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The Journal of Neuroscience, August 27, 2003, 23(21):7863-7872
Previous Article | Next Article
Interaction between
-Melanocyte-Stimulating Hormone and Corticotropin-Releasing Hormone in the Regulation of Feeding and Hypothalamo-Pituitary-Adrenal Responses
Xin-Yun Lu,1 Gregory S. Barsh,2 Huda Akil,1 andStanley J. Watson1 1University of Michigan School of Medicine, Mental Health Research Institute, Ann Arbor, Michigan 48109, and 2Departments of Pediatrics and Genetics, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305
Abstract
Top
Abstract
Introduction
Both central-melanocyte-stimulating hormone and corticotropin-releasing hormone (CRH) have been implicated in feeding and neuroendocrine mechanisms. The anatomical overlap and functional similarities between these two neurotransmitter systems led to the hypothesis that CRH might act as one of the mediators of the central actions of the melanocortin system. By double-labeling in situ hybridization, a subpopulation of CRH neurons in the paraventricular nucleus of the hypothalamus (PVN) were shown to contain the melanocortin-4 receptor (MC4R), concentrated in the ventromedial part of the parvicellular PVN (up to 33%). Intracerebroventricular injection of melanocortin agonist MTII to conscious and freely moving rats induced a rapid induction of CRH gene transcription in the PVN. This effect was accompanied by a rise in plasma corticosterone levels in a dose- and time-dependent manner, with the maximum response observed 30 min after MTII injection. MTII (0.5 nmol)-induced increase in plasma corticosterone was attenuated by the selective MC4R antagonist HS014 (0.25-1.0 nmol) and nonselective CRH receptor antagonist
Key words: melanocortin-4 receptors; MTII; corticotropin-releasing hormone;
Introduction
The central melanocortin system is defined by the agonist; Krieger, 1979
The central melanocortin system has become the focus of much attention in recent years because of its critical role in the control of eating and related behaviors. For example, intracerebroventricular infusions of the agonists
Although the neural mechanisms by which melanocortins control feeding and regulate the HPA axis remain mostly unknown, evidence suggests that CRH may be one of the candidates relaying melanocortin signaling. First, like
Materials and Methods
Animals
Adult male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 250-300 gm were housed in plastic cages. Animals had ad libitum access to water and food (Rodent Chow 5008; Ralston Purina, St. Louis, MO) and were maintained on a 12 hr light schedule (on 6:00 A.M. to 6:00 P.M.), with constant temperature and humidity. Animals were allowed to acclimate to these housing conditions for 1 week before experiments began. All procedures described were approved by the University of Michigan Committee on Use and Care of Animals.Surgery
After 1 week of habituation to the housing conditions, rats were anesthetized with sodium pentobarbital (50 mg/100 gm body weight, i.p.) for stereotaxic surgery. Animals were mounted on the stereotaxic instrument. To achieve the flat skull position, the incisor bar was adjusted until the heights of lambda and bregma were equal. Stainless-steel guide cannulas (26 gauge; Plastics One, Roanoke, VA) were implanted into the lateral ventricles, +1.5 mm lateral and -0.8 mm posterior to bregma, and 3.5 mm ventral to the surface of the skull. Guide cannulas were fixed to the skull with three screws and dental cement. A dummy cannula was placed into the guide cannula, protruding 1 mm below the tip of the guide cannula, to prevent blockage. After intracerebroventricular cannulation, rats were housed in individual cages. Animals were handled daily and habituated to the injection procedure for a minimum of 10 d to minimize stress. Placement of cannulas in the lateral ventricle was functionally confirmed by injection of 1 µl of angiotensin II (10 µM) on the fifth or sixth day after surgery. Only rats that drank >5 ml of tap water within 10 min after injection were used for the microinjection experiments.Microinjection
Melanocortin agonist MTII (Phoenix Pharmaceuticals, Belmont, CA), MC4 receptor selective antagonist HS 014 (Phoenix Pharmaceuticals), and CRH receptor antagonistExperimental protocol
Experiment 1: Expression of MC4R mRNA in CRH-containing neurons in the PVN. Three animals that were naive to any experimental procedure were used to determine the degree of colocalization of MC4R mRNA and CRH mRNA in the PVN. Animals were killed by decapitation in the early light cycle. Brains were rapidly removed and frozen in isopentane-dry ice bath at -40°C and stored at -80°C. Sections (10 µm) were cut on a cryostat through the hypothalamus. Tissue sections were stored at -80°C until processing for dual in situ hybridization.Experiment 2: Effects of melanocortin agonist MTII on CRH gene transcription. Four groups of rats (n = 5-6 per group) were used. Two groups received intracerebroventricular saline injection, and the other two groups were injected with 1 nmol of MTII. Intracerebroventricular injections were performed in the early light cycle (8:00-10:30 A.M.). Animals were killed by decapitation at 15 or 30 min after injection. Brains were removed, frozen, sectioned, and stored as described above until processing for intronic in situ hybridization.
Experiment 3: Effects of melanocortin agonist MTII on circulating corticosterone levels. For the time course of corticosterone in response to central administration of MTII, 44 rats were used. Forty animals received intracerebroventricular injection of saline (n = 20) or 1 nmol of MTII (n = 20). Injections were made in the early light cycle (8:30-11:00 A.M.). This time was chosen because in the morning, basal activity of the hypothalamo-pituitary-adrenal axis is low, and the HPA is also highly responsive to both activating and inhibitory stimuli (Dallman et al., 1994
The dose-effect on circulating corticosterone was determined by injecting 0, 0.1, 0.5, or 1.0 nmol doses of MTII between 8:30 and 11:00 A.M. On the basis of the time course data, a blood sample was taken by tail-nick 30 min after intracerebroventricular injection. Animals were mildly restrained while tail-nick was conducted. A tail vein was cut with a razor blade, and blood was collected in a heparinized capillary tube for the determination of plasma corticosterone levels.
Experiment 4: Effects of a CRH receptor antagonist and a selective melanocortin MC4R antagonist on MTII-induced plasma corticosterone response. In our initial experiments, we used 1 nmol of MTII to induce an increase in circulating corticosterone levels. This high-dose effect, however, could not be attenuated with several doses of the nonselective CRH antagonist
Experiment 5: Effects of a CRH receptor antagonist on MTII-induced suppression of food intake. For the feeding study, spontaneous food intake and body weight were measured for each rat 2 d before injection. These two parameters were counterbalanced across different treatment groups. Food was removed 1 hr before the dark cycle. Intracerebroventricular injections were performed at 15-20 min before the dark cycle (between 5:40 and 6:00 P.M.). To examine whether
Single-labeling in situ hybridization for CRH heteronuclear RNA
Antisense 35S-labeled cRNA probes for rat CRH heteronuclear RNA (hnRNA) (530 mer, complementary to the intron sequence) were generated with 35S-UTP and 35S-CTP using the standard transcription system. Brain sections were fixed in 4% paraformaldehyde for 1 hr and rinsed in 2x SSC (300 mM NaCl, 30 mM Na citrate, pH 7.2). Sections were acetylated in 0.1 M triethanolamine, pH 8.0, with 0.25% acetic anhydride (for 10 min) and dehydrated through a graded series of alcohol (50-100%). 35S-labeled cRNA probes were diluted to 3x 104 cpm/µl in 50% hybridization buffer (50% formamide, 10% dextran sulfate, 3x SSC, 50 mM sodium phosphate buffer, pH 7.4, 1x Denhardt's solution, 0.1 mg/ml yeast tRNA, and 10 mM DTT). Brain sections were hybridized with 70 µl of the diluted probes at 55°C overnight. Sections were rinsed in 2x SSC and incubated in RNase A buffer containing 200 µg/ml RNase A for 1 hr at 37°C followed by a series of washes of increasing stringency (2x, 1x, 0.5x, and 0.1x SSC, for 5 min each at room temperature). Finally, the sections were placed in 0.1x SSC at 65°C for 1 hr, rinsed in distilled water, and dehydrated in a graded series of alcohol. Brain sections were exposed to x-ray film for 14 d.Levels of CRH hnRNA were evaluated by analyzing film autoradiography. Films were visualized under a CCD camera (Model XC-77; Sony, Tokyo, Japan), and brain section images were captured and analyzed with the AIS image analysis system (Imaging Research, Ontario, Canada). Signals were expressed as optical density levels above threshold. The threshold level was defined as 3.5 SDs above the mean optical density of a fiber tract region. Results were expressed as integrated optical density, which is the product of the signal intensity and number of pixels above the threshold within the defined brain region. Equivalent planes of coronal brain sections through the PVN were ensured for analysis between animals.
Double-labeling in situ hybridization for colocalization of MC4R mRNA and CRH mRNA
cRNA probes complementary to either the rat MC4R mRNA (1040 mer; courtesy of Ira Gantz, University of Michigan, Ann Arbor, MI) or the rat CRH mRNA were labeled with 35S-UTP and 35S-CTP (for the MC4R probe) or digoxigenin (dig)-UTP (for the CRH probe) using standard transcription methods. Brain sections were hybridized with a mixture of 35S-MC4R and dig-CRH probes at 55°C overnight. Sections were rinsed in 2x SSC, treated with RNase A (200 µg/ml) for 1 hr at 37°C, and washed in 2x, 1x, 0.5x, and 0.1x SSC (for 5 min each). Sections were placed in 0.1x SSC at 65°C for 1 hr followed by immunohistochemical staining for visualization of digoxigenin-labeled CRH probe. Brain sections were treated with a blocking solution (0.1 M phosphate buffer containing 0.5% Triton X-100 and 0.25% carageenan, pH 7.5) for 4 hr, and then incubated overnight with an antibody against digoxigenin and conjugated to alkaline phosphatase (sheep anti-dig-AP, and Fab fragments, Boehringer Mannheim, Indianapolis, IN), diluted 1:15,000. After rinsing twice in both 0.1 M phosphate buffer and 0.1 M Tris buffer (30 min each), sections were incubated with color reaction buffer containing 0.45% nitroblue tetrazolium chloride (Boehringer Mannheim), 0.35% 5-bromo-4-chloro-3-indoylphosphate 4-toluidine salt (Boehringer Mannheim), 5% polyvinyl alcohol, and 0.24% levamizole. Color reaction was completed in 3 hr. Sections were rinsed in water and incubated with 0.1 M glycine buffer, pH 2.2, containing 0.5% Triton X-100 for 10 min. Finally, sections were fixed in 2.5% glutaraldehyde for 2 hr. After rinsing in water and dehydrating in a graded series of alcohol, sections were dipped in liquid emulsion (Ilford KD-5; Polysciences, Warrington, PA), air-dried, and stored in a dark box at 4°C. After 14 d of exposure to emulsion, sections were developed, fixed, dehydrated, and coverslipped in a xylene-based mounting medium (Permount; Fisher Scientific, Houston, TX). CRH mRNA labeled with nonradioactive dig-probe was visualized as a purple-blue precipitate, and MC4R mRNA labeled with radioactive probe was visualized as silver grains. For evaluation of the colocalization of MC4R mRNA and CRH mRNA, six consecutive sections through the PVN were analyzed. Signal specificity was ensured either by hybridization with sense-strand probes or pretreatment of brain sections with RNase A (200 µg/ml at 37°C for 60 min).Plasma corticosterone analysis
Plasma corticosterone was assayed using a highly specific corticosterone antibody developed in our laboratory. Briefly, 10 µl duplicate samples of plasma were heated at 70°C for 30 min to denature corticosterone-binding protein and incubated overnight with corticosterone antibody and [3H] corticosterone (Amersham, Arlington Heights, IL). Free and bound corticosterone were separated by incubation with charcoal for 15 min.Statistical analysis
All results were analyzed by a one-way or two-way ANOVA followed by Bonferroni-Dunn post hoc testing. Significance levels were taken as p < 0.05.
Results
Colocalization of MC4R and CRH in the PVN
The PVN consists of a number of distinct subdivisions, including five parvicellular and three magnocellular subdivisions (Swanson and Kuypers, 1980
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Figure 1. Dark-field emulsion autoradiograms showing the distribution of MC4R mRNA in the PVN of the rat hypothalamus. In situ hybridizations were performed using an MC4R mRNA probe on a series on rostral (A) to caudal (F) sections through the PVN. The sections are 100 µm apart. Locations of the PVN are indicated with dashed lines. MC4R mRNA-expressing cells are distributed in the anterior, dorsal, ventral, medial, and posterior parvicellular subdivisions of the PVN. Scattered MC4R-containing neurons were also noted in the magnocellular subdivision of the PVN.
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Figure 2. Colocalization of MC4R mRNA and CRH in the PVN. A, Microscopic images of dual in situ hybridization histochemistry of MC4R mRNA (35S-labeled riboprobe, clusters of green grains) and CRH mRNA (digoxigenin-labeled riboprobe, dark purple cells). Subdivisions of the PVN: dp, dorsal parvicellular; mpd, medial parvicellular, dorsal aspect; mpv, medial parvicellular, ventral aspect; pm, posterior magnocellular. B, High magnification of microscopic images showing double-labeled cells. Black arrows indicate cells double-labeled for MC4R mRNA and CRH mRNA. White arrows indicate cells labeled for MC4R mRNA only.
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Figure 3. Schematic drawing indicating the distribution of CRH neurons that express MC4R mRNA in the paraventricular nucleus of the hypothalamus (A-F in a rostrocaudal sequence). Open circles indicate cells single labeled for CRH mRNA, and closed circles represent cells double labeled for both MC4R and CRH mRNA. ap, Anterior parvicellular; lp, lateral parvicellular; mp, medial parvicellular; mpd, medial parvicellular, dorsal aspect; mpv, medial parvicellular, ventral aspect; pm, posterior magnocellular.
Melanocortin signaling and CRH gene expression
To determine whether activation of the MC4R stimulated CRH gene transcription, we used a CRH hnRNA probe to detect its intronic sequence. Introns are removed rapidly post-transcriptionally before translation, so they will report on levels of de novo gene transcription. Levels of CRH hnRNA in the PVN were determined after infusion of melanocortin agonist MTII or vehicle into the lateral ventricle of conscious rats. We found that CRH hnRNA levels increased rapidly and dramatically after intracerebroventricular infusion of 1 nmol MTII, whereas animals injected with vehicle exhibited low expression of CRH hnRNA (Fig. 4A,B). An eightfold induction in CRH hnRNA was observed 15 min after injection of MTII (58.5 ± 15.8 vs 460 ± 187.6; vehicle vs MTII; p < 0.01). This induction appeared to be short-lived lived because CRH hnRNA levels declined 30 min after injection (56 ± 26.9 vs 297 ± 139.0; vehicle vs MTII; p < 0.05).
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Figure 4. Changes in CRH heteronuclear RNA levels in the PVN after intracerebroventricular (i.c.v.) infusion of melanocortin agonist MTII (1 nmol) or vehicle into the lateral ventricle of conscious, freely moving rats. CRH hnRNA levels were detected using an intronic in situ hybridization. A, Dark-field film autoradiographs showing induction of CRH hnRNA by melanocortin agonist MTII (bottom) compared with the vehicle treatment (top). B, Induction of CRH hnRNA after intracerebroventricular injection of 1 nmol of MTII (n = 5-6 per group). *p < 0.05, **p < 0.01 versus vehicle-treated controls. IOD, Integrated optical density.
Functional interaction between melanocortin and CRH systems
Evidence from our colocalization and expression data described above (Results) suggests a functional link between CRH and melanocortin systems. We therefore decided to investigate consequences of melanocortin-induced activation of CRH expression with respect to two physiological functions, namely the hypothalamo-pituitary-adrenal axis activity and food intake.Melanocortin signaling and corticosterone secretion
It is well established that stimulation of CRH expression in the PVN and peptide release in the median eminence lead to HPA axis activation (Rivest et al., 1989
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Figure 5. A, Time course of plasma corticosterone levels in response to intracerebroventricular (i.c.v.) injection of MTII (1 nmol) or vehicle to conscious, freely moving rats. Corticosterone levels increased rapidly after infusion of MTII and reached the peak at 30 min after injection. A smaller, short-lived increase in corticosterone levels was also observed after vehicle injection. Data are expressed as the mean ± SEM (n = 5-6 per group). B, Dose-effect of MTII. MTII dose-dependently increased plasma corticosterone levels. Data are expressed as mean ± SEM (n = 6 for vehicle; n = 4 for 0.1 nmol of MTII; n = 5 for 0.5 nmol of MTII; n = 6 for 1.0 nmol of MTII). **p < 0.01, ***p < 0.001 versus vehicle-treated controls.
The dose-response for MTII is shown in Figure 5B. One-way ANOVA revealed that MTII dose-dependently increased circulating corticosterone concentrations at 30 min after injection compared with the vehicle condition (F(3,17) = 12.12; p < 0.0005). MTII at 0.5 and 1.0 nmol doses significantly increased plasma corticosterone levels. The magnitude of the increase induced by 1 nmol MTII was comparable with the time course study with the same dose at the 30 min time point. On the basis of our preliminary observations, we chose to use a 0.5 nmol dose of MTII for additional experiments. Figure 6A shows the effect of pretreatment with the selective MC4 receptor antagonist HS014 on the MTII-induced rise in circulating corticosterone levels. ANOVA revealed a significant effect of treatment (F(5,36) = 11.462; p < 0.0001). Post hoc analyses indicated that HS014 at 0.25, 0.5, and 1.0 nmol significantly antagonized the MTII (0.5 nmol)-induced increase in plasma corticosterone levels (0.25 nmol, p < 0.01; 0.5 nmol, p < 0.05; 1.0 nmol, p < 0.0001). HS014 alone had no effect compared with the vehicle-vehicle condition (p = 0.269). Figure 6B shows the effect of pretreatment with different doses of
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Figure 6. Effects of blockade of MC4Rs and CRH receptors on MTII-elicited effects on circulating corticosterone levels. A, Blockade of MC4Rs with the selective MC4R antagonist HS014. The treatment groups were as follows: vehicle (Veh) plus vehicle (n = 16), 0.5-1.0 nmol of HS014 plus vehicle (n = 6), vehicle plus 0.5 nmol of MTII (n = 8), 0. 25 nmol of HS014 plus 0.5 nmol of MTII (n = 4), 0.5 nmol of HS014 plus 0.5 nmol of MTII (n = 4), 1.0 nmol of HS014 plus 0.5 nmol of MTII (n = 6). B, Blockade of CRH receptors with the nonselective CRH receptor antagonist
Melanocortin signaling and feeding
Because both the CRH and melanocortin systems regulate feeding behavior, we examined whether the endogenous CRH system was involved in the feeding effects induced by melanocortin signaling. As shown in Figure 7, injections of MTII at 0.1 and 1.0 nmol doses resulted in significant decreases in food intake over 2, 4, and 24 hr periods, compared with the vehicle condition. A moderate dose of MTII (0.5 nmol) was used to induce anorexia, and the role of CRH in MTII-induced anorexia was further characterized (Fig. 8). We found that MTII (0.5 nmol) significantly decreased spontaneous food intake over 1, 2, and 24 hr periods. Although the CRH receptor antagonist
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Figure 7. Effect of MTII on spontaneous food intake. Central administration of MTII suppresses food intake (n = 5 per group). *p < 0.05; **p < 0.01 versus the vehicle-treated group.
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Figure 8. Effect of
Discussion
We have described a range of evidence for a functional link between the central melanocortin and CRH systems. First, we showed that a subpopulation of CRH neurons contained MC4R in the PVN. Second, central administration of the melanocortin agonist MTII was observed to stimulate CRH gene transcription. Third, we confirmed and extended previous reports of the anorectic and neuroendocrine effects elicited by melanocortin agonists. We demonstrated that MTII activated the HPA axis, which appeared to be mediated by the MC4R, and it inhibited food intake in a time-related and dose-dependent manner. These effects were significantly attenuated by pretreatment with CRH antagonistInteraction between melanocortin and CRH systems
Using double-labeling in situ hybridization, we have demonstrated for the first time that MC4R is expressed in a subset of CRH neurons in the PVN. These neurons are concentrated in the ventromedial part of the parvicellular PVN, in accordance with the prominent projections ofIn the present study, we used a heteronuclear RNA probe, which detected the intron sequence of CRH hnRNA, thus providing a reliable indicator of changes in de novo CRH gene transcription. CRH hnRNA levels increased dramatically 15 min after central administration of MTII and declined with increasing time. Considering that a subpopulation of CRH neurons contains MC4R, MTII may activate CRH gene transcription directly through the MC4R on these neurons. However, in contrast to the subdivision-specific colocalization pattern of MC4R and CRH, MTII-induced activation of CRH gene transcription in the PVN displayed a generalized effect without notable subdivisional preference. Consistent with our findings, Sarkar and colleagues (2002
Physiological correlates of melanocortin-CRH interaction
Central injections of the melanocortin agonist MTII to conscious, freely moving rats induced anorexia and increased the plasma concentrations of corticosterone in a time-related and dose-dependent manner. Although the MTII-induced anorexia has been thought to be mediated by the MC4R because of the inability of MTII to suppress food intake in MC4R-/- mice (Marsh et al., 1999CRH has been well characterized as a primary factor regulating basal and stress-induced activity of the HPA axis (Rivest et al., 1989
CRH is not only a major regulator of neuroendocrine responses, but it produces anorectic effects when administered centrally (Richard et al., 2002
The biological actions of CRH are mediated via two receptor subtypes, CRH1 and CRH2. These two receptor subtypes exhibit distinct anatomical distribution and pharmacological specificity (Richard et al., 2002
Alteration of the CRH system and correlated feeding and neuroendocrine responses elicited by melanocortin agonist MTII, as indicated in the present study, raise a question. Is the feeding effect of MTII dependent on its effect on the HPA axis, or vice versa, or are they independent? The experiments here were not designed to address this question, because we chose different optimal times to investigate the responses of the HPA axis and food intake. We examined plasma corticosterone levels in response to central administration of MTII in the early light cycle, when basal activity of the HPA axis is low and the responsiveness of the HPA axis is maximal to a given stimulus, whereas the experiments for feeding were performed after the onset of the dark cycle, when animals normally eat. In view of the ability of
Implications for eating and affective disorders
Hyperactivity of the HPA axis and overproduction of CRH have been implicated in anorexia nervosa and affective disorders such as stress, depression, and anxiety (Licinio et al., 1996In conclusion, the present study delineates an anatomical and functional relationship between the central melanocortin and CRH systems. Our observations provide direct evidence that melanocortin agonism functions as a rapid enhancer of CRH synthesis and that melanocortin agonists may mediate their effects on both feeding and HPA functions via activation of the CRH system.
Footnotes
Received Feb 20, 2003; revised June 25, 2003; accepted June 30, 2003.This study was supported by a pilot feasibility grant from the University of Michigan Gastrointestinal Peptide Research Center, National Institutes of Health Grant P30-DK-34933 (X.Y.L.), and National Institute of Mental Health Grant MH-42251 (S.J.W.). We are grateful to Audrey Seasholtz and John Stead for helpful comments, and to Jim Stewart for his assistance in collecting blood samples.
Correspondence should be addressed to Xin-Yun Lu, Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229. E-mail: xylu{at}umich.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/237863-10$15.00/0
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