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Endocrinology Vol. 140, No. 5 2387-2397
Copyright © 1999 by The Endocrine Society

ARTICLES

Physiological and Anatomical Circuitry between Agouti-Related Protein and Leptin Signaling1

Brent D. Wilson2, Didier Bagnol2, Christopher B. Kaelin, Michael M. Ollmann3, Ira Gantz, Stanley J. Watson and Gregory S. Barsh4

Departments of Pediatrics and Genetics and the Howard Hughes Medical Institute, Stanford University School of Medicine (B.D.W., C.B.K., M.M.O., G.S.B.), Stanford, California 94305-5428; the Mental Health Research Institute (D.B., S.J.W.) and the Department of Surgery (I.G.), University of Michigan School of Medicine, Ann Arbor, Michigan 48109-0682

Address all correspondence and requests for reprints to: Dr. Greg Barsh, Beckman Center B271A, Stanford University School of Medicine, Stanford, California 94305-5323. E-mail: gbarsh{at}cmgm.stanford.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Agouti-related protein (AGRP) is an orexigenic neuropeptide that acts via central melanocortin receptors, and whose messenger RNA (mRNA) levels are elevated in leptin-deficient mice. Fasting associated with a decline in circulating leptin normally causes a 15-fold elevation of hypothalamic Agrp mRNA levels but has no effect in leptin-deficient mice. Chronic hyperleptinemia associated with the tubby and Cpefat mutations has no effect on Agrp mRNA levels, but short term leptin administration causes a 17% reduction of Agrp mRNA levels in nonmutant mice and a 700% reduction in leptin-deficient mice. In young nonobese animals, melanocortin receptor blockade associated with the Ay mutation causes complete resistance to leptin-induced weight loss. Dual in situ hybridization reveals that Agrp-expressing neurons in the medial portion of the arcuate nucleus constitute a subpopulation different from Pomc-expressing neurons, and that a significant proportion of Agrp-expressing neurons (10–25%) coexpresses the leptin receptor, Lepr-b. Immunocytochemistry confirms distinct locations of AGRP- and POMC-expressing cell bodies, but reveals an overlapping distribution of their terminal fields in the arcuate nucleus, the paraventricular hypothalamus, and the dorsomedial hypothalamus. These results suggest that in the fed state, AGRP is normally suppressed by leptin, and that release of this suppression during fasting leads to increased ingestive behavior.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MOUSE OBESITY mutations have provided insight into pathways that normally regulate energy balance (reviewed in Refs. 1, 2). Certain mutations of the Agouti coat color gene, such as lethal yellow (Ay), cause obesity and increased linear growth due to ubiquitous expression of Agouti protein, a paracrine signaling molecule that is normally restricted to the skin (3, 4). The effects of Agouti protein on body weight probably represent its ability to mimic Agouti-related protein (AGRP), which is normally expressed in the hypothalamus and whose levels are elevated 8- to 10-fold in obesity caused by deficiency of leptin or its receptor (5, 6). Ubiquitous expression of AGRP and Agouti have identical effects on weight gain and growth (5, 7); intraventricular injection of an AGRP fragment increases feeding (8), but little is known about factors that normally regulate AGRP expression.

Agouti protein and AGRP act by binding to and inhibiting melanocortin receptors (Mcr) (9), a family of G protein-coupled receptors identified by their ability to activate adenylate cyclase in response to melanocortin derivatives of POMC such as {alpha}-melanocyte-stimulating hormone ({alpha}MSH) (reviewed in Ref. 10). Elevation of hypothalamic Agrp RNA levels in Lepob/Lepob or Leprdb/Leprdb mice suggests that AGRP may be negatively regulated by leptin, a circulating hormone produced by adipocytes that controls feeding, metabolism, and neuroendocrine function (reviewed in Refs. 2, 11, 12). Supraphysiological levels of leptin cause short term reduced feeding and weight loss, but the primary role for this hormone system may be during starvation, when a rapid decline in leptin levels triggers a series of behavioral and endocrine responses to conserve energy and stimulate feeding behavior (13, 14). Many, if not all, of the central effects of leptin are mediated by an isoform, Lepr-b, expressed prominently in the arcuate nucleus and ventromedial hypothalamus (15, 16, 17).

Several observations suggest a connection between leptin and melanocortinergic pathways. Lepr-b and Pomc messenger RNA (mRNA) are coexpressed in the arcuate nucleus (18), Pomc RNA levels are reduced in leptin-deficient animals, and leptin administration causes increased expression of Pomc mRNA (19, 20, 21). In addition, a synthetic melanocortin receptor antagonist blocks the effects of leptin on feeding (22). However, the view that melanocortinergic neurons mediate central anorexigenic effects of leptin has been challenged by studies of animals doubly mutant for Lepob and Ay, in which leptin deficiency and Mc4r blockade caused by ubiquitous expression of Agouti were found to have an additive effect on weight gain (23).

AGRP and POMC are just two of several neuropeptides implicated in the control of feeding behavior (reviewed in Ref. 24), and there may be considerable redundancy and/or cross-talk between hypothalamic circuits that respond to circulating hormones. Here we describe a series of gene expression, physiological, and anatomical studies that clarify the role of AGRP in central control of feeding. Our results point to a causal link between alterations in leptin signaling and AGRP action and suggest that increased AGRP expression plays a key role in the response of feeding behavior to nutrient deprivation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
C57BL/6J-a/a, C57BL/6J-Ay/a, C57BL/6J-Lepob/Lepob, C57BLKS/J, and C57BLKS/J-Cpefat/Cpefat mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Tissues from C57BL/6J-tub/tub mice were a generous gift from Drs. Patsy Nishina and Jurgen Naggert, The Jackson Laboratory. In situ hybridization and immunocytochemistry experiments were carried out on 300- to 350-g male Sprague-Dawley rats obtained from Charles River Laboratories, Inc. (Wilmington, MA). All animal experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with approval from local animal care review committees.

Determination of Agrp mRNA expression
After animals were euthanized with carbon dioxide, the hypothalamus was immediately dissected, and total RNA was prepared using guanidinium isothiocyanate after first extracting excess lipid with chloroform. For each Northern hybridization experiment, identical amounts of RNA, between 2.5–15 µg depending on the experiment, from three to six pairs of mutant and nonmutant animals were electrophoresed on the same agarose gel and transferred to a nylon filter. Hybridization with radiolabeled probe in the presence of 10% dextran sulfate was carried out according to standard procedures (25). A 228-bp probe from exon 4 of mouse Agrp was prepared by PCR using the oligonucleotide primers 5'-TGCTAGATCCACAGAACCGC-3' and 5'-CGATCCTTTATTCTCATCCC-3' and was subcloned into a plasmid vector. After hybridization and autoradiography, the filter was rehybridized to a control ß-actin probe, and the ratio of Agrp RNA to Actb RNA was determined in a 6- to 12-h exposure using a phosphorimaging device (Molecular Dynamics, Inc., Sunnyvale, CA). For individual experiments, the ratio of Agrp RNA to Actb RNA exhibited very little variation among replicate animals (see Tables 1–3GoGoGo). However, valid comparisons could not be made between different experiments, in part because animals of different ages were used, and also because the specific activity of the hybridization probes varied.


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  		Table 1. Hypothalamic Agrp mRNA levels in mouse obesity mutants

 

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  		Table 2. Effect of food deprivation on hypothalamic Agrp mRNA levels

 

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  		Table 3. Effect of leptin administration on hypothalamic Agrp mRNA levels

 
Food deprivation and leptin experiments
For fasting experiments, animals were deprived of food at the beginning of the daily 12-h dark cycle. During fasting, animals were allowed free access to water. For leptin injection experiments, mice were housed individually and allowed free access to water and chow for 7 days before the initiation of leptin injections. During this time food consumption and body weight were measured daily. Mice were given this acclimatization period to avoid experimental variation secondary to fluctuations in food consumption and body weight that sometimes occur immediately after singly housing mice. Purified recombinant mouse leptin (R&D Systems, Minneapolis, MN) at a dose of 12.5 µg/g body mass in PBS or PBS alone was administered by ip injection every 12 h. Circulating leptin levels were measured on serum obtained by cardiac puncture after avertin anesthesia using a commercially available kit (Linco Research, Inc., St. Charles, MO).

RNA in situ hybridization studies
Animals were killed by rapid decapitation 1–2 h after initiation of the light cycle. Brains were removed, frozen in isopentane (-40 C), and stored at -80 C. Serial 10-µm sections from the hypothalamus were prepared on a cryostat, thaw-mounted onto polylysine-subbed slides, and stored at -80 C until processing.

The rat Agrp probe is a 345-bp fragment corresponding to residues 50–395 and was isolated by RT-PCR. The rat Pomc probe is a 936-bp fragment corresponding to the entire open reading frame. The long isoform of the rat LepR is a 938-bp fragment corresponding to residues 2655–3593 that was provided by Dr. J. Krause. Sense and antisense 35S-labeled complementary RNA probes were generated from 1 µg linearized plasmid using T7 or T3 polymerase (Life Technologies, Gaithersburg, MD), 75 µCi [{alpha}-35S]UTP [>1000 Ci/mmol; 20 mCi/ml; Amersham (Arlington Heights, IL) and Pharmacia (Piscataway, NJ)], and 100 µCi [{alpha}-35S]CTP (800 Ci/mmol; 40 mCi/ml; Amersham). Digoxigenin-labeled probes were generated in a similar fashion, but with 140–320 µM digoxigenin-UTP (Boehringer Mannheim, Indianapolis, IN); the reaction was supplemented with unconjugated UTP to a final concentration of 400 µM. RNA probes were separated from free nucleotides on Sephadex G-50 columns. The specificity of hybridization was confirmed by control experiments using sense probes or tissue that had been pretreated with ribonuclease A (200 µg/ml) for 1 h at 37 C before hybridization with antisense probes. No specific hybridization was observed for any of the controls.

The method used for double in situ hybridization was adapted from that described by Curran and Watson (26). Before hybridization, sections were air dried for 15 min and fixed for 1 h at 22 C in 4% paraformaldehyde in PBS. The sections were rinsed three times in 2 x SSC (300 mM sodium chloride and 30 mM sodium citrate, pH 7.2) and once in distilled water (5 min/rinse), then treated with 0.1 M triethanolamine, pH 8.0, and acetic anhydride (0.25%) for 10 min at 22 C. Sections were rinsed in water, dehydrated through graded alcohols, and allowed to air-dry.

Radiolabeled and digoxigenin-labeled probes were diluted together in hybridization buffer (50% formamide, 10% dextran sulfate, 3 x SSC, 50 mM sodium phosphate buffer (pH 7.4), 1 x Denhardt’s solution, 0.1 mg/ml yeast transfer RNA, and 10 mM dithiothreitol) to yield an approximate concentration of 2–2.5 x 106 cpm/70 µl. Appropriate dilutions for nonradioactive probes were estimated from pilot experiments. Diluted probe (70 µl) was placed on each slide, and the sections were coverslipped. Slides were placed in plastic trays lined with filter paper dampened with 50% formamide-50% water. Trays were sealed and incubated at 55 C for 16 h. Coverslips were floated off in 2 x SSC, and sections were rinsed three times in 2 x SSC, incubated in ribonuclease A (200 µg/ml) for 60 min at 37 C, then rinsed in 2, 1, 0.5, and 0.1 x SSC. Sections were washed to a final stringency of 0.1 x SSC at 70 C for 60 min, then allowed to cool to room temperature.

After hybridization and washing, sections were processed for detection of the digoxigenin-labeled probe by rinsing in 0.1 M phosphate buffer, pH 7.4, and incubating in blocking solution (0.25% carageenan, 0.5% Triton X-100, and 0.1 M phosphate buffer, pH 7.4) for 2–4 h at room temperature. Sections were incubated overnight at room temperature with sheep antidigoxigenin antibody conjugated to alkaline phosphatase (Fab fragment, Boehringer Mannheim), diluted 1:20,000 in blocking solution. Sections were then washed three times in 0.1 M phosphate buffer, twice in Tris-buffered saline, and once in alkaline substrate buffer (100 mM Tris, 50 mM NaCl, and 50 mM MgCl2, pH 9.5) before carrying out the color reaction in alkaline substrate buffer containing 5% polyvinyl alcohol, 0.025% levamisole, 0.45% 4-nitro blue tetrazolium chloride, and 0.35% 5-bromo-4-chloro-3-indoyl-phosphate, 4-toluidine salt. Sections were incubated in the dark for 12–24 h at room temperature and examined under a microscope to determine reaction completion. Slides were then washed extensively in water, incubated in 0.1 M glycine and 0.5% Triton X-100, pH 2.2, for 10 min at room temperature to remove the antibody, and then washed in water. Finally, sections were fixed in 2.5% glutaraldehyde for 1 h, washed in water, and air-dried. (The glycine and glutaraldehyde steps helped prevent the increase in color background observed after development of emulsion-dipped slides.) Sections were initially exposed to x-ray film for 1–7 days, then dipped in emulsion (KD-5, Ilford, Paramus, NJ) and stored in light-tight boxes for 7–35 days at 4 C. After development (Kodak D-19, Eastman Kodak Co., Rochester, NY) of dipped slides, sections were generally dehydrated in alcohols and mounted in a xylene-based mounting medium (Permount, Fisher Scientific, Fairlawn, NJ) for photomicrography.

Antisera and immunocytochemistry
Polyclonal antibodies to recombinant full-length (Ala21-Thr132) human AGRP or to synthetic {gamma}MSH (Lys-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-Pro-Arg-Asn-Ser-Ser-Ser-Ala-Gly-Gly-Ser-Ala-Gln) coupled to thyroglobulin with glutaraldehyde were generated in rabbits by standard procedures (27) using complete Freund’s adjuvant.

For tissue preparation, animals were anesthetized, then perfused with 250 ml 0.9% NaCl and 2.2% sodium nitrite, followed by 500 ml Zamboni’s fixative. Brains were removed, incubated in the same fixative for 2 h at 4 C, then cryoprotected with a 20% phosphate-buffered saccharose solution overnight at 4 C. Sections prepared by cryostat (25 µm) were stored in a cryopreservative solution (30% sucrose and 30% ethylene glycol in 50 mM potassium PBS) at -20 C until use.

For immunocytochemistry, free-floating sections were washed in 50 mM potassium PBS (KPBS), incubated with 0.3% hydrogen peroxide (30 min), rinsed in 50 mM KPBS, and incubated with blocking solutions (Vector Laboratories, Inc., Burlingame, CA) for 15 min at a 1:5 dilution. Sections were incubated in antibody diluent composed of 50 mM KPBS, 0.4% Triton X-100, 1% BSA, and 1% normal goat serum for 30 min at 22 C, then transferred to AGRP antiserum diluted 1:30,000 or {gamma}MSH antiserum diluted 1:20,000. After 48 h at 4 C, tissues were washed in 50 mM KPBS with 0.02% Triton X-100, incubated with biotinylated goat antirabbit IgG (Vector Laboratories, Inc.) diluted 1:1,000 for 1 h at 22 C, then incubated with avidin-biotin complex coupled to horseradish peroxidase diluted 1:1000 for 1 h at 22 C. The horseradish peroxidase reaction product was visualized with 0.04% 3,3'-diaminobenzidine tetrahydrochloride, 2.5% nickel chloride, and 0.01% H2O2, dissolved in 0.1 M sodium acetate. The reaction was terminated by two consecutive 0.9% NaCl washes, after which free floating tissues were mounted on gelatin-coated slides. Finally, the sections were treated with graded alcohol and xylene, and then coverslipped with Permount. Immunocytochemical staining with antiserum against {gamma}MSH corresponds closely to what we have previously described. As controls for specificity of AGRP immunostaining, antiserum was preadsorbed or coincubated, respectively, with 4 or 1.2 µM recombinant AGRP.

Photomicrography and image analysis
Sections were viewed with a Zeiss Axiophot (Carl Zeiss, New York, NY) or a Leica Corp. (Leitz DMR, Rockleigh, NJ) microscope, and double exposure dual in situ hybridization photomicrographs were acquired with Kodak Tmax-100 or Kodak Ektachrome Tungsten film (Eastman Kodak). Images were converted to digital form using Photoshop 5.0 (Adobe Systems, San Jose, CA). The distribution of Pomc and Lepr type b mRNAs within Agrp mRNA-containing cells was determined using a Leica Corp. (Leitz DMR) microscope. Nonradioactive riboprobes were visualized under brightfield as a purple precipitate, and radioactive probes were visualized under darkfield by silver grain distribution. To avoid counting the same cell more than once in different sections, a series of 10-µm sections spaced 100 µm apart was analyzed for each set of probes. No attempt was made to determine the total number of cells in the arcuate nucleus; the cell counts shown in Figs. 4Go and 6Go represent the total number of digoxigenin- or 35S-labeled cells per section and therefore provide a relative guide to the distribution of Pomc-, Agrp-, and Lepr-b-expressing cells rather than their absolute number. Neuroanatomical boundaries were determined according to the methods of Kruger et al. (28) and Paxinos and Watson (29), using white matter tracts as a guide.



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  		Figure 4. Distribution of Agrp-expressing and Pomc-expressing cells along the rostral-caudal axis of the arcuate. Cell counts were made on 24 serial sections spaced every 100 µm apart through the arcuate nucleus (bregma -1.8 to bregma -4.2) as indicated. The Agrp-expressing cells are more highly concentrated in the caudal arcuate, whereas the Pomc-expressing cells are more highly concentrated in the rostral arcuate, suggesting that the two subtypes constitute distinct populations.

 


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  		Figure 6. Distribution of Agrp-expressing cells that also express Lepr-b along the rostral-caudal axis of the arcuate. Cell counts were made on 24 serial sections spaced every 100 µm apart through the arcuate nucleus (bregma -1.8 to bregma -4.2), and the percentage of cells was calculated across four groups of six sections each as indicated.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Agrp in mouse obesity mutations
Deficiency of leptin or its receptor is associated with an 8- to 10-fold elevation in hypothalamic levels of Agrp mRNA (5, 6). To investigate whether these findings are specific for defects in leptin signaling or are a nonspecific secondary response to obesity, we used Northern blot hybridization to measure hypothalamic levels of Agrp mRNA in Cpefat/Cpefat and tub/tub mice, genetic causes of obesity thought to be distinct from the leptin pathway (30, 31). For each experiment, a phosphorimager was used to determine the ratio of Agrp mRNA to ß-actin (Actb) mRNA in mutant and matched nonmutant control animals. This value varied from 0.01–0.50 depending on age of the animals and the specific activity of the two probes in different experiments, but, in general, was remarkably consistent among replicate animals (Table 1Go). There was no detectable change in hypothalamic Agrp mRNA levels in either Cpefat/Cpefat or tub/tub animals (Fig. 1AGo and Table 1Go); thus, the increased expression of Agrp mRNA observed previously in animals deficient for leptin signaling does not represent a nonspecific response to obesity.



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  		Figure 1. Levels of hypothalamic Agrp mRNA expression as determined by Northern blotting. As described in Materials and Methods, total hypothalamic RNA was isolated from animals of the indicated genotype, fractionated on formaldehyde gels, and transferred to a nylon filter before sequential hybridization with Agrp or Actb probes. Results are shown for two of the three to six pairs of animals used in each experiment. A, Mice homozygous for the Cpefat or tub mutations have identical levels of Agrp mRNA compared with nonmutant controls. The Cpefat mutation is on a C57BLKS background (BKS); the tub mutation is on a C57BL/6J background (C57). B, Fasting causes an increase in Agrp mRNA levels in nonmutant, but not in leptin-deficient (ob/ob), animals. C, Leptin-deficient or nonmutant control animals were administered leptin as described in Materials and Methods for 5–6 days, and Agrp mRNA levels were determined 12 h after the last injection.

 
Increased Agrp expression in response to short term fasting: requirement for leptin signaling
In both humans and mice, fasting leads to a rapid reduction in plasma leptin concentrations that is disproportionate to the reduction in adipose tissue that results from the fast (reviewed in Refs. 11, 13, 14, 32). To further investigate the relationship between leptin signaling and Agrp expression, we asked whether food deprivation altered steady state levels of hypothalamic Agrp mRNA. In C57BL/6J animals deprived of food commencing with onset of the dark cycle, we found a steady and progressive increase in hypothalamic levels of Agrp mRNA (Fig. 1BGo and Table 2Go), from 5-fold at 12 h of fasting to 15-fold by 48 h. To determine whether this increase was mediated by the decline in leptin levels triggered by fasting, we carried out the same experiment in leptin-deficient animals. At 48 or even 72 h of fasting, Agrp mRNA levels remained unchanged from those before food deprivation (Fig. 1BGo and Table 2Go).

Effect of leptin administration on Agrp expression and weight loss
Administration of exogenous leptin by ip injection causes reduced food intake and weight loss, although leptin-deficient animals are more sensitive than their nonmutant counterparts (33, 34). To determine whether these effects are accompanied by alterations in Agrp expression, Lepob/Lepob or normal animals were treated every 12 h with a dose of leptin (12.5 µg/g BW) sufficient to induce weight loss in nonmutant animals (Fig. 2Go), and hypothalamic levels of Agrp mRNA expression were measured after 6–7 days. Leptin treatment caused a large reduction (7-fold) of Agrp mRNA levels in Lepob/Lepob animals, but only a modest, albeit significant, reduction (17%; P = 0.047) in nonmutant animals (Fig. 1CGo and Table 3Go).



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  		Figure 2. Response of Ay/a, +/+, and Lepob/Lepob animals to peripheral leptin administration. Leptin in PBS (12.5 µg/g body mass; solid symbols) or PBS alone (open symbols) was injected into the peritoneal cavity every 12 h, and food intake (not shown) and body weight were measured at the indicated times. Leptin-deficient mice (ob/ob), 1.5 months of age, displayed a robust response, C57BL/6J animals, 1 month of age, displayed a modest response, but Ay/a animals, 1 month of age and with body weights and leptin levels (Table 3Go) similar to those of their nonmutant counterparts, were completely resistant to exogenous leptin.

 
Halaas et al. (35) have previously reported that Ay/a mice, in which obesity and hyperleptinemia are caused by chronic melanocortinergic blockade, are resistant to leptin when administered peripherally or centrally. These results are consistent with a pathway in which the effects of leptin on feeding are mediated via central melanocortin receptors, but could also reflect a long term secondary adaptation to obesity and hyperleptinemia, especially since other obesity models also are associated with leptin resistance (35, 36). To investigate this possibility, we measured the response to high dose leptin administration in 4-week-old Ay/a animals, which exhibit body weights and leptin levels similar to those in age-matched nonmutant controls (Table 1Go). Using the same protocol that caused a 12% reduction of body weight in control animals, 4-week-old Ay/a animals were completely resistant to administration of peripheral leptin (Fig. 2Go).

Location and projections of Agrp-expressing neurons
All of the experiments described above suggest that leptin is a negative regulator of Agrp mRNA expression in free feeding animals. To examine a possible anatomical basis for this regulation, we asked whether Agrp-expressing neurons also express the type b mRNA isoform of the leptin receptor, Lepr-b, that mediates most, if not all, of the effects of receptor activation and contains a long intracellular domain. Shutter et al. (6) have previously reported that Agrp mRNA is found predominantly in the arcuate nucleus, and several groups have suggested that Pomc-expressing neurons in the arcuate serve as a relay station for leptin signaling (18, 19, 20, 21, 22). We first compared the distribution of Agrp mRNA to that of Pomc mRNA using double label in situ hybridization (Fig. 3Go). In the retrochiasmatic nucleus and rostral arcuate nucleus, the distributions of Agrp- and Pomc-expressing cells occasionally overlapped, particularly in the medio-basal portion of the nucleus and the internal layer of the median eminence. In the remainder of the arcuate nucleus, however, Pomc-expressing cells extended more laterally and became clearly distinct from, although closely apposed to, Agrp-expressing cells. Quantitative analysis of 24 sections that span the arcuate nucleus demonstrated that Pomc-expressing neurons are more numerous than Agrp- expressing neurons and that the 2 cell types constituted populations clearly distinct from each other (Fig. 4Go).



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  		Figure 3. Dual in situ hybridization studies of Agrp and Pomc mRNA. Frontal sections through the rat arcuate nucleus were obtained and processed as described in Materials and Methods. The green signal represents silver grains corresponding to the 35S-labeled Pomc antisense probe; the blue signal represents the alkaline phosphatase reaction product corresponding to the digoxigenin-labeled Agrp antisense probe. Control sense probes gave no signal (not shown). Individual cells positive for Agrp (white arrows) or Pomc (black arrows) are easily distinguishable. Although both cell types are in the medio-basal area of the arcuate nucleus, very few cells are positive for both probes in this or other (not shown) sections examined. Scale bars are 50 µm in A and 25 µm in B and C. 3V, Third ventricle.

 
We next compared the distribution of Agrp-expressing neurons to that of neurons that express mRNA for Lepr-b using a probe that corresponds to the intracellular portion of the Lepr. Consistent with earlier reports (16, 17), Lepr-b mRNA was broadly expressed in multiple hypothalamic nuclei. At the level of the arcuate nucleus, we found a medio-basal population of cells with a low level of expression of Lepr-b, whereas another population localized more laterally displayed a high level of Lepr-b mRNA (Fig. 5Go). Both populations correspond to the area where POMC neurons are found. Overall, the number of Lepr-b-expressing cells was higher in the caudal compared with the rostral portion of the arcuate nucleus (data not shown). Double-label in situ hybridization histochemistry revealed a substantial number of cells in the medio-basal portion of the arcuate nucleus that expressed both Lepr-b and Agrp mRNAs (Fig. 5Go). Quantitative analysis of serial sections revealed that the proportion of Agrp-expressing neurons that also expressed Lepr-b varied from 10–25% (Fig. 6Go), but did not correlate with the rostral to caudal increase in the total number of Lepr-b-expressing cells (data not shown).



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  		Figure 5. Dual in situ hybridization studies of Agrp and Lepr type b mRNA. Frontal sections through the rat arcuate nucleus were obtained and processed as described in Materials and Methods. The green signal represents silver grains corresponding to the 35S-labeled Lepr-b antisense probe; the blue signal represents the alkaline phosphatase reaction product corresponding to the digoxigenin-labeled Agrp antisense probe. Control sense probes gave no signal (not shown). Individual cells positive for Lepr-b only (black arrows) are more widely distributed than those positive for Agrp only (white arrows) or doubly positive cells (red arrows); the latter were generally located in the medial portion of the arcuate. Scale bars are 50 µm in A and 25 µm in B and C. 3V, Third ventricle.

 
Although Agrp-expressing cells are clearly distinct from Pomc-expressing cells throughout most of the arcuate nucleus, one might expect their projection fields to overlap, as they encode alternative ligands for the same receptor class. To test this hypothesis, we raised a polyclonal antiserum against recombinant AGRP and carried out immunocytochemistry on adjacent sections stained with either antiserum against AGRP (Fig. 7Go, A, C, and E) or antiserum against {gamma}MSH, a POMC derivative (Fig. 7Go, B, D, and F). Consistent with the results obtained for mRNA in situ hybridization (Fig. 3Go), AGRP-immunoreactive cell bodies were generally located in a more medial position, closer to the wall of the third ventricle, than {gamma}MSH-immunoreactive cell bodies. In addition, AGRP-immunoreactive cell bodies were smaller (9.4 ± 0.35 µm) than {gamma}MSH-immunoreactive cell bodies (13.8 ± 0.3 µm). However, AGRP- and {gamma}-MSH-immunoreactive projection fields exhibited a significant degree of overlap, particularly in the arcuate and paraventricular nuclei (Fig. 7Go) where melanocortinergic neurons are thought to play important roles in regulating feeding behavior.



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  		Figure 7. Distribution of AGRP-immunoreactive and {gamma}MSH-immunoreactive cells and terminal fields in the region of the third ventricle (3V). Adjacent sections (25 µm) of the arcuate nucleus (A–D) and the paraventricular nucleus (E and F) of the rat hypothalamus were stained with polyclonal antiserum against AGRP (A, C, and E) or against {gamma}MSH (B, D, and F) as described in Materials and Methods. Antisera preadsorbed or coincubated with antigen showed no staining in control experiments (not shown). In the arcuate, AGRP-immunoreactive neurons (A and C) are more numerous and smaller than those immunoreactive for {gamma}MSH (B and D) and are found in a more medial position. Very dense nerve fibers immunoreactive for both antigens are apparent in the arcuate nucleus and the paraventricular nucleus of the hypothalamus, and the distribution is very similar for the two antibodies. Some AGRP- and {gamma}MSH-immunoreactive fibers are apparent in the internal layer of the median eminence (ME). Scale bars are 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The number of neuropeptide systems implicated in hypothalamic regulation of feeding behavior and energy balance has increased dramatically in the last several years, and potential pathways for integrating these systems with each other as well as with upstream and downstream components has become increasingly complex (reviewed in Refs. 24, 37). The work described here provides strong evidence that the expression of AGRP, an orexigenic peptide produced primarily in the arcuate nucleus, is negatively regulated by leptin, a circulating hormone released by adipocytes that serves as a critical indicator of fat stores and energy deprivation. Besides its role in regulating feeding behavior and efficiency of energy utilization, leptin has several other physiological roles; furthermore, the hypothalamic effects of leptin are likely to involve several pathways that act in parallel. These considerations notwithstanding, our results indicate that AGRP plays a key role in mediating the orexigenic and metabolic effects of leptin signaling and have important implications for understanding and treating disorders of energy balance.

Relationship of Agrp to other neuropeptides implicated in feeding behavior
Like several other hypothalamic neuropeptides implicated in the regulation of feeding, endogenous AGRP exhibits altered expression in response to food deprivation or leptin deficiency, and artificially increased expression causes changes in ingestive behavior. Neuropeptide Y (NPY) (reviewed in Ref. 38), melanin-concentrating hormone (39), and orexins/hypocretins (40, 41) all display effects similar to those of AGRP, whereas melanocortin agonists (42) and cocaine- and amphetamine-regulated transcript (CART) (43) exhibit the opposite pattern and are proposed to act as naturally occurring anorexigenic or satiety factors. Neuroanatomical studies have helped to provide an initial guide to the circuitry for these systems. For example, the effects on feeding of NPY, POMC, CART, and AGRP are thought to be mediated primarily via their expression in the arcuate nucleus, whereas orexins/hypocretins and melanin-concentrating hormone are expressed primarily in the lateral hypothalamus. POMC and AGRP terminals project to the paraventricular nucleus, and it will be interesting to conduct more detailed mapping studies to examine other areas of the hypothalamus. In addition, there is considerable heterogeneity within individual nuclei; for example, the distribution of CART-expressing neurons in the arcuate has been reported to be distinct from that of both POMC and NPY (43). As described here, AGRP-expressing neurons constitute a separate population from POMC-expressing neurons in the arcuate nucleus; a finding consistent with the idea that the two cell types send opposing types of signals. By contrast, AGRP and NPY are both orexigenic, and their pharmacological actions, inhibition of a Gs-coupled receptor for AGRP and activation of a Gi-coupled receptor for NPY, should reinforce one another. Furthermore, a scenario in which AGRP and NPY represent redundant components of a parallel circuit between the arcuate and paraventricular nuclei may help to explain why NPY-deficient animals show only minor defects in eating behavior and leptin signaling (44). In support of this idea, Schwartz and colleagues have recently shown that AGRP and NPY are coexpressed in the medial arcuate nucleus, and that expression of both genes is increased by food deprivation (45).

Regardless of the redundancy or lack thereof between AGRP and NPY in control of feeding, NPY clearly has additional roles in central nervous system (CNS) function, as indicated both by its expression in multiple areas of the brain outside the hypothalamus and by the diversity of NPY receptor subtypes. By contrast, Agrp-expressing neurons lie mainly within the hypothalamus. In vitro, AGRP is a potent antagonist of the Mc3r, whose expression is also limited to the hypothalamus, and the Mc4r, which is widely expressed in multiple areas of the brain (5, 46, 47). However, Mc4r-deficient animals exhibit increased feeding, linear growth, and obesity, which suggests that at least some, if not all, of the effects of AGRP on feeding behavior are mediated by the Mc4r (48). Additional insight into these questions may come from neuroanatomical studies that directly compare AGRP or melanocortinergic terminals with Mc3r- and Mc4r-expressing cells and from phenotypic analysis of Mc3r-deficient animals. Sites of Mc4r expression that lie outside AGRP terminal fields may be responsible for many of the behavioral effects ascribed to melanocortin agonists, including learning, sexual behavior, and neural regulation of the response to inflammatory cytokines (49). If so, pharmacological agents targeted to inhibit AGRP production or action may be more specific than Mc4r agonists.

In addition to AGRP, studies from several groups (19, 20, 21) indicate that POMC is also a CNS effector of leptin. As described here, cells that express Agrp and Pomc constitute different subpopulations within the arcuate nucleus, yet project to similar locations in the paraventricular and arcuate nuclei. More detailed mapping studies will be required to determine the extent to which the projection fields of AGRP overlap with those of POMC in other areas of the brain, but at least in the hypothalamus, these two types of neurons could serve to integrate different afferent signals into a common efferent pathway.

Regulation of Agrp expression by leptin signaling
The realization that many types of obesity are associated with elevated circulating levels of leptin has led to increasing interest in understanding the events that lie downstream of leptin receptor signaling in the CNS. Halaas et al. (35) demonstrated that chronic melanocortinergic blockade in obese hyperleptinemic Ay mice can inhibit or mask both the peripheral and central nervous system response to leptin. Our results demonstrate that leptin resistance is not the result of long term changes secondary to obesity and hyperleptinemia, as young Ay mice with body weights and circulating leptin levels identical to those of nonmutant animals were completely resistant to supraphysiological doses of peripheral leptin. In a different paradigm that yielded similar results, Seeley et al. (22) reported that CNS administration of SHU9119, a nonselective Mc4r antagonist, prevented or masked the subsequent behavioral response to leptin, but not that to the anorexigenic peptide glucagon-like peptide-1. While all of these observations are consistent with the idea that leptin-induced changes in food intake and body weight are mediated via melanocortin receptors, they do not completely exclude the possibility that the two systems act via separate parallel pathways that can overcome each other’s effects. Indeed, Boston et al. (23) suggested that obesity caused by leptin deficiency was independent of that mediated through melanocortin receptors, because Ay and Lepob had an additive effect on weight gain in adrenalectomized animals. However, an important caveat to this conclusion is the underlying assumption that ubiquitous expression of Agouti in Ay animals is equivalent to complete melanocortinergic blockade. In particular, expression of Agrp is probably up-regulated in Ay/a;Lepob/Lepob compared with Ay/a;+/+ animals, and the combined effects of Agouti and AGRP are likely to exceed those of Agouti alone. Thus, the phenotypic additivity between Ay and Lepob observed by Boston et al. (23) could simply reflect a greater level of melanocortinergic blockade in Ay/a;Lepob/Lepob compared with Ay/a;+/+ animals.

Strong evidence for a linear pathway in which AGRP lies downstream of leptin is apparent from the observation that food deprivation has no effect on Agrp mRNA levels in Lepob/Lepob mice, but causes a 15-fold increase in Agrp mRNA levels in nonmutant mice. Thus, the increase in Agrp mRNA induced by fasting is completely dependent on the presence of leptin. An alternative explanation, that long term adaptation to high levels of Agrp expression in Lepob/Lepob mice prevents a further increase in response to fasting, is unlikely by analogy to studies of NPY. Npy mRNA levels in the hypothalamus are much greater than those of Agrp, and in Leprdb/Leprdb mice, Npy mRNA levels are further elevated approximately 2-fold by fasting (19). Additional insight into these questions should come from analysis of animals doubly mutant for Lep and the Mc4r or Agrp.

An underlying theme of the observations reported here is that decreased leptin (in fasted nonmutant mice or in Lepob/Lepob mice) causes a dramatic change in Agrp expression, whereas increased leptin (in nonmutant animals treated with leptin or in Cpefat/Cpefat or tub/tub mice) causes little or no change. An exception is the large (7-fold) reduction in Agrp expression caused by administration of leptin to Lepob/Lepob animals. However, chronic deficiency for leptin leads to a secondary hypersensitivity, possibly via up-regulation of the Lepr (50). In fact, a combination of leptin resistance caused by the Ay mutation and leptin hypersensitivity caused by the Lepob mutation may account for the normal response to exogenous leptin observed in Ay/a;Lepob/Lepob animals (23). Taken together, our results suggest that in the fed state, expression of Agrp is tonically down-regulated by physiological levels of leptin, and the orexigenic effects of increased Agrp expression occur primarily in response to the decline in leptin levels that accompanies food deprivation. Nonmutant animals refed after food deprivation exhibit increased food intake that can be suppressed by administration of exogenous leptin (14). Our findings suggest that increased expression of Agrp triggered by fasting plays a key role in this process.

As shown here, the majority of Agrp-expressing neurons do not express Lepr-b. However, our neuroanatomical studies were carried out with rats fed ad libitum, whereas the Northern hybridization experiments were carried out with mice, and inferences regarding the anatomic basis for regulation of Agrp expression by leptin should be tempered by the possibility that the exact circuitry may vary among species and according to nutritional status. For example, if food deprivation causes an increased number of Agrp-expressing cells to activate expression of Lepr-b or vice versa, transcription of Agrp might be directly controlled (in a negative manner) by Lepr signaling. Indeed, Schwartz and colleagues (45) reported that fasting increases the number of detectable Agrp-expressing cells, which is consistent with the possibility of direct regulation.

Concluding remarks
The physiological studies described here indicate that changes in Agrp expression are mediated by leptin signaling. However, the relatively wide hypothalamic distribution of the Lepr-b isoform implies that the effects of leptin on feeding and body weight are mediated through multiple downstream effectors, and additional work will be required to determine the relative contributions and potential redundancy of each of these effectors. Regardless, a view of leptin signaling in which AGRP represents one of several downstream arms has physiological and pharmacological implications very different from a view in which the two pathways are independent. The work described here demonstrates the utility of combining physiological and anatomical studies; similar studies carried out on animals with genetic defects in individual signaling components should lead to a more detailed understanding of the circuitry between peripheral stimuli and neuropeptide effectors.


   Acknowledgments
 
We are grateful to Julie Kerns for help with hypothalamus dissections, to Heidi Day and Xinyun Lu for their help and criticism, and to Robert Pavlic and Sharon Burke for excellent technical support. We thank Drs. Patsy Nishina and Jurgen Naggert for providing tissues, and Dr. J. Krause for providing a leptin receptor probe.


   Footnotes
 
1 This work was supported in part by grants from the NIH, MH-4251 (to S.J.W.) and DK-28506 (to G.S.B.), the Pritzker Depression Research Network, and Medical Scientist Trainee Grant GM-07365 from the NIH (to B.D.W.). Back

2 These authors contributed equally to this work. Back

3 Current address: Exelixis Pharmaceuticals, 260 Littlefield Avenue, South San Francisco, California 94080. Back

4 Associate Investigator with the Howard Hughes Medical Institute. Back

Received October 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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