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Endocrinology Vol. 147, No. 12 5893-5900
Copyright © 2006 by The Endocrine Society

Hyperphagia Alters Expression of Hypothalamic 5-HT2C and 5-HT1B Receptor Genes and Plasma Des-Acyl Ghrelin Levels in Ay Mice

Katsunori Nonogaki, Kana Nozue and Yoshitomo Oka

Center of Excellence, Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Miyagi 980-8575, Japan

Address all correspondence and requests for reprints to: Katsunori Nonogaki M.D., Ph.D., Associate Professor, Center of Excellence, Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, 2-1 Seiryou-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. E-mail: knonogaki-tky{at}umin.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The central melanocortin (MC) pathway is suggested to mediate satiety signaling downstream of serotonin (5-HT)2C receptors. 5-HT2C receptor mutant mice consume more food, which leads to late-onset obesity and impaired glucose tolerance. Ay mice with ectopic expression of the agouti peptide, which leads to a perturbation of the central MC pathway, develop obesity and diabetes, associated with low levels of plasma total ghrelin. Here, we report that 5-wk-old Ay mice consumed more food in association with decreases in levels of plasma des-acyl ghrelin, but not active ghrelin, and increases in hypothalamic 5-HT2C and 5-HT1B receptor gene expression compared with wild-type mice matched for age and body weight. These alterations were also observed in 8-wk-old obese Ay mice. Restricted feeding significantly decreased hypothalamic 5-HT2C and 5-HT1B receptor gene expression in association with a reversal of the decreases in plasma des-acyl ghrelin levels in 5-wk-old Ay mice. Moreover, restricted feeding reduced body weight, hyperinsulinemia, and hyperglycemia in association with increases in plasma des-acyl ghrelin levels in 8-wk-old obese Ay mice. Administration of m-chlorophenylpiperazine and fenfluramine, both of which induce anorexic effects via 5-HT2C receptors and/or 5-HT1B receptors, suppressed food intake in 5- and 8-wk-old Ay mice, whereas the anorexic effects were attenuated in food-restricted Ay mice. These findings suggest that the agouti peptide down-regulates hypothalamic 5-HT2C and 5-HT1B receptor gene expression under restricted feeding conditions, whereas chronic hyperphagia increases the expression of these genes and decreases plasma des-acyl ghrelin levels in Ay mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPERPHAGIA IS A risk factor for obesity and type 2 diabetes mellitus, both of which are increasing at alarming rates in industrialized countries, including Japan and the United States. Management of eating behavior is an important therapeutic strategy for obesity and type 2 diabetes.

Brain serotonin (5-hydroxytryptamine; 5-HT), systems contribute to regulate eating behavior and energy homeostasis (1, 2, 3, 4). 5-HT2C receptors and/or 5-HT1B receptors contribute to mediate the appetite-suppressing effects of 5-HT drugs, such as m-chlorophenylpiperazine (mCPP) and d-fenfluramine (2, 5, 6, 7). 5-HT2C receptor mutant mice display leptin-independent hyperphagia that leads to late-onset obesity and impaired glucose tolerance (3).

The central melanocortin (MC) pathway is suggested to mediate satiety signaling downstream of 5-HT2C receptors (8). Ay mice have dominant alleles at the agouti locus (A), which produces ectopic expression of the agouti peptide, an antagonist of the hypothalamic MC4 receptors and MC3 receptors (9, 10, 11), and display hyperphagia and obesity. Ay mice are reportedly resistant to the anorexic effects induced by leptin and fenfluramine (8, 12, 13). In addition, obese Ay mice have lower levels of plasma ghrelin, an orexigenic peptide secreted from the stomach, than wild-type mice (14).

Ghrelin stimulates GH secretion and food intake (15). Fasting increases plasma ghrelin levels, whereas feeding reduces plasma ghrelin levels in normal animals and humans (15). Plasma ghrelin levels are also higher in patients with anorexia nervosa and lower in obese subjects than in normal individuals (15). It is therefore reasonable that plasma total ghrelin levels are lower in obese Ay mice (14). Ghrelin has two forms, however: active n-octanoyl-modified ghrelin and nonactive des-acyl ghrelin. Plasma active and des-acyl ghrelin levels during the development of obesity in Ay mice have not been evaluated.

To determine the effects of the agouti peptide on plasma active and des-acyl ghrelin levels and 5-HT signaling of satiety, we examined plasma levels of active ghrelin and des-acyl ghrelin and hypothalamic 5-HT2C and 5-HT1B receptor gene expression in food-unrestricted and food-restricted Ay mice and wild-type mice matched for age. In addition, to determine the role of hyperphagia in obesity and diabetes in Ay mice, we examined the effects of restricted feeding on obesity and diabetes in Ay mice. Moreover, to determine whether Ay mice are sensitive to 5-HT2C and/or 5-HT1B receptor stimulation-induced feeding suppression, we examined the effects of mCPP, a 5-HT2C receptor agonist, and fenfluramine, a 5-HT reuptake inhibitor and releaser that induces anorexic effects via 5-HT2C receptors and/or 5-HT1B receptors, on food intake in Ay mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
General procedures
Animals were purchased from Japan CLEA (Tokyo, Japan). Mice were housed in individual cages with free access to water and chow pellets in a light-controlled (12 h on/12 h off; lights off at 2000 h) and temperature-controlled (20–22 C) environment.

In the first experiment, age- and body-weight-matched male 5-wk-old wild-type mice (25.9 ± 0.4 g) and Ay mice (25.8 ± 0.4 g),and age-matched 8-wk-old wild-type mice (31.5 ± 0.5 g) and Ay mice (37.9 ± 0.9 g) were used. Daily food intake of the mice was measured. Animals were decapitated and blood was collected between 1000 and 1100 h. The hypothalamus, cerebral cortex, and medulla oblongata were removed to determine mRNA levels, and plasma active ghrelin and des-acyl ghrelin levels were measured. Plasma was immediately frozen and stored at –80 C until assayed for active and des-acyl ghrelin. Plasma des-acyl ghrelin levels were also measured after a 24-h fast in 5-wk-old wild-type mice (23.3 ± 0.3 g) and in 5-wk-old Ay mice (22.4 ± 0.3 g).

In the second experiment, plasma active ghrelin and des-acyl ghrelin levels and hypothalamic 5-HT2C and 5-HT1B receptor gene expression were measured in 5-wk-old Ay mice (19.7 ± 0.2 g) and wild-type mice (19.8 ± 0.4 g), which were provided 3.5 g chow pellets daily for 5 d before the experiment. Animals were decapitated, and blood was collected between 1000 and 1100 h. Plasma was immediately frozen and stored at –80 C until assayed for active and des-acyl ghrelin.

In the third experiment, 5-wk-old male Ay mice and wild-type littermates, which were provided 3.5 g chow pellets daily for 5 d before the experiment, were ip injected with saline or mCPP (5 mg/kg). Chow pellets were provided 30 min later. Intake of chow pellets was measured for the next hour. The experiment was performed between 1000 and 1200 h.

In the fourth experiment, body weight, blood glucose, plasma active ghrelin, and des-acyl ghrelin levels were measured in 8-wk-old Ay mice, which were provided 3.5 g chow pellets daily for 3 d, and freely fed Ay mice. Animals were decapitated, and blood was collected between 1000 and 1100 h. Blood glucose was measured using a glucose strip (Freestyle, Kissei, Japan). Plasma was immediately frozen and stored at –80 C until assayed for insulin and active and des-acyl ghrelin.

In the fifth experiment, 5-wk-old male Ay mice and wild-type littermates were ip injected with saline or mCPP (5 mg/kg) or fenfluramine (3 mg/kg) 30 min before the onset of the dark cycle. The intake of chow pellets was measured for the next hour after the onset of the dark cycle.

Finally, 8-wk-old male Ay mice and wild-type littermates were ip injected with saline or mCPP (5 mg/kg) twice daily (at 1000 and 1800 h) for 3 d. Daily food intake and body weight were measured.

The doses of mCPP (5 mg/kg) and fenfluramine (3 mg/kg) were selected based on the evidence that mCPP or fenfluramine-induced hypophagia was attenuated by a genetic blockade of 5-HT2C receptors (2, 5).

Plasma ghrelin and insulin assays
Plasma active ghrelin levels were measured by ELISA (active ghrelin ELISA kit and des-acyl ghrelin ELISA kit; Mitsubishi Kagaku Iatron Inc., Tokyo, Japan). For the ELISA of active ghrelin, 1 N hydrogen chloride was added to the samples at a final concentration of 0.1 N immediately after plasma separation. Plasma insulin levels were measured using a rat insulin RIA kit (Linco, St. Louis, MO).

The animal studies were conducted under protocols in accordance with the institutional guidelines for animal experiments at Tohoku University Graduate School of Medicine.

Real-time quantitative RT-PCR
Total RNA was isolated from mouse hypothalamic tissue using the RNeasy Midi kit (QIAGEN, Hilden, Germany) according to the manufacturer’s directions, and cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System for RT-PCR Kit (Invitrogen, Rockville, MD) using 1 µg total RNA. cDNA synthesized from total RNA was evaluated in a real-time PCR quantitative system (LightCycler Quick System 350S; Roche Diagnostics, Mannheim, Germany). The primers used were as follows: mouse proopiomelanocortin (POMC), sense, 5'-ATA GAT GTG TGG AGC TGG TG-3', and antisense, 5'-GGC TGT TCA TCT CCG TTG-3'; for mouse cocaine- and amphetamine-regulated transcript (CART), sense, 5'-CTG GAC ATC TAC TCT GCC GTG G-3', and antisense, 5'-GTT CCT CGG GGA CAG TCA CAC AGC-3; for mouse neuropeptide Y (NPY), sense, 5'-GCT TGA AGA CCC TTC CAT TGG TG-3', and antisense, 5'-GGC GGA GTC CAG CCT AGT GG-3'; for mouse agouti-related peptide (AGRP), sense, 5'-CAG ACC GAG CAG AAG AAG-3', and antisense, 5'-GAC TCG TGC AGC CTT ACA-3'; for mouse orexin, sense, 5'-CTC CTT CAG GCC AAC GGT A-3', and antisense, 5'-GTG GTA GTT ACG GTC GGA CA-3'; for mouse 5-HT1B receptor, sense, 5'-TGC CTG CTG GTT TCA CAT-3', 5'-ATA GAT GTG TGG AGC TGG TG-3', and antisense, 5'-GCG CAC TTA AAG CGT ATC A-3'; 5-HT2C receptor, sense, 5'-CTG AGG GAC GAA AGC AAA G-3', and antisense, 5'-CAC ATA GCC AAT CCA AAC AAA C-3'; and for mouse ß-actin, sense, 5'-TTG TAA CCA ACT GGG ACG ATA TGG-3', and antisense, 5'-GAT CTT GAT CTT CAT GGT GCT AGG-3'. The relative amount of mRNA was calculated using ß-actin mRNA as the invariant control. The data are shown as the fold change of the mean value of the control group, which received saline.

Data are presented as the mean values ± SEM (n = 5–8). Comparisons between two groups were performed using two-tailed unpaired Student’s t test. A P value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma active and des-acyl ghrelin levels in Ay mice
To exclude the effects of differences in body weight, we evaluated plasma active and des-acyl ghrelin levels in 5-wk-old Ay mice and wild-type mice matched for age and body weight. There were no genotypic differences in plasma active ghrelin levels, whereas plasma des-acyl ghrelin levels in Ay mice were lower than those in wild-type mice (Fig. 1Go, A and B). Five-week-old Ay mice consumed more food than wild-type mice (Fig. 1CGo). In addition, plasma des-acyl ghrelin levels in Ay mice were lower than those in wild-type mice after a 24-h fast (Fig. 1DGo).


Figure 1
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  		FIG. 1. Plasma active ghrelin (A) and des-acyl ghrelin levels (B) and daily food intake (C) in male 5-wk-old wild-type (WT) mice and Ay mice matched for body weight (wild-type mice, 25.9 ± 0.4 g; Ay mice, 25.8 ± 0.4 g) as described in Materials and Methods. Plasma des-acyl ghrelin levels were measured after a 24-h fast in 5-wk-old wild-type mice (23.3 ± 0.3 g) and in 5-wk-old Ay mice (22.4 ± 0.3 g) (D). Data are presented as the mean values ± SEM (n = 7–8). *, P < 0.05.

 
There were decreased plasma des-acyl ghrelin levels in 8-wk-old obese Ay mice compared with wild-type mice (Fig. 2BGo), whereas there were no genotypic differences in plasma active ghrelin levels between 8-wk-old obese Ay mice and wild-type mice (Fig. 2AGo). Eight-week-old Ay mice consumed more food and had significantly higher body weight compared with age-matched wild-type mice (Fig. 2Go, C and D).


Figure 2
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  		FIG. 2. Plasma active ghrelin (A) and des-acyl ghrelin levels (B), daily food intake (C), and body weight (D) in 8-wk-old Ay mice and wild-type (WT) mice as described in Materials and Methods. Data are presented as the mean values ± SEM (n = 7–8). *, P < 0.05.

 
Alterations of hypothalamic neuropeptide mRNA levels in Ay mice
To determine the relationship between plasma des-acyl ghrelin levels and the expression of hypothalamic neuropeptide genes involved in regulating feeding and energy homeostasis, we examined the gene expression of hypothalamic NPY, AGRP, orexin (orexigenic peptides), POMC, and CART (anorexigenic peptides), which are major neuropeptides involved in the regulation of feeding behavior (16). There were no significant differences in hypothalamic NPY and POMC mRNA levels between 5-wk-old Ay mice and wild-type mice matched for body weight, whereas hypothalamic CART, AGRP, and orexin mRNA levels were significantly decreased (0.66-, 0.73-, and 0.76-fold, respectively) (Table 1Go). There were no significant differences in the gene expression of these peptides between 8-wk-old Ay mice and wild-type mice (Table 1Go).


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  		TABLE 1. Hypothalamic neuropeptide mRNA levels in 5- and 8-wk-old Ay mice and wild-type mice as described in Materials and Methods

 
Alterations of hypothalamic 5-HT2C and 5-HT1B receptor mRNA levels in Ay mice
To determine the effects of agouti peptide on hypothalamic 5-HT2C and 5-HT1B receptor gene expression, we examined the expression of hypothalamic 5-HT2C and 5-HT1B receptor genes in 5- and 8-wk-old Ay mice and wild-type mice. Hypothalamic 5-HT2C and 5-HT1B receptor mRNA levels were significantly increased in 5-wk-old nonobese Ay mice compared with wild-type mice (5-HT2C receptor mRNA, 1.5-fold; 5-HT1B receptor mRNA, 2-fold) (Fig. 3AGo). The increases in hypothalamic 5-HT2C and 5-HT1B receptor mRNA levels were also observed in 8-wk-old obese Ay mice (5-HT2C receptor mRNA, 1.4-fold; 5-HT1B receptor mRNA, 1.4-fold; Fig. 3BGo). Next, we examined 5-HT2C and 5-HT1B receptor mRNA levels in the cerebral cortex and medulla oblongata where the agouti peptide is not translated. There were no significant differences in 5-HT2C and 5-HT1B receptor mRNA in the cerebral cortex and medulla oblongata between 5-wk-old Ay mice and wild-type mice (Fig. 3Go, C and D).


Figure 3
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  		FIG. 3. 5-HT2C receptor (A) and 5-HT1B receptor (B) mRNA levels in the hypothalamus were evaluated in freely fed 5-wk-old and 8-wk-old Ay mice and wild-type (WT) mice. 5-HT2C receptor mRNA levels in cerebral cortex and medulla oblongata (C) and 5-HT1B receptor mRNA levels in cerebral cortex and medulla oblongata (D) were evaluated in freely fed 5-wk-old Ay mice and wild-type mice as described in Materials and Methods. Data are presented as the mean values ± SEM (n = 5). *, P < 0.05.

 
Effects of restricted feeding on plasma active and des-acyl ghrelin levels, hypothalamic 5-HT1B and 5-HT2C receptor mRNA levels, and the responsiveness to mCPP in 5-wk-old Ay mice
To determine whether the alterations of plasma des-acyl ghrelin and hypothalamic 5-HT2C and 5-HT1B receptor mRNA levels result from direct effects of the agouti peptide or to secondary effects induced by hyperphagia, we examined the effects of restricted feeding (3.5 g/d for 5 d) on plasma active and des-acyl ghrelin levels, hypothalamic 5-HT1B and 5-HT2C receptor mRNA levels, and the responsiveness to mCPP in 5 wk-old Ay mice and wild-type mice. There were no significant differences in plasma active and des-acyl ghrelin levels between the food-restricted 5 wk-old Ay mice and wild-type mice (Fig. 4Go, A and B). In addition, hypothalamic 5-HT2C and 5-HT1B receptor mRNA levels were significantly decreased in food-restricted Ay mice compared with wild-type mice (Fig. 4CGo). Both Ay mice and wild-type mice consumed 3.5 g of food during the light cycle for 5 d and were fasted during the dark cycle. After a 15-h fast, mCPP (5 mg/kg) had no effect on food intake in food-restricted Ay mice, whereas it significantly suppressed food intake in food-restricted wild-type mice (Fig. 4DGo).


Figure 4
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  		FIG. 4. Effects of restricted feeding on plasma active ghrelin (A) and des-acyl ghrelin levels (B) and hypothalamic 5-HT2C and 5-HT1B receptor mRNA levels (C) and effects of mCPP on food intake (D) in 5-wk-old Ay mice and wild-type (WT) mice. Plasma active and des-acyl ghrelin levels and hypothalamic 5-HT2C and 5-HT1B receptor mRNA levels were measured, and the drug was administered as described in Materials and Methods. Data are presented as the mean values ± SEM (n = 6). *, P < 0.05.

 
Effects of restricted feeding on body weight, blood glucose, plasma insulin, and active and des-acyl ghrelin levels in 8-wk-old Ay mice
Restricted feeding (3.5 g/d for 3 d) significantly increased plasma des-acyl ghrelin levels in Ay mice (Fig. 5BGo), whereas it had no effect on plasma active ghrelin levels (Fig. 5AGo). In addition, restricted feeding significantly reduced body weight and normalized hyperglycemia in 8-wk-old Ay mice (Fig. 5Go, C and D). The blood glucose levels in food-restricted 8-wk-old Ay mice were significantly lower than those in nonobese 5-wk-old Ay (food-restricted 8-wk-old Ay mice, 127 ± 5 mg/dl; nonobese 5-wk-old Ay mice, 185 ± 8 mg/dl; n = 6 for each group; P < 0.05). In addition, the blood glucose levels in food-restricted 8-wk-old Ay mice were significantly lower than those in 8-wk-old wild-type mice (8-wk-old wild-type mice, 166 ± 6 mg/dl; n = 6; P < 0.05). Moreover, restricted feeding significantly reduced plasma insulin levels in 8-wk-old Ay mice (freely fed Ay mice, 10.8 ± 1.6 ng/ml; restricted-fed Ay mice, 2.3 ± 0.4 ng/ml; n = 6 for each group; P < 0.05).


Figure 5
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  		FIG. 5. Effects of restricted feeding on plasma active (A) and des-acyl ghrelin levels (B), body weight (C), and blood glucose levels (D) in 8-wk-old male Ay mice. Plasma active and des-acyl ghrelin levels and blood glucose levels were measured as described in Materials and Methods. Each column and bar represents the mean value ± SEM of eight mice. Ff, Freely feeding; Rf, restricted feeding. *, P < 0.05.

 
Effects of mCPP and fenfluramine on food intake in Ay mice
To characterize hyperphagia in Ay mice, we examined the responsiveness to 5-HT drugs such as mCPP and fenfluramine, which cause anorexic effects via 5-HT2C receptors and/or 5-HT1B receptors, in Ay mice. Both mCPP and fenfluramine significantly suppressed food intake in Ay mice and wild-type mice (Fig. 6Go, A and B). In addition, chronic treatment with mCPP for 3 d decreased daily food intake (Fig. 7Go, A and B) and increased weight loss (Fig. 7Go, C and D) in both Ay mice and wild-type mice.


Figure 6
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  		FIG. 6. Effects of mCPP (A) or fenfluramine (Fen) (B) on food intake in 5-wk-old male Ay mice and wild-type (WT) mice after onset of the dark cycle. Drugs were administered as described in Materials and Methods. Each column and bar represents the mean value ± SEM of seven to eight mice. *, P < 0.05.

 

Figure 7
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  		FIG. 7. Effects of chronic treatment with mCPP on food intake and body weight in 8-wk-old Ay mice (A and C) and wild-type (WT) mice (B and D). Each column and bar represents the mean value ± SEM of seven to nine mice. Basal body weight for wild-type, saline controls (white bars) was 29.0 ± 0.7 g; for the mCPP treatment group (black bars), 29.8 ± 0.3 g; for Ay mice, saline controls (white bars), 37.7 ± 0.5 g; and for the mCPP treatment group (black bars), 37.6 ± 0.5 g. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results indicate that plasma des-acyl ghrelin levels, but not active ghrelin levels, are decreased in nonobese Ay mice that consume more food. The low plasma des-acyl ghrelin levels were observed not only in the fed state but also in a 24-h fasted state of nonobese Ay mice. Moreover, decreases in plasma des-acyl ghrelin levels were observed in 8-wk-old obese Ay mice. Thus, decreases in plasma des-acyl ghrelin, but not active ghrelin levels, preceded the development of obesity in Ay mice. In addition, restricted feeding reversed the decrease in plasma des-acyl ghrelin levels in nonobese Ay mice and increased plasma des-acyl ghrelin levels in obese Ay mice. These findings suggest that chronic hyperphagia induced by the agouti peptide, rather than the direct effects of the agouti peptide, contributed to the decreases in des-acyl ghrelin levels, leading to an increased ratio of active/des-acyl ghrelin. Active ghrelin is an endogenous ligand for the GH secretagogue receptor 1a, whereas des-acyl ghrelin exhibits its bioactivities via a mechanism independent of the GH secretagogue receptor 1a (15). It is therefore unlikely that des-acyl ghrelin competes with active ghrelin for binding sites.

Because the acylation of ghrelin is assumed to be essential for its actions, des-acyl ghrelin, which lacks the fatty acid modification of ghrelin, is assumed to be devoid of biological effects (15). Des-acyl ghrelin does not stimulate GH release (15). Several recent studies, however, indicate that des-acyl ghrelin has biological effects on cell proliferation, survival, and metabolism of cardiomyocytes, endothelial cells, adipocytes, myocytes, and myelocytes and on food intake (15, 17, 18, 19, 20, 21, 22, 23, 24), although the effects of des-acyl ghrelin remain controversial. Toshinai et al. (20) reported that central des-acyl ghrelin increases food intake, whereas peripheral des-acyl ghrelin has no effects on food intake in rats and C57BL6J and ddy mice. Asakawa et al. (21) and Chen et al. (22) reported that either central or peripheral administration of des-acyl ghrelin suppresses food intake in ddy mice and rats.

In addition, Thompson et al. (23) reported that peripheral des-acyl ghrelin directly promotes adipogenesis in the bone marrow of GH-deficient dwarf (dw/dw) rats in vivo, whereas Zhang et al. (24) reported that des-acyl ghrelin inhibits adipogenesis by stimulating cell proliferation in 3T3-L1 cells. Moreover, Asakawa et al. (21) reported that transgenic mice overexpressing des-acyl ghrelin have reduced food intake, body weight, and fat pad mass weight accompanied by decreased linear growth, whereas Ariyasu et al. (25) reported that similar transgenic mice did not reduce food intake and body fat mass despite reduced longitudinal growth and body weight. Thus, the results of studies of the effects of des-acyl ghrelin on feeding and adipogenesis are not consistent. On the other hand, knockout of the ghrelin gene in mice has no effect on food intake or body weight changes during growth (26, 27). Because of the low plasma des-acyl ghrelin levels in pre-obese and obese Ay mice, it is unlikely that des-acyl ghrelin promotes food intake and adiposity in Ay mice.

In the hypothalamus, ghrelin neurons contact the cell bodies and dendrites of NPY/AGRP and POMC neurons in the arcuate nucleus (28) and orexin neurons in the lateral hypothalamus (20, 29). The present study, however, demonstrates that alterations of the expression of hypothalamic NPY, AGRP, POMC, CART, and orexin genes were not proportional to the alterations of plasma des-acyl ghrelin and the ratio of active and des-acyl ghrelin. Ay mice have an agouti peptide-induced disturbance of the {alpha}-MSH ligand for MC-4 receptors that leads to hyperphagia (9, 10). These neuropeptides, therefore, are unlikely to contribute to hyperphagia, decreases in plasma des-acyl ghrelin levels, or the ratio of plasma active/des-acyl ghrelin in Ay mice.

We previously reported that fasting increases hypothalamic 5-HT2C and 5-HT1B receptor gene expression (30). In the present study, 5-HT2C and 5-HT1B receptor gene expression was increased in nonobese hyperphagic Ay mice and decreased by food restriction. The agouti peptide might therefore have an inhibitory role in hypothalamic 5-HT2C and 5-HT1B receptor gene expression under restricted feeding conditions. The increase in hypothalamic 5-HT2C and 5-HT1B receptor gene expression might be a compensatory response to hyperphagia induced by the agouti peptide in Ay mice, because 5-HT2C and 5-HT1B receptors have an inhibitory role in feeding (2, 3, 4, 5, 6, 7). The present results also demonstrate that low plasma des-acyl ghrelin levels are not associated with alterations of hypothalamic NPY, AGRP, orexin, POMC, and CART gene expression in nonobese Ay mice. The increases in hypothalamic 5-HT2C and 5-HT1B receptor gene expression preceded the development of obesity in Ay mice, indicating that the induction of hypothalamic 5-HT2C and 5-HT1B receptor gene expression is not a secondary response to obesity and is independent of hypothalamic NPY, AGRP, orexin, POMC, and CART gene expression in Ay mice. Hypothalamic 5-HT2C and 5-HT1B receptor gene expression is likely to be inversely proportional to alterations of plasma des-acyl ghrelin in Ay mice and is altered by the feeding conditions.

Other investigators previously reported that Ay mice are resistant to the anorexic effects of fenfluramine (8). The present study, however, demonstrates that hyperphagic Ay mice are responsive to mCPP- and fenfluramine-induced appetite suppression. mCPP and fenfluramine increase hypothalamic POMC gene expression and POMC neuronal activity in vivo and in vitro (8, 30). 5-HT drugs such as mCPP and fenfluramine interact with POMC neurons in the hypothalamus and might stimulate POMC neurons to release enough {alpha}-MSH to overcome agouti blockade of MC receptors, because Ay mice are sensitive to MC agonist-induced feeding suppression (10). In addition, mCPP and fenfluramine increase hypothalamic CART gene expression (30). CART neurons have a different downstream pathway from MC (31, 32). POMC as well as CART might contribute to the anorexic effects of mCPP and fenfluramine.

The discrepancy between our results and previous results by other investigators might be because of the different methods used. Heisler et al. (33) examined the effects of fenfluramine on food intake for 50 min after ip fenfluramine injection. On the other hand, we administered the drugs ip, and 30 min later food was provided, and then we measured food intake for the next 1 h, because it usually takes about 30 min for drugs to act systemically after ip injection. The feeding state of mice might also contribute to the discrepancy. The present study demonstrates that the anorexic effects induced by stimulation of 5-HT2C and 5-HT1B receptors were attenuated in food-restricted Ay mice, but not in hyperphagic Ay mice. In a pharmacological study, Heisler et al. (33) used 4-wk-old Ay and wild-type mice, which might not have differed in food intake before the experiment. Restricted feeding for 3 d markedly reduced body weight, whereas treatment with mCPP slightly reduced body weight in Ay mice despite the suppression of feeding. mCPP-induced decreases in locomotor activity (33, 34) might contribute to this difference, because decreases in locomotor activity decrease energy expenditure.

In summary, these results suggest that the agouti peptide down-regulates hypothalamic 5-HT2C and 5-HT1B receptor gene expression under restricted feeding conditions and that chronic hyperphagia contributes to decreases in plasma des-acyl ghrelin levels and increases in hypothalamic 5-HT2C and 5-HT1B receptor gene expression in Ay mice. Hyperphagia predisposes Ay mice to obesity, hyperinsulinemia, and hyperglycemia.


   Footnotes
 
This work was supported by a Grant in-Aid for Scientific Research (C2) and Human Science Research (KH21016).

The authors have nothing to disclose.

First Published Online September 14, 2006

Abbreviations: AGRP, Agouti-related peptide; CART, cocaine- and amphetamine-regulated transcript; 5-HT, serotonin (5-hydroxytryptamine); MC, melanocortin; mCPP, m-chlorophenylpiperazine; NPY, neuropeptide Y; POMC, proopiomelanocortin.

Received April 3, 2006.

Accepted for publication September 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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