus; melanocortin system; proopiomelanocortin; neuropeptide Y

(N-PCT) was originally described as a 57-amino acid neuroendocrine peptide derived from the aminoterminal half of rat prohormone procalcitonin with bone cell mitogenic properties (6, 7). N-PCT derives from the calcitonin-I gene (CALC-I), which is localized in chromosome 11 (21, 50), a chromosome associated with body metabolism and obesity in humans (8). N-PCT, a highly conserved amino acid peptide with ⬎85% amino acid identity in human and rodents (41), belongs to the calcitonin (CT) peptide family, which is made up by CT, CT gene-related peptide (CGRP), amylin, adrenomedullin, intermedin, and CT receptor-stimulating protein (3). These peptides function as ligands for a complex family of G protein-coupled receptors consisting of the CT receptor (CT-R), CT receptor-like receptor, and three receptor-

AMINOPROCALCITONIN

Address for reprint requests and other correspondence: F. J. Miñano, Dept. of Pharmacology, Pediatrics, and Radiology, Seville Univ. Faculty of Medicine, 41009 Seville, Spain (e-mail: [email protected]). http://www.ajpendo.org

associated modified proteins (40). CALC-I gene products and their receptors are widely distributed in the central nervous system (CNS), and several neural functions have been ascribed to these molecules, including hormone secretion regulation and energy homeostasis, among others (3, 4, 42, 60). Although CT has been considered as an enigmatic hormone (19), and there is not a true ligand for CT-R, N-PCT has the potential to be an endogenous ligand for central CT-R. Recent evidence indicates that N-PCT can function as a ligand for the CT-R and may act as an anorectic signaling molecule in the CNS (47, 54, 55). The CT-R has been cloned and characterized in different cell types and species (40). Two CT-R isoforms, CTa and CTb, have been described in the rat brain with an overlapping distribution pattern particularly in hypothalamic nuclei involved in feeding behavior, including the arcuate nucleus (ARC) and the paraventricular nucleus (PVN) (4, 23, 48). RT-PCR analysis in combination with laser capture microdissection, in situ hybridization, and immunocytochemistry has demonstrated that a subpopulation of proopiomelanocortin (POMC) neurons and corticotropin-releasing hormone (CRH) neurons selectively expressed the CTa isoform, whereas the CTb isoform was expressed in neuropeptide Y (NPY) neurons (23, 24). These findings suggest that the anorectic effects of N-PCT may be mediated via a possible interaction with CT-R expressed in ARC POMC/NPY neurons. Intracerebroventricular (icv) administration of N-PCT causes a dose-dependent reduction in food intake and body weight gain in rats via a mechanism unlikely to involve taste aversion (55). N-PCT-immunoreactive cells have been found in brain areas closely associated with food intake and body weight, including the ARC and PVN (34 –36). This distribution coincides with that of CT-R, which is expressed abundantly in these hypothalamic nuclei (4, 37). Recent findings show that N-PCT colocalized with CRH in parvocellular neurons of the PVN (54). Moreover, c-Fos expression, a marker of neuronal activity, was observed primarily within the ARC and PVN after icv injection of N-PCT (54, 57). In vitro and in vivo studies suggest that N-PCT, similarly to other neuropeptides of the CT family, like amylin and CT gene-related peptides, can function as a ligand for the CT-R (47, 55, 57). Together, these findings suggest that the anorectic actions of N-PCT likely occur within the hypothalamus. However, the neural pathways underlying these actions of N-PCT are not well understood. One possibility is that N-PCT, like other anorexigenic peptides, alters melanocortin (MC) signaling in the ARC. The MC signaling in the hypothalamus plays a pivotal role in the integration of signals regulating appetite and energy balance (12). Regulation through this pathway is exerted by two functionally opposing neuronal populations in the ARC that coexpress NPY and agouti-related peptide (AgRP) to

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stimulate food intake and POMC and cocaine- and amphetamine-regulated transcript (CART) to induce anorexia (1, 9, 33, 45). Activation of POMC neurons decreases food intake and increases energy expenditure through activation of target neurons expressing melanocortin-3/4 receptors (MC3/4-R) via the release of ␣-MSH, a posttranslational cleavage product of POMC (30). Activation of neighboring NPY/AgRP neurons counteracts the activity of POMC neurons (10) in part through the release of AgRP, a competitive inhibitor of MC3/4-R (38). Because N-PCT is found together with CT-R in the ARC, CT-Rs are expressed in hypothalamic POMC neurons, and ARC POMC neurons form an integral part of the MC system regulating feeding behavior within the CNS (9), we hypothesized that hypothalamic N-PCT is an anorectic peptide and that this function is mediated via the MC system. Therefore, we investigated the effect of nutritional status on hypothalamic CALC-I gene expression. We also characterized cell types expressing N-PCT in the ARC and explored the colocalization of N-PCT with hypothalamic POMC neurons. Changes in feeding behavior after central immunoneutralization of N-PCT were also studied. Subsequently, we investigated the potential mechanism of action by determining sites of neuronal activation and interaction between N-PCT and other hypothalamic neuropeptides regulating energy homeostasis after icv administration of N-PCT. Finally, using a MC3/4-R antagonist, we established a potential link between N-PCT and the MC system in modulating feeding behavior. MATERIALS AND METHODS

Materials. N-PCT and the MC3/4-R antagonist SHU 9119 [acetyl(Nle4,Asp5,D-2-Nal7,Lys10)-cyclo-␣-MSH (4 –10) amide] were purchased from Bachem (Bubendorf, Switzerland). The monoclonal antibody directed against rat N-PCT was synthesized by Abcam (Cambridge, MA), and the isotype-matched nonimmune mouse IgG2a was purchased from Biogenex (San Ramon, CA). All drugs were dissolved in rat artificial cerebrospinal fluid (aCSF; Harvard Apparatus, Holliston, MA), which served as the control vehicle. All solutions were passed through 0.22-␮m pore size Millipore filters and stored at ⫺80°C. The same batches and solutions were used for all experiments and contained an endotoxin level of ⬍0.1 ng/ml protein (i.e., below the detection limit of endotoxin assay), as determined by the limulus lysate assay (ICN Biomedicals, Costa Mesa, CA). Fresh aliquots were thawed before each experiment, briefly sonicated, and warmed to 37°C just before use. Animals. Adult male Wistar rats (Harlan Iberica, Barcelona, Spain) initially weighing 250 –300 g were used. The rats were initially housed in group cages (3/cage) in a room with temperature controlled at 22 ⫾ 1°C and a 12:12-h light-dark cycle (lights on at 0900). Standard rodent chow (2014S Teklad; Harlan) and tap water were provided ad libitum throughout the experiments, except where indicated. At least 2 wk before the experiments, the rats were placed in individual cages and housed in a separate room under a reversed 12:12-h light-dark cycle (lights off at 0900) with temperature controlled at 25 ⫾ 1°C, in or near the thermoneutral ambient temperature range for rats. Experiments began after 2 wk of acclimatization. Experiments were carried out during the dark phase (except where noted), the physiological feeding time for rodents (51). All experiments were performed in accordance with the Council of the European Communities’ Directive (86/609/EEC) and Spanish regulations (BOE/67:8509/1988) for the use of experimental animals. The protocol was approved by the Animal Care and Use Committee of the University of Seville.

Surgical procedures: icv cannulation and injection. The detailed procedure for implantation of guide cannulas into the rat lateral cerebral ventricle has been described previously (55). Briefly, animals were anesthetized with an intraperitoneal injection of a ketamine (100 mg/kg)-xylazine (5 mg/kg) cocktail. A 22-gauge stainless-steel guide cannula was stereotaxically implanted into the lateral ventricle. The coordinates used were 0.8 mm posterior to the bregma, 1.5 mm lateral to the medline, and 2.5 mm ventral to the skull surface (39). The cannula was attached to the skull with dental cement and bone screws, and a nonsteroidal anti-inflammatory drug, meloxicam (Metacam, 1 mg/kg; Boehringer Ingelheim, Barcelona, Spain), was injected subcutaneously. Animals were allowed ⱖ7 days recovery before commencement of experimentation. The animals were housed individually after surgery for ⱖ1 wk and were handled and habituated to the injection procedure daily. Test substances were injected icv by gravity flow (5.0-␮l volume) in conscious, lightly restrained rats. Correct cannula positioning was confirmed postmortem by histological examination. For feeding experiments, cannulated rats were isolated in individual cages without litter. Food intake of each rat was measured for ⱖ3 days to establish a baseline. On test days, ad libitum-fed rats were weighed, and food was removed 2 h before the beginning of the dark cycle. Other rats were fasted for 24 h. Fasted animals received water ad libitum, but food was removed from the cages 24 h before lights off. This fasting duration resulted in ⬃15% weight loss. Rats in both conditions received an icv injection of the drugs tested immediately before the dark phase (except where noted). Immediately after injections, a preweighed amount of food was presented to the animals. Food consumption was measured 1, 2, and 4 h later (except where noted). Real-time RT-PCR analysis. Four hours after the beginning of the dark phase, rats were euthanized by decapitation. The brains were immediately removed and placed in a brain matrix (Harvard Apparatus) on dry ice, with the ventral surface on top under a dissecting microscope. To remove the hypothalamus, we used the optic chiasm and rostral edge of mammillary bodies as rostral and caudal limits, respectively; the hypothalamic sulci were used as lateral limits. Brain slices containing the whole hypothalamus were prepared, and the entire ARC was excised from the left and right sides. We removed the ARC by cutting between the rostral and caudal limits of the median eminence and 0.5 mm to each lateral side of the median eminence (see Fig. 1) (39). In general, the entire ARC with minimal surrounding tissue was taken for analysis. Hypothalamic blocks containing the ARC were preserved immediately in an RNA stabilization solution (Qiagen, Hilden, Germany) and stored at ⫺80°C until RNA extraction and RT-PCR analysis. RT-PCR was performed essentially as described previously for CALC-I and CRH gene expression in hypothalamic rat tissue (54, 56). Total RNA was isolated using Tripure reagent (Roche Diagnostics, Basel, Switzerland) and then reverse-transcribed into cDNA using the Quantitect Enzyme Reverse Transcriptase Kit (Qiagen) according to the manufacturer’s instructions. SYBR Green real-time RT-PCR was performed with the Stratagene Mx3005P sequence detection system (Agilent Technologies) using SensiMix SYBR Low-ROX (Bioline, Luckenwalde, Germany). Primers for CALC-I were designed using the software Beacon Designer (Premier Biosoft, Palo Alto, CA) according to the rat CALC-I sequence NM_017338 (National Center for Biotechnology Information Database). The primers were 5=CGCTGCCCAGATCAAGAGTC-3= (forward) and 5=-AGATGCTGACAACCAGGAAAGG-3= (reverse) (Eurogentec, Liege, Belgium). The primers for the target rat genes (POMC: Rn_Pomc2_1_SG; NPY: Rn_Npy_1_SG; AgRP: Rn_Agrp_1_SG) and the reference gene glyceraldehyde-3-phosphate dehydrogenase (rat GADPH; Rn_Gapd_1_SG) were predesigned and validated by Qiagen (QuantiTect Primer Assay). Threshold cycle values were determined automatically using the software supplied by Stratagene (MxPro Software 3.20). Primer specificity was verified by melt curve analysis. Relative gene expres-

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Fig. 1. Schematic figure demonstrating the brain region dissected for the real-time RT-PCR experiment. ARC, arcuate nucleus; DM, dorsomedial nucleus; f, fornix; OX, optic chiasm; M, mammillary bodies; ME, median eminence; VM, ventromedial nucleus; 3V, 3rd ventricle.

sion was determined using the 2⌬⌬CT method (27). All values for gene expression following PCR analysis are expressed as relative to GAPDH mRNA content and referred to as relative expression. Tissue preparation for immunohistochemistry. Rats were deeply anesthetized with a ketamine (100 mg/kg)-xylazine (5 mg/kg) cocktail and transcardially perfused with PBS (pH 7.4), followed by 4% paraformaldehyde in 0.1 M PBS for fixation. The brains were removed and postfixed in the same fixative for 2 h, cryopreserved in 30% sucrose-PBS for 48 h at 4°C, and stored at ⫺80°C until sectioning. Coronal (30-␮m-thick) sections containing the ARC were cut on a freezing microtome and stored in cryoprotectant at ⫺20°C until they were required for immunohistochemistry. Multiple fluorescence immunohistochemistry. To characterize cell types expressing N-PCT, hypothalamic sections were subjected to double-labeling immunohistochemistry for a neuronal nuclei (NeuN) or a glial marker [glial fibrillary acidic protein (GFAP)], as described previously (54). To determine whether POMC neurons express NPCT, double-labeling immunohistochemistry was performed using a rabbit anti-POMC antibody (57). Briefly, after quenching of autofluorescence with 0.3 M glycine and saturation of nonspecific sites with 3% normal donkey serum (Jackson Laboratories, West Grove, CA), sections were incubated for 72 h at 4°C with a mouse anti-N-PCT monoclonal antibody (1:100; Abcam) or incubated overnight at 4°C with rabbit anti-NeuN antibody (1:500; Millipore), anti-GFAP antibody (1:500; Dakocytomation, Carpinteria, CA), or anti-POMC antibody (1:1,000; Phoenix Pharmaceuticals). The specificity of the anti-N-PCT antibody used in this study was determined previously in the rat hypothalamus by Western blot and immunohistochemistry experiments (54). After washing, the sections were incubated with FITC-conjugated donkey anti-mouse IgG (1:200; Jackson Laboratories) and TRITC-conjugated donkey anti-rabbit IgG (1:200; Jackson Laboratories). Omission of primary antibodies and substitution of the primary antibody with specific blocking peptides confirmed antibody specificity. Immunohistochemistry was performed on sections be-

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tween ⫺1.80 and ⫺3.60 from the bregma (39). Sections were mounted on slides, coverslipped with anti-fading mounting medium (Vectashield; Vector Laboratories), and examined under a fluorescent Olympus BX41 microscope. Sections were postfixed on glass slides under the same conditions to avoid the possibility of varied staining. Colocalization pictures were the combination of each corresponding fluorescent signals. Image-editing software (Adobe Photoshop) was used to combine photomicrographs into plates. Double-stained sections were examined with a laser-scanning confocal microscope, as described below. Laser-scanning confocal microscopy and quantitative colocalization analysis. Images were acquired using a confocal microscope (Zeiss LSM 7 DUO) in sequential mode with a ⫻40 (NA 0.95) objective, 12-bit resolution and two lines averaging. The conditions for FITC channel were excitation at 488 nm, emission 493–558 nm, pinhole at 1.03 Airy units, and section thickness of 1.1 ␮m. For TRITC channel, we used the following conditions: excitation at 561 nm, emission 566 –585 nm, pinhole at 0.75 Airy units, and section thickness of 1.0 ␮m. We chose the most intense signal plane between several z-axis positions of the slice as the most representative for the analysis. Twelve images were stitched to reconstruct the whole ARC in each slice. For quantitative colocalization analysis, we used the corresponding ZEN 2010 imaging software module (Carl Zeiss). Three square regions of 400 ⫻ 400 pixels were located randomly in each nucleus of every image, and the Manders’ overlap coefficient (29), which indicates quantitative colocalization on a scale of 0 (no overlap) to 1 (100% overlap), was calculated for statistical analysis. Double-labeling immunohistochemistry for c-Fos and POMC. Immunohistochemical staining for c-Fos and POMC was performed by using a sequential two-color avidin-biotin immunoperoxidase complex technique according to a previously described method (16, 49, 57). Briefly, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. Nonspecific binding was prevented by using 10% normal goat serum in PBS for 1 h. Then, sections were washed and incubated with rabbit anti-c-Fos antiserum (1:3,000, sc-52; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-POMC antiserum (1:2,000; Phoenix Pharmaceuticals) for 48 h at 4°C. The secondary antibody was goat anti-rabbit IgG conjugated to biotin (Merck Millipore, Darmstadt, Germany). The commercially available avidin-biotin immunoperoxidase complex system (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) was used to visualize bound antibody. The color reaction was produced with nickel-intensified diaminobenzidine (Ni-DAB; Sigma) in the presence of 0.01% hydrogen peroxide. This results in a black reaction product in singly stained sections. For double labeling, brain sections were processed for the localization of c-Fos, as described above. Subsequently, we performed the second immunostaining, incubating the tissue in the POMC antiserum to label POMC neurons. Sections were processed using only DAB (without nickel) as chromogen, resulting in brown staining. Cells with dark blue nuclear staining were c-Fos positive, and cells with brown cytoplasmic staining were POMC immunoreactive. Image analyses and cell counting. For data acquisition and quantification of c-Fos and/or POMC-labeled cells in the ARC, positive cells were counted on an Olympus BX41 microscope fitted with a grid reticule, with the investigator blinded to study conditions. Using the rat brain atlas of Paxinos and Watson (39) as an anatomic guide, the ARC was divided into two areas (defined as rostral and caudal ARC) of approximately equal length. Rostral ARC included the retrochiasmatic area, marking the most rostral extent of the distribution of POMC-immunoreactive neurons, at ⬃1.8 mm posterior to the bregma and continued caudally to the appearance of the dorsomedial hypothalamic nucleus ⬃2.56 mm posterior to the bregma. Caudal ARC corresponded to a region from ⬃2.8 to 3.6 mm posterior to the bregma and included the most caudal region of the ARC and disappearance of POMC-immunoreactive neurons. For each region, four representative matched sections were examined bilaterally in each rat to determine

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the total number of c-Fos- or POMC-immunoreactive cells and double-labeled cells. Colocalization was expressed as a percentage of the total number of POMC-immunoreactive cells expressing c-Fos. Study 1: effect of nutritional status on hypothalamic CALC-I mRNA expression. Two groups of rats (ad libitum fed and fasted for 24 h, n ⫽ 16) were euthanized 4 h after the beginning of the dark phase. Whole hypothalami were dissected, and the entire ARC was removed, preserved in an RNA stabilization solution, and stored at ⫺80°C until it was processed for RT-PCR analysis as described above. Study 2: characterization of cell types expressing N-PCT. To determine cell types expressing N-PCT, rats (n ⫽ 4) were anesthetized and transcardially perfused 90 min after the beginning of the dark phase. Brains were removed and processed for double-labeling immunohistochemistry for N-PCT, and NeuN or GAFP was performed on sections, including the ARC, as described previously (54). Study 3: effect of nutritional status on N-PCT expression in POMCexpressing neurons. For this study, rats were either fed ad libitum (n ⫽ 4) or fasted for 24 h (n ⫽ 6). Ninety minutes after the beginning of the dark phase, rats were anesthetized and fixed, and hypothalamic sections, including the ARC, were prepared for double-labeling immunohistochemistry for N-PCT and POMC, as described above. Study 4: effect of icv administration of anti-N-PCT antibody on food intake. Ad libitum-fed rats (n ⫽ 8/group) were injected icv with either control IgG (2 ␮g protein/rat) or anti-N-PCT antibody (0.5, 1, and 2 ␮g protein/rat) in the early light phase, when rats normally eat very little. Food intake was measured 60 min after injections. Study 5: effect of icv administration of anti-N-PCT antibody on N-PCT-induced suppression of food intake in ad libitum-fed and fasted rats. Two groups of rats, ad libitum fed and fasted for 24 h (n ⫽ 10 –12/group), were injected into the lateral ventricle with either 1) aCSF ⫹ aCSF, 2) anti-N-PCT antibody (2 ␮g protein/rat) ⫹ aCSF, 3) aCSF ⫹ N-PCT (1 nmol/rat, 5 ␮g/rat), or 4) anti-N-PCT antibody (2 ␮g protein/rat) ⫹ N-PCT (1 nmol/rat). This dose of N-PCT has been shown to suppress food intake in rats in preliminary doseresponse studies (55). The first injection occurred 30 min before the beginning of the dark phase and was followed 30 min later by the second injection. After the second injections, preweighed food was returned to all subjects. Food intake was measured 1, 2, and 4 h later. Each rat received only one combination of treatments. Study 6: effect of icv injection of N-PCT on c-Fos in ARC POMC neurons. Two weight-matched groups, ad libitum fed and fasted for 24 h (n ⫽ 10/group), were injected into the lateral ventricle with either control aCSF or 1 nmol N-PCT at the beginning of the dark phase. To prevent the induction of c-Fos expression due to feeding, food was immediately removed from the cages of injected rats. Ninety minutes later, rats were deeply anesthetized and transcardially perfused. Brains were removed and processed for double-labeling immunohistochemistry for c-Fos and POMC, as described above. Study 7: effect of icv N-PCT on the gene expression of hypothalamic neuropeptides known to affect appetite. Rats were separated into two weight-matched groups (n ⫽ 10/group); one was allowed ad libitum access to food and the other was fasted for 24 h before icv injections. This fasting duration is effective to demonstrate coexpression of orexigenic and anorexigenic neuropeptides in fasting-activated hypothalamic neurons (2, 15, 44). On the day of the experiment, each rat received an icv injection of either control aCSF or 1 nmol N-PCT immediately before the onset of the dark cycle. Rats were euthanized by decapitation 4 h after injections. The brain was immediately removed from the skull, and the ARC was dissected out from the whole hypothalamus, as described above. Hypothalamic blocks containing the ARC were preserved in an RNA stabilization solution (Qiagen) and stored at ⫺80°C until processing for RT-PCR analysis. Study 8: effect of melanocortin receptor antagonism on N-PCTelicited suppression of food intake in ad libitum-fed and fasted rats. Two weight-matched groups of rats, ad libitum fed and fasted for 24 h (n ⫽ 10 –12/group), were injected icv with either 1) aCSF ⫹ aCSF, 2) SHU 9119 (0.1 nmol/rat) ⫹ aCSF, 3) aCSF ⫹ N-PCT (1 nmol/rat),

or 4) SHU 9119 (0.1 nmol/rat) ⫹ N-PCT (1 nmol/rat). The dose of SHU 9119 has been reported to inhibit reductions in feeding induced by icv injection of the MC receptor agonist MTII in rats but does not affect food intake or body weight alone (13). The first injection occurred 30 min before dark phase and was followed 30 min later by the second injection. Immediately after the second injection, a preweighed amount of food was presented to the animals. Food intake was measured 1, 2, and 4 h later. Every rat was subjected to each treatment, with ⱖ5 days between tests. Statistical analysis. Data were analyzed using parametric statistics (Sigma Plot 11; Systat Software, San Jose, CA). Two-way repeatedmeasures ANOVA, followed by Student-Newman-Keuls test, was used for analysis of food intake. For other comparisons, i.e., mRNA levels, either a one-way ANOVA (for more than a 2-group comparison) followed by Student-Newman-Keuls test or an unpaired Student’s t-test (for 2-group comparison) was used. P values ⬍0.05 were considered statistically significant. RESULTS

Expression of CALC-I gene is reduced during fasting. The expression levels of hypothalamic neuropeptides that reduce appetite are often elevated in states of positive energy balance and reduced in states of negative energy balance (32, 33). Hypothalamic CALC-I gene expression, the gen precursor of N-PCT, was significantly reduced by 80% in rats fasted for 24 h (Fig. 2A). This is consistent with the hypothesis that N-PCT is an endogenous anorectic hypothalamic neuropeptide. Characterization of cell types expressing N-PCT in the ARC. To characterize cell types expressing N-PCT in the ARC, we used multiple immunofluorescence labeling. The immunohistochemical analysis of hypothalamic sections indicates that N-PCT-immunoreactive cells were astrocytes and neurons and localized the rostral ARC (Fig. 3, A–F). Colocalization of N-PCT and GFAP immunoreactivities confirmed N-PCT expression by astrocytes (Fig. 3C). N-PCT was also colocalized with NeuN, a marker of neuronal cell bodies and nuclei in the ARC, particularly in the rostral ARC, where the majority of POMC neurons reside (10, 46) (Fig. 3F). We also found N-PCT-immunoreactive cells in other hypothalamic regions surrounding the ARC and involved in energy homeostasis, including the median eminence, the supraoptic and dorsomedial nuclei, and the ventromedial and lateral hypothalamic areas (data not shown). Confocal immunofluorescence microscopy showed the expression of N-PCT on ARC cells in all examined samples. Quantitative colocalization analysis by Manders’ coefficient showed that 68% (overlap coefficient: 0.68 ⫾ 0.01) of N-PCT ligands (green) overlap with GAFP ligands (red), implying that N-PCT is expressed in astrocytes. The analysis also indicated that 55% (overlap coefficient: 0.55 ⫾ 0.01) of N-PCT ligands overlap with NeuN, suggesting that N-PCT is also expressed in ARC neurons. Fasting markedly reduced the degree of overlap between N-PCT and GAFP (28% decrease) or NeuN (16% decrease) (overlap coefficients: fed, 0.49 ⫾ 0.01; fasted, 0.46 ⫾ 0.01; P ⬍ 0.001 vs. ad libitum-fed rats, respectively). These findings suggest a potential role for N-PCT on ARC cells and open the possibility that POMC neurons may mediate, at least in part, the anorexigenic effects of N-PCT. N-PCT is colocalized with POMC in the ARC. To corroborate this hypothesis, we next performed colocalization studies of N-PCT and POMC, using double-immunofluorescent staining. We also determined the effect of nutritional status on

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Fig. 2. Fasting reduces hypothalamic calcitonin-I gene expression, and immunoblockade of endogenous aminoprocalcitonin (N-PCT) increases food intake in the light phase. A: hypothalamic expression of calcitonin-I mRNA in the ARC of ad libitum-fed and 24-h-fasted rats (n ⫽ 8/group) is shown. Results are expressed as means ⫾ SE. ***P ⬍ 0.001 vs. fed group. B: blocking the action of endogenous N-PCT in the brain with a specific monoclonal antibody (N-PCT Ab; icv; 0.5–2 ␮g protein) induces a dose-dependent increase in food intake even during the light phase. Data are means ⫾ SE; n ⫽ 8/group. *P ⬍ 0.05; **P ⬍ 0.01 vs. control (2 ␮g of protein isotype IgG2 mouse).

hypothalamic N-PCT expression in ARC POMC neurons. We observed a prominent presence of N-PCT protein in the ARC (Fig. 3G). Figure 3, G–L, depicts a representative section with colocalization of POMC and N-PCT immunoreactivity in the rostral ARC at bregma ⫺2.30 mm, where most of the population of POMC neurons resides (46). We found that in both ad libitum-fed (Fig. 3, G–I) and fasted conditions (Fig. 3, J–L), N-PCT was expressed by neurons and by cells that present glial morphology. We also found that the number of cells expressing N-PCT (Fig. 3, G and J) or POMC (Fig. 3, H and K) in the rostral ARC was markedly reduced in fasted rats (N-PCT: fed 71.8 ⫾ 9.1 vs. fasted 39.1 ⫾ 1.2; POMC: fed 57.1 ⫾ 4.8 vs. fasted 29.8 ⫾ 6.0; P ⬍ 0.01). Quantitative double labeling revealed that the percentage of ARC POMC neurons that express N-PCT was reduced significantly in rats fasted for 24 h (fed 41.8 ⫾ 2.4% vs. fasted 32.9 ⫾ 1.5%, P ⬍ 0.01). However, the percentage of N-PCT cells expressing POMC was increased slightly (fed 40.7 ⫾ 3.6% vs. fasted 45.7 ⫾ 4.1%; P ⬎ 0.05). These results indicate that N-PCT is produced locally within the ARC, including within POMC neu-

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rons, astrocytes, and possibly other neurons. These data also suggest that N-PCT, acting via a paracrine or neurocrine effect, plays an important role in feeding regulation. Intracerebroventricular administration of anti-N-PCT antibody stimulated food intake and attenuated the anorectic effect of N-PCT. To determine whether endogenous N-PCT signaling influences food intake, we administered anti-N-PCT antibody icv to ad libitum-fed rats in the early light phase. Rats injected with anti-N-PCT antibody ate significantly more than rats injected with control IgG antibody, suggesting that endogenous N-PCT may restrain appetite (Fig. 2B). No adverse reactions such as sedation or ataxia were observed. A subsequent experiment determined whether anti-N-PCT antibody could specifically block N-PCT anorectic action. As depicted in Fig. 4, icv administration of N-PCT alone (1 nmol) decreased food intake significantly in both ad libitum-fed (Fig. 4A) and 24 h-fasted (Fig. 4B) conditions, and this effect was attenuated by the antibody at 1, 2, and 4 h. Furthermore, icv administration of anti-N-PCT antibody to ad libitum-fed and fasted rats increased food intake in the early light phase. N-PCT activates POMC neurons in the ARC. To determine whether icv administration of N-PCT resulted in activation of arcuate POMC neurons, we performed colocalization studies of c-Fos and POMC in ad libitum-fed and 24-h-fasted rats after icv administration of N-PCT. Intracerebroventricular injection of N-PCT in fed animals decreased feeding significantly over the 90-min test period (vehicle: 3.8 ⫾ 0.2 g; N-PCT: 0.9 ⫾ 0.1 g; P ⬍ 0.001 vs. control). Figure 5 shows a quantitative analysis of c-Fos- and POMC-like immunoreactive cells in the rat ARC after icv administration of either vehicle (aCSF) or N-PCT in ad libitum-fed (Fig. 5A) and fasted rats (Fig. 5B). Both groups of N-PCT-treated rats showed a significant increase in the number of c-Fos, POMC, and POMC neurons expressing c-Fos in the ARC (P ⬍ 0.001, P ⬍ 0.05, and P ⬍ 0.05, respectively, vs. control aCSF). Representative images of the effects of N-PCT on the expression of c-Fos in POMCimmunoreactive neurons in the ARC are summarized in Fig. 6 (ad libitum-fed: Fig. 6, A and B; fasted: Fig. 6, D and E). The increased activation of arcuate POMC neurons induced by N-PCT was associated with a significant increase in the percentage of rostral POMC-expressing neurons exhibiting c-Fos immunoreactivity in both ad libitum-fed (Fig. 6C) and 24-hfasted rats (Fig. 6F). However, N-PCT did not cause significant changes in c-Fos expression in POMC neurons within the caudal ARC compared with their respective controls (Fig. 6, C and F). These results suggest that POMC neurons are a target site for the anorectic effect of N-PCT. Intracerebroventricular administration of N-PCT prevented fasting-induced changes in CALC-I, POMC, NPY, and AgRP mRNA levels in the ARC. To examine this possibility and to determine whether N-PCT alters the expression of other neuropeptides associated with energy homeostasis, hypothalamic CALC-I, POMC, AgRP, and NPY mRNA levels were measured in N-PCT-treated rats. Intracerebroventricular administration of N-PCT to rats fed ad libitum increased POMC and CALC-I mRNA levels significantly (P ⬍ 0.001; Fig. 7, A and B, respectively) but did not alter AgRP or NPY mRNA levels in the ARC compared with aCSF-treated rats (Fig. 7, C and D, respectively). To assess whether the changes in gene expression associated with food deprivation could be influenced by N-PCT, fasted rats were infused icv with either N-PCT or

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Fig. 3. Characterization of cell types expressing NPCT in the rat ARC. Representative examples of single- and double-immunofluorescent staining for N-PCT and glial fibrillary acidic protein (GFAP; an astrocyte marker) (A–C), neuronal nucleus (NeuN; a neuronal marker) (D–F), or proopiomelanocortin (POMC) neurons (G–I) in the rat ARC of ad libitumfed rats. J–L: effects of fasting on the coexpression of N-PCT in arcuate POMC neurons. Fasting for 24 h reduced the expression of N-PCT and POMC as well as the coexpression of N-PCT in neurons expressing POMC in the rostral ARC. Green fluorescence reveals N-PCT presence (A, D, G, and J); red fluorescence represents GFAP (B), NeuN (E), or POMC immunoreactivities (H and K). Colocalization appears as yellow (C, F, I, and L); n ⫽ 6 rats/group. Bregma, ⫺2.30. Scale bars, 50 ␮m.

aCSF. In normal male rats, a period of 24 h of fasting reduced POMC and CALC-I mRNA levels by 40 and 80% (Fig. 7, A and B, respectively) and increased NPY and AgRP mRNA levels by 60 and 150% in the ARC (Fig. 7, C and D, respectively) compared with ad libitum-fed controls. However, icv administration of N-PCT reversed the fasting-induced increase in NPY and AgRP mRNA levels and the decrease in POMC and CALC-I mRNA levels such that there was no significant difference between the levels in these fasted rats and those that were fed ad libitum (Fig. 7, A–D). These findings suggest a hypothalamic autocrine/paracrine role for N-PCT in energy homeostasis. N-PCT-induced reduction of food intake was attenuated by central blockade of MC3/4 receptors. N-PCT significantly stimulates arcuate POMC neurons, suggesting that icv N-PCT may mediate part of its anorectic effect via the MC system. If so, then blockade of downstream MC receptors should either block or attenuate the influence of N-PCT on food intake. To evaluate this possibility, a subthreshold dose (0.1 nmol) of SHU 9119, a MC3/4-R antagonist, was injected icv immediately before icv injection of N-PCT in both ad libitum-fed and 24-h-fasted rats. As depicted in Fig. 8, icv injection of SHU 9119 alone did not produce a reliable effect on food intake, whereas N-PCT (1 nmol) reduced 0- to 1-, 0- to 2-, and 0- to 4-h food intake significantly in ad libitum-fed and fasted rats. However, when the two injections were combined, SHU 9119 abolished the anorectic effect of N-PCT, implying that an interaction occurs between N-PCT and the MC system with regard to feeding regulation by the brain.

DISCUSSION

The neuroendocrine peptides N-PCT and CT-R have been localized in rat hypothalamic structures involved in the control of feeding behavior, including the PVN and ARC (34 –36). Indeed, N-PCT expression is downregulated in the hypothalamus of fasted animals (54, 55). Intracerebroventricular injection of N-PCT results in a significant decrease in food intake and body weight. In addition, N-PCT induces Fos expression in these hypothalamic nuclei (54, 55) and stimulates the hypothalamic-pituitary-adrenal (HPA) axis (22). Altogether, these observations strongly suggest that N-PCT plays a role in energy homeostasis by regulating appetite and energy expenditure. However, the mechanism by which N-PCT regulates food intake remains unknown. The presence of a high density of N-PCT in the ARC prompted us to examine whether N-PCT may interact with the NPY/POMC system to exert its anorexigenic activity. The present data show that fasting strongly reduces expression of the N-PCT precursor gene CALC-I in the ARC and that central immunoneutralization of N-PCT increases food intake, supporting the hypothesis that hypothalamic N-PCT may act as an endogenous anorectic signal. The observation that icv administration of N-PCT reduced food intake in fed and fasted rats and that its anorectic effect was attenuated by immunoneutralization with an anti-N-PCT antibody further supports that the effect is specific to N-PCT. To characterize cell types expressing N-PCT in the ARC, we examined the cellular localization of N-PCT in the rat ARC in

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terminals make synaptic contacts with POMC neurons in the ARC. These findings suggest that N-PCT may function in a paracrine manner to exert its anorectic effect in part by modulation of POMC neuron activity at sites outside of the ARC. However, the existence of morphofunctional interactions between N-PCT and POMC neurons depends on the possibility that ARC POMC neurons express CT-R. Which neurons/cells carry the N-PCT receptor that may mediate the interaction between N-PCT and the melanocortin system? Two major rat CT-R isoforms, CTa and CTb, have been described in rat ARC (23). The CTa isoform is expressed specifically in POMC neurons, whereas the CTb isoform is expressed in NPY neurons. Altogether, these observations suggest that the feeding response evoked by N-PCT may be directly or indirectly modulated by CT-R (23, 42, 55). However, there is no reliable means of pharmacologically distinguishing these receptors, and a selective antagonist for the CT-R is lacking. The present study shows that N-PCT is expressed in neurons and astrocytes surrounding POMC neurons, suggesting that N-PCT may act as a paracrine-signaling molecule in the hypothalamus. The relevance of these findings is unclear but

Fig. 4. Central administration of anti-N-PCT antibody increases feeding during the dark phase and selectively blocks N-PCT-induced reduction of food intake in ad libitum-fed and fasted rats. Intracerebroventricular (icv) administration of anti-N-PCT antibody (N-PCT Ab; 2 ␮g) increases dark phase food intake, whereas icv administration of N-PCT (1 nmol) potently decreases dark phase food intake in both freely fed (A) and 24-h-fasted rats (B). Anti-N-PCT was administered icv 30 min before dark in rats chronically implanted with cannula into the lateral brain ventricle. N-PCT or vehicle was injected 30 min later at the beginning of the dark phase. Data are means ⫾ SE for n ⫽ 10 –12/group. Statistical significance of between-group comparisons during each interval is indicated in the figure. Symbols immediately above bars indicate significance of comparison with artificial cerebrospinal fluid (aCSF) ⫹ aCSF control group, and a bracket spanning respective bars indicates significance of comparisons between other groups. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001; ⫹⫹P ⬍ 0.01; ⫹⫹⫹P ⬍ 0.001.

fed and fasted rats. N-PCT-immunoreactive fibers and cells have been observed in several areas of the adult rat brain but are found the in highest concentrations in appetite control hypothalamic nuclei expressing high density of CT-R, including the ARC and PVN (4, 34, 37, 54, 57). In the present study, we found that N-PCT is produced locally within the ARC, including within POMC neurons, astrocytes, and possibly other neurons. We also found that fasting reduces the coexpression of N-PCT and POMC, thus providing anatomic and functional evidence for a possible role of N-PCT in feeding. We also found N-PCT fibers and/or terminals in contact with or in close proximity to POMC neurons, indicating that N-PCT axon

Fig. 5. N-PCT increases the number of c-Fos- and POMC-immunoreactive neurons in the ARC of ad libitum-fed and fasted rats. Rats received a single icv injection of either control aCSF vehicle or N-PCT (1 nmol) at the beginning of the dark phase. Bar graph shows the number of c-Fos, POMC, and POMC-immunoreactive neurons expressing c-Fos in the ARC of rats fed ad libitum (A) or fasted for 24 h (B). Each bar represents the mean ⫾ SE of 4 – 6 rats/group. The rostral and caudal regions of the ARC correspond to the following atlas level designations described by Paxinos and Watson (39): rostral, 25–27; caudal, 30 –32. Sections were anatomically matched and counted blind to the treatment. For each region, 8 sections were counted bilaterally, and mean (⫾ SE) values were calculated. *P ⬍ 0.05 and ***P ⬍ 0.001 vs. respective control group treated with aCSF by 1-way ANOVA.

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Fig. 6. N-PCT activates rostral ARC POMC neurons. Photomicrographs of ARC coronal sections immunolabeled for c-Fos and POMC. Ad libitum-fed (A) and 24 h-fasted rats (D) were injected icv with control aCSF vehicle (A and D) or N-PCT (1 nmol/rat) (B and E) at the beginning of the dark phase and euthanized 90 min later. N-PCT increased the number of c-Fos-positive neurons (black) in the ARC of ad libitum-fed (B and C) and fasted rats (E and F). The higher magnification (boxed areas in B and E) showed that a proportion of these activated neurons colocalized with rostral POMC neurons (gray). Scale bars (A, B, D, and E), 100 ␮m; scale bars in B and E, insets, 50 ␮m. Coronal sections were cut at 30 ␮m. Bregma: ⫺2.30. C and F: percentage of rostral and caudal POMC neurons containing c-Fos immunoreactivity (ir) in ad libitum-fed (C) and fasted rats (F). Each bar represents the mean ⫾ SE of 4 – 6 rats/group. *P ⬍ 0.05 and **P ⬍ 0.05 vs. control group treated with aCSF.

emphasizes the potential role of N-PCT in energy homeostasis (34). In the hypothalamus, astrocytes regulate the secretory activity of neuroendocrine neurons via release of prostaglandins in response to cell-cell signaling initiated by neurons and glial cells (58). The activation of astrocytes leads to the release of gliotransmitters that trigger rapid responses in neighboring cells and thus contribute to the region-specific homeostatic regulation of neuronal function (18). Consistent with these

findings, we have shown previously that inhibition of prostaglandin synthesis attenuates the effects of N-PCT on food intake (57). Therefore, we speculate that astrocytes are involved, at least in part, in the anorexigenic effect of N-PCT. The ARC is believed to be an important center for integrating circulating energy signals (9). Neuroanatomic studies have shown that a population of caudal ARC neurons is able to synthesize both an agonist for an anabolic neuroendocrine

Fig. 7. N-PCT regulates POMC, calcitonin-I (CALC-I), neuropeptide Y (NPY), and agouti-related peptide (AgRP) mRNA expression in the ARC. Levels of POMC, CALC-I, AgRP, and NPY mRNA in the ARC were determined by RT-PCR in rats fed ad libitum (n ⫽ 10) or fasted for 24 h (n ⫽ 10). N-PCT (1 nmol) or an equivalent volume (2 ␮l) of aCSF was administered icv immediately before the dark phase (n ⫽ 5/group); icv administration of N-PCT prevented fastinginduced upregulation of hypothalamic AgRP and NPY mRNAs as well as downregulation of POMC and CALC-I mRNAs. ***P ⬍ 0.001 vs. aCSF-treated ad-libitum fed rats; ⫹⫹⫹P ⬍ 0.001 vs. aCSF-treated fasted rats.

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the organism (17, 25, 28). In particular, the expression of orexigenic peptides NPY and AgRP is potently upregulated by fasting (5- to 10-fold increase compared with basal) (5, 32). In opposition, the levels of anorexigenic POMC are only modestly decreased with fasting (20 –50% decrease compared with basal) (31, 44). N-PCT, injected icv, stimulates POMC mRNA in fed and fasted rats, whereas it reduces the expression of NPY and AgRP only in fasted rats, in which those mRNAs are normally elevated. Altogether, these data strongly suggest N-PCT exerts its anorectic action by enhancing activity of the MC system. The fact that POMC neurons express the CTa isoform, whereas NPY neurons express the CTb isoform (23), suggests that N-PCT may exert complex effects on POMC and NPY neuron activity, depending, for instance, on the relative affinity of each receptor subtype. It is thus tempting to speculate that N-PCT may modulate, via an autocrine and/or paracrine effect, the production of these neuropeptides in the ARC according to the variations of energetic status of the animal. It is also interesting to note that the effect of N-PCT on POMC, NPY, and AgRP mRNA levels is accompanied by a significant decrease in CALC-I gene expression. These findings suggest a model in which N-PCT upregulates its own expression via an endogenous/autoregulatory loop. However, the detailed molecular mechanism of how N-PCT increases CALC-I gene expression remains unknown. Whether these findings have an impact on the feeding response to N-PCT will require further investigation. Finally, using a MC3/4-R antagonist, we established a potential link between N-PCT and the MC system in modulating feeding behavior. We hypothesized that blockade of downstream MC receptors will block or attenuate the anorectic action of N-PCT. Intracerebroventricular pretreatment with the Fig. 8. Central melanocortin-3/4 receptor (MC3/4-R) signaling is required for N-PCT-induced feeding inhibition in fed and fasted rats. Intracerebroventricular administration of the MC3/4-R antagonist SHU 9119 (0.1 nmol) at the beginning of the dark period attenuated the anorectic effect of N-PCT (1 nmol) in both ad libitum-fed (A) and 24-h-fasted rats (B). N-PCT was injected 30 min after SHU 9119 at the beginning of the dark phase. Data are means ⫾ SE; n ⫽ 10 –12/group. Statistical significance of between-group comparisons computed and indicated as in Fig. 2. **P ⬍ 0.01; ***P ⬍ 0.001; ⫹P ⬍ 0.05; ⫹⫹⫹P ⬍ 0.001.

system (NPY) and an antagonist for a catabolic system (AgRP). In contrast, anorexigenic POMC-expressing neurons appeared to form a separate population in the rostral ARC (14, 46). To identify specific sites in the ARC that are activated by N-PCT, we used c-Fos as a marker of neuronal activation (20). Intracerebroventricular injection of N-PCT to ad libitum-fed and fasted rats causes neuronal activation of a subpopulation of rostral ARC POMC neurons. In contrast, our neuroanatomic studies revealed that N-PCT has no significant effect in neurons located in the caudal portion of the ARC, indicating that ARC neurons exhibit regional sensitivity to N-PCT. Therefore, these findings suggest that POMC neurons in the hypothalamus may mediate N-PCT’s signaling via POMC-derived peptides in the CNS. To further investigate cellular mechanisms involved in the anorectic effect of N-PCT, the expressions of several key neuropeptides implicated in regulating feeding were measured in the ARC of ad libitum-fed and 24-h-fasted rats. Compelling evidence indicates that expression of feeding-related neuropeptides in ARC neurons is regulated by the energetic condition of

Fig. 9. A simplified model of the feeding/regulatory signaling of N-PCT. N-PCT stimulates the POMC anorexigenic pathway and inhibits the NPY/ AgRP orexigenic pathway, resulting in reduced food intake. Fasting decreases N-PCT expression, leading to the activation of the orexigenic pathway. This response might be important for the adaptation to fasting. PVN, paraventricular nucleus; CART, cocaine- and amphetamine-regulated transcript; CRH, corticotropin-releasing hormone; CT-R, calcitonin receptor.

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MC3/4-R antagonist SHU 9119, at a dose that has no effect on its own, attenuated the intake-suppressive effect of N-PCT in both fed and fasted rats. Altogether, our results suggest that N-PCT-induced suppression of food intake is mediated through activation of the hypothalamic MC system. POMC neurons in the ARC are known to be primary recipients of circulating nutritional information, and their output to the PVN via the MC4-R can strongly inhibit food intake (53). These neurons project to feeding-related areas of the CNS, including the PVN (11). Although melanocortin receptors are expressed in a number of distinct regions in the CNS that could mediate the effects of ␣-MSH on energy balance, there is substantial evidence to implicate the hypothalamic PVN as a major center for the action of the MC signaling system on the regulation of appetite and satiety. Microinjections of ␣-MSH or ␣-MSH agonists directly into the PVN fully replicate reduced feeding responses observed after icv ␣-MSH administration (61). Conversely, injection of the ␣-MSH antagonist SHU 9119 into the PVN has a potent effect to increase feeding (26). Recently, a role of N-PCT as an activator of the HPA axis has been documented (54). Intracerebroventricular administration of N-PCT stimulates CRH mRNA expression in the PVN, releases CRH, and increases ACTH and glucocorticoid plasma levels in rats. However, it is not yet clear whether N-PCT’s effects on the HPA axis occur via direct regulation of CRH-producing neurons or via indirect pathways. Thus, it is also possible that N-PCT will mediate part of its anorectic effect via activation of ARC POMC neurons, which in turn project and activate MC receptors expressed in anorexigenic parvocellular PVN neurons (52). Altogether, our data support a mechanistic model in which N-PCT suppresses food intake via activation of CT-R in the hypothalamus. This model suggests that ARC POMC expression is regulated in an autocrine and/or paracrine manner via presynaptic CTa receptors present in POMC neurons and acting to increase POMC expression. During fasting, N-PCT inhibits the expression of the orexigenic neuropeptides NPY and AgRP via presynaptic CTb receptors present in NPY/AgRP neurons (see Fig. 9). Although further research is required to verify this hypothesis, our observations add neuroanatomic and functional support to the concept that N-PCT is involved in the hypothalamic control of energy homeostasis. The present data also suggest that N-PCT interacts with the hypothalamic melanocortin system in ways similar to leptin. Leptin is hypothesized to affect food intake not only by stimulating catabolic effector pathways such as the MC system but also by inhibiting anabolic signaling systems such as NPY/AgRP neurons in the ARC. Leptin receptors are expressed particularly in the ARC, particularly in neurons expressing POMC and CART, and in neurons expressing NPY and AgRP (2, 43, 59). Intracerebroventricular injection of leptin results in reduced expression of NPY and AgRP and increased expression of POMC (14). In addition, administration of a MC4-R-blocking agent prevents the ability of leptin to inhibit food intake acutely (46). The apparent redundancies in the effects of N-PCT and leptin on peptidergic neurons involved in the control of energy balance suggest that leptin and N-PCT may interact functionally to regulate food intake. However, in contrast to leptin, which is a circulating hormone produced outside the ARC, N-PCT is produced locally within the ARC, including within POMC neurons, astrocytes, and

possibly other neurons. Further studies to assess whether leptin and N-PCT would act synergistically or additively when coadministered into the ventricular system of the brain are necessary. In conclusion, the present report shows that hypothalamic N-PCT is involved in control of food intake and that its anorexigenic effects are mediated through the MC system. Our data also suggest that N-PCT-containing cells in the ARC may be part of the hypothalamic neuropeptide circuitry involved in control of energy balance. Characterization of the central effect of N-PCT on feeding should stimulate further research into the physiological functions of this peptide, and mechanistic understanding mediating the anorectic function of N-PCT may lead to the development of new treatments for metabolic disorders such as obesity. ACKNOWLEDGMENTS We are grateful to Ana Maria Garcia and Pablo Martinez-Aguado for technical support. Some experiments were performed in the Biology and Microscopy Central Services of the Universidad de Sevilla. We thank Dr. Juan Luis Ribas for help with the confocal microscope. GRANTS This study was supported in part by grants from the Andalusia Government (293/2010) and the Spanish Ministry of Health (FIS 09/1563 and 12/01074). DISCLOSURES The authors declare no conflicts of interest, financial or otherwise. AUTHOR CONTRIBUTIONS E.T. and F.J.M. contributed to the conception and design of the research; E.T. and R.M. performed the experiments; E.T., R.M., and F.J.M. analyzed the data; E.T. and F.J.M. interpreted the results of the experiments; E.T., R.M., and F.J.M. prepared the figures; E.T., R.M., and F.J.M. approved the final version of the manuscript; F.J.M. drafted the manuscript; E.T. and F.J.M. edited and revised the manuscript. REFERENCES 1. Ahima RS, Antwi DA. Brain regulation of appetite and satiety. Endocrinol Metab Clin North Am 37: 811–823, 2008. 2. Basking DG, Breininger JF, Schwartz MW. Leptin receptor mRNA identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat hypothalamus. Diabetes 48: 828 –833, 1999. 3. Becker KL, Muller B, Nylen ES, Cohen R, Silva OL, RH S. Calcitonin gene family of peptides. In: Principles and Practice of Endocrinology and Metabolism, edited by Becker KL. Philadelphia, PA: J. B. Lippincott, 2001, p. 520 –534. 4. Becskei C, Riediger T, Zünd D, Wooley P, Lutz TA. Immunohistochemical mapping of calcitonin receptors in the adult rat brain. Brain Res 1030: 221–233, 2004. 5. Brady LS, Smith MA, Gold PW, Herkenham M. Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinology 52: 441–447, 1990. 6. Burns DM, Birnbaum RS, Roos BA. A neuroendocrine peptide derived from the amino-terminal half of rat procalcitonin. Mol Endocrinol 3: 140 –147, 1989. 7. Burns DM, Forstrom JM, Friday KE, Howard GA, Roos BA. Procalcitonin’s amino-terminal cleavage peptide is a bone-cell mitogen. Proc Natl Acad Sci USA 86: 9519 –9523, 1989. 8. Comuzzie AG, Allison DB. The search for human obesity genes. Science 280: 1374 –1377, 1998. 9. Cone RD. Anatomy and regulation of the central melanocortin system. Nat Neurosci 8: 571–578, 2005.

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CENTRAL MELANOCORTIN SIGNALING IN N-PCT-INDUCED ANOREXIA 10. Cowley MA. Hypothalamic melanocortin neurons integrate signals of energy state. Eur J Pharmacol 480: 3–11, 2003. 11. Dube D, Lissitzky JC, Leclerc R, Pelletier G. Localization of ␣-melanocyte-stimulating hormone in rat brain and pituitary. Endocrinology 102: 1283–1291, 1978. 12. Garfield AS, Lam DD, Marston OJ, Przydzial MJ, Heisler LK. Role of central melanocortin pathways in energy homeostasis. Trends Endocrinol Metab 20: 203–215, 2009. 13. Gotoh K, Liu M, Benoit SC, Clegg DJ, Davidson WS, D’Alessio D, Seeley RJ, Tso P, Woods SC. Apolipoprotein A-IV interacts synergistically with melanocortins to reduce food intake. Am J Physiol Regul Integr Comp Physiol 290: R202–R207, 2006. 14. Gropp E, Shanabrough M, Borok E, Xu AW, Janoschek R, Buch T, Plum L, Balthasar N, Hampel B, Waisman A, Barsh GS, Horvath TL, Brüning JC. Agouti-related peptide-expressing neurons are mandatory for feeding. Nat Neurosci 8: 1289 –1291, 2005. 15. Hahn TM, Breininger JF, Baskin DG, Schwartz MW. Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1: 271–272, 1998. 16. Hancock MB. Two-color immunoperoxidase staining: visualization of anatomic relationships between immunoreactive neural elements. Am J Anat 175: 343–352, 1986. 17. Harrold JA, Williams G, Widdowson PS. Changes in hypothalamic agouti-related protein (AGRP), but not MSH or pro-opiomelanocortin concentrations in dietary-obese and food-restricted rats. Biochem Biophys Res Commun 258: 574 –577, 1999. 18. Haydon PG, Carmignoto G. Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev 86: 1009 –1031, 2006. 19. Hirsch PF, Lester GE, Talmage RV. Calcitonin, an enigmatic hormone: does it have a function? J Musculoskelet Neuronal Interact 1: 299 –305, 2001. 20. Hoffman GE, Smith MS, Vervalis JG. c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems. Front Neuroendocrinol 14: 173–213, 1993. 21. Jacobs JW, Lund PK, Potts JT Jr, Bell NH, Habener JF. Procalcitonin is a glycoprotein. J Biol Chem 256: 2803–2807, 1981. 22. Jacobson L. Hypothalamic-pituitary-adrenocortical axis regulation. Endocrinol Metab Clin North Am 34: 271–292, 2005. 23. Ji A. Pathway-specific expression of calcitonin receptors in hypothalamic and brain stem nuclei regulating food intake and transcriptomic changes in hypothalamic orexin neurons after fasting. In: Medizin Anatomie und Zellbiologie. Märburg, Germany: Universität Märburg, 2005, p. Xx–XX. 24. Ji A, Li Y, Peter M, Weihe E, Schafer MK. Cell-specific expression of calcitonin receptor isoforms in hypothalamic neurons regulating feeding behaviour. In: Program No. 4917. Washington, DC: Society for Neuroscience, 2003, p. X-X. 25. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS. Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 20: 68 –100, 1999. 26. Kask A, Schioth HB. Tonic inhibition of food intake during inactive phase is reversed by the injection of the melanocortin receptor antagonist into the paraventricular nucleus of the hypothalamus and central amygdala of the rat. Brain Res 887: 460 –464, 2000. 27. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2[⫺Delta Delta C(T)] Method. Methods 25: 402–408, 2001. 28. Low MJ. Role of proopiomelanocortin neurons and peptides in the regulation of energy homeostasis. J Endocrinol Invest 27: 95–100, 2004. 29. Manders EM, Verbeek FJ, Aten JA. Measurement of co-localization of objects in dual-colour confocal images. J Microsc 169: 375–382, 1993. 30. Millington GW. The role of proopiomelanocortin (POMC) neurones in feeding behavior. Nutr Metab (Lond) 4: 18 –33, 2007. 31. Mizuno TM, Kleopolus SP, Bergen HT, Roberts JL, Priest CM, Mobbs CV. Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47: 294 –297, 1998. 32. Mizuno TM, Makimura H, Silverstein J, Roberts JL, Lopingco T, Mobbs CV. Fasting regulates hypothalamic neuropeptide Y, agoutirelated peptide, and proopiomelanocortin in diabetic mice independent of changes in leptin or insulin. Endocrinology 140: 4551–4557, 1999. 33. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight. Nature 443: 289 –295, 2006.

E1261

34. Ojeda ML, Ambrosiani J, Maldonado R, Tavares E, Miñano FJ. Identification and localization of procalcitonin-like immunoreactivity in the rat hypothalamus. Neurosci Lett 408: 40 –45, 2006. 35. Ojeda ML, Ambrosiani J, Maldonado R, Tavares E, Miñano FJ. N-Procalcitonin-like immunoreactivity in the rat brain after lipopolysaccharide challenge (Abstract). J Physiol Biochem 61: A136, 2005. 36. Ojeda ML, Maldonado R, Tavares E, Miñano FJ. Dynamics and immunolocalization of procalcitonin in the hypothalamus of the rat during fever induced by endotoxin (Abstract). Meth Find Exptl Clin Pharmacol 25: A181, 2003. 37. Olgiati VR, Guidobono F, Netti C, Pecile A. Localization of calcitonin binding sites in rat central nervous system: evidence of its neuroactivity. Brain Res 265: 209 –215, 1983. 38. Ollmann MM, Wilson BD, Yang YK, Kerns JA, Chen Y, Gantz I, Barsh GS. Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science 278: 135–138, 1997. 39. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. San Diego, CA: Academic, 1997. 40. Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W, Muff R, Fischer JA, Foord SM. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol Rev 54: 233–246, 2002. 41. Russwurm S, Wiederhold M, Oberhoffer M, Stonans I, Zipfel PF, Reinhart K. Molecular aspects and natural source of procalcitonin. Clin Chem Lab Med 37: 787–797, 1999. 42. Sawada H, Yamaguchi H, Shimbara T, Toshinai K, Mondal MS, Date Y, Murakami N, Katafuchi T, Minamino N, Nunoi H, Nakazato M. Central effects of calcitonin receptor-stimulating peptide-1 on energy homeostasis in rats. Endocrinology 147: 2043–2205, 2006. 43. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98: 1101–1106, 1996. 44. Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG. Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46: 2119 – 2123, 1997. 45. Schwartz MW, Woods SC, Porte DJ, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 404: 661–671, 2000. 46. Seeley RJ, Yagaloff KA, Fisher SL, Burn P, Thiele TE, van Dijk G, Baskin DG, Schwartz MW. Melanocortin receptors in leptin effects. Nature 390: 349, 1997. 47. Sexton PM, Christopoulos G, Christopoulos A, Nylen ES, Snider RH Jr, Becker KL. Procalcitonin has bioactivity at calcitonin receptor family complexes: potential mediator implications in sepsis. Crit Care Med 36: 1637–1640, 2008. 48. Sexton PM, Houssami S, Hilton JM, O’Keeffe LM, Center RJ, Gillespie MT, Darcy P, Findlay DM. Identification of brain isoforms of the rat calcitonin receptor. Mol Endocrinol 7: 815–821, 1993. 49. Smith DW, Day TA. Neurochemical identification of fos-positive neurons using two-colour immunoperoxidase staining. J Neurosci Methods 47: 73–83, 1993. 50. Steenbergh PH, Hoppener JW, Zandberg J, Visser A, Lips CJ, Jansz HS. Structure and expression of the human calcitonin/CGRP genes. FEBS Lett 209: 97–103, 1986. 51. Stütz AM, Staszkiewicz J, Ptitsyn A, Argyropoulos G. Circadian expression of genes regulating food intake. Obesity (Silver Spring) 15: 607–615, 2007. 52. Swanson LW, Sawchenko PE. Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31: 410 –417, 1980. 53. Tao YX. The melanocortin-4 receptor: physiology, pharmacology, and pathophysiology. Endocr Rev 31: 506 –543, 2010. 54. Tavares E, Maldonado R, Garcia-Martinez A, Miñano FJ. Central administration of aminoprocalcitonin inhibits food intake and stimulates the hypothalamic-pituitary-adrenal axis in rats via the corticotrophin-releasing factor system. J Neuroendocrinol 24: 1040 –1054, 2012. 55. Tavares E, Maldonado R, Miñano FJ. N-procalcitonin: central effects on feeding and energy homeostasis in rats. Endocrinology 148: 1891– 1901, 2007.

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00590.2012 • www.ajpendo.org

E1262

CENTRAL MELANOCORTIN SIGNALING IN N-PCT-INDUCED ANOREXIA

56. Tavares E, Miñano FJ. Immunoneutralization of the aminoprocalcitonin peptide of procalcitonin protects rats from lethal endotoxaemia: neuroendocrine and systemic studies. Clin Sci (Lond) 119: 519 –534, 2010. 57. Tavares E, Miñano FJ. Procalcitonin N-terminal peptide causes catabolic effects via the hypothalamus and prostaglandin-dependent pathways. Neuroendocrinology 88: 316 –326, 2008. 58. Theodosis DT, Poulain DA, Oliet SH. Activity-dependent structural and functional plasticity of astrocyte-neuron interactions. Physiol Rev 88: 983–1008, 2008.

59. van Houten M, Posner BI, Kopriwa BM, Brawer JR. Insulin-binding sites in the rat brain: in vivo localization to the circumventricular organs by quantitative radioautography. Endocrinology 105: 666 –673, 1979. 60. Wimalawansa SJ. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily. Crit Rev Neurobiol 11: 167– 239, 1997. 61. Wirth MM, Olszewski PK, Yu C, Levine AS, Giraudo SQ. Paraventricular hypothalamic alpha-melanocyte-stimulating hormone and MTII reduce feeding without causing aversive effects. Peptides 22: 129 –134, 2001.

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00590.2012 • www.ajpendo.org

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