Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic growth hormone secretagogues (GHSs), ghrelin specifically releases growth hormone (GH) after intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. Recently, we showed that a single central administration of ghrelin increased food intake and hypothalamic agouti-related protein (AGRP) gene expression in rodents, and the orexigenic effect of this peptide seems to be independent of its GH-releasing activity. However, the effect of chronic infusion of ghrelin on food consumption and body weight and their possible mechanisms have not been elucidated. In this study, we determined the effects of chronic intracerebroventricular treatment with ghrelin on metabolic factors and on neuropeptide genes that are expressed in hypothalamic neurons that have been previously shown to express the GHS-R and to regulate food consumption. Chronic central administration of rat ghrelin (1 g/rat every 12 h for 72 h) significantly increased food intake and body weight. However, it did not affect plasma insulin, glucose, leptin, or GH concentrations. We also found that chronic central administration of ghrelin increased both neuropeptide Y (NPY) mRNA levels (151.0 ؎ 10.1% of saline-treated controls; P < 0.05) and AGRP mRNA levels (160.0 ؎ 22.5% of saline-treated controls; P < 0.05) in the arcuate nucleus. Thus, the primary hypothalamic targets of ghrelin are NPY/AGRPcontaining neurons, and ghrelin is a newly discovered orexigenic peptide in the brain and stomach. Diabetes 50:2438 -2443, 2001 E nergy intake and expenditure are tightly regulated in mammals (1). Several neuronal populations, particularly in the hypothalamic arcuate nucleus (ARC), are involved in the regulation of energy homeostasis and have been implicated as possible targets of orexigenic peptides (1). These include neuropeptide Y (NPY)/agouti-related protein (AGRP) coexpressing neurons, pro-opiomelanocortin (POMC) neurons, and growth hormone-releasing hormone (GHRH) neurons, which are known to be stimulated (NPY and AGRP) or suppressed (POMC and GHRH) by starvation (2-10). However, central administration of NPY, AGRP, and GHRH increased food intake in rats, whereas ␣-melanocyte-stimulating hormone (␣-MSH), an end product of POMC processing, decreased food intake when injected centrally (11-13).The growth hormone secretagogues (GHSs) are synthetic peptide and nonpeptide compounds that stimulate the pulsatile release of growth hormone (GH) after intravenous administration (14). Central administration of GHSs also stimulates feeding behavior (15). GHS receptor (GHS-R) mRNA is expressed in the pituitary gland and in several areas of the brain, including the hypothalamus (16). In the hypothalamus, GHS-R mRNA is expressed in NPY/AGRP-, POMC-, and GHRH-containing neurons (17)(18)(19). Ghrelin, an end...

IntroductionGrowth hormone secretagogues (GHSs), which were developed from met-enkephalin based on conformational energy calculations, peptide chemistry, and biological activity, stimulate GH secretion via a specific receptor (1). An intracerebroventricular (ICV) injection of GHS also stimulates food intake in freely feeding rats (2). The GHS receptor (GHS-R) was cloned and found to be a member of the G protein-coupled receptor superfamily (3). The expression of GHS-R mRNA is observed by in situ hybridization or an RNase protection assay mainly in the arcuate nucleus (Arc) and ventromedial nucleus of the hypothalamus and in the pituitary (4). Ghrelin, an endogenous ligand for GHS-R, has recently been isolated from stomach extracts of rats and subsequently cloned in rats and humans (5). Ghrelin-producing cells are found in the hypothalamus as well as in the stomach (5). As expected, ICV administration of ghrelin stimulated GH secretion and food intake in rats (6, 7). Daily peripheral administration of ghrelin caused weight gain by reducing fat utilization in mice and rats (8). However, the physiological role of endogenous ghrelin in the hypothalamus is still unknown.To attenuate GHS-R expression in vivo, we attempted to create transgenic (Tg) rats with impaired GHS-R function in the hypothalamus, especially in the Arc. For this purpose, we used a construct that expresses GHS-R-specific antisense RNA under the control of the promoter for tyrosine hydroxylase (TH). TH is the rate-limiting enzyme in catecholamine biosynthesis and is a marker for the dopaminergic neurons in the hypothalamus. TH-like immunoreactivity is present in most neurons in the ventral part of the Arc that contain GH-releasing hormone (GHRH) (9). GHS-R mRNA hybridizing cells show an extensive overlap with GHRH-expressing neurons (10). These results suggest that a certain number of GHS-R-expressing neurons in the Arc also contain TH. Tg mice bearing a fusion gene containing the TH promoter and the coding region of the human GH gene have been generated, and these Tg mice showed human GH-like immunoreactivity in all the catecholaminergic neurons in the hypothalamus (11). Based on this report, an antisense GHS-R mRNA under the control of the TH promoter would be expected to suppress GHS-R expression in the Arc. Therefore, in the present study we have generated Tg rats that express an antisense GHS-R mRNA under the control of the TH promoter to determine the physiological role of the ghrelin/GHS-R system in the hypothalamus. MethodsGeneration of Tg rats. To construct the antisense GHS-R fusion gene, a synthetic 108-nucleotide DNA fragment spanning the 5′ extracellular region of GHS-R was cloned in the antisense orientation into the vector 4.5THpAL+ (kindly provided by Dona Chikaraishi), which contains 4.5 kb of the upstream region of the rat TH gene (11). The antisense orientation of the cloned fragments was verified by DNA sequencing. A 4.8-kb Growth hormone secretagogues (GHSs) stimulate GH secretion and food intake. GHS receptor (GHS-R) mRNA has b...

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Neonatal female and male rats of the Sprague-Dawley strain were injected subcutaneously with a daily dose of MSG (2.2–4.2 mg/g body wt.) beginning on the 2nd day of life. The rats were autopsied at 40 and 110 days of age. At 40 days of age, the body weight and nasoanal lengths were significantly reduced in the MSG-treated rats. At 110 days, the body lengths of MSG-treated rats were approximately 10–12% shorter than those of control rats, and calculation of the ‘Lee’ index indicated a significant increase in carcass fat. Food consumption studies, carried out at 75 days of age, showed a significant hypophagia in some MSG-treated rats. Upon autopsy at 40 days of age, the absolute weights of the thyroid and adrenal glands of both sexes were significantly decreased from those of control rats; but when corrected for body weight, these differences were marginal. At 110 days, the absolute weights of the adrenal and thyroid glands of both sexes were significantly reduced from their respective control levels. Gonadal weights of MSG-treated rats were significantly reduced at 40 and 110 days of age. Size and weight of the anterior pituitary glands of both male and female MSG-treated rats were significantly reduced from control values. There was a marked decrease in growth hormone and luteinizing hormone content in the anterior pituitaries of male and female MSG-treated rats at 40 days. However, thyrotropin content of the anterior pituitary of MSG-treated male rats did not show any significant change from control levels at 40 days of age.

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic GHSs, ghrelin specifically releases GH following intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. However, the central actions of ghrelin have not been elucidated. Here, we used radioactive in situ hybridization histochemistry to examine the effects of central administration of rat ghrelin on neuropeptide genes that are expressed in hypothalamic neurons that were previously shown to express GHS-R. We found that central administration of ghrelin increased both agouti-related protein (AGRP) mRNA levels (245.8 +/- 28.3% of the saline-treated controls; p < 0.01) in the hypothalamus and food intake (5.7 +/- 0.9 g ghrelin vs. 1.9 +/- 0.5 g saline; p < 0.05). On the other hand, 1 microg of rat ghrelin central administration did not alter the episodic GH release of freely moving adult male rats. Thus, ghrelin has an alternative role in stimulating food intake via an increase of AGRP rather than the release of GH from the pituitary.