In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish

We studied the in vitro and in vivo effects of octanoylated goldfish ghrelin peptides (gGRL-19 and gGRL-12) on luteinizing hormone (LH) and growth hormone (GH) release in goldfish. gGRL-19 and gGRL-12 at picomolar doses stimulated LH and GH release from dispersed goldfish pituitary cells in perifusion and static incubation. Incubation of pituitary cells for 2 h with 10 nM gGRL-12 and 1 or 10 nM gGRL-19 increased LH-beta mRNA expression, whereas only 10 nM gGRL-19 increased GH mRNA expression. Somatostatin-14 abolished the stimulatory effects of ghrelin on GH release from dispersed pituitary cells in perifusion and static culture. The GH secretagogue receptor antagonist d-Lys(3)-GHRP-6 inhibited the ghrelin-induced LH release, whereas no effects were found on stimulation of GH release by ghrelin. Intracerebroventricular injection of 1 ng/g body wt of gGRL-19 or intraperitoneal injection of 100 ng/g body wt of gGRL-19 increased serum LH levels at 60 min after injection, whereas significant increases in GH levels were found at 15 and 30 min after these treatments. Our results indicate that, in addition to its potent stimulatory actions on GH release, goldfish ghrelin peptides have the novel function of stimulating LH release in goldfish.     

Effects of homologous ghrelins on the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus

Ghrelin is a gut-brain peptide synthesized mainly in the oxyntic mucosal cells of the stomach, and has potent growth hormone (GH)-releasing and orexigenic activities. Recently, two forms of ghrelin, ghrelin-C8 and -C10, were identified in the Mozambique tilapia (Oreochromis mossambicus). The present study describes in vitro and in vivo effects of these endogenous ghrelins on the GH/insulin-like growth factor-I (IGF-I) axis. Ghrelin-C8 (100 nM) stimulated GH release from primary cultures of pituitary cells after 4 and 8 h of incubation, whereas no effect was seen on prolactin (PRL) release. Stimulatory effects of ghrelin-C8 and -C10 (100 nM) on GH release during 6 h of incubation were blocked by pre-incubation with GHS receptor antagonist, [D-Lys(3)]-GHRP-6 (10 microM). Intraperitoneal injection of ghrelin-C8 (1 ng/g body weight) and -C10 (0.1 and 1 ng/g body weight) significantly increased plasma GH levels after 5 h. Significant increases were observed also in hepatic expression of IGF-I and GH receptor (GHR) mRNA following injections of both forms of ghrelin (0.1 and 1 ng/g body weight), although there was no effect on plasma levels of IGF-I. In the next experiment, both forms of ghrelin (1 ng/g body weight) significantly increased plasma IGF-I levels 10 h after the injection. No significant effect of either ghrelin was observed on plasma PRL levels. Both forms of GHS receptor (GHSR-1a and -1b) were found in the pituitary, clearly indicating that tilapia ghrelins stimulate primarily GH release through the GHS receptor. Stimulation of hepatic expression of IGF-I and GHR suggests metabolic roles of ghrelin in tilapia.     

The influence of mammalian and teleost somatostatins on the secretion of growth hormone from goldfish (Carassius auratus L.) pituitary fragments in vitro

The effect of various vertebrate somatostatins (SRIF) on basal growth hormone (GH) secretion from goldfish pituitary fragments was studied using an in vitro perifusion system. SRIF-14 caused a rapid and dose-dependent decrease in the rate of GH release from goldfish pituitary fragments. The half-maximal effective dose (ED50) of SRIF-14 was calculated as 1.3 nM following exposure to two minute pulses of increasing concentrations of SRIF-14, whereas the ED50 of SRIF-14 calculated after continuous exposure to sequentially increasing doses of SRIF-14 was 65 nM. This difference suggests that the pituitary fragments were less responsive to SRIF-14 in the latter experiment, possibly as a result of previous exposure to SRIF-14. SRIF-28 was found to be equipotent with SRIF-14 in decreasing basal GH secretion from the goldfish pituitary. In contrast, catfish SRIF-22, a uniquely teleost SRIF isolated from catfish pancreatic islets, did not alter GH secretion. These results provide further support for the hypothesis that SRIF-14 or a very similar molecule functions as a GH release-inhibiting factor in teleosts, indicating that this action of SRIF-14 has been fully conserved throughout vertebrate evolution.     

Somatostatins and their receptors in fish

Somatostatin (SRIF) is a multigene family of peptides. SRIF-14 is conserved with identical primary structure in species across the vertebrates. The presence of multiple SRIF genes has been demonstrated in a number of fish species. Notably, three distinct SRIF genes have been identified in goldfish. One of these genes, which encodes [Pro(2)]SRIF-14, has also been identified in sturgeon and African lungfish, and is closely associated with the amphibian [Pro(2),Met(13)]SRIF-14 gene and mammalian cortistatin gene. The main neuroendocrine role of SRIF-14 peptide that has been determined in fish is the inhibition of pituitary growth hormone secretion. The functions of SRIF-14 variant or larger forms of SRIF peptide and the regulation of SRIF gene expression remain to be explored. Type one and two SRIF receptors have been identified from goldfish and type three SRIF receptor from an electric fish. Fish SRIF receptors display considerable homology to mammalian counterparts in terms of primary structure and negative coupling to adenylate cyclase. The identification of the multiple gene family of SRIF peptides and multiple types of SRIF receptors in fish opens a new avenue for the study of physiological roles of SRIF, and the molecular and cellular mechanisms of SRIF actions in fish.     

The effect of somatostatins (SRIF-14, 25 and 28), galanin and anti-SRIF on plasma growth hormone levels in Coho salmon (Oncorhynchus kisutch, Walbaum)

Porcine galanin, somatostatins (SRIF-25 and SRIF-28) and invariant SRIF-14, known to have inhibitory-stimulatory actions on growth hormone (GH) secretion in higher vertebrates, were tested for their ability to affect plasma GH levels in coho salmon. Peptides were administered by intraperitoneal injection of 10 or 100 ng g⁻¹ body weight. All three SRIFs decreased plasma GH concentrations, their activity following the order SRIF-14 > SRIF-28 > SRIF-25. Galanin and an anti-SRIF produced pronounced, although transient increases in plasma GH.     

Somatostatin family of peptides and its receptors in fish

Somatostatin (SRIF or SS) is a phylogenetically ancient, multigene family of peptides. SRIF-14 is conserved with identical primary structure in species of all classes of vertebrates. The presence of multiple SRIF genes has been demonstrated in a number of fish species and could extend to tetrapods. Three distinct SRIF genes have been identified in goldfish. One of these genes, which encodes [Pro2]SRIF-14, is also present in sturgeon and African lungfish, and is closely associated with amphibian [Pro2,Met13]SRIF-14 gene and mammalian cortistatin gene. The post-translational processing of SRIF precursors could result in multiple forms of mature SRIF peptides, with differential abundance and tissue- or cell type-specific patterns. The main neuroendocrine role of SRIF-14 peptide that has been determined in fish is the inhibition of pituitary growth hormone secretion. The functions of SRIF-14 variant or larger forms of SRIF peptide and the regulation of SRIF gene expression remain to be explored. Type 1 and type 2 SRIF receptors have been identified from goldfish and a type 3 SRIF receptor has been identified from an electric fish. Fish SRIF receptors display considerable homology with mammalian counterparts in terms of primary structure and negative coupling to adenylate cyclase. Although additional types of receptors remain to be determined, identification of the multiple gene family of SRIF peptides and multiple types of SRIF receptors opens a new avenue for the study of physiological roles of SRIF, and the molecular and cellular mechanisms of SRIF action in fish.     

Absence of effects of short-term fasting on plasma ghrelin and brain expression of ghrelin receptors in the tilapia, Oreochromis mossambicus

Ghrelin is an important endocrine peptide that links the gastrointestinal system and brain in the regulation of food intake and energy expenditure. In human, rat, and goldfish plasma levels of ghrelin and GH are elevated in fasted animals, suggesting that ghrelin is an orexigenic signal and a driving force behind the elevated plasma levels of GH during fasting. Ghrelin's orexigenic action is mediated by the ghrelin receptor (GHS-R1a and GHS-R1b) which is localized on neuropeptide Y (NPY) neurons in the brain. Studies were undertaken to investigate the effect of short-term fasting on plasma ghrelin and brain expression of GHS-R1a, GHS-R1b, and NPY in the tilapia. Fasting for 7 days had no effect on plasma ghrelin concentrations, whereas significant increases in plasma levels of GH were observed on day 3. Fasting significantly reduced plasma levels of IGF-I on days 3 and 7, and of glucose on days 3, 5, and 7. Brain expression of ghrelin and GHS-R1b were significantly elevated in fasted fish on day 3, but were significantly reduced on day 5. This reduction was likely due to a significant increase in the expression in the fed controls on day 5 compared to day 0. No change was detected in the expression of GHS-R1a or NPY in the brain. These results indicate that ghrelin is not acting as a hunger signal in short-term fasted tilapia and is not responsible for the elevated levels of plasma GH.     

Gastrin releasing peptide reduces meal size in rats

The satiety-eliciting effect of gastrin-releasing peptide (GRP), a putative mammalian counterpart of bombesin (BBS), was examined in mildly food-deprived rats. Intraperitoneal injections of GRP resulted in a significant decrease of 30-minute food intake at 2, 4, 8 and 16 μg/kg, while 1 μg/kg had no reliable effect. Intraperitoneal GRP at 4 and 8 μg/kg did not suppress 30-minute water consumption by thirsty rats. When the dose-effect curves of GRP and BBS are compared on a molar scale, GRP is approximately 30% less potent than BBS in suppressing food intake. The two dose-effect curves are similar in shape and their regression lines have parallel slopes. These data lend further support to the hypothesis that GRP is a mammalian counterpart of BBS and strengthen the argument that they may function as endogenous satiety factors.     

Ghrelin--not just another stomach hormone

Growth hormone (GH) secretagogues (GHSs) are non-natural, synthetic substances that stimulate GH secretion via a G-protein-coupled receptor called the GHS-receptor (GHS-R). The natural ligand for the GHS-R has been identified recently; it is called ghrelin. Ghrelin and its receptor show a widespread distribution in the body; the greatest expression of ghrelin is in stomach endocrine cells. Administration of exogenous ghrelin has been shown to stimulate pituitary GH secretion, appetite, body growth and fat deposition. Ghrelin was probably designed to be a major anabolic hormone. Ghrelin also exerts several other activities in the stomach. The findings that ghrelin is produced in mucosal endocrine cells of the stomach and intestine, and that ghrelin is measurable in the general circulation indicate its hormonal nature. A maximal expression of ghrelin in the stomach suggests that there is a gastrointestinal hypothalamic-pituitary axis that influences GH secretion, body growth and appetite that is responsive to nutritional and caloric intakes.     

Plasma ghrelin levels in patients with end-stage renal disease

Ghrelin is an acylated peptide stimulating secretion of the growth hormone (GH). It was originally isolated from the rat stomach as an endogenous ligand for the growth hormone secretagogue receptor. Although being predominantly produced by endocrine cells of the gastric fundus, its secretion has been found in various tissues including the kidney. To study the influence of renal failure on plasma ghrelin levels we examined 16 patients with end-stage renal disease (ESRD) receiving hemodialysis (8 men and 8 women) and 19 controls (10 men and 9 women). Both groups were comparable in age and BMI. In all subjects we assessed plasma levels of ghrelin, leptin, soluble leptin receptor, insulin, IGF-I, IGFBP-1, IGFBP-3 and IGFBP-6. Ghrelin levels were significantly higher in the group of dialyzed patients (4.49+/-0.74 vs. 1.79+/-0.15 ng/ml; p<0.001). These patients had significantly higher levels of GH, IGFBP-1, IGFBP-6, leptin and percentage of body fat (p<0.05). In the group of patients with ESRD plasma ghrelin levels positively correlated with IGFBP-1 (p<0.01). In the control group, ghrelin positively correlated with GH concentrations (p<0.01) and negatively correlated with the levels of insulin and creatinine (p<0.05). In conclusion, patients with ESRD have higher ghrelin concentrations, which might be caused by a decreased excretion/metabolism of ghrelin in the kidney during renal failure.     

Bombesin-like peptides stimulate growth hormone secretion mediated by the gastrin-releasing peptide receptor in cattle

This study was designed to determine the effects of bombesin-like peptides (BLPs) on the secretion of growth hormone (GH) and to characterize the receptor subtypes mediating these effects in cattle. Four experiments were conducted: (1) six steers were randomly assigned to receive intravenous (IV) bolus injections of 0, 0.2, 1.0, 12.5 and 50.0μg/kg neuromedin C (NMC); (2) seven pre-weaned calves were IV injected with 1.0μg/kg NMC; (3) six steers were IV injected with 2.5μg/kg bovine gastrin-releasing peptide (GRP), 1.0μg/kg NMC combined with 20.0μg/kg [d-Lys(3)]-GHRP-6 (an antagonist for the GH secretagogue receptor type 1a [GHS-R1a]), 1.0μg/kg NMC combined with 20.0μg/kg N-acetyl-GRP(20-26)-OCH(2)CH(3) (N-GRP-EE, an antagonist for the GRP receptor), 20.0μg/kg N-GRP-EE alone, 1.0μg/kg neuromedin B (NMB); and (4) four rats were IV injected 1.0μg/kg NMC. A serial blood sample was collected before and after injection. Plasma GH levels dose-dependently increased at 5min after NMC injection and the minimal effective dose was 1.0μg/kg. Plasma GH level was elevated by GRP, but not by NMB. The NMC-induced elevation of GH was completely blocked by N-GRP-EE. The administration of NMC elevated GH level in pre-weaned calves but not in rats. Ghrelin level was unaffected by any treatments; and [d-Lys(3)]-GHRP-6 did not block the NMC-induced elevation of GH. The results indicate BLP-induced elevation of GH levels is mediated by the GRP receptor but not through a ghrelin/GHS-R1a pathway in cattle.     

Effects of ovine LH, GH and prolactin, and testosterone on serum testosterone and estradiol-17 beta levels, and seminal vesicle and testicular activity in the catfish Clarias batrachus (L.)

Intraperitoneal administrations of testosterone (0.5 microgram/g body wt), and ovine LH (1.0 microgram/g body wt), GH (5 micrograms/g body wt) and prolactin (10 micrograms/g body wt) daily for 7 days during early prespawning phase (May) in C. batrachus produced varied effects on seminal vesicle (SVSI) and testicular (GSI) weights and biochemical correlates. Testosterone and LH treatments significantly increased serum testosterone level and concentrations of total proteins, fructose, hexosamines and sialic acid in both seminal vesicles and testis. Serum E2 levels increased significantly only after testosterone treatment. GH treatment increased significantly serum testosterone level and only the concentrations of SV hexosamines and testicular protein. Prolactin, however, significantly lowered serum testosterone level and concentrations of total protein, hexosamines in both SV and testis, and testicular fructose and sialic acid levels. The results show that the stimulating effect of LH and GH on SV and testicular activity is mediated through the increased secretion of testosterone and the inhibitory effect of prolactin by decreased testosterone secretion.     

GHRP-6 mimics ghrelin-induced stimulation of food intake and suppression of locomotor activity in goldfish

Ghrelin was first identified and characterized from rat stomach as an endogenous ligand for the growth hormone secretagogue (GHS) receptor (GHS-R). Ghrelin also acts as an orexigenic factor and regulates energy balance in rodents. In goldfish, native ghrelin consists of 11 molecular variants, the major form being a 17-residue peptide with n-octanoic acid modification (n-octanoyl ghrelin17), and intraperitoneal (IP) administration of n-octanoyl ghrelin17 induces central actions such as stimulation of food intake and suppression of locomotor activity through capsaicin-sensitive afferents. Four types of GHS-Rs (1a-1, 1a-2, 2a-1 and 2a-2) have been identified in goldfish, and one GHS, GHRP-6, can activate only GHS-R2a-1 in vitro. However, there is no information about the effect of GHRP-6 on food intake and locomotor activity in goldfish in vivo. Therefore, in the present study, we examined whether IP-administered GHRP-6 would mimic the orexigenic action of n-octanoyl ghrelin17 and its suppression of locomotor activity. IP administration of GHRP-6 at 1pmol/g body weight (BW) stimulated food intake, and was equipotent to the orexigenic action of n-octanoyl ghrelin17 at 10 pmol/g BW. IP-injected GHRP-6 at 1 pmol/g BW also induced a significant decrease of locomotor activity, as was the case for IP-injected n-octanoyl ghrelin17 at 10 pmol/g BW. The action of GHRP-6 was blocked by IP-preinjected capsaicin at 160 nmol/g BW. These results suggest that the central action of GHRP-6 might be mediated via the GHS-R2a-1-signaling pathway, and subsequently through capsaicin-sensitive afferents in goldfish.     

Central effects of a novel acylated peptide, ghrelin, on growth hormone release in rats

Ghrelin, a novel growth-hormone-releasing acylated peptide, was recently isolated from rat stomach by the search of an endogenous ligand to an "orphan" G-protein-coupled-receptor. Ghrelin neuron is present in the arcuate nucleus of rat hypothalamus, but its central effect on growth hormone (GH) release has yet to be clarified. We determined the plasma GH concentration and GH mRNA level in the pituitary in response to central administration of ghrelin. A single intracerebroventricular (ICV) administration of ghrelin to rats increased the plasma GH concentration dose-dependently. A continuous ICV administration of ghrelin via osmotic pump for 12 days increased the plasma GH concentration on day 6, but did not keep the high GH concentration on day 12. The GH mRNA levels in both groups of single and continuous administration of ghrelin were not significantly different from those of controls. A single administration of growth-hormone secretagogue also did not stimulate GH synthesis. Central ghrelin stimulated GH release but did not augment GH synthesis. In addition to gastric ghrelin, hypothalamic ghrelin functions to regulate GH release.     

Growth hormone receptor deficiency results in blunted ghrelin feeding response, obesity, and hypolipidemia in mice

We have previously shown that growth hormone (GH) overexpression in the brain increased food intake, accompanied with increased hypothalamic agouti-related protein (AgRP) expression. Ghrelin, which stimulates both appetite and GH secretion, was injected intracerebroventricularly to GHR-/- and littermate control (+/+) mice to determine whether ghrelin's acute effects on appetite are dependent on GHR signaling. GHR-/- mice were also analyzed with respect to serum levels of lipoproteins, apolipoprotein (apo)B, leptin, glucose, and insulin as well as body composition. Central injection of ghrelin into the third dorsal ventricle increased food consumption in +/+ mice, whereas no change was observed in GHR-/- mice. After ghrelin injection, AgRP mRNA expression in the hypothalamus was higher in +/+ littermates than in GHR-/- mice, indicating a possible importance of AgRP in the GHR-mediated effect of ghrelin. Compared with controls, GHR-/- mice had increased food intake, leptin levels, and total and intra-abdominal fat mass per body weight and deceased lean mass. Moreover, serum levels of triglycerides, LDL and HDL cholesterol, and apoB, as well as glucose and insulin levels were lower in the GHR-/- mice. In summary, ghrelin's acute central action to increase food intake requires functionally intact GHR signaling. Long-term GHR deficiency in mice is associated with high plasma leptin levels, obesity, and increased food intake but a marked decrease in all lipoprotein fractions.     

Deterministic construct of amplifying actions of ghrelin on pulsatile growth hormone secretion

Ghrelin is a native ligand for the growth hormone secretagogue (GHS) receptor that stimulates pulsatile GH secretion markedly. At present, no formal construct exists to unify ensemble effects of ghrelin, GH-releasing hormone (GHRH), somatostatin (SRIF), and GH feedback. To model such interactions, we have assumed that ghrelin can stimulate pituitary GH secretion directly, antagonize inhibition of pituitary GH release by SRIF, oppose suppression of GHRH neurons in the arcuate nucleus (ArC) by SRIF, and induce GHRH secretion from ArC. The dynamics of such connectivity yield self-renewable GH pulse patterns mirroring those in the adult male and female rat and explicate the following key experimental observations. 1) Constant GHS infusion stimulates pulsatile GH secretion. 2) GHS and GHRH display synergy in vivo. 3) A systemic pulse of GHS stimulates GH secretion in the female rat at any time and in the male more during a spontaneous peak than during a trough. 4) Transgenetic silencing of the neuronal GHS receptor blunts GH pulses in the female. 5) Intracerebroventricular administration of GHS induces GH secretion. The minimal construct of GHS-GHRH-SRIF-GH interactions should aid in integrating physiological data, testing regulatory hypotheses, and forecasting innovative experiments.     

Stimulatory effect of n-octanoylated ghrelin on locomotor activity in the goldfish, Carassius auratus

Ghrelin is implicated in growth and feeding regulation in fish. The influence of ghrelin on behavior has not been well studied and the physiological role of des-fatty acid modification of this peptide is unclear. Therefore, the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic n-octanoylated (acyl) goldfish ghrelin and des-n-octanoylated (des-acyl) ghrelin on locomotor and orexigenic activity in the goldfish were examined. ICV administration of acyl ghrelin at doses of 1 and 2 pmol/g body weight (BW) and IP administration at 16 pmol/g BW both induced significant increases in locomotor activity during for 45-60 min after treatment. Cumulative food intake was significantly increased by ICV injection of acyl ghrelin at doses of 1 and 2 pmol/g BW and IP injection at 8 and 16 pmol/g BW during the 60-min post-treatment observation period. In contrast, ICV and IP administration of des-acyl ghrelin produced no changes in locomotor and orexigenic activity. We also analyzed fasting-induced changes in the expression of ghrelin mRNA in the brain and intestine using a real-time PCR method. The level of ghrelin mRNA in the intestine, but not in the brain, obtained from fish fasted for 7 days was significantly higher than that in fish that had been fed normally. These results suggest that, in the goldfish, acyl ghrelin, but not des-acyl ghrelin, stimulates locomotor activity and enhances food intake via central and peripheral pathways.     

Plasma levels of total and active ghrelin in acromegaly and growth hormone deficiency

Ghrelin is an endogenous growth hormone (GH) secretagogue recently isolated from the stomach. Although it possesses a strong GH releasing activity in vitro and in vivo, its physiological significance in endogenous GH secretion remains unclear. The aim of this study was to characterize plasma ghrelin levels in acromegaly and growth hormone deficiency (GHD). We investigated plasma total and active ghrelin in 21 patients with acromegaly, 9 patients with GHD and 24 age-, sex- and BMI-matched controls. In all subjects, we further assessed the concentrations of leptin, soluble leptin receptor, insulin, IGF-I, free IGF-I and IGFBP-1, 2, 3 and 6. Patients with acromegaly and GHD as well as control subjects showed similar levels of total ghrelin (controls 2.004+/-0.18 ng/ml, acromegalics 1.755+/-0.16 ng/ml, p=0.31, GHD patients 1.704+/-0.17 ng/ml, p=0.35) and active ghrelin (controls 0.057+/-0.01 ng/ml, acromegalics 0.047+/-0.01 ng/ml, p=0.29, GHD patients 0.062+/-0.01 ng/ml, p=0.73). In acromegalic patients plasma total ghrelin values correlated negatively with IGF-I (p<0.05), in GHD patients active ghrelin correlated with IGF-I positively (p<0.05). In the control group, total ghrelin correlated positively with IGFBP-2 (p<0.05) and negatively with active ghrelin (p=0.05), BMI (p<0.05), WHR (p<0.05), insulin (p=0.01) and IGF-I (p=0.05). Plasma active ghrelin correlated positively with IGFBP-3 (p=0.005) but negatively with total ghrelin and free IGF-I (p=0.01). In conclusion, all groups of the tested subjects showed similar plasma levels of total and active ghrelin. In acromegaly and growth hormone deficiency plasma ghrelin does not seem to be significantly affected by changes in GH secretion.     

Effects of ghrelin administration on decreased growth hormone status in obese animals

Obesity is characterized by markedly decreased ghrelin and growth hormone (GH) secretion. Ghrelin is a GH-stimulating, stomach-derived peptide that also has orexigenic action. Ghrelin supplement may restore decreased GH secretion in obesity, but it may worsen obesity by its orexigenic action. To reveal effects of ghrelin administration on obese animals, we first examined acute GH and orexigenic responses to ghrelin in three different obese and/or diabetic mouse models: db/db mice, mice on a high-fat diet (HFD mice), and Akita mice for comparison. GH responses to ghrelin were significantly suppressed in db/db, HFD, and Akita mice. Food intake of db/db and Akita mice were basally higher, and further stimulation of food intake by ghrelin was suppressed. Pituitary GH secretagogue receptor mRNA levels in db/db and HFD mice were significantly decreased, which may partly contribute to decreased GH response to ghrelin in these mice. In Akita mice for comparison, decreased hypothalamic GH-releasing hormone (GHRH) mRNA levels may be responsible for decreased GH response, since maximum GH response to ghrelin needs GHRH. When ghrelin was injected into HFD mice with GHRH coadministrated, GH responses to ghrelin were significantly emphasized. HFD mice injected with low-dose ghrelin and GHRH for 10 days did not show weight gain. These results indicate that low-dose ghrelin and GHRH treatment may restore decreased GH secretion in obesity without worsening obesity.