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Endocrine Unit, Faculty of Medicine, Imperial College, Hammersmith Hospital, London W12 ONN, United Kingdom
Address all correspondence and requests for reprints to: Prof. S. R. Bloom, Endocrine Unit, Faculty of Medicine, Imperial College, 6th Floor Commonwealth Building, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: s.bloom{at}ic.ac.uk.
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Abstract |
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-melanocyte stimulating hormone, cocaine- and amphetamine-related transcript, corticotropin-releasing hormone, and arginine vasopressin from hypothalamic explants to 127 ± 19%, 148 ± 10%, 158 ± 17%, and 198 ± 21% of basal levels, respectively (P < 0.05 vs. basal), but did not alter the release of either neuropeptide Y or agouti-related protein. Hypothalamic CGRP mRNA levels in 24-h fasted rats were increased to 130 ± 8% of control levels [CGRP mRNA (arbitrary units), 4.75 ± 0.4; controls, 3.65 ± 0.34; P < 0.05]. Our data suggest that CGRP administered to the PVN inhibits food intake and stimulates the HPA axis. |
Introduction |
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To investigate the hypothalamic site and mechanism of action of CGRP on food intake and the HPA axis we investigated the effects of PVN administration of CGRP on food intake and the HPA axis in rats, the effect of CGRP on the release of hypothalamic neuropeptides known to influence food intake and the HPA axis from hypothalamic explants, and hypothalamic CGRP mRNA expression in 24-h fasted and ad libitum-fed rats.
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Materials and Methods |
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was used throughout these studies because previous studies have used this form of CGRP, therefore enabling us to compare our results to other published work) and CGRP8–37 were purchased from Bachem (St. Helens, UK). Cannulation materials were purchased from Plastic One, Inc. (Roanoke, VA). Reagents for basal hypothalamic explant experiments were purchased from BDH (Poole, UK).
Animals
Male Wistar rats (specific pathogen free; Imperial College School of Medicine, London, UK), weighing 250–300 g, were maintained in individual cages under controlled temperature (21–23 C) and light (12-h light, 12-h dark cycle; lights on at 0700 h) with ad libitum access to food (RM1 diet, SDS Ltd., Witham, UK) and water. Animal procedures were approved by the British Home Office Animals Scientific Procedures Act 1986 (Project License 90/1077).
Intraparaventricular (iPVN) cannulation and injection
Animals were anesthetized with a mixture of ketamine HCl (60 mg/kg; Ketalar, Parke-Davis, Pontypool, UK) and xylazine (12 mg/kg; Rompun, Bayer Corp., Bury St. Edmunds, UK). Prophylactic antibiotics, flucloxacillin (37.5 mg/kg), and amoxicillin (37.5 mg/kg) were administered before surgery. Animals were implanted with 26-gauge stainless steel guide cannula projecting immediately above the paraventricular nucleus (PVN; coordinates; 1.8 mm posterior to the bregma, 0.5 mm laterally, and implanted 7 mm below the outer surface of the skull) as previously described (5). Briefly, a Kopf stereotactic frame (David Kopf, Tujunga, CA) was used, and the coordinates were calculated using the rat brain atlas of Paxinos and Watson (6). Three stainless steel screws were inserted into the cranium, and the cannula was fixed to these with dental cement. After surgery, the animals were given 5 ml 0.9% saline for circulatory support and buprenorphine (45 µg/kg; Schering-Plough Corp., Welwyn Garden City, UK) for analgesia postoperatively. The animals were allowed 1 wk of recovery after surgery. They were then accustomed to handling on a daily basis.
After a 7-d recovery period, animals received one sham injection and two 0.9% saline injections to habituate them to the injection procedure. All compounds were dissolved in 0.9% saline and administered in a volume of 1 µl by a stainless steel injector projecting 0.5 mm into the PVN (iPVN) over 1 min. All feeding studies were performed after a 24-h fast with the animals allowed ad libitum access to drinking water. To investigate the effect of iPVN CGRP on the HPA axis (study 3), rats were killed by decapitation 10 and 30 min post injection. Trunk blood was collected in plastic lithium heparin tubes containing 0.6 mg aprotinin (Bayer Corp., Haywards Heath, UK) for corticosterone analysis and in plastic EDTA bottles for ACTH analysis. Plasma was separated by centrifugation, frozen on dry ice, and stored at -20 C until assayed.
Cannula placement was verified by histological examination of the animal brains at the end of study 3 as previously described (5). Briefly, after decapitation, animals were injected with India ink, and the brains were rapidly removed and snap-frozen in liquid nitrogen using isopentane as a cryopreservative. A freezing cryostat (Bright, Huntington, UK) was used to take 15-µm sections, and every fourth section was counterstained with Cressyl Violet to allow anatomical localization. Cannula placement was assessed by an observer blinded to the intended cannula placement and was considered acceptable if the hypothalamic nucleus was identifiable and the cannula tract was seen in the nucleus. Animals with cannula placement more than 0.5 mm away from the coordinates used to identify the PVN were excluded from all data analysis after identification of cannula placement. Using these criteria, it is likely that peptides injected through the cannula were administered directly into the PVN. However, it is possible that the peptides could be acting elsewhere.
Study 1: effect of iPVN administration of CGRP on food intake
Groups of rats fasted for 24 h (n = 10–12) were injected with CGRP (0.03, 0.1, 0.3, or 1 nmol) or 0.9% saline in the early light phase (0800–1100 h). The animals were returned to their home cages with a preweighed amount of rat chow. The food remaining in the cage food dispenser was reweighed 1, 2, 4, 8, and 24 h later. In previous studies it has been shown that intranuclear injection increases the sensitivity, such that 1/10th of the effective icv dose results in significant effects when administered iPVN (7, 8, 9). The iPVN dose range of CGRP used in this study was 1/10th of the dose range known to inhibit food intake when administered icv (1); therefore, it is very unlikely that iPVN CGRP administered in our study reaches the third cerebral ventricle.
Study 2: effect of iPVN injection of CGRP and CGRP8–37 on food intake
Groups of rats fasted for 24 h (n = 10–12) were given iPVN injections 0 and 60 min later in the early light phase. The injection regimen in the four groups was as follows: saline/saline, saline/CGRP (0.3 nmol), CGRP8–37 (10 nmol)/saline, and CGRP8–37 (10 nmol)/CGRP (0.3 nmol). Food was presented after the second iPVN injection, and food intake was measured 1, 2, 4, 8, and 24 h later.
The 0.3 nmol dose of CGRP was chosen based on the results of the dose-response obtained in study 1. This was the minimally effective dose of iPVN CGRP that significantly reduced food intake compared with that in saline controls.
Study 3: effect of iPVN administration of CGRP on plasma ACTH and corticosterone
Groups of ad libitum-fed rats (n = 10–12) were injected with saline or CGRP (1 nmol) in the early light phase and decapitated 10 and 30 min after injection, and trunk blood was collected for plasma ACTH and corticosterone measurements. The study was carried out in the early light phase (0800–1100 h) in ad libitum-fed animals to minimize basal plasma ACTH and corticosterone levels. This dose of CGRP was chosen because it potently reduced food intake compared with saline controls in study 1. The time points were chosen from previous studies in which central nervous system administration of peptides increased plasma ACTH and corticosterone levels (10).
Study 4: effect of CGRP on the release of other hypothalamic neuropeptides known to influence food intake and the HPA axis from hypothalamic explants in vitro
The static incubation system used was described previously (11). Briefly, ad libitum-fed male Wistar rats were killed by decapitation, and the whole brain was immediately removed. The brain was mounted with the ventral surface uppermost and was placed in a vibrating microtome (Microfield Scientific Ltd., Dartmouth, UK). A 1.7-mm slice was taken from the basal hypothalamus and blocked lateral to the circle of Willis to include the PVN. The hypothalamic slice was incubated in individual chambers containing 1 ml artificial cerebrospinal fluid (aCSF; 20 mM NaHCO3, 126 mM NaCl, 0.09 mM Na2HPO4, 6 mM KCl, 1.4 mM CaCl2, 0.09 mM MgSO4, 5 mM glucose, 0.18 mg/ml ascorbic acid, and 100 µg/ml aprotinin) equilibrated with 95% O2 and 5% CO2.
The tubes were placed on a platform in a water bath maintained at 37 C. After an initial 2-h equilibration period, the hypothalami were incubated for 45 min in 600 µl aCSF (basal period), before being challenged with CGRP (at doses of 1, 10, and 100 nM) in 600 µl aCSF for 45 min. The viability of the tissue was verified by 45 min of exposure to aCSF containing 56 mM KCl. Hypothalamic explants that failed to show peptide release above the basal level in response to aCSF containing 56 mM KCl were excluded from the data analysis. Isotonicity was maintained by substituting K+ for Na+. Each experiment was repeated three times with 8–12 hypothalamic slices used for each dose of peptide administered. At the end of each period, aCSF was collected and stored at -20 C until measurement of MSH, cocaine- and amphetamine-related transcript (CART), neuropeptide Y (NPY), agouti-related protein (Agrp), CRH, and arginine vasopressin (AVP) by RIA. These neuropeptides are known to be important in the regulation of food intake and the HPA axis. MSH and CART have been shown to reduce food intake, and NPY and Agrp have been shown to increase food intake in rats. These neuropeptides are all synthesized in the arcuate nucleus of the hypothalamus (for a review, see Ref. 12). CRH and AVP are synthesized in the PVN and are major ACTH secretagogues (13, 14). CRH injection into the PVN has also been shown to reduce food intake in rats (15).
Study 5: measurement of CGRP mRNA in 24-h fasted and ad libitum-fed rats
One group of rats was fasted for 24 h, and the other group was fed ad libitum (n = 10/group). The rats were then killed by decapitation, and hypothalami were removed. RNA was extracted from individual frozen hypothalami using Tri-Reagent (Helena Biosciences, Sunderland, UK) according to the manufacturer’s protocol. Quantification of CGRP mRNA was performed as previously described (16) using a ribonuclease protection assay kit (Ambion, Inc., Austin, TX) with conditions optimized within our laboratory. The CGRP riboprobe corresponded to nucleotides 131–397 of the rat CGRP cDNA sequence containing the coding region for CGRP (accession no. M11597). Rat ß-actin (Ambion, Inc.) was used as an internal control to correct for RNA loading. Briefly, 5 µg RNA were hybridized overnight at 42 C with 1.3 x 103 Bq [32P]CTP-labeled riboprobe. Reaction mixtures were digested with ribonuclease A/T1, and the protected fragments were precipitated and separated on a 4% polyacrylamide gel. The dried gel was exposed to a PhosphorImager screen overnight, and bands were quantified by image densitometry using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
RIA
RIAs for MSH immunoreactivity (IR), CART-IR, NPY-IR, Agrp-IR, CRH-IR, and AVP-IR were measured using established methods (5, 17, 18, 19, 20, 21). CGRP did not cross-react with any of the antibodies used. The intra- and interassay coefficients of variation were, respectively, 7% and 8% for the MSH RIA, 6% and 9% for the CART55–102 RIA, 12% and 15% for the NPY RIA, 9% and 8% for the Agrp83–132 RIA, less than 10% for the CRH RIA, and 11% and 20% for the AVP RIA.
Plasma corticosterone was measured using an RIA kit from ICN Biomedicals, Inc. (Costa Mesa, CA), for which the intra- and interassay coefficients of variation were less than 10% and 7%, respectively. Plasma ACTH was measured by immunoradiometric assay purchased from Euro-Diagnostica B.V. (Arnhem, The Netherlands). The intra- and interassay coefficients of variation were both less than 4%.
Statistics
Data are presented as the mean ± SEM. For the PVN studies, groups were compared by one-way ANOVA, followed by post hoc Fisher’s least significant difference test (Systat, Evanston, IL). Data from hypothalamic explant release experiments were compared by paired t test between the basal period and the test period. Data from CGRP mRNA experiments were compared by unpaired t test between 24-h fasted and ad libitum-fed rats. In all cases P < 0.05 was considered statistically significant.
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Results |
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]. Only animals receiving the 1-nmol dose of CGRP still had reduced food intake compared with saline-injected controls 2 h post injection (1–2 h food intake: saline, 1.4 ± 0.2 g; 1 nmol CGRP, 0.5 ± 0.1 g; P < 0.01). Administration of 1 nmol CGRP significantly increased food intake compared with saline-injected animals 2–4 h post injection. Thus, animals receiving the 1-nmol dose of CGRP ate less than animals receiving lower doses of CGRP or saline in the first 2 h post injection but then ate more in the 2- to 4-h period (2–4 h food intake: saline, 1.5 ± 0.3 g; 1 nmol CGRP, 3.1 ± 0.3 g; P < 0.05). There was no significant difference in food intake between rats injected with CGRP and saline controls at any other time point. The 0.3-nmol dose of CGRP was used in study 2 because this was the lowest dose of CGRP that significantly reduced food intake.
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].
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]. CGRP administration into the PVN resulted in a significant increase in plasma corticosterone 30 min post injection [corticosterone 10 min post injection, 262.8 ± 16.9 ng/ml; saline, 222.0 ± 18.5 ng/ml (P = 0.15); corticosterone 30 min post injection: CGRP, 311.7 ± 26.5 ng/ml; saline, 155.8 ± 30.7 ng/ml (P < 0.001); Fig. 4B].
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MSH, CART, CRH, and AVP from hypothalamic explants, but did not affect the release of NPY or Agrp (Table 1).
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mRNA in 24-h fasted and ad libitum-fed rats mRNA levels in 24-h fasted rats were increased to 130 ± 8% of levels observed in ad libitum-fed rats (CGRP mRNA: 24-h fasted rats, 4.75 ± 0.4 arbitrary units; controls, 3.65 ± 0.34 arbitrary units; P < 0.05; Fig. 5).
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Discussion |
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It has been demonstrated that the hypophagic effects of icv CGRP can be completely blocked by prior administration of icv CGRP8–37 (23). In contrast, in our studies CGRP8–37 did not completely reverse the effects of iPVN CGRP, despite being given at 30 times the dose of CGRP. In rat L6 myocytes that express high affinity receptors for CGRP, the 50% effective concentration of rat CGRP is similar to the Kd of CGRP8–37 (24). In our studies because CGRP8–37 was given at a much higher dose than CGRP, one would have expected to completely block the effects of iPVN administered CGRP being mediated by the CGRP1 receptor. The anorectic effect of iPVN CGRP was significantly attenuated, but not reversed, by CGRP8–37. It is possible that the anorectic effect of iPVN CGRP is partly mediated directly via CGRP1 receptors, but also partly through another mechanism. Thus, iPVN CGRP may be inhibiting food intake partly through the CGRP1 receptor (an effect attenuated by CGRP8–37), but part of the anorectic effect of iPVN CGRP may be mediated through the release of other anorectic neuropeptides (an effect that may not be attenuated by CGRP8–37). CGRP8–37, as well as acting as a CGRP1 receptor antagonist, has also been reported to act as a partial agonist on the calcitonin receptor (25). There are calcitonin receptors in the PVN (26), and calcitonin causes a potent reduction in food intake when administered iPVN (27). Thus, it is possible that CGRP8–37 did not completely reverse the anorectic effect of iPVN CGRP because it was acting as a partial agonist at the calcitonin receptor to reduce food intake. However, this is unlikely because iPVN injection of CGRP8–37 alone did not have any effect on food intake compared with that in saline-injected animals.
Our in vitro studies indicate that CGRP stimulates the release of CRH, MSH, and CART from hypothalamic explants. CRH is synthesized in the PVN, and studies in the rat have suggested that the PVN is the likely site of the anorectic effect of CRH (15). Thus, it is possible that iPVN CGRP inhibits food intake through the release of CRH from the PVN. MSH and CART are synthesized in the arcuate nucleus, and the majority of CART-containing neurons in the arcuate nucleus also contain proopiomelanocortin mRNA (28). Both MSH and CART have been reported to inhibit food intake when administered into the central nervous system (29, 30). Thus, part of the anorectic effect of iPVN CGRP may be mediated through the stimulation of MSH and CART release. The PVN is an area rich in MSH- and CART-immunoreactive neurons (31, 32), and CGRP may be acting within this nucleus to effect changes in neuropeptide release. However, the hypothalamic explant method does not localize peptide release, and CGRP may be acting in other hypothalamic nuclei; for instance, the arcuate nucleus, where the cell bodies are located. The ability of iPVN injection of neuropeptides to affect the release of arcuate neuropeptides has previously been demonstrated by Kim et al. (33). They showed that iPVN injection of MSH decreased proopiomelanocortin gene expression in the arcuate nucleus.
We have shown that iPVN administration of CGRP causes a significant increase in plasma ACTH and corticosterone. Our in vitro studies demonstrate that CGRP stimulates the release of CRH and AVP from hypothalamic explants. Our data suggest that central nervous system administration of CGRP stimulates the HPA axis by acting through the PVN of the hypothalamus, and this may involve the release of CRH and AVP that result in the release of ACTH.
Fasting in rats is known to decrease the hypothalamic expression of anorectic peptides. For example, fasting reduces hypothalamic expression of the MSH precursor, proopiomelanocortin, and CART (34, 35). Therefore, one might expect hypothalamic CGRP expression to be reduced during fasting. However, fasting also causes a stimulation of the HPA axis with raised plasma ACTH and corticosterone levels (36). Although CRH and AVP mRNA expression in the PVN is reduced during fasting (37, 38), CRH and AVP content in the neurointermediate lobe of the pituitary is increased (39, 40). We found that hypothalamic CGRP mRNA is increased in rats fasted for 24 h compared with ad libitum-fed controls. Thus, during fasting, elevated hypothalamic CGRP may stimulate CRH and AVP release from the PVN that activates the HPA axis. Therefore, the role of hypothalamic CGRP during fasting could be stimulation of the HPA axis rather than control of food intake.
Our data suggest that CGRP administered into the PVN inhibits food intake and stimulates the HPA axis.
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Footnotes |
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Abbreviations: aCSF, Artificial cerebrospinal fluid; Agrp, agouti-related protein; AP/NTS, area postrema/nucleus of the tract solitarus; AVP, arginine vasopressin; CART, cocaine- and amphetamine-related transcript; CGRP, calcitonin gene-related protein; HPA, hypothalamo-pituitary-adrenal; icv, intracerebroventricular; iPVN, intraparaventricular nucleus; IR, immunoreactivity; NPY, neuropeptide Y; PVN, paraventricular nucleus.
Received August 28, 2002.
Accepted for publication December 31, 2002.
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-MSH antagonize feeding behavior stimulated by kappa opiate agonists. Peptides 7:843–848[CrossRef][Medline]
-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression. J Neurosci 20:1550–1558-MSH, but not ß-endorphin, into the PVN decreases POMC gene expression in the ARC. NeuroReport 13:497–500[CrossRef][Medline]
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, Saline-injected animals; 
, CGRP-injected animals. Significance values are indicated: ***, P < 0.001 (vs. saline; n = 10–12/group).
, saline/CGRP (0.3 nmol); 
, CGRP8–37 (10 nmol)/CGRP (0.3 nmol); 
, CGRP8–37 (10 nmol)/saline. Significance values for each group are indicated: *, P < 0.05; ***, P < 0.001 (between groups indicated; n = 10–12/group).
