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Endocrinology Vol. 148, No. 2 638-646
Copyright © 2007 by The Endocrine Society

Importance of Melanocortin Signaling in Refeeding-Induced Neuronal Activation and Satiety

Praful S. Singru, Edith Sánchez, Csaba Fekete and Ronald M. Lechan

Tupper Research Institute and Department of Medicine (P.S.S., E.S., C.F., R.M.L.), Division of Endocrinology, Diabetes, and Metabolism, Tufts-New England Medical Center, Boston, Massachusetts 02111; Department of Endocrine Neurobiology (C.F.), Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest H-1083 Hungary; and Department of Neuroscience (R.M.L.), Tufts University School of Medicine, Boston, Massachusetts 02111

Address all correspondence and requests for reprints to: Ronald M. Lechan, M.D., Ph.D., Professor of Medicine, Division of Endocrinology, Box No. 268, New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111. E-mail: rlechan{at}tufts-nemc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To identify regions in the hypothalamus involved in refeeding and their regulation by {alpha}-MSH, adult rats were subjected to a 3-d fast, and 2 h after refeeding, the distribution of c-Fos-immunoreactive neurons was elucidated. Compared with fed and fasted animals, a significant increase (P < 0.001) in the number of c-Fos-immunoreactive cells was identified in refed animals in the supraoptic nucleus, magnocellular and ventral parvocellular subdivisions of the hypothalamic paraventricular nucleus (PVNv), and the dorsal and ventral subdivisions of the dorsomedial nucleus (DMNd and DMNv, respectively). Refeeding shifted the location of c-Fos-labeled neurons from the medial to lateral arcuate where c-Fos was induced in 88.7 ± 2.2% of {alpha}-MSH-containing neurons. {alpha}-MSH-containing axons densely innervated the PVNv, DMNd, and DMNv and organized in close apposition to the majority of refeeding-activated c-Fos-positive neurons. To test whether the melanocortin system is involved in induction of c-Fos in these regions, the melanocortin 3/4 receptor antagonist, agouti-related protein (AGRP 83–132), was administered to fasting animals just before refeeding. Compared with artificial cerebrospinal fluid, a single intracerebroventricular bolus of agouti-related protein (5 µg/5 µl) not only significantly increased the total amount of food consumed within 2 h but also nearly abolished refeeding-induced c-Fos expression in the PVNv and DMNd and partially reduced c-Fos immunoreactivity in the DMNv. We conclude that refeeding activates a subset of neurons in the PVN and DMN as a result of increased melanocortin signaling and propose that one or more of these neuronal populations mediate the potent anorexic actions of {alpha}-MSH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROOPIOMELANOCORTIN (POMC)-DERIVED anorexigenic peptide, {alpha}-MSH, plays a crucial role in central regulation of energy homeostasis (1, 2). Targeted deletion of POMC or the type 4 melanocortin receptor (MC4-R), the primary {alpha}-MSH receptor in the central nervous system (CNS), produces an obesity syndrome characterized by hyperphagia, hyperinsulinemia, and reduced energy expenditure (3, 4). Similarly, in humans, mutations that interfere with the functions of the MC4-R, POMC gene, or processing enzymes necessary to generate mature {alpha}-MSH result in severe obesity (5, 6, 7, 8).

The production of {alpha}-MSH in neurons of the hypothalamic arcuate nucleus is stimulated by peripheral anorexigenic hormones such as leptin and insulin and inhibited by fasting (1, 9, 10). These neurons project to feeding-related areas of the CNS, including the hypothalamic paraventricular nucleus (PVN) (11). Although melanocortin receptors are expressed in a number of distinct regions in the CNS that could mediate the effects of {alpha}-MSH on energy balance (12, 13), there is substantial evidence to implicate the hypothalamic PVN as a major center for the action of the melanocortin signaling system on the regulation of appetite and satiety. Focal injections of {alpha}-MSH or {alpha}-MSH agonists directly into the PVN fully replicate reduced feeding responses observed after intracerebroventricular (icv) {alpha}-MSH administration (14). Conversely, injection of the {alpha}-MSH antagonist, SHU9119, into the PVN has a potent effect to increase feeding (15, 16). The most compelling evidence for the importance of the PVN in the control of food intake by melanocortin signaling, however, was recently reported by Balthasar et al. (17), showing that in MC4-R null mice, reactivation of the MC4-R in the PVN prevented hyperphagia that is characteristic of these mice.

Recently, Timofeeva et al. (18, 19) demonstrated that refeeding induces neuronal activation in the ventral parvocellular subdivision of the PVN (PVNv). Because central administration of {alpha}-MSH increases cAMP response element-binding protein (CREB) phosphorylation in the majority of neurons in the PVNv (20), {alpha}-MSH-containing fibers (11) and MC-4R (12, 21) are highly enriched in this region of the PVN, and the PVNv shows expression of MC4-R mRNA (21), we hypothesized that activation of the PVNv contributes to the refeeding-induced satiety. To test this hypothesis, we examined whether refeeding activates the {alpha}-MSH-synthesizing neurons in the hypothalamic arcuate nucleus, which of the refeeding-activated neuronal populations in the hypothalamus are innervated by {alpha}-MSH-containing fibers, and if central administration of the endogenous melanocortin receptor antagonist (22), agouti-related protein (AGRP), alters meal size during refeeding and prevents refeeding-induced neuronal activation of {alpha}-MSH-innervated neuronal groups.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The experiments were performed on adult, male Sprague Dawley rats (Taconic Farms, Germantown, NY). After being acclimatized to standard environmental conditions (lights on between 0600 and 1800 h; temperature, 22 ± 1 C; rat chow and water ad libitum) for 1 wk before the beginning of the experiment, the animals were housed individually in cages during the course of the experiment. Fasted animals received water ad libitum, but food was removed from the cages. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Tufts-New England Medical Center and Tufts University School of Medicine.

Tissue preparation for immunocytochemistry
Animals weighing 210–230 g were divided into three groups and given free access to food (fed group, n = 5), fasted for 65 h (fasted group, n = 5), or fasted for 65 h and then given free access to food for 2 h (n = 5) or 4 h (n = 5) (refed group) before perfusion. This fasting duration resulted in approximately 15% weight loss (222.4 ± 3.5 g vs. 191.2 ± 4.2 g). The animals were deeply anesthetized with an overdose of pentobarbital (50 mg/kg; Ovation Pharmaceuticals, Inc., Deerfield, IL) and perfused transcardially with 20 ml heparinized PBS (pH 7.4) for 10–20 sec followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10–15 min. The brains were removed from the calvarium, postfixed by immersion in the same fixative, and immersed in 20% sucrose in 0.01 M PBS (pH 7.4) at 4 C for 1 d. Series of 25-µm-thick coronal sections were cut through the rostrocaudal extent of the hypothalamus on the cryostat (Leica Microsystems GmbH, Wetzlar, Germany; CM 3050S) and collected in PBS (pH 7.4).

Identification of hypothalamic neurons activated by refeeding
To study the effects of refeeding on hypothalamic neurons, sections of fed, fasted, and refed animals were processed for c-Fos immunohistochemistry. The sections were treated with 0.5% H2O2 in PBS for 15 min to remove endogenous peroxidase activity and then with 0.5% Triton X-100 in PBS for 20 min to improve the antibody penetration. After preincubation in 10% normal horse serum for 30 min, the sections were incubated in the rabbit primary antibody against c-Fos, diluted 1:50,000 (Ab5; Oncogene Science, Cambridge, MA) for 2 d at 4 C with continuous agitation on a rotary shaker. The primary antisera dilution was prepared in 1% normal horse serum in PBS containing 0.08% sodium azide and 0.2% PhotoFlo (Kodak, Rochester, NY). After rinsing in PBS, sections were incubated in biotinylated goat antirabbit IgG (1:400; Vector Laboratories, Burlingame, CA) for 2 h. The sections were washed in PBS and immersed in avidin-biotin-peroxidase complex (ABC, Vector Elite Kit, 1:100) for 2 h at room temperature, rinsed in PBS, and then developed in 0.025% diaminobenzidine/0.15% nickel ammonium sulfate/0.0036% H2O2 in 0.05 M Tris buffer (pH 7.6) for 5 min. The reaction was stopped by immersion of tissue in 0.05 M Tris buffer. The sections were mounted on Superfrost slides (Fisher Scientific, Pittsburgh, PA), air dried, dehydrated in ascending series of alcohol, cleared in Histosol, and then coverslipped with DPX histology mounting media (Fluka, Buchs, Switzerland).

Double-labeling immunofluorescence for c-Fos-immunoreactive nuclei and {alpha}-MSH-immunoreactive cells in the arcuate nucleus
To determine whether refeeding induces c-Fos in {alpha}-MSH-containing neurons of the arcuate nucleus, sections of fasted and refed animals through the arcuate nucleus were incubated in a mixture of rabbit antiserum against c-Fos at 1:10,000 dilution and sheep antiserum against {alpha}-MSH (gift from Dr. Jefrey B. Tatro, Tufts-New England Medical Center, Boston, MA) at 1: 25,000 for 2 d at 4 C. The sections were rinsed in PBS, incubated in biotinylated goat antirabbit IgG (1:400; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 2 h, followed by ABC at 1:100 dilution for 1 h. The immunoreaction product was amplified using the TSA kit according to the manufacturer’s instructions (NEN Life Science Products, Boston, MA) for 10 min. Finally, the sections were incubated in a mixture of Texas Red-Avidin D (1:250; Vector) and fluorescein isothiocyanate (FITC)-conjugated donkey antisheep IgG (1:40; Jackson ImmunoResearch) overnight, mounted on Superfrost/Plus slides, and coverslipped with Vectashield mounting medium (Vector).

Double-labeling immunocytochemistry of {alpha}-MSH-containing fibers and c-Fos-expressing neurons in the PVN and DMN
Double-labeling immunofluorescence was performed using the same method as described above, except that Texas Red-Avidin D was replaced with 7-amino-4-methyl-coumarin-3-acetic acid-Avidin D (Vector) to yield blue immunofluorescence in c-Fos-activated nuclei. Because the boundary of the c-Fos-containing cells could not be visualized with the double-labeling immunofluorescence method, we employed double-labeling immunocytochemistry for c-Fos and {alpha}-MSH and counterstained the sections with cresyl violet to visualize the cytoplasm. The sections were incubated in rabbit antiserum against c-Fos at 1:50,000 dilution for 2 d at 4 C, washed in PBS, incubated in biotinylated goat antirabbit IgG (1:400; Vector) for 2 h, followed by ABC (Vector) at 1:100 dilution for 2 h. After three washes in PBS and a rinse in Tris buffer (pH 7.6), the color reaction was developed in 0.025% diaminobenzidine and 0.0027% H2O2 for 5 min to yield a dark brown labeling in the cell nucleus. The sections were washed several times in PBS and incubated in sheep antiserum against {alpha}-MSH at 1:100,000 for 2 d at 4 C. After rinses in PBS, sections were incubated in biotinylated rabbit antisheep IgG (1:400; Vector) for 2 h at room temperature. The sections were then washed in PBS and immersed in ABC (Vector) at 1:100 for 2 h at room temperature, rinsed in PBS, and developed in 0.025% diaminobenzidine/0.15% nickel ammonium sulfate/0.0036% H2O2 in 0.05 M Tris buffer (pH 7.6) for 5 min to yield a purple color in axons. The reaction was stopped by immersion of the tissue in 0.05 M Tris buffer. The sections were mounted on Superfrost/Plus slides, air dried, and stained in cresyl violet for 5 min. The sections were washed in distilled water and dehydrated in graded series of ethanol followed by two changes in Histosol and coverslipped in DPX.

Effect of icv AGRP administration on food intake and neuronal activation during refeeding
Eight days before experimentation, animals were anesthetized with ketamine (80 mg/kg body weight ip; Phoenix Pharmaceuticals Inc., St. Joseph, MO) and xylazine (9 mg/kg body weight ip; Phoenix Pharmaceuticals). A 22-gauge stainless steel guide cannula (Plastic One, Roanoke, VA) was placed into the lateral cerebral ventricle under stereotaxic control (coordinates from bregma, anteroposterior, –0.8 mm; lateral, –1.2 mm; and ventral, 3.5 mm) (23) through a burr hole in the skull. The cannula was secured to the skull with three stainless steel screws and dental cement and temporarily occluded with a dummy cannula. The rats were made accustomed to handling by daily mock injections consisting of removal of the dummy cannula and connecting to an empty cannula connector for at least 1 wk before experimentation to reduce stress by acclimatizing the animals to handling. Animals weighing 265–291 g were divided into four groups. After 65 h fasting, the first two groups (n = 10) were administered 5 µl artificial cerebrospinal fluid (aCSF) (140 mM NaCl; 3.35 mM KCl; 1.15 mM MgCl2; 1.26 mM CaCl2; 1.2 mM Na2HPO4; and 0.3 mM NaH2PO4, pH 7.4) containing 0.05% BSA (Sigma Chemical Co., St. Louis, MO) icv, the remaining two groups (n = 10) received 5 µg AGRP (Phoenix Pharmaceuticals, Belmont, CA) in 5 µl aCSF icv. The fasting duration resulted in approximately 13% weight loss (275.2 ± 3 vs. 240.6 ± 3.5 g). All the icv injections were made in freely moving animals through 28-gauge needle that extended 1 mm below the guide cannula. The needle was connected by polyethylene tubing to a 1-ml GlassPak syringe, and injections were made over 2 min by a microprocessor-controlled infusion pump (Bee Electronic Minipump; Bioanalytical Systems, West Lafayette, IN). In two groups (aCSF and AGRP), food was introduced into the cages 15 min after the injections, and the animals were allowed to eat ad libitum for 2 h. The remaining two groups continued to fast for an additional 2 h. At the completion of the experiment, all animals were overdosed with pentobarbital and perfused through the ascending aorta, postfixed in 4% paraformaldehyde, and cut on cryostat. The accuracy of the icv cannula placement was determined during sectioning, and only animals in which the cannula penetrated the lateral ventricle were studied. The brain sections were processed for c-Fos immunolabeling according to the procedures described above. The 2-h food intake of the refed animals was measured.

Images were captured using a Spot digital camera and adjusted for size, contrast, and brightness using Adobe Photoshop CS 8.0 and CorelDraw 12 software.

Image analyses
For semiquantitative analysis of c-Fos-labeled nuclei in the supraoptic nucleus (SON), PVN, and DMN, distinct subdivisions of the SON, PVN, and DMN were identified based on the rat brain atlas of Paxinos and Watson (23) projected from a Zeiss Axioplan microscope (Carl Zeiss Microimaging Inc., Thornwood, NY) equipped with a COHU video camera (San Diego, CA) onto the monitor of a Macintosh computer using IMAGE 1.54 software (NIH). All nuclei with intense or medium-intensity c-Fos labeling in nuclear regions of the hypothalamus showing the most prominent c-Fos activation were counted bilaterally in four rostrocaudal serial sections on each side for each animal. The data from all the animals in each group were pooled separately and the mean ± SEM calculated. The data were statistically analyzed using one-way ANOVA followed by the Newman-Keuls test using Prism 4 software (GraphPad Software, Inc., San Diego, CA). A probability of P < 0.05 was considered significant. Similarly, the percentage of c-Fos-labeled cells in the PVNv and dorsal and ventral subdivisions of the dorsomedial nucleus (DMNd, and DMNv, respectively) that were contacted by {alpha}-MSH-containing fibers was analyzed.

To determine the percentage of {alpha}-MSH-containing neurons of the arcuate nucleus that contain c-Fos, three fluorescent-labeled sections of the arcuate nucleus from each animal were analyzed under a Zeiss Axioplan 2 epifluorescence microscope using a dual filter set for Texas Red and FITC (Texas Red excitation 560–585 nm, bandpass 585 nm, emission 600–652 nm; FITC excitation 490–505 nm, bandpass 510 nm, emission 515–545 nm; Chroma Technology Corp., Brattleboro, VT) such that the immunofluorescence for both c-Fos and {alpha}-MSH could be visualized simultaneously. The percentage of {alpha}-MSH neurons containing c-Fos labeling in fasted and 2-h refed animals and c-Fos-labeled neurons that do not contain {alpha}-MSH in the arcuate nucleus of refed animals were determined and the mean ± SEM calculated. Data were statistically analyzed by t test using Prism 4 software (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of c-Fos-expressing cells in the hypothalamus after refeeding
In the fed state, only isolated c-Fos-containing cells were observed throughout the hypothalamus (Figs. 1Go, A–E, 2Go, and 3AGo), and little difference was observed in the fasting animals (Figs. 1Go, F–J, and 2Go) except in the arcuate nucleus where fasting induced c-Fos expression in cells of the medial part of the arcuate nucleus (Fig. 3BGo). Two hours after refeeding, a dramatic and statistically significant increase (P < 0.001) in the number of c-Fos-containing cells were observed in the SON (Figs. 1KGo and 2Go), posterior magnocellular subdivision of the PVN (PVNpm) (Figs. 1LGo and 2Go), PVNv (Figs. 1MGo and 2Go), DMNd (Figs. 1NGo and 2Go), and DMNv (Fig. 1OGo and 2Go). The increase in c-Fos-containing cells in the SON, PVNv, DMNd, and DMNv were particularly pronounced, increasing more than 10-fold in each subregion (Figs. 1Go, K–O, and 2Go). A less prominent increase in c-Fos immunoreactivity was observed in the medial parvocellular subdivision of the paraventricular nucleus (PVNmp) (Fig. 1MGo) and in the lateral hypothalamus. Although the c-Fos-containing cells were diffusely distributed in the DMNd in the refed animals (Fig. 1NGo), c-Fos-containing cells in the DMNv were organized into at least two different populations. These included an intensely immunostained group of neurons that were densely clustered in the medial part of the DMNv and a second population of more diffusely distributed cells in lateral portions (Fig. 1OGo). Refeeding altered the distribution of c-Fos-immunoreactive cells in the arcuate nucleus, which diminished in the medial portion of the nucleus and shifted into more laterally located cells (Fig. 3CGo). In both the PVN and DMN, c-Fos immunostaining was sustained even at 4 h after refeeding (Fig. 4Go, A–C).


Figure 1
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  		FIG. 1. Immunocytochemical demonstration of c-Fos immunoreactivity in the SON (A, F, and K), PVNpm (B, G, and L), PVNv (C, H, and M), DMNd (D, I, and N), and DMNv (E, J, and O) in fed (A–E) and fasted (F–J) animals and fasted animals 2 h after refeeding (K–O). Although few immunoreactive cells are seen in the fed and fasted states, a dramatic increase in c-Fos expression is apparent in the refed animals. OC, Optic chiasma; III, third ventricle. Scale bar, 200 µm.

 

Figure 2
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  		FIG. 2. Semiquantitative analysis of the c-Fos-immunoreactive cells in the SON, PVNpm, PVNv, DMNd, and DMNv of fed, fasted, and 2 h after refeeding fasting animals. *, Significantly different (P < 0.001) compared with fed and fasted.

 

Figure 3
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  		FIG. 3. Effect of fasting and refeeding on expression of c-Fos immunoreactivity in the arcuate nucleus of fed (A), fasted (B), and fasted animals refed for 2 h (C). Refeeding is associated with a shift in c-Fos-immunoreactive cells from medial to lateral portions of the nucleus. III, Third ventricle. Scale bar, 200 µm.

 

Figure 4
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  		FIG. 4. Immunolabeling of c-Fos in the PVNv (A), DMNd (B), and DMNv (C) 4 h after refeeding. Note the distribution and presence of intense c-Fos-immunolabeling in each subregion similar to that shown in Fig. 1Go. III, Third ventricle. Scale bar, 200 µm.

 
Effect of refeeding on c-Fos expression in {alpha}-MSH-containing neurons of the arcuate nucleus
By double-labeling immunocytochemistry, none of the {alpha}-MSH-containing neurons showed evidence of c-Fos expression when the animals were fasting (Fig. 5AGo). However, 2 h after refeeding, there was a dramatic increase in c-Fos expression in {alpha}-MSH-immunoreactive neurons such that 88.7 ± 2.2% of {alpha}-MSH-containing neurons were c-Fos positive (Fig. 5BGo). Only 27.7 ± 1.2% of c-Fos cells in the arcuate nucleus of the refed animals did not contain {alpha}-MSH immunoreactivity.


Figure 5
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  		FIG. 5. Effect of fasting and refeeding on c-Fos expression in {alpha}-MSH-containing neurons of the arcuate nucleus (A and B) and neuroanatomical associations between refed activated c-Fos-immunoreactive cells and {alpha}-MSH-containing axons in the PVNv, DMNd, and DMNv (C–H). No c-Fos immunofluorescence (red nuclei) is present in {alpha}-MSH-containing neurons (green cells) during fasting (A). However, with refeeding, the majority of {alpha}-MSH-containing neurons show c-Fos immunoreactivity (arrows) (B). In refed animals, intense {alpha}-MSH immunofluorescent axons (green) in the PVNv (C), DMNd (D), and DMNv (E) are closely associated with the c-Fos-containing cells (blue). In each of these subregions, {alpha}-MSH-containing axons are closely juxtaposed to the c-Fos-containing cells (arrows) (F–H). III, Third ventricle. Scale bars, 50 µm (A and B), 200 µm (C–E), and 25 µm (F–H).

 
Neuroanatomical associations between {alpha}-MSH-containing fibers and c-Fos-immunoreactive cells in refeeding animals
Refeeding-activated neurons contacted by {alpha}-MSH-containing fibers are shown in Fig. 5Go, C–E. In double-labeled preparations counterstained with cresyl violet, {alpha}-MSH-containing axons were observed in close proximity to the vast majority of c-Fos-containing cells in the PVNv and both subdivisions of the DMN (Fig. 5Go, F–H). Semiquantitative analysis revealed that 79.0 ± 1.0% of the c-Fos-containing neurons in the PVNv, 76.3 ± 0.8% in the DMNd, 62.1 ± 0.3% in the medial part of the DMNv, and 7.2 ± 1% in the lateral part of the DMNv were juxtaposed by {alpha}-MSH-containing axon terminals. No apparent associations of {alpha}-MSH-containing fibers were observed with magnocellular neurons in the PVN and SON where marked c-Fos activation was also observed after refeeding.

Effect of icv AGRP on cumulative food intake and refeeding-induced c-Fos expression in the PVNv, DMNd, and DMNv
In keeping with the previous observations, only few, isolated c-Fos-containing neurons were seen in the PVN or DMN after icv aCSF administration in fasting animals (Figs. 6Go, A–C, and 7Go), but c-Fos immunoreactivity dramatically increased 2 h after refeeding: PVNv, fast 29 ± 7 vs. refed 215 ± 15; DMNd, fast 59 ± 13 vs. refed 625 ± 31; and DMNv, fast 58 ± 15 vs. refed 537 ± 51 (P < 0.001) (Figs. 6Go, D–F, and 7Go). The administration of AGRP to fasting animals had no significant effects on c-Fos expression in the PVNv (Figs. 6GGo and 7Go), DMNd (Figs. 6HGo and 7Go), or DMNv (Figs. 6IGo and 7Go). However, compared with the aCSF-treated refed animals (Figs. 6Go, D–F, and 7Go), AGRP significantly reduced c-Fos expression after refeeding in the PVNv (aCSF refed 215 ± 15 vs. AGRP refed 38 ± 6; P < 0.001) (Figs. 6JGo and 7Go) and DMNd (aCSF refed 625 ± 31 vs. AGRP refed 114 ± 21; P < 0.001) (Figs. 6KGo and 7Go) and partially inhibited c-Fos expression in the medial and lateral parts of the DMNv (aCSF refed 537 ± 51 vs. AGRP refed 205 ± 26) (Figs. 6LGo and 7Go). In both the PVNv and DMNd, the number of c-Fos-containing cells in these nuclear subregions in refeeding animals treated with AGRP was not significantly different from the number of c-Fos-containing cells in fasting animals receiving either aCSF or AGRP (P > 0.05). No effect of AGRP on c-Fos immunoreactivity was observed in magnocellular neurons in the PVN (aCSF refed 89 ± 3 vs. AGRP refed 93 ± 4) and SON (aCSF refed 267 ± 13 vs. AGRP refed 281 ± 18).


Figure 6
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  		FIG. 6. Comparison of the effects of icv administration of aCSF or AGRP on c-Fos immunoreactivity in the PVN, DMNd, and DMNv of fasting animals or fasting animals that have been refed. Refeeding significantly increased c-Fos immunoreactivity in the PVNv, DMNd, and DMNv in aCSF-treated animals compared with fasting animals (compare A–C with D–F). AGRP had no effect in the fasting animals compared with aCSF (compare A–C with G–I) but inhibited the refeeding response in the PVNv (compare D and J) and DMNd (compare E and K) and partially inhibited c-Fos immunoreactivity in DMNv (compare F and L). Scale bar, 200 µm.

 

Figure 7
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  		FIG. 7. Semiquatitative analysis of number of c-Fos-containing cells in the PVNv, DMNd, and DMNv in fasting and refed animals pretreated with aCSF or AGRP. *, Significantly different compared with aCSF and AGRP fasted animals; +, significantly different (P < 0.05) from aCSF and AGRP fasted animals.

 
The effect of icv administration of AGRP on food intake is shown in Fig. 8Go. In refed animals, AGRP significantly increased food intake compared with aCSF-infused animals.


Figure 8
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  		FIG. 8. Effect of central AGRP administration on cumulative food intake in animals after a prolonged fast. AGRP administration significantly increased food intake during refeeding when compared with aCSF-treated animals. *, Significantly different (P < 0.05) compared with aCSF.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Refeeding is characterized by an early phase of hyperphagia followed by a period of satiety that may persist for up to 6 h before increased food intake resumes (24, 25, 26). In these studies, we provide evidence that {alpha}-MSH, a peptide known to exert important regulatory effects on energy homeostasis by inhibiting food intake and increasing energy expenditure (1, 2), may contribute to the development of satiety during the early phase of refeeding after a prolonged fast by direct effects on discrete populations of neurons in the PVN and DMN.

The role of melanocortins in the regulation of refeeding is suggested by the activation of {alpha}-MSH neurons in the arcuate nucleus within 2 h after reintroducing food to fasted animals. In contrast to the fasted animals in which c-Fos was confined to the medial arcuate nucleus where orexigenic neurons reside (9), refeeding shifted the location of c-Fos-immunoreactive cells to the lateral arcuate nucleus where anorexigenic neurons reside (1). Indeed, in refed animals, c-Fos immunoreactivity was present in approximately 90% of immunohistochemically identifiable {alpha}-MSH-producing neurons, indicating that these neurons respond to refeeding signals as a nearly homogeneous population. The importance of melanocortin signaling in the regulation of meal size after a prolonged fast is further supported by the effect of AGRP, a melanocortin receptor antagonist (1), to increase food intake during the first 2 h of refeeding.

The mechanism whereby melanocortin neurons induce satiety during refeeding may be through activation of neurons in the PVN and DMN. Refeeding resulted in a marked increase in c-Fos expression in the PVNv, DMNd, and DMNv. These regions are known to be heavily inundated with {alpha}-MSH-containing fibers (11, 27) and in double-labeling immunocytochemical studies, {alpha}-MSH-immunoreactive axons established close juxtapositions with the majority of c-Fos-expressing neurons in each of the three subregions. In addition, antagonizing melanocortin signaling by the administration of AGRP prevented refeeding-induced activation of neurons in the PVNv and DMNd and substantially reduced activation in the DMNv.

Despite the well known effects of melanocortins on energy expenditure and food intake, energy expenditure increases only 24 h after the start of refeeding (28, 29, 30, 31), whereas attenuation of food intake occurs within 2 h (24, 25, 26). Presumably, this is an important homeostatic mechanism to allow fasting animals to restore depleted energy stores while restraining powerful eating responses developed during fasting to prevent excessive feeding and overdistension of the gastrointestinal tract. Thus, refeeding may provide a unique model in which brain regions involved in mediating the effects of {alpha}-MSH on satiety can be differentiated from those mediating the effects of {alpha}-MSH on energy expenditure.

The PVN is organized into several subnuclei with distinct physiological functions that include a magnocellular division and a parvocellular division (32, 33). Whereas refeeding induced an increase in c-Fos in both the magnocellular and parvocellular subdivisions, the absence of an {alpha}-MSH innervation of magnocellular neurons and apparent ineffectiveness of AGRP to prevent activation of these neurons suggests that they do not have an important role in melanocortin signaling. Rather, activation of these neurons may be mediated through the organum vasculosum of the lamina terminalis (OVLT), as demonstrated by the substantial reduction in c-Fos expression in magnocellular neurons by transections that separate the OVLT from the PVN (19). Because the OVLT has an important role in osmoregulation (34), activation of magnocellular neurons may promote the increase in water consumption during refeeding (28, 35). In contrast, neurons in the PVNv would appear to be part of the melanocortin signaling pathway. These neurons also express melanocortin receptors (12) and show a marked increase in CREB phosphorylation after the central administration of {alpha}-MSH (20) consistent with the coupling of melanocortin receptors in a stimulatory fashion to cAMP generation (36). The large increase in c-Fos expression in the PVNv, therefore, may implicate this region as a satiety center within the PVN, mediating the anorexigenic actions of {alpha}-MSH.

Compared with the substantial increase in c-Fos expression in the PVNv, a relatively low number of c-Fos-containing neurons were observed in the PVNmp where hypophysiotropic TRH neurons reside (37). This observation is somewhat surprising given that icv administration of {alpha}-MSH markedly increases CREB phosphorylation in TRH neurons in the PVN (20) and prevents fasting-induced inhibition of TRH gene expression (38). The minimal increase in c-Fos immunoreactivity in the PVNmp and observations that less than 10% of TRH neurons in this region contain c-Fos 2 h after refeeding (our personal observations), however, are in keeping with the hypothesis proposed above that in the early phase of refeeding, the action of {alpha}-MSH is primarily on satiety and not energy expenditure. One mechanism to explain the differential effects of {alpha}-MSH in PVNv and PVNmp may be modulation of the action of {alpha}-MSH by endogenous AGRP. AGRP fibers innervate both the PVNv and PVNmp, but the number of axons containing {alpha}-MSH in the PVNv substantially exceeds that observed in the PVNmp (39, 40). Studies by Swart et al. (41) have shown that even 6 h of refeeding is not sufficient to decrease the fasting-induced increase in AGRP mRNA in the arcuate nucleus. Therefore, it is possible that increased AGRP tone may supercede any activating effects of {alpha}-MSH released in the PVNmp, preventing early activation of TRH neurons, but insufficient to prevent the satiety effects of {alpha}-MSH released from the substantially higher number of {alpha}-MSH terminals in the PVNv. In addition, studies demonstrating an acute increase in energy expenditure after the central administration of {alpha}-MSH or melanocortin receptor agonists (42) that could act diffusely on melanocortin receptors may not reflect what occurs physiologically during refeeding when only discrete populations of neurons expressing melanocortin receptors are activated as a result of their innervation by {alpha}-MSH-producing neurons.

The significance of refeeding-induced c-Fos expression in the DMNd and DMNv is uncertain. However, the DMN is well known to be involved in the regulation of food intake (43). After ablation of the DMN, animals become hypophagic but also show increased food intake compared with intact, control animals during the first hour of refeeding after a 24-h fast (44, 45). Similar observations have been made after disconnection of the arcuate nucleus from the DMN (46). These observations indicate that the DMN may contain both orexigenic and anorexigenic neuronal populations. Given the increase in c-Fos expression in the DMNd and DMNv with refeeding, we presume that DMN neurons are also involved in the mediation of satiety by melanocortin signaling. Alternatively, {alpha}-MSH regulation of DMNd and/or DMNv neurons may mediate the autonomic effects of melanocortins through descending projections of the DMN to the brain stem (47). However, evidence for delayed increase in energy expenditure with refeeding (28, 29, 30, 31) make this possibility less likely.

Although leptin is a well-known regulator of POMC neurons in the arcuate nucleus (1, 9), it is unlikely that leptin is responsible for mediating the early activating effects of refeeding on {alpha}-MSH neurons. This is based on the observation that circulating levels of leptin begin to rise only 4 h after the start of refeeding (48), whereas {alpha}-MSH neurons are already activated 2 h after the reintroduction of food to fasted animals. In addition, the systemic administration of leptin has no effect on c-Fos immunoreactivity in the arcuate nucleus of fasted animals or on food intake during the early phase of refeeding (26, 49, 50), suggesting the presence of leptin resistance. The mechanisms and importance of this brief leptin-resistant period in the early phase of refeeding is unclear but may contribute to the restoration of energy stores by preventing the potent stimulatory effect of leptin on energy expenditure.

In contrast to leptin, insulin increases rapidly after refeeding, rising 30 min after the start of food intake (48). The action of insulin on arcuate nucleus neurons in fasting animals shares many similarities to that of refeeding, because both insulin and refeeding activate POMC neurons (51, 52) and have a delayed inhibitory effect on neuropeptide Y mRNA (25), and neither insulin nor short-term refeeding affect AGRP gene expression (41, 51). In addition, the effects of insulin on energy expenditure in fasting animals is delayed for 24 h after administration (30), similar to the delay in energy expenditure observed after refeeding (28, 29, 30, 31). These data together with the known potent anorexigenic effect of insulin (53, 54) raises the possibility that insulin contributes to the termination of food intake during the early phase of refeeding by activating the arcuate nucleus melanocortin signaling system.

Other peptides that have been implicated in the generation of satiety including peptide YY and cocaine- and amphetamine-regulated transcript, may also contribute to the regulation of {alpha}-MSH in the arcuate nucleus during refeeding. Peptide YY has been shown to activate c-Fos in small number of POMC-containing neurons of the arcuate nucleus (1, 55, 56). Cocaine- and amphetamine-regulated transcript, which has emerged as potent inhibitor of food intake (57), is coexpressed in approximately 90% of POMC-expressing neurons (58), but evidence would suggest that its effects are mediated independent of melanocortin signaling (59).

In summary, we propose that {alpha}-MSH of arcuate nucleus origin participates in regulation of food intake during the early phase of refeeding. These effects may be mediated through the effects of {alpha}-MSH on the PVNv and/or DMNd and DMNv that may serve as satiety centers in the hypothalamus.


   Footnotes
 
This work was supported by grants from the National Institutes of Health DK-37021 and the Sixth EU Research Framework Program (contact LSHM-CT-2003-503041).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 26, 2006

Abbreviations: aCSF, Artificial cerebrospinal fluid; AGRP, agouti-related protein; CNS, central nervous system; CREB, cAMP response element-binding protein; DMN, dorsomedial nucleus; DMNd, dorsal subdivision of the DMN; DMNv, ventral subdivision of the DMN; FITC, fluorescein isothiocyanate; icv, intracerebroventricular(ly); MC4-R, melanocortin 4 receptor; OVLT, organum vasculosum of the lamina terminalis; POMC, proopiomelanocortin; PVN, paraventricular nucleus; PVNmp, medial parvocellular subdivision of the PVN; PVNpm, posterior magnocellular subdivision of the PVN; PUN, ventral parvocellular subdivision of the PVN; SON, supraoptic nucleus.

Received September 7, 2006.

Accepted for publication October 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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