Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

We have identified ghrelin and cDNA encoding precursor protein from the stomach of a euryhaline tilapia, Oreochromis mossambicus. The sequence of 20-amino acid tilapia ghrelin is GSSFLSPSQKPQNKVKSSRI. The third serine residue was modified by n-decanoic acid. The carboxyl-terminal end of the peptide possessed an amide structure. RT-PCR analysis revealed high levels of gene expression in the stomach and low levels in the brain, kidney and gill. Tilapia ghrelin stimulated growth hormone (GH) and prolactin (PRL) release from the organ-cultured tilapia pituitary at a dose of 10 nM. Thus, a novel regulatory mechanism of GH secretion by gastric ghrelin seems to be conserved in the tilapia. Stimulation of PRL release by homologous ghrelin has been reported in human, bullfrog and eel, and suggests the presence of growth hormone secretagogue receptor not only on somatotrophs but also on PRL cells of the tilapia pituitary.     

Rat ghrelin stimulates growth hormone and prolactin release in the tilapia,Oreochromis mossambicus

Recently, ghrelin (Ghr), a new peptide which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL(177) and PRL(188)) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL(177) and PRL(188) in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL(188), but significantly increased PRL(177) content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.     

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

Effects of fasting on the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis were examined in the tilapia (Oreochromis mossambicus) acclimated to fresh water. Fasting for 2 weeks resulted in significant reductions in body weight, specific growth rate and hepatosomatic index in both males and females. Significant reductions in specific growth rates were observed after 1 and 2 weeks in both sexes, although the decrease in body weight was not significant in the female. A significant reduction was also seen in the condition factor of females after 2 weeks. No change was seen in the gonadosomatic index in either sex. Two weeks of fasting also produced a significant reduction in plasma IGF-I but not in plasma GH, prolactin (PRL(188)) or cortisol. Significant reductions in the hepatic IGF-I mRNA were seen in both sexes. On the other hand, a significant increase was observed in cortisol receptor mRNA in the female liver. Plasma IGF-I levels were correlated significantly with specific growth rate, condition factor and hepatosomatic index, indicating that plasma IGF-I is a good indicator of growth in the tilapia. No change was seen in plasma glucose or osmolality after 2 weeks of fasting. During fasting, tilapia appears to convert metabolic energy from growth to basal metabolism including maintenance of ion and water balance.     

Endocrine and orexigenic actions of growth hormone secretagogues in rainbow trout (Oncorhynchus mykiss)

The effects of growth hormone secretagogues (GHSs) on the teleost somatotropic axis are poorly understood, particularly with respect to insulin-like growth factor-I (IGF-I) and the IGF-binding proteins (IGFBPs). To assess the endocrine and orexigenic responses of rainbow trout (Oncorhynchus mykiss) to GHS treatment, animals were injected with human GHRH(1-29)-amide, KP-102 or rat ghrelin at 0, 1 or 10 pmol/g body mass. Feed intake was tested at 2 and 5 h post-injection and plasma levels of growth hormone (GH), IGF-I and the IGFBPs were determined at 3, 6 and 12 h post-injection. Feed intake was significantly elevated by all of the GHSs tested at both post-injection time points. All GHSs elevated plasma GH levels in a time-dependent manner. Plasma IGF-I levels were elevated by all GHSs at 3 h post-injection, whereas those animals treated with KP-102 and ghrelin exhibited depressions at 6 h. Four IGFBPs were identified in the plasma by western blotting. Levels of the 20 kDa IGFBP decreased over the sampling time. Levels of the 32 kDa IGFBP were significantly depressed by all GHSs tested. Levels of the 42 kDa IGFBP were significantly elevated by all GHSs tested. Plasma levels of the 50 kDa IGFBP was decreased in some treatment groups at 3 h, but elevated by 6 h in the ghrelin-treated groups and elevated in all treatment groups by 12 h post-injection. The endocrine and orexigenic responses demonstrate that GHSs influence the teleost neuroendocrine system beyond short-term actions (<3 h post-injection) on GH release and the responses of the IGFBPs to GHS treatment support this notion and clarify their identification as functional homologues to mammalian IGFBPs.     

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.     

Interleukin-1 inhibits the induction of insulin-like growth factor-I by growth hormone in CWSV-1 hepatocytes

Sepsis results in hepatic "growth hormone (GH) resistance" with reductions in plasma IGF-I despite a two- to fourfold increase in circulating GH. In this study, we examine the effects of IL-1 on GH receptor (GHR) expression, GH signaling (via the JAK/STAT and MAPK pathways), and the induction of gene expression [IGF-I mRNA and serine protease inhibitor (Spi) 2.1] by GH in CWSV-1 hepatocytes. Incubation of cells with IL-1beta (10 ng/ml, 24 h) had no effect on the relative abundance of GHR or signaling proteins JAK2, STAT5b, and ERK1/2 in cell lysates. Baseline phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 was minimal. After GH stimulation, tyrosine phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 increased 2- to 10-fold. However, neither the time course nor the magnitude of GHR, JAK2, and ERK1/2 phosphorylation by GH were significantly altered by IL-1. The GH-induced translocation of STAT5b to the nucleus was not prevented by IL-1. Although phosphorylated STAT5 in nuclear extracts from GH + IL-1 cells was decreased by 24% (vs. controls) 15 min after GH stimulation, this did not result in reduced STAT5-DNA binding activity. Pretreatment with IL-1 did not significantly decrease IGF-I mRNA stability. We conclude that IL-1 only minimally affects the time course of JAK2/STAT5 and MAPK signaling by GH. Therefore, an inhibitory effect of IL-1 on IGF-I and Spi 2.1 mRNA synthesis by GH represents the most likely mechanism for IL-1-mediated GH resistance.     

Glucose regulates ghrelin, neuropeptide Y, and the GH/IGF-I axis in the tilapia, Oreochromis mossambicus

In general, a fish's ability to clear glucose is sluggish in relation to mammals, which has lead to the idea that fish are glucose intolerant. It has been reported that circulating glucose levels do fluctuate in response to environmental challenges. Recent reports suggest that glucose may function as a metabolic signal regulating ‘glucosensors’ in the brain in fish, as has been reported in mammals. The current study was designed to investigate the effect of glucose on ghrelin and neuropeptide Y (NPY) signaling in the brain, and on the growth hormone/insulin-like growth factor-I (GH/IGF-I) in the tilapia, Oreochromis mossambicus. Glucose treatment significantly increased plasma and stomach mRNA levels of ghrelin. In the brain, mRNA levels of the ghrelin receptor (GRLN-R) were significantly reduced, whereas NPY mRNA levels were significantly elevated; suggesting that NPY containing neurons may be a “glucosensor” as reported in mammals. Glucose treatment resulted in changes in the GH/IGF-I axis. Liver mRNA levels of both GH receptors (GHR1 and GHR2) were significantly elevated, whereas liver IGF-I mRNA were unaltered by glucose treatment. No change in plasma or pituitary mRNA levels of GH was observed. Glucose significantly reduced plasma IGF-I levels. These data show that glucose regulates endocrine factors involved in appetite, growth, and possibly energy homeostasis, and suggests that glucose may be acting as a signal of metabolic status in fish.     

Insights into a role of GH secretagogues in reversing the age-related decline in the GH/IGF-I axis

Growth hormone (GH) secretion and serum insulin-like growth factor-I (IGF-I) decline with aging. This study addresses the role played by the hypothalamic regulators in the aging GH decline and investigates the mechanisms through which growth hormone secretagogues (GHS) activate GH secretion in the aging rats. Two groups of male Wistar rats were studied: young-adult (3 mo) and old (24 mo). Hypothalamic growth hormone-releasing hormone (GHRH) mRNA and immunoreactive (IR) GHRH dramatically decreased (P < 0.01 and P < 0.001) in the old rats, as did median eminence IR-GHRH. Decreases of hypothalamic IR-somatostatin (SS; P < 0.001) and SS mRNA (P < 0.01), and median eminence IR-SS were found in old rats as were GHS receptor and IGF-I mRNA (P < 0.01 and P < 0.05). Hypothalamic IGF-I receptor mRNA and protein were unmodified. Both young and old pituitary cells, cultured alone or cocultured with fetal hypothalamic cells, responded to ghrelin. Only in the presence of fetal hypothalamic cells did ghrelin elevate the age-related decrease of GH secretion to within normal adult range. In old rats, growth hormone-releasing peptide-6 returned the levels of GH and IGF-I secretion and liver IGF-I mRNA, and partially restored the lower pituitary IR-GH and GH mRNA levels to those of young untreated rats. These results suggest that the aging GH decline may result from decreased GHRH function rather than from increased SS action. The reduction of hypothalamic GHS-R gene expression might impair the action of ghrelin on GH release. The role of IGF-I is not altered. The aging GH/IGF-I axis decline could be rejuvenated by GHS treatment.     

LHRH-A对尼罗罗非鱼生长及生长轴相关基因表达的影响

促性腺激素释放激素(GnRH)的主要作用是刺激脑垂体促性激素(GtH)的释放, 亦可促进鱼类生长激素(GH)的释放。促黄体素释放激素类似物(LHRH-A)是哺乳类GnRH的类似物, 为了分析LHRH-A对尼罗罗非鱼生长调节的作用, 设计了长期和短期2个实验, 采用腹腔注射(剂量0.1 μg/g体重)方法, 分析LHRH-A对尼罗罗非鱼绝对生长率、特定生长率、肝体系数和肥满度的影响, 并应用荧光实时定量PCR方法检测在注射LHRH-A后不同时段(6h、12h、24h、2周)尼罗罗非鱼垂体GH、肝脏GHR和肝脏IGF-I基因的表达变化。结果表明, LHRH-A组尼罗罗非鱼的绝对生长率、特定生长率、肝体系数、肥满度均显著高于对照组(P<0.05); 注射LHRH-A后12h、24h垂体GH mRNA表达水平均显著升高(P<0.05), 2周后恢复到对照组水平; 注射LHRH-A后24h和2周肝脏GHR mRNA表达水平显著上升(P<0.05); 注射LHRH-A后6h肝脏IGF-I mRNA表达水平显著升高(P<0.05), 12h、24h和2周恢复到对照组水平。以上结果提示, LHRH-A可显著上调尼罗罗非鱼生长轴相关基因的表达, 从而促进鱼类的生长。     

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 long-term treatment with growth hormone-releasing peptide-2 in the GHRH knockout mouse

Growth hormone (GH) secretagogues (GHS) stimulate GH secretion in vivo in humans and in animals. They act on the ghrelin receptor, expressed in both the hypothalamus and the pituitary. It is unknown whether GHSs act predominantly by increasing the release of hypothalamic GH-releasing hormone (GHRH) or by acting directly on the somatotroph cells. We studied whether a potent GHS could stimulate growth in the absence of endogenous GHRH. To this end, we used GHRH knockout (GHRH-KO) mice. These animals have proportionate dwarfism due to severe GH deficiency (GHD) and pituitary hypoplasia due to reduced somatotroph cell mass. We treated male GHRH-KO mice for 6 wk (from week 1 to week 7 of age) with GH-releasing peptide-2 (GHRP-2, 10 microg s.c. twice a day). Chronic treatment with GHRP-2 failed to stimulate somatotroph cell proliferation and GH secretion and to promote longitudinal growth. GHRP-2-treated mice showed an increase in total body weight compared with placebo-treated animals, due to worsening of the body composition alterations typical of GHD animals. These data demonstrate that GHRP-2 failed to reverse the severe GHD caused by lack of GHRH.     

Periprandial changes in growth hormone release in goldfish: role of somatostatin, ghrelin, and gastrin-releasing peptide

In goldfish, growth hormone (GH) transiently rises 30 min after meals, returning to baseline at 1 h postmeal. Somatostatin (SRIF) is the major inhibitor of GH release. Three cDNAs encoding pre-pro-SRIF (PSS) have been previously cloned from goldfish brain: PSS-I, which encodes SRIF-14; PSS-II, which is potentially processed into gSRIF-28 that has [Glu(1),Tyr(7)(,)Gly(10)]SRIF-14 at the COOH terminus; and PSS-III, which encodes [Pro(2)]SRIF-14 at its COOH terminus. In goldfish, bombesin (BBS), mimicking the endogenous gastrin-releasing peptide (GRP), acutely suppresses food intake and also stimulates GH release. Ghrelin was recently characterized in goldfish as a GH secretagogue and an orexigen. In this paper, we studied the changes in SRIF mRNA levels during feeding and analyzed the influences of BBS and ghrelin peptides on forebrain PSS expression. The results showed a 60% reduction in PSS-II mRNA after meals, but no changes in the expression of PSS-I and PSS-III were found. Intraperitoneal injections of 100 ng/g body wt of BBS increased GH secretion and decreased PSS-I and PSS-II gene expression. Intraperitoneal injection of goldfish ghrelin (100 ng/g body wt) transiently increased the serum GH levels and increased PSS-I, while decreasing PSS-II mRNA levels. Ghrelin (50 ng/g body wt) blocked the effects of BBS (100 ng/g body wt) on PSS-I but not on PSS-II expression. Coadministration of BBS and ghrelin decreased only the PSS-II gene expression. We conclude that the interactions between BBS/GRP and ghrelin can account for the postprandial variations in serum GH levels and the forebrain expression of PSS-II. Furthermore, we demonstrate that intraperitoneal administration of BBS reduces the ghrelin expression levels in the gut. Thus the inhibition of production of ghrelin in the gut may contribute to the satiety effects of BBS/GRP peptides.     

Trp3, Arg5]-ghrelin(1-5) stimulates growth hormone secretion and food intake via growth hormone secretagogue (GHS) receptor

Ghrelin, a 28 amino acid peptide identified as an endogenous ligand for growth hormone secretagogue (GHS) receptor, stimulates food intake and growth hormone (GH) secretion. We designed low molecular weight peptides with affinity for the GHS receptor based on the primary structure of ghrelin. We found that [Trp3, Arg5]-ghrelin(1-5) (GSWFR), a novel pentapeptide composed of all L-amino acids, had affinity for the GHS receptor (IC50 = 10 microM). GSWFR stimulated GH secretion after intravenous or oral administration. Centrally administered GSWFR increased food intake in non-fasted mice. The orexigenic action of GSWFR was inhibited by a GHS receptor antagonist, [D-Lys3]-GH-releasing peptide-6, suggesting that GSWFR stimulated food intake through the GHS receptor. The orexigenic action of GSWFR was also inhibited by a neuropeptide Y (NPY) Y1 receptor antagonist, BIBO3304. These results suggest that the GSWFR-induced feeding is mediated by the NPY Y1 receptor.     

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.     

Effects of leptin replacement on hypothalamic-pituitary growth hormone axis function and circulating ghrelin levels in ob/ob mice

Leptin-deficient obese mice (ob/ob) have decreased circulating growth hormone (GH) and pituitary GH and ghrelin receptor (GHS-R) mRNA levels, whereas hypothalamic GH-releasing hormone (GHRH) and somatostatin (SST) expression do not differ from lean controls. Given the fact that GH is suppressed in diet-induced obesity (a state of hyperleptinemia), it remains to be determined whether the absence of leptin contributes to changes in the GH axis of ob/ob mice. Therefore, to study the impact of leptin replacement on the hypothalamic-pituitary GH axis of ob/ob mice, leptin was infused for 7 days (sc), resulting in circulating leptin levels that were similar to wild-type controls (approximately 1 ng/ml). Leptin treatment reduced food intake, body weight, and circulating insulin while elevating circulating n-octanoyl ghrelin concentrations. Leptin treatment did not alter hypothalamic GHRH, SST, or GHS-R mRNA levels compared with vehicle-treated controls. However, leptin significantly increased pituitary GH and GHRH-R expression and tended to enhance circulating GH levels, but this latter effect did not reach statistical significance. In vitro, leptin (1 ng/ml, 24 h) did not affect pituitary GH, GHRH-R, or GHS-R mRNA but did enhance GH release. The in vivo effects of leptin on circulating hormone and pituitary mRNA levels were not replicated by pair feeding ob/ob mice to match the food intake of leptin-treated mice. However, leptin did prevent the fall in hypothalamic GHRH mRNA and circulating IGF-I levels observed in pair-fed mice. These results demonstrate that leptin replacement has positive effects on multiple levels of GH axis function in ob/ob mice.