Hitoshi Sugihara

Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding, and adiposity

Growth hormone secretagogues (GHSs) stimulate GH secretion and food intake. GHS receptor (GHS-R) mRNA has been identified mainly in the arcuate nucleus (Arc) and ventromedial nucleus of the hypothalamus and in the pituitary. Ghrelin, an endogenous ligand for GHS-R, has recently been purified from rat stomach. Although ghrelin is also expressed in the hypothalamus, the physiological significance of the ghrelin/GHS-R system is still unknown. We have created transgenic (Tg) rats expressing an antisense GHS-R mRNA under the control of the promoter for tyrosine hydroxylase (TH), thus selectively attenuating GHS-R protein expression in the Arc. Tg rats had lower body weight and less adipose tissue than did control rats. Daily food intake was reduced, and the stimulatory effect of GHS treatment on feeding was abolished in Tg rats. GH secretion and plasma insulin-like growth factor-I levels were reduced in female Tg rats. These results suggest that GHS-R in the Arc is involved in the regulation of GH secretion, food intake, and adiposity.

Effects of insulin, leptin, and glucagon on ghrelin secretion from isolated perfused rat stomach☆

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, was originally purified from the rat stomach. Although ghrelin has been recognized as an important regulator of energy metabolism, the regulation of the ghrelin secretion is largely unknown. Here, we examined the direct effects of insulin, leptin, and glucagon on the release of ghrelin from the isolated rat stomach. The isolated pancreas-spleen-duodenum deprived preparation of rat stomach was used. After a baseline control infusion into the left gastric artery, insulin, leptin, or glucagon were infused for 15 min at concentrations of 0.1, 1, and 10 nM. The levels of immunoreactive ghrelin in the venous effluents were measured with a radioimmunoassay. Insulin and leptin inhibited ghrelin secretion dose-dependently (total amount of ghrelin release: insulin at 1 nM, 73.5+/-7.3% of the control infusion; leptin at 1 nM, 81.8+/-2.5% of the control infusion; n=5, P<0.05), while glucagon increased it dose-dependently (total amount of ghrelin released at 10 nM was 143.9+/-19.3% of the control infusion; n=5, P<0.01). These results indicate that the ghrelin responses observed in vivo could be due to direct effects of multiple hormonal signals on the stomach.

Generation of polyclonal antiserum against the growth hormone secretagogue receptor (GHS-R): evidence that the GHS-R exists in the hypothalamus, pituitary and stomach of rats.

Growth hormone (GH) secretagogues (GHSs), which stimulate GH secretion, are synthetic compounds that act through the GHS receptor (GHS-R) which has been recently cloned. We raised an antiserum in a rabbit against a synthetic peptide corresponding to amino acid residues 248-260 of the third intracellular loop of the rat GHS-R. A competitive immunoassay showed that the antiserum had a specific affinity for the target peptide. To confirm the specificity of the antiserum, the GHS-R cDNA was stably expressed in COS-7 cells. In Western blot analysis, the band was detected at 44 kDa in the extracts from COS-7 cells expressing GHS-R (COS-7/tf3-2) but not in those from wild-type COS-7 cells. Furthermore, while COS-7/tf3-2 cells were strongly immunostained for GHS-R, no GHS-R-like immunoreactivity was observed in wild-type COS-7 cells. Immunoreactive bands were also observed at approximately 46 kDa in the extracts from rat hypothalamus, pituitary and stomach by Western blot analysis. These studies are the first to show the existence of GHS-R protein in the stomach. The antiserum for the GHS-R is sensitive and specific, and it would be useful for clarifying the roles of GHS/ghrelin.

Glucocorticoids Regulate Pituitary Growth Hormone Secretagogue Receptor Gene Expression

Glucocorticoids regulate growth hormone (GH) secretion by modulating both hypothalamic and pituitary function. At the level of the pituitary, glucocorticoids increase GH and GH‐releasing hormone receptor (GHRH‐R) gene expression. To test if glucocorticoids might also regulate the pituitary expression of the recently identified GH secretagogue (GHS) receptor, GHS‐R; adult male rats were adrenalectomized or sham operated, and treated with the synthetic glucocorticoid (dexamethasone, 200 µg/day) or vehicle for 8 days. Pituitary GHS‐R mRNA levels were assessed by reverse transcriptase polymerase chain reaction (RT‐PCR). Adrenalectomy decreased pituitary GHS‐R mRNA to 45% of vehicle‐treated, sham‐operated rats (P < 0.05). Administration of dexamethasone increased GHS‐R mRNA levels in sham‐operated as well as in adrenalectomized rats (199 ± 24% (P < 0.05) and 369 ± 48% (P < 0.01) of vehicle‐treated controls). Addition of dexamethasone to primary rat pituitary cell cultures increased GHS‐R mRNA levels in a dose‐ and time‐dependent manner while the transcriptional inhibitor, actinomycin D, completely blocked the stimulatory action of dexamethasone. Taken together, these results suggest glucocorticoids directly increase pituitary GHS‐R mRNA levels by stimulating GHS‐R gene transcription.

Central glucoprivation evoked by administration of 2-deoxy-D-glucose induces expression of the c-fos gene in a subpopulation of neuropeptide Y neurons in the rat hypothalamus.

Central glucoprivation evoked by the intracerebroventricular administration of 2-deoxy-D-glucose (2DG) induces eating and suppresses growth hormone (GH) secretion in rats. To elucidate the hypothalamic mechanism of these phenomena, the induction of c-fos gene expression was examined by in situ hybridization using rats with centrally administered 2DG. Autoradiography on X-ray film showed that c-fos gene expression was transiently induced in discrete hypothalamic regions; namely the paraventricular nucleus, arcuate nucleus (ARC), the surrounding regions of the third ventricle dorsal to the ARC, and the periventricular nucleus (PeV). The time course of the expression was different in these nuclei. Double-label in situ hybridization for c-fos mRNA and neuropeptide Y (NPY) or somatostatin mRNAs revealed that 20% of the NPY neurons in the ARC expressed the c-fos gene, while a small population of somatostatin neurons (6.1% in the ARC and 2.6% in the PeV) expressed the c-fos gene following 2DG administration. Since NPY is an orexigenic neuropeptide and has an inhibitory effect on GH secretion, the data suggest that the activation of a subpopulation of NPY neurons in the ARC contributes, in part, to the increased food intake and suppression of GH secretion after central glucoprivation evoked by 2DG.

ACTH independent Cushing's syndrome occurring in siblings

Familial Cushings syndrome due to ACTH independent bilateral macronodular adrenocortical hyperplasia occurring in siblings is reported. The proband was a 69‐year‐old woman who presented with a typical Cushingoid appearance. The serum cortisol level was elevated, with a loss of diurnal rhythm, and the plasma ACTH level was undetectable. Dynamic testing showed no suppression of urinary 17‐OHCS by high dose dexamethasone and no stimulation by metyrapone. An abdominal CT scan showed bilateral adrenal enlargement. The patient died of a subarachnoid haemorrhage, and autopsy revealed a massively thickened adrenal cortex composed of nodules up to 3.5 cm in diameter. A pituitary adenoma was not found. We learned that the patients elder brother was also diagnosed at 59 years of age with Cushings syndrome due to bilateral macronodular adrenocortical hyperplasia. His plasma cortisol levels were not suppressed by high dose dexamethasone and the plasma ACTH level was undetectable. Screening of the available family members by administering 1 mg dexamethasone at midnight and performing abdominal CT scan revealed impaired suppressibility of serum cortisol associated with enlarged bilateral adrenal glands in a 64‐year‐old sister and a 54‐year‐old brother. The 64‐year‐old sister was considered as a possible ‘affected’ case in the early stages of development, because the basal level of ACTH was not suppressed and hyperplasia of the bilateral adrenal glands as revealed by CT scan was less evident.

Hypothalamic Neuropeptide Y/Y1 Receptor Pathway Activated by a Reduction in Circulating Leptin, but Not by an Increase in Circulating Ghrelin, Contributes to Hyperphagia Associated with Triiodothyronine-Induced Thyrotoxicosis

Food intake is regulated by hypothalamic neuropeptides which respond to peripheral signals. Plasma ghrelin and leptin levels reflect peripheral energy balance and regulate hypothalamic neuropeptides such as neuropeptide Y (NPY), pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and orexins. Thyroid hormone stimulates food intake in humans and rodents. However, the mechanisms responsible for this stimulation have not been fully elucidated. To investigate the hyperphagic response to triiodothyronine (T3)-induced thyrotoxicosis, adult male rats were studied 7 days after daily intraperitoneal injections of T3 or vehicle. T3-treated rats were markedly hyperphagic. During this hyperphagia, plasma leptin levels were markedly decreased. However, the expression of the ghrelin gene in the stomach and the plasma ghrelin concentrations did not differ between the 2 groups. Hypothalamic NPY mRNA levels were significantly increased and associated with a marked decreased in both hypothalamic POMC and CART mRNA levels in the T3-treated rats. Hypothalamic MCH and orexin mRNA levels did not differ between the 2 groups. In addition, hyperphagia was partially reversed by intracerebroventricular administration of the NPY Y1 receptor antagonist BIBO3304. Therefore, the decreased plasma leptin levels could contribute to hyperphagia in T3-induced thyrotoxicosis. However, plasma ghrelin levels did not contribute to this hyperphagia.

Thyroid Hormones Regulate Pituitary Growth Hormone Secretagogue Receptor Gene Expression

Thyroid hormones regulate growth hormone (GH) secretion by actions both at the hypothalamus and at the pituitary gland. At the level of the pituitary, thyroid hormones increase GH and GH‐releasing hormone receptor (GHRH‐R) mRNA expression. To test if thyroid hormones might also regulate the pituitary expression of mRNA for the recently identified GH secretagogue (GHS) receptor, GHS‐R, primary pituitary cell cultures from adult male rats were treated with triiodothyronine (T3) and GHS‐R mRNA levels were assessed by reverse transcriptase‐polymerase chain reaction. T3 increased pituitary GHS‐R mRNA levels in a dose‐ and time‐dependent manner. The stimulatory action of T3 on GHS‐R mRNA levels was also observed in the presence of the RNA synthesis inhibitor, actinomycin D, indicating that gene transcription is not required. Closer examination of the decay rates of GHS‐R mRNA in the presence of actinomycin D revealed T3 extended the half‐life of the GHS‐R mRNA from 8 h (basal) to15 h, demonstrating that T3 increases GHS‐R mRNA levels in vitro by increasing message stability.

Expression of growth hormone receptor gene in rat hypothalamus.

Growth hormone receptor (GHR) mRNA‐expressing cells in the hypothalamus were observed using hybridization histochemistry in adult male rats. Digoxigenin‐labeled cRNA corresponding to the extracellular part of rat GHR was used as a probe. Northern blotting analysis of hypothalamic total RNA from adult male rats revealed that the 4.5 kilobase (kb) transcript of the GHR gene corresponding to the GHR messenger RNA (mRNA) predominated over the 1.2 kb transcript corresponding to GH‐binding protein mRNA. GHR mRNA‐containing cells were observed in the arcuate nucleus (ARC), the periventricular nucleus (PeV), ventrolateral region of the ventromedial nucleus, the paraventricular nucleus and the supraoptic nucleus. To further understand the significance of the GHR gene expression in the hypothalamus, the effect of in vivo manipulation of GH on the somatostatin (SS) gene expression in the ARC and PeV, and the GRF gene expression in the ARC was observed among adult male rats using in situ hybridization histochemistry. Ten days after hypophysectomy, the SS mRNA level in the ARC as well as PeV was significantly lower than that in the respective nuclei of sham‐operated control rats, while the GRF mRNA level in the ARC was significantly higher than that in the ARC of control animals. Subcutaneous injection of recombinant human GH (0.33 mg) to hypophysectomized rats every 12 h for 5 days restored the SS mRNA level in the ARC and PeV, and reduced the GRF mRNA level in the ARC to that of control animals. The data suggest that GH directly acts on the hypothalamic PeV and ARC, and alters the gene expression of SS and GRF.

Genetic suppression of ghrelin receptors activates brown adipocyte function and decreases fat storage in rats.

To clarify the role of ghrelin and its receptor (GHS-R) in the regulatory mechanism of energy metabolism, we analyzed transgenic (Tg) rats expressing an antisense GHS-R mRNA under the control of the tyrosine hydroxylase (TH) promoter. Tg rats showed lower visceral fat weight and higher O(2) consumption, CO(2) production, rectal temperature, dark-period locomotor activity, brown adipose tissue (BAT) weight and uncoupling protein 1 expression compared with wild-type (WT) rats on a standard diet. A high-fat diet for 14days significantly increased body weight, visceral fat weight, and the sizes of white and brown adipocytes in WT rats but not in Tg rats compared with the corresponding standard-diet groups. Antisense GHS-R mRNA was expressed and GHS-R expression was reduced in TH-expressing cells of the vagal nodose ganglion in Tg rats. Ghrelin administered intravenously suppressed noradrenaline release in the BAT of WT rats, but not in Tg rats. These results suggest that ghrelin/GHS-R plays an important role in energy storage by modifying BAT function and locomotor activity. As our previous study showed that peripheral ghrelin-induced noradrenaline release suppression in BAT is blocked by vagotomy, the present findings also suggest that vagal afferents transmit the peripheral ghrelin signal to the sympathetic nervous system innervating BAT.