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Endocrinology Vol. 143, No. 10 3905-3915
Copyright © 2002 by The Endocrine Society

ARTICLE

Diurnal Rhythm of Agouti-Related Protein and Its Relation to Corticosterone and Food Intake

Xin-Yun Lu, Kun-Ruey Shieh, Mohamed Kabbaj, Gregory S. Barsh, Huda Akil and Stanley J. Watson

University of Michigan School of Medicine, Mental Health Research Institute (X.-Y.L., K.-R.S., M.K., H.A., S.J.W.), Ann Arbor, Michigan 48109; and Departments of Pediatrics and Genetics, Howard Hughes Medical Institute, Stanford University (G.S.B.), Stanford, California 94305

Address all correspondence and requests for reprints to: Dr. Xin-Yun Lu, Mental Health Research Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, Michigan 48109-0720. E-mail: xylu{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study we examined the diurnal patterns of agouti-related protein (AGRP) and proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus and their relation to circulating glucocorticoids and food intake. Animals were killed at 4-h intervals throughout the 24-h diurnal cycle, and the expression of AGRP and POMC mRNA was evaluated by semiquantitative in situ hybridization analysis. We observed a significant diurnal rhythm in AGRP mRNA expression, with a marked peak at 2200 h (4 h after lights off) and a trough at 1000 h (4 h after lights on), consistent with the overall day-night rhythm of food intake. In contrast, POMC mRNA levels did not show a significant fluctuation across the diurnal cycle, although there was a tendency for levels to decrease after the onset of the dark cycle. Corticosterone secretion temporally coincided with the rising phase of AGRP mRNA expression. Depletion of corticosterone by adrenalectomy abolished the AGRP diurnal rhythm by suppressing the nighttime expression, but did not alter the feeding rhythm. Exposure of adrenalectomized rats to constant corticosterone replacement (10 or 50 mg continuous release corticosterone pellet) resulted in fixed AGRP mRNA expression throughout the 12-h light, 12-h dark cycle. A relatively high level of corticosterone (50 mg) significantly increased AGRP mRNA expression, with a positive correlation between these two measures. These results indicate that 1) the diurnal expression of AGRP mRNA is regulated by corticosterone independently of the light/dark cue; and 2) a normal endogenous corticosterone rhythm is required for generating the diurnal AGRP rhythm.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EMERGING EVIDENCE has indicated an important role for the central melanocortinergic system in the regulation of feeding and body weight. Melanocortins, including {alpha}-, ß-, and {gamma}-MSH, are derived from posttranslational processing of the precursor protein proopiomelanocortin (POMC). {alpha}-MSH is one of the major end products of POMC processing in the brain. {alpha}-MSH and its analogs suppress feeding and body weight gain through activation of central melanocortin 3 (MC3R) and melanocortin 4 receptors (MC4R) (1, 2, 3, 4, 5, 6, 7). Target disruption of the POMC gene and the MC4R gene produces hyperphagia and obesity in mice (8, 9). Disruption of the MC3R gene in mice results in higher feeding efficiency, leading to increased fat storage (10, 11). In addition, genetic defects in the POMC gene and the MC4R gene in humans have been identified as being associated with obesity (12, 13, 14, 15, 16, 17, 18, 19).

Agouti-related protein (AGRP) is a naturally occurring antagonist of MC3 and MC4 receptors (20). It is normally expressed in the arcuate nucleus of the hypothalamus, within the vicinity of hypothalamic POMC cells (21, 22), and has been implicated in the regulation of food intake, body weight, and energy homeostasis. Overexpression of AGRP gene in mice results in hyperphagic and obese phenotypes, similar to those seen in POMC and MC4R knockout mice (20). Conversely, defects in the AGRP gene are found to be associated with anorexia nervosa and weight loss (23). Central administration of the C-terminal fragment of AGRP stimulates food intake and blocks the reduction in food intake elicited by {alpha}-MSH in rodents and primates (24, 25, 26, 27, 28, 29). On the other hand, levels of AGRP mRNA in the arcuate nucleus increase, whereas levels of POMC mRNA decrease, in response to food restriction or food deprivation, thereby leading to stimulation of appetite to regain body weight (30, 31). Moreover, AGRP and POMC mRNA expressions respond inversely to photoperiodic manipulations in the context of seasonal appetite and body weight regulation (32, 33). In view of these antagonizing effects elicited by AGRP and POMC-derived {alpha}-MSH on food intake and opposing changes in AGRP and POMC mRNA in relation to energy balance, it would seem reasonable to speculate that under physiological conditions melanocortin receptor-mediated feeding behavior may reflect a balance between the activity of stimulatory AGRP and inhibitory POMC circuits in the brain. Whether AGRP and POMC would fluctuate coordinately in an inverse relationship across the 24-h light/dark cycle in an association with spontaneous food intake, however, is unknown.

A role for adrenal glucocorticoids in the regulation of feeding and the development of obesity has been recognized. The peak and nadir of glucocorticoid diurnal secretion over 24 h coincide with the initiation and termination, respectively, of the active feeding period and locomotor activities (34, 35, 36). Exogenous glucocorticoids stimulate food intake when administered centrally and promote obesity, whereas adrenalectomy decreases food consumption and prevents the development of obesity (37, 38, 39, 40, 41). The functions of glucocorticoids are mediated through two steroid receptor subtypes, type I receptors [i.e. mineralocorticoid receptors (MRs)] and type II receptors (i.e. glucocorticoid receptors (GRs)]. MR is tonically activated by low basal levels of glucocorticoids, whereas the activation of GR requires higher levels of glucocorticoids, usually occurring at times of diurnal surge or stress conditions (42). MR and GR have distinct distributions in the brain. In particular, mRNA immunoreactivity and binding sites for GR are abundant in the arcuate nucleus, overlapping with the distribution of AGRP and POMC neurons (43, 44, 45, 46 46A 46B ). Furthermore, it has been demonstrated that POMC neurons and neuropeptide Y (NPY) neurons in the arcuate nucleus contain GR (44, 45, 46). Given that AGRP is heavily colocalized with NPY in the arcuate nucleus (47), often being referred to the same subset of neurons, these findings raise the possibility that the activities of AGRP and POMC neurons in this brain region might be under the modulation of glucocorticoids.

The goal of the present study was to examine the diurnal profiles of AGRP and POMC mRNA expression in the arcuate nucleus and their relation to circulating glucocorticoids and food intake. The involvement of glucocorticoids in entraining the diurnal expression of AGRP mRNA was further investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Adult male Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA), weighing 280–330 g, were housed in groups of three on a 12-h light, 12-h dark cycle, with lights on at 0600 h. Food and water were available ad libitum. Animals were allowed to acclimate to these housing conditions for 1 wk before experiments were started. All procedures described were approved by the University of Michigan committee on use and care of animals.

Experimental protocol
For the determination of diurnal patterns of AGRP mRNA, POMC mRNA, circulating corticosterone levels, and food intake, 48 animals (6 rats/group) were used. One group of animals (n = 6) was housed individually to allow for measuring of daily food intake. In this group, food was weighed every 2 h over 2 consecutive days. A safe light was used at the time of food measurement during the dark phase. The rest of the animals were housed in a separate room in groups of 3/cage and were killed by decapitation at 4-h intervals for 24 h starting from 1800 h. Trunk blood was collected into siliconized tubes containing heparin and was kept on ice until centrifugation. Plasma was separated, frozen on dry ice, and stored at -80 C. Brains were rapidly removed on ice to minimize degradation of mRNAs, frozen in an isopentane-dry ice bath (-40 C), and stored at -80 C. Coronal brain sections (10 µm) were cut through the hypothalamus in a cryostat, thaw-mounted onto polylysine-subbed slides, and stored at -80 C until processing for in situ hybridization as described previously (48). The time taken to kill 3 animals in each cage was less than 30 sec, and all animals at each time point (n = 6) were killed within 10 min. Safe lights were used during the dark cycle to avoid exposure to light that might alter the diurnal rhythm.

To determine the effect of corticosterone on the diurnal feeding rhythm and diurnal variations in the gene expression, 82 rats were sham-operated or adrenalectomized by a dorsolateral approach to remove the adrenal glands bilaterally. Initial body weight and presurgery daily food intake were balanced between animal groups undergoing sham operation or adrenalectomy. In addition, a placebo or a 10- or 50-mg corticosterone pellet (21-d release; Innovative Research of America, Toledo, OH) was implanted sc under the dorsal neck skin to produce constant corticosterone levels in adrenalectomized rats. Surgery was performed in the morning when normal corticosterone levels are low. Drinking water for all adrenalectomized rats was replaced with 0.9% saline. Body weight changes and food intake were recorded daily after surgery. To determine the effect of adrenalectomy on the feeding rhythm, food consumed during the 12-h light cycle and that consumed during the 12-h dark cycle were measured on the seventh and eighth days postsurgery for 1 group of adrenalectomized animals (n = 14) and 1 group of sham controls (n = 11). All other animals were killed by decapitation at 1000 h and 2200 h, 7 d after surgery. Trunk blood was collected, and brains were quickly removed as described above. Coronal brain sections (10 µm) were cut through the hypothalamus and stored at -80 C.

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 binding protein and were incubated overnight with corticosterone antibody. [3H]Corticosterone (Amersham Pharmacia Biotech, Arlington Heights, IL) was used as a radioactive tracer. Free and bound corticosterone were separated by incubating with charcoal for 15 min. Corticosterone concentrations were calculated using an equation derived from a standard curve.

In situ hybridization
cDNA fragments complementary to rat AGRP (345 bp; courtesy of Dr. Ira Gantz, University of Michigan) and POMC (936 bp) were subcloned into pBluescript SK vector. To generate sense and antisense 35S-labeled cRNA probes, the linearized plasmid was incubated at 37 C for 2 h in 20 µl reaction mixture consisting of 1x transcription buffer (Life Technologies, Inc., Gaithersburg, MD), 75 µCi [{alpha}-35S]UTP (>1000 Ci/mmol; 20 mCi/ml; Amersham Pharmacia Biotech), 100 µCi [{alpha}-35S]CTP (800 Ci/mmol; 40 mCi/ml), 150 µM ATP, 150 µM GTP, 10 mM dithiothreitol, 20 U ribonuclease (RNase) inhibitor, and 6 U T7, T3, or SP6. The radioactively labeled cRNA probes were separated from free nucleotides on a Sephadex G-50/50 column.

Tissue sections were removed from the -80 C freezer, fixed in 4% paraformaldehyde for 1 h, and rinsed twice in 2x SSC (300 mM sodium chloride and 30 mM sodium citrate, pH 7.2). Brain sections were then acetylated in 0.1 M triethanolamine (pH 8.0) with 0.25% acetic anhydride (10 min), rinsed in distilled water, dehydrated through a graded series of alcohol (50–100%, 30 sec each), and subsequently air-dried. 35S-Labeled cRNA probes were diluted to 2 x 106/70 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 30 mM dithiothreitol]. Diluted probes (70 µl) were placed on each slide, and the sections were coverslipped. Tissue slides were placed in plastic trays moistened with 50% formamide. Hybridization was performed in an incubator at 55 C overnight. The following day, coverslips were lifted with 2x SSC, and slides were rinsed three times in 2x SSC, then incubated in RNase A (200 µg/ml) for 1 h at 37 C. Slides were then washed in 2x SSC, 1x SSC, 0.5x SSC, and 0.1x SSC (5 min each at room temperature). Finally, the sections were placed in 0.1x SSC at 70 C for 1 h, then rinsed in distilled water and dehydrated in a graded series of alcohols. Sections were exposed to x-ray film (BioMax MR, Eastman Kodak, Rochester, NY) or dipped in liquid emulsion (Ilford KD-5, Polysciences, Warrington, PA). The specificity of hybridization was assured by hybridization with sense strand probes or pretreatment with RNase (200 µg/ml at 37 C for 60 min) before hybridization.

Image analysis
Sixteen tissue sections (100 µm apart) were selected for the arcuate nucleus from each animal, corresponding to Bregma -2.1 mm to Bregma -3.6 mm. Levels of AGRP and POMC mRNA were analyzed by computer-assisted optical densitometry. Digital images of brain sections were captured from x-ray films in the linear range of the gray levels using a CCD camera. The relative OD of the mRNA expressed in the arcuate nucleus was determined using Analysis of Imaging System (Imaging Research, Inc., Ontario, Canada). Briefly, OD measures representing in situ hybridization signals were defined as being 3.5 SD above background and were multiplied by the area sampled, yielding integrated OD units. Mean values for each animal were determined from eight sections though the arcuate nucleus. The arcuate nucleus boundaries on the digitized images were determined using cresyl violet counterstained slides and were compared with the rat brain atlas of Paxinos and Watson (49).

Statistical analysis
Data were analyzed by one-way (the time-course data of AGRP and POMC mRNA expression) or two-way (the adrenalectomy data) ANOVA, followed by Newman-Keuls multiple comparisons. Trend analysis was conducted for the diurnal expression data of AGRP and POMC mRNA. The average AGRP mRNA expressions at night and during the day were compared by t test. Results were expressed as the mean ± SEM, and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of AGRP and POMC mRNA in the arcuate nucleus
The distribution of AGRP and POMC mRNA on adjacent coronal brain sections through the arcuate nucleus is shown in Fig. 1Go. AGRP and POMC cells are present throughout the rostrocaudal extent of the arcuate nucleus. AGRP mRNA-expressing cells are concentrated in the medial sector of the arcuate nucleus (Fig. 1Go, A–F), whereas POMC-expressing cells are spread more laterally (Fig. 1Go, A'–F'). At caudal levels, AGRP cells extend from the medial to ventrolateral portion of the arcuate nucleus. Note that the inner layer of the median eminence exhibits sparse, but strong, AGRP labeling. Interestingly, POMC signals were also observed in the ventral portion of the third ventricle lining.



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  		Figure 1. Darkfield microphotographs of emulsion autoradiograms showing the expression of AGRP mRNA (A–F) and POMC mRNA (A'–F') in coronal brain sections arranged in a rostrocaudal sequence. Counterstaining with cresyl violet shows brain structures. Arc, Arcuate nucleus. Scale bar, 2 mm.

 
Diurnal expression of AGRP and POMC mRNA in the arcuate nucleus
Figure 2AGo shows the diurnal expression of AGRP mRNA at six time points across the light-dark cycle. The greatest magnitude of difference in AGRP mRNA expression was noted between 1000 and 2200 h (Fig. 2BGo). Analysis of AGRP mRNA expression data revealed a diurnal rhythm with a single peak. Statistical analysis by one-way ANOVA indicated significant effects of time of day on AGRP mRNA expression in the arcuate nucleus (Fig. 3AGo; n = 6/time point; by one-way ANOVA, P < 0.05). Trend analysis by polynomial fit revealed a quadratic relationship between AGRP expression and time of day (P < 0.05). The nadir value of AGRP mRNA expression occurred at 1000 h, 4 h after lights on, after which levels of AGRP mRNA increased and reached a peak at 2200 h, 4 h after lights off (P < 0.01 vs. 1000 h; P < 0.05 vs. 0600 h). Subsequently, AGRP mRNA levels gradually decreased before lights on and returned to the trough expression at 1000 h in the light cycle (0200 h vs. 1000 h, P < 0.05). The average expression of AGRP mRNA in the dark cycle (from 1800–0600 h) was significantly greater than that in the light cycle (from 0600–1800 h; by t test, P < 0.01). Levels of AGRP mRNA at 2200 h in the dark cycle differed significantly from those at 0600 h (P < 0.05), 1000 h (P < 0.001), and 1400 h (P < 0.01) in the light cycle.



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  		Figure 2. A, Darkfield photographs of film autoradiograms showing the diurnal expression of AGRP mRNA in the arcuate nucleus at each time of death over a 24-h period. B and C, Representative emulsion-dipped sections showing cellular expression of AGRP mRNA in the arcuate nucleus at 1000 h in the light cycle (B) and at 2200 h in the dark cycle (C). Emulsion slides were counterstained with cresyl violet to facilitate evaluation of cellular localization of the silver grains. Note the increases in both numbers of silver grain clusters and numbers of silver grains per cluster in the arcuate nucleus (Arc) at 2200 h compared with 1000 h. Scale bars, 2 mm in A; 1 mm in B.

 


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  		Figure 3. The diurnal profiles of AGRP mRNA (A) and POMC mRNA (B) in the arcuate nucleus. Data are expressed as the mean ± SEM (n = 6/time point). By one-way ANOVA of the diurnal expression of AGRP mRNA, P < 0.05. Additional trend analysis was performed for AGRP expression across time of day, showing a quadratic function (P < 0.05). The average expression of AGRP mRNA in the dark cycle from 1800–0200 h is significantly greater than that in the light cycle from 0600–1400 h (by t test, P < 0.01). Levels of AGRP mRNA at 2200 h in the dark cycle differed significantly from those at 0600, 1000, and 1400 h in the light cycle. IOD, Integrated optical density. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. 2200 h).

 
In contrast, although levels of POMC mRNA expression in the arcuate nucleus exhibited a gradual decreasing trend after lights on and fell to their lowest level at 0200 h, ANOVA did not reveal a significant diurnal fluctuation (Fig. 3BGo; by one-way ANOVA, P = 0.11).

Comparison of AGRP mRNA, food intake, and plasma corticosterone diurnal rhythms
In Fig. 4Go, the diurnal rhythm of AGRP mRNA expression is superimposed on curves reflecting spontaneous daily food intake and plasma corticosterone levels. Food intake was measured every 2 h throughout the 24-h light/dark cycle (shown in Fig. 4AGo). Total food consumption within 24 h was 35 ± 1.4 g. Food intake in the light phase was low, only 15.4% of their total daily food consumption, varying from 0.83–1.83 g/2 h. A dramatic increase in food intake was observed after the onset of the dark cycle (1800 h), and active food intake was maintained from 1800–2400 h. Subsequently, the amount of food ingestion declined until 0400 h. A sharp rise in food intake, however, was observed between 0400–0600 h, the 2-h period immediately preceding the onset of the light cycle. Animals consumed 23% of total daily food intake during this 2-h period. Note that the overall AGRP mRNA expression during the day and night coincides with the day-night rhythm of food intake (Fig. 4AGo).



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  		Figure 4. Relation of the AGRP diurnal rhythm to spontaneous food intake and corticosterone. A, Food intake data ({square}) and AGRP mRNA data ({blacksquare}). Note that the trough and peak seen at 1000 and 2200 h in AGRP mRNA rhythm appear to coincide with the day-night rhythm of food intake. B, Plasma corticosterone data ({square}) and AGRP mRNA data ({blacksquare}). Note that a sharp rise in corticosterone appears to correspond to the rising phase of AGRP mRNA and that the peak of circulating corticosterone precedes the peak AGRP mRNA expression. IOD, Integrated optical density.

 
In rats housed under 12-h light, 12-h dark illumination conditions with food available ad libitum, circulating corticosterone concentrations exhibited a diurnal rhythm (Fig. 4BGo). Plasma corticosterone levels were low in the morning (from 0600–1000 h), ranging from 0–1.22 µg/dl. Then, corticosterone levels exhibited an increase from trough levels to 6.45 ± 2.17 µg/dl 4 h before the dark cycle and a further rise to reach a peak at the light/dark transition phase (1800 h; 20.63 ± 2.45 µg/dl). Subsequently, plasma corticosterone levels declined to 4.38 ± 1.46 µg/dl 4 h before returning to the nadir at 0600 h. Plasma corticosterone levels and AGRP mRNA expression across the light-dark cycle were determined in the same animals. Note that a sharp rise in corticosterone preceded the rising phase of AGRP mRNA expression in the night (Fig. 4BGo). The peak of AGRP mRNA expression lagged 4 h behind the peak of plasma corticosterone.

Effect of adrenalectomy and constant corticosterone levels on the diurnal expression of feeding and AGRP mRNA in the arcuate nucleus
Adrenalectomy resulted in a reduction in body weight (-10.9 ± 4.13% from the presurgery initial body weight; n = 14), whereas sham controls gained 16.2 ± 1.28% from their presurgery initial body weight in 7 d (n = 11; P < 0.0001 vs. adrenalectomized rats; upper panel in Fig. 5AGo). The effect of adrenalectomy on body weight can be reversed by a relatively low dose of corticosterone (10 mg) replacement (data not shown). Daily food intake was measured for 7 d starting from the day before surgery. Adrenalectomized rats ate significantly less than sham controls (P < 0.001, by ANOVA with repeated measures; bottom panel in Fig. 5AGo). As reported previously (50, 51), however, the general feeding rhythm was not altered by adrenalectomy (Fig. 5BGo). Actually, the magnitude of the feeding rhythm in the adrenalectomy group appeared to be greater than that in the sham control group due to a more evident reduction of food intake during the day.



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  		Figure 5. Effect of adrenalectomy on body weight and food intake. A, Dynamic changes in body weight gain (upper panel) and daily food intake (lower panel) in rats sham-operated or adrenalectomized compared with their presurgery state. B, Food intake during the 24-h period or the 12-h light and dark cycles. As noted, the feeding rhythm is unaffected by adrenalectomy. n = 14 for the sham-operated group; n = 11 for the adrenalectomized group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (adrenalectomy vs. sham operation).

 
The effect of corticosterone on the diurnal expression of AGRP mRNA was examined in rats sham-adrenalectomized or adrenalectomized given placebo or 10- or 50-mg corticosterone pellets. In sham-operated rats, plasma corticosterone showed a normal diurnal variation, i.e. low at 1000 h (0.75 ± 0.33 µg/d) and high at 2200 h (8.88 ± 3.48 µg/d; Fig. 6AGo). Corticosterone was undetectable in rats adrenalectomized with placebo pellet. Adrenalectomized rats given 10- and 50-mg corticosterone pellets had plasma corticosterone levels elevated over the sham control values at 1000 h, but fixed throughout the day (Fig. 6AGo). Levels of circulating corticosterone for rats given the 10-mg corticosterone pellet were 1.52 ± 0.59 µg/d at 1000 h and 1.52 ± 0.71 µg/d at 2200 h. Levels of circulating corticosterone for rats given the 50 mg corticosterone pellet were 5.50 ± 1.37 µg/d at 1000 h and 5.09 ± 0.30 µg/dl at 2200 h.



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  		Figure 6. Effect of adrenalectomy and constant corticosterone levels on the diurnal expression of AGRP mRNA in the arcuate nucleus. Rats were sham-adrenalectomized or adrenalectomized with a placebo or 10- or 50-mg corticosterone continuous release sc pellets. Results were pooled from two experiments. A, Plasma corticosterone concentrations. B, Darkfield photomicrographs of emulsion-dipped sections showing AGRP mRNA expression at 1000 and 2200 h in sham-operated and adrenalectomized rats. Corticosterone and AGRP mRNA expression were measured at 1000 h ({square}) and at 2200 h ({blacksquare}). C, Levels of AGRP mRNA expression, which were standardized to the expression observed in the sham control rats at 1000 h (100%). AGRP mRNA exhibits significant down-regulation at 2200 h in response to adrenalectomy. Relatively high, fixed levels of corticosterone significantly increase AGRP mRNA expression across the light/dark cycle. ADX, Adrenalectomy. Values represent the mean ± SEM (n = 9–14 for sham controls; n = 7–9 for ADX; n = 4 for 10-mg corticosterone pellet replacement; n = 10 for 50-mg corticosterone pellet replacement). **, P < 0.01, sham at 2200 h vs. sham at 1000 h. {blacktriangledown}{blacktriangledown}{blacktriangledown}, P < 0.001, ADX vs. sham at 2200 h. {ddagger}, P < 0.05, ADX vs. 10 mg at 2200 h. {diamondsuit}{diamondsuit}{diamondsuit}, P < 0.001, 50 mg vs. sham, ADX, and 10 mg at 1000 h. +, P = 0.053, 50 mg vs. sham. {blacktriangleup}{blacktriangleup}{blacktriangleup}, P < 0.001, 50 mg vs. ADX. #, P < 0.05, 50 vs. 10 mg at 2200 h. Scale bar in B, 2 mm.

 
Sham controls maintained the diurnal rhythm of AGRP mRNA expression as observed in intact rats (Fig. 6Go, B and C), with significantly higher levels of AGRP mRNA expression in the evening (2200 h) than in the morning (1000 h; P < 0.01, by t test; Fig. 6CGo). Removal of endogenous glucocorticoids by adrenalectomy had no significant effect on the 1000 h expression; however, adrenalectomy significantly decreased the 2200 h expression by 43 ± 4.5% relative to that in sham-operated rats (P < 0.001), thereby eliminating the diurnal rhythm of AGRP mRNA expression (Fig. 6Go, B and C). The diurnal variation in AGRP mRNA expression was also abolished in rats with constant corticosterone levels (Fig. 6CGo). A relatively low dose of corticosterone replacement (10-mg pellet) did not significantly alter the expression at either 1000 or 2200 h compared with that in sham controls at 1000 h. A moderately high constant level of corticosterone (50-mg pellet) significantly increased AGRP mRNA expression with a higher magnitude in the morning than in the evening compared with sham controls, resulting in flat levels of AGRP mRNA expression throughout the day (Fig. 6CGo). Moreover, levels of AGRP mRNA were significantly correlated with corticosterone concentrations at the time of death (Fig. 7Go; by Pearson analysis: r = 0.71; P < 0.001; r2 = 0.504).



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  		Figure 7. Pearson correlation analysis between plasma corticosterone concentrations and levels of AGRP mRNA in the arcuate nucleus in rats adrenalectomized with placebo or 10- and 50-mg corticosterone pellet replacement. The analysis showed a positive correlation between levels of AGRP mRNA and plasma corticosterone concentrations. Pearson correlation coefficient, r = 0.71; P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates the expression of AGRP and POMC mRNAs in the arcuate nucleus in response to time of day. Although levels of AGRP mRNA exhibit a diurnal rhythm in parallel to the overall feeding rhythm, POMC mRNA expression did not show a significant fluctuation across the diurnal cycle. The natural corticosterone surge was observed to precede the active feeding phase and the peak expression of AGRP mRNA. Adrenalectomy eliminated the AGRP mRNA rhythm via decreasing the night AGRP mRNA levels; however, the feeding rhythm was not affected by adrenalectomy, although adrenalectomized rats ate significantly less than sham controls. Adrenalectomized rats with constant corticosterone replacement exhibited no diurnal variation in AGRP mRNA expression. However, a relatively high dose of corticosterone significantly increased AGRP mRNA expression, with a greater magnitude in the morning than in the evening, resulting in flat levels of AGRP mRNA expression across the light/dark cycle. Furthermore, circulating corticosterone concentrations were positively correlated with levels of AGRP mRNA expression in the arcuate nucleus.

Several studies have suggested that the diurnal pattern of feeding behavior is evoked by appetite-regulating neuropeptides (52 – 54). AGRP, an endogenous antagonist of POMC-derived {alpha}-MSH at MC3R and MC4R, is a potent appetite-stimulating neuropeptide. Central administration of AGRP and {alpha}-MSH induces, respectively, an increase or a decrease in food intake (1, 2, 3, 4, 5, 6, 7, 24, 25, 26, 27, 28, 29). Relationships between levels of endogenous AGRP and POMC and possible association between these two molecules and spontaneous food intake are unknown. We showed here that levels of AGRP mRNA expression displayed a significant diurnal rhythm with a nadir at 1000 h in the light cycle and a peak at 2200 h in the dark cycle. The diurnal expression of AGRP mRNA coincides with the overall day-night feeding rhythm in rats, rising before the active phase of feeding, and peaking 4 h after dark onset. This pattern is similar to the report on NPY in the hypothalamus (55). Given the fact that AGRP and NPY colocalize in the arcuate nucleus neurons, endogenous AGRP and NPY might act in concert to regulate diurnal food intake. However, it is worth noting that AGRP mRNA levels do not always parallel the feeding pattern, as rats exhibited binge eating during the 2 h before lights on (23% daily food intake), when AGRP levels diminished.

The mechanism by which AGRP mRNA expression fluctuates diurnally is not clear; however, the increasing trend in AGRP mRNA before the onset of the dark cycle may suggest that the diurnal rhythm of AGRP mRNA is unlikely to be light-entrained. Analysis of diurnal events that precede the rising phase of AGRP mRNA expression may predict the regulatory factors of diurnal expression of AGRP mRNA. One candidate is corticosterone, which exhibits a diurnal rhythm, consisting of a single peak occurring just before the active feeding period (the dark cycle). The trough of secretion was observed in the early light cycle, which is characterized by very low levels of corticosterone. In comparison with the diurnal pattern of AGRP mRNA expression, the natural corticosterone surge appeared to anticipate the rising phase of AGRP mRNA expression, implying that corticosterone might act as a causitive factor of the evening AGRP mRNA expression. This idea was supported by anatomical evidence showing that GRs are likely to colocalize with AGRP in the arcuate nucleus (44, 45, 46). We speculate that the evening AGRP mRNA expression might be positively regulated by the diurnal secretion of corticosterone, and that the time lag between the corticosterone peak and the AGRP mRNA expression peak might be required for changes in AGRP mRNA to become evident. Alternatively, the diurnal surge of corticosterone might induce an increase in AGRP peptide release, anticipating the increase in AGRP mRNA expression; the subsequent peak of AGRP mRNA in the evening, therefore, may represent a mechanism by which the peptide reservoir is replenished. If so, in either case, the evening peak of AGRP mRNA expression subsequent to the diurnal surge of corticosterone should be blunted by removal of corticosterone. This hypothesis was supported by the finding that adrenalectomy totally abolished the diurnal rhythm of AGRP mRNA expression due to a significant decrease in AGRP mRNA levels at a time corresponding to the sham diurnal peak (at 2200 h). Interestingly, adrenalectomy had no significant effect on AGRP mRNA expression in the morning (at 1000 h), corresponding to the sham diurnal trough when circulating glucocorticoid levels are low. These results may suggest that the basal level of AGRP mRNA expression (i.e. at the nadir) is maintained independently of glucocorticoids, but the high evening expression may be glucocorticoid sensitive.

Given the fact that GR occupancy is increasing in the late afternoon and evening with rising plasma corticosterone, these observations render it tempting to speculate that the evening peak of AGRP synthesis involves GR regulation rather than MR regulation. It has been reported that the high affinity receptor, MR, is activated by low basal levels of corticosterone (0.5–2 µg/dl), whereas the activation of the low affinity receptor, GR, requires a high level of corticosterone (2–10 µg/dl) (42). In an attempt to achieve these two concentration ranges, we successfully clamped the plasma corticosterone levels at an average of 1.5 or 5.3 µg/dl with continuous release 10- and 50 mg corticosterone pellets, respectively. We found that constant corticosterone replacement in adrenalectomized rats resulted in fixed levels of AGRP mRNA expression across the 12-h light, 12-h dark cycle. Although low circulating levels of corticosterone (10-mg pellets) produced no significant effect on AGRP mRNA expression, a higher dose of corticosterone (50-mg pellets) up-regulated AGRP mRNA expression, suggesting that there exists a threshold level for corticosterone to exert a regulatory effect on the AGRP mRNA expression. This threshold level may reflect the involvement of GR activation, although interactive effects of GR and MR cannot be ruled out based on the data in the present study. Taken together, these observations suggest that glucocorticoid secretion rather than the light/dark cycle are involved in entraining the diurnal rhythm of AGRP mRNA expression. We propose that an endogenous corticosterone rhythm is necessary for generating the AGRP mRNA rhythm. However, as the corticosterone replacement employed in this study represents a chronic constant state, secondary mechanisms may occur and contribute to the diurnal regulation of AGRP mRNA expression. Therefore, we plan in future studies to evaluate the effects of acutely induced pulsatile corticosterone on AGRP mRNA expression and further investigate whether GR-selective antagonists will abolish and selective agonists will reinstate the AGRP diurnal rhythm.

As demonstrated in previous studies (50, 51), despite the association of corticosterone with feeding behavior, adrenalectomized rats exhibit a normal feeding rhythm. With 12-h light, 12-h dark cycle, adrenalectomy resulted in a reduction in total food intake in rats, but did not alter the ratio of food consumption during 12-h periods compared with that in sham controls. In fact, the diurnal feeding rhythm in adrenalectomized rats appeared to be more evident, because food intake in the light cycle was more affected by adrenalectomy. This finding is in agreement with the report by Bellinger et al. (50), but in contrast to the findings of Kumar et al. (58), who observed that food intake in the dark cycle was more affected by adrenalectomy. These discrepancies may be due to the procedure, age, and strain of the animals or the time postsurgery when food intake was measured. Moreover, although the diurnal rhythm of AGRP mRNA coincided with the overall day-night feeding rhythm, maintaining the feeding rhythm seemingly does not require a normal AGRP rhythm, as adrenalectomized rats exhibit fixed levels of AGRP mRNA expression throughout the day. Nonetheless, the possibility still remains that AGRP might influence the micropattern of food intake within the light or the dark cycle. In fact, feeding and macronutrient selection patterns in the early dark cycle have been reported to be selectively affected by adrenalectomy (58, 59). This may be attributed at least in part to the adrenalectomy-induced suppression of AGRP synthesis, as levels of AGRP mRNA expression in intact rats are high during the early dark cycle.

In contrast with AGRP mRNA, we failed to show a significant diurnal variation in POMC mRNA levels, although a tendency for POMC mRNA expression to fall during the late phase of the dark cycle, at 0200 h, was observed. This is in contrast to two previous reports in which diurnal rhythms in POMC mRNA levels were described; however, the diurnal patterns of POMC mRNA expression demonstrated in these two studies were not in agreement with each other (52, 60). Using in situ hybridization, Steiner et al. (60) observed a peak in POMC mRNA expression in the anterior 25% of the arcuate nucleus neurons at the time of onset of the light cycle, 0600 h (55), whereas Xu et al. (52), by analyzing the isolated entire arcuate nucleus using RNase protection assays, demonstrated that POMC mRNA levels were lowest at this circadian time point (50). In the present study we determined mRNA levels by measuring the integrated OD of the outlined arcuate area of in situ hybridization film autoradiograms, which represents a summation of mRNA content per cell and the total number of cells expressing POMC mRNA per section. We further conducted sample analyses of POMC mRNA in the rostral vs. caudal portions of the arcuate nucleus. Neither of these analyses, however, gave statistically significant results. The reason for these discrepancies is unclear, but may be due to the strain of animals, the sensitivity of the assay methods, and the photoperiods (32, 33). Collectively, it appears that the modulation of AGRP gene expression across the 24-h light/dark cycle may be the stronger variable in altering the AGRP/POMC balance.

Nonetheless, it is noteworthy that the posttranslational processing of POMC precursor could produce peptides with very different biological activities in terms of appetite control (61, 62), although the pharmacological and genetic evidence implies that the dominant function of the POMC gene and its products is to induce anorectic effects. Intraneuronal enzymatic processing of POMC protein in brain produces MSH and ß-endorphin, whose biological properties can be further markedly altered by subsequent N-acetylation (2, 63, 64). Although the deacetylated form of MSH has no effect on food intake after central infusions, the acetylated form of MSH, i.e. {alpha}-MSH, is a potent anorectic peptide (2). In contrast, ß- endorphin (the deacetylated form) is known to reinforce feeding (65, 66), whereas N-acetylation can eliminate all effects of ß-endorphin on food intake (2). It has been believed that these peptides may be coreleased from neuronal terminals. Thus, the ultimate behavior expression is due not only to transcriptional regulation of the POMC gene, but also to translational regulation of the POMC mRNA as well as further posttranslational processing of POMC-derived peptides. Whether multiple forms of active peptides also exist for the endogenous antagonist AGRP and whether there are additional changes in AGRP or POMC peptide processing or secretion across the light/dark cycle over 24 h remain to be determined.

In summary, we have demonstrated that physiological fluctuations in the synthesis of AGRP occur diurnally. The diurnal rhythm of AGRP mRNA expression can be abolished by depletion of glucocorticoids or constant glucocorticoid replacement. A relatively high dose of glucocorticoid stimulates AGRP mRNA expression. The present study provided evidence that glucocorticoids are involved in entraining the diurnal rhythm of AGRP mRNA expression. The precise mechanisms by which corticosterone and AGRP interact across the diurnal cycle and how they act in concert to regulate feeding behavior under physiological conditions remain to be determined.


   Acknowledgments
 
We thank Fu-Min Lei and Adam Dezure for their assistance with collecting brain tissue.


   Footnotes
 
This work was supported by a pilot feasibility grant from the University of Michigan GI Peptide Research Center (NIH Grant P30-DK-34933, to X.-Y.L.) and NIMH Grant MH-42251 (to S.J.W.).

Abbreviations: AGRP, Agouti-related protein; GR, glucocorticoid receptor; MC3R, melanocortin 3 receptor; MC4R, melanocortin 4 receptor; MR, mineralocorticoid receptor; NPY, neuropeptide Y; POMC, proopiomelanocortin; RNase, ribonuclease.

Abbreviations: AGRP, Agouti-related protein; GR, glucocorticoid receptor; MC3R, melanocortin 3 receptor; MC4R, melanocortin 4 receptor; MR, mineralocorticoid receptor; NPY, neuropeptide Y; POMC, proopiomelanocortin; RNase, ribonuclease; 2x SSC, 300 mM sodium chloride and 30 mM sodium citrate, pH 7.2.

Received February 7, 2002.

Accepted for publication June 24, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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