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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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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