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.     

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.     

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.     

Hypophysectomy Prevents Ghrelin-Induced Adiposity and Increases Gastric Ghrelin Secretion in Rats

Objective: The novel gastric hormone ghrelin has recently been identified as an important modulator of energy homeostasis. Leptin-responsive hypothalamic neuropeptide Y/Agouti-related protein neurons are believed to mediate afferent ghrelin signals. Little is known, however, about ghrelin-induced efferent signals. We therefore investigated if hypothalamic-pituitary axes have a role in transferring ghrelin-induced changes of energy balance to the periphery. Research Methods and Procedures: We subcutaneously injected hypophysectomized, as well as adrenalectomized, thyroidectomized, and sham-operated control rats with GH secretagogues [ghrelin, growth hormone (GH)-releasing peptide] for 1 week. Body weight, food intake, and body composition (chemical carcass analysis) were analyzed and compared with vehicle-treated controls. In addition, we quantified circulating levels of endogenous ghrelin in hypophysectomized and GH–treated normal rats. Results: GH-secretagogue treatment of sham-operated control rats dose-proportionally increased food intake, body weight, and fat mass compared with vehicle-injected controls (p < 0.01). These effects, however, were not observed in ghrelin-treated hypophysectomized, thyroidectomized, or adrenalectomized rats, indicating an essential role for the pituitary axis in ghrelin-induced adiposity. Circulating levels of endogenous ghrelin were reduced by administration of GH in normal rats and were about 3-fold higher in hypophysectomized rats (n = 20, p = 0.001), suggesting a regulatory feedback loop involving the stomach and the pituitary to regulate gastric ghrelin secretion. Discussion: According to these results, the endocrine pituitary is mediating ghrelin-induced changes toward a positive energy balance and is involved in the regulation of ghrelin secretion through a gastro-hypophyseal feedback loop.     

Changes in the hypothalamic-pituitary somatotropic function of infant hypothyroid rats

The effects of the perturbation of the pituitary-thyroid axis induced during development on the functional activity of the growth hormone (GH) regulatory neuronal systems, GH-releasing hormone (GHRH), and somatostatin (SS) were studied in 14- and 21-day-old rats made hypothyroid by giving dams propylthiouracil in the drinking water since the day of parturition. Infant hypothyroid rats, both at 14 and 21 days of life, had elevated plasma thyroid-stimulating hormone levels and decreased pituitary and plasma GH levels. Simultaneous determination of hypothalamic GHRH/SS-like immunoreactivity (LI) and GHRH/SS mRNA levels did not reveal any difference in 14-day-old hypothyroid rats when compared with age-matched controls. In contrast, 21-day-old hypothyroid rats had decreased GHRH-LI content and a striking rise in GHRH mRNA levels, whereas SS-LI content and SS gene expression remained unaltered. These data indicate that in infant hypothyroid rats, changes in the functional activity of the GHRH neuronal system occur later than changes in GH secretion and are probably dependent on the GH deficiency. The functional activity of SS neurons was apparently unaltered in these hypothyroid rats, pointing to a lesser sensitivity of this system to the perturbation of the pituitary-thyroid axis.     

Neither intravenous nor intracerebroventricular administration of obestatin affects the secretion of GH, PRL, TSH and ACTH in rats

To examine the effect of obestatin, a recently identified peptide derived from preproghrelin, on pituitary hormone secretion, obestatin was administered in anesthetized male rats. Intravenous administration of obestatin did not show any effect on plasma GH, PRL, ACTH and TSH levels. Since obestatin has been reported to have opposite effects of ghrelin in regulating food intake, gastric emptying and intestinal contractility, GH suppressive effect, which is opposite effect of ghrelin, was tested. Intravenous administration of GHRH or GHRP-2, a ghrelin receptor ligand, resulted in a marked plasma GH elevation. However obestatin did not show any effect on GHRH- or GHRP-2-induced GH rise. Furthermore intracerebroventricular administration of obestatin also did not influence plasma GH, PRL, ACTH and TSH levels. These findings suggest that obestatin has no effect on pituitary hormone secretions despite the presence of GPR39, a receptor for obestatin, in the pituitary.     

Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia-reperfusion injury in rat hearts in vivo

Ghrelin is a novel gut-brain peptide that binds to the growth hormone secretagogue receptor (GHS-R), thereby functioning in the regulation of growth hormone (GH) release and food intake. Ghrelin-producing cells are most abundant in the oxyntic glands of the stomach. The regulatory mechanism that governs the biosynthesis and secretion of ghrelin has not been clarified. We report that ghrelin mRNA expression in the gastric fundus was increased, but that ghrelin peptide content decreased after a 48-h fast. Both values returned to control levels after refeeding. The ghrelin plasma concentration in the gastric vein and systemic venous blood increased after 24- and 48-h fasts. Furthermore, des-octanoylated ghrelin and n-octanoylated ghrelin were found in rat stomach, with the ratio of des-octanoylated ghrelin to n-octanoylated ghrelin markedly increased after fasting. The ghrelin mRNA level in the stomach also increased after administration of insulin and leptin. Conversely, db/db mice, which are deficient in the leptin receptor, had lower ghrelin mRNA levels than control mice. These findings suggest that this novel gastrointestinal hormone plays a role in the regulation of energy balance.     

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.     

Hypothalamic-pituitary somatotropic function in prepubertal hypothyroid rats: effect of growth hormone replacement therapy.

The effect of thyroid hormone deficiency and growth hormone (GH) treatment on hypothalamic GH-releasing hormone (GHRH)/somatostatin (SS) concentrations, GHRH/SS mRNA levels, and plasma GH and somatomedin-C (IGF-I) concentrations were studied in 28- and 35-day-old rats made hypothyroid by giving dams propylthiouracil in the drinking water since the day of parturition. Hypothyroid rats, at both 28 and 35 days of life, had decreased hypothalamic GHRH content and increased GHRH mRNA levels, unaltered SS content and SS mRNA levels, and reduced plasma GH and IGF-I concentrations. Treatment of hypothyroid rats with GH for 14 days completely restored hypothalamic GHRH content and reversed the increase in GHRH mRNA, but did not alter plasma IGF-I concentrations. These data indicate that, in hypothyroid rats, the changes in hypothalamic GHRH content and gene expression are due to the GH deficiency ensuing from the hypothyroid state. Failure of the GH treatment to increase plasma IGF-I indicates that the feedback regulation on GHRH neurons is operated by circulating GH and/or perhaps tissue but not plasma IGF-I concentrations. Presence of low plasma IGF-I concentrations would be directly related to thyroid hormone deficiency.     

Importance of melanin-concentrating hormone receptor for the acute effects of ghrelin

The hypothalamic peptide melanin-concentrating hormone (MCH) and the gastric hormone ghrelin take part in the regulation of energy homeostasis and stimulate food intake. In the present study, ghrelin was administered centrally to MCH-receptor knockout (MCHr KO) mice. MCHr KO mice and wild type (WT) controls both consumed more food when treated with ghrelin. After ghrelin administration, the serum levels of insulin increased only in WT mice whereas the serum levels of corticosterone increased both in WT and MCHr KO mice. The level of growth hormone (GH) mRNA in the pituitary gland was markedly increased in response to ghrelin injection in the WT mice but was unaffected in the MCHr KO mice. The different ghrelin responses could not be explained by a difference in growth hormone secretagogue receptor expression between MCHr KO and WT mice in the pituitary or hypothalamus. In summary, the MCHr is not required for ghrelin induced feeding. However, the MCHr does play a role for the effect of ghrelin on GH expression in the pituitary and serum insulin levels.     

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 regulation of growth hormone-releasing hormone gene expression

Slot-blot hybridization technique was used to evaluate growth hormone-releasing hormone (GHRH) mRNA levels in the hypothalamus of long-term (14 days) hypophysectomized (HPX) rats treated or not with 125 micrograms hGH/rat, twice daily IP, since the first day postsurgery. In addition, mRNA levels were determined in the hypothalamus of short-term (4 days) GH-treated (250 micrograms hGH/rat, twice daily IP) intact rats. GHRH mRNA levels were increased in HPX rats, and GH treatment partially counteracted this rise. Short-term administration of GH decreased GHRH mRNA levels in intact rats. These results, evaluated together with previous findings showing decreased hypothalamic GHRH-like immunoreactivity in both HPX rats and intact rats given GH (6, 7, 9), indicate that GH exerts a negative feedback action on the synthesis and release of GHRH.     

睡眠中间断低氧对大鼠下丘脑-垂体-肾上腺轴和生长激素的影响

目的:探讨睡眠中间断低氧对大鼠下丘脑-垂体-肾上腺轴和生长激素水平的影响.方法:大鼠分别给予吸入空气,持续低氧和间断低氧气体,在1 d,3 d,7 d和30 d后测定下丘脑促肾上腺皮质激素释放激素(CRH)和生长激素释放激素(GHRH)mRNA水平,并测定30d后血浆CRH,GHRH,促肾上腺皮质激素(ACTH)和皮质酮水平,分析其间的变化关系.结果:与对照组比较,在低氧后1 d,3 d,7 d后大鼠下丘脑CRH mRNA升高,GHRH mRNA降低,在30 d后,间断低氧组下丘脑CRH mRNA升高,GHRH mRNA降低,而持续低氧组则接近正常.间断低氧30 d后,血浆CRH、ACTH,皮质酮均升高,GHRH降低,而生长激素没有明显变化.结论:大鼠睡眠中慢性间断低氧可以引起下丘脑-垂体-肾上腺轴激素水平升高,反馈调节紊乱,可引起GHRH分泌抑制.     

Evidence that endogenous SST inhibits ACTH and ghrelin expression by independent pathways

Corticosterone and total ghrelin levels are increased in somatostatin (SST) knockout mice (Sst-/-) compared with SST-intact controls (Sst+/+). Because exogenous ghrelin can increase glucocorticoids, the question arises whether elevated levels of ghrelin contribute to elevated corticosterone levels in Sst-/- mice. We report that Sst-/- mice had elevated mRNA levels for pituitary proopiomelanocortin (POMC), the precursor of adrenocorticotropic hormone (ACTH), whereas mRNA levels for hypothalamic corticotropin-releasing hormone (CRH) did not differ from Sst+/+ mice. Furthermore, SST suppressed pituitary POMC mRNA levels and ACTH release in vitro independently of CRH actions. In contrast, it has been reported that ghrelin increases glucocorticoids via a central effect on CRH secretion and that n-octanoyl ghrelin is the form of ghrelin that activates the GHS-R1a and modulates CRH neuronal activity. Consistent with elevations in total ghrelin levels, Sst-/- mice displayed an increase in stomach ghrelin mRNA levels, whereas hypothalamic and pituitary expression of ghrelin was not altered. Despite the increase in total ghrelin levels, circulating levels of n-octanoyl ghrelin were not altered in Sst-/- mice. Because glucocorticoids and ghrelin increase in response to fasting, we examined the impact of fasting on the adrenal axis and ghrelin in Sst+/+ and Sst-/- mice and found that endogenous SST does not significantly contribute to this adaptive response. We conclude that endogenous SST inhibits basal ghrelin gene expression in a tissue specific manner and independently and directly inhibits pituitary ACTH synthesis and release. Thus endogenous SST exerts an inhibitory effect on ghrelin synthesis and on the adrenal axis through independent pathways.     

Ghrelin Stimulation of Growth Hormone-Releasing Hormone Neurons Is Direct in the Arcuate Nucleus

Background

Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin.

Principal Findings

Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by γ-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate.

Conclusion

Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.     

Induction of growth hormone (GH) mRNA by pulsatile GH-releasing hormone in rats is pattern specific

Growth hormone-releasing hormone (GHRH) is a main inducer of growth hormone (GH) pulses in most species studied to date. There is no information regarding the pattern of GHRH secretion as a regulator of GH gene expression. We investigated the roles of the parameters of exogenous GHRH administration (frequency, amplitude, and total amount) upon induction of pituitary GH mRNA, GH content, and somatic growth in the female rat. Continuous GHRH infusions were ineffective in altering GH mRNA levels, GH stores, or weight gain. Changing GHRH pulse amplitude between 4, 8, and 16 microg/kg at a constant frequency (Q3.0 h) was only moderately effective in augmenting GH mRNA levels, whereas the 8 microg/kg and 16 microg/kg dosages stimulated weight gain by as much as 60%. When given at a 1.5-h frequency, GHRH doubled the amount of GH mRNA, elevated pituitary GH stores, and stimulated body weight gain. In the rat model, pulsatile but not continuous GHRH administration is effective in inducing pituitary GH mRNA and GH content as well as somatic growth. These studies suggest that the greater growth rate, pituitary mRNA levels, and GH stores seen in male compared with female rats are likely mediated, in part, by the endogenous episodic GHRH secretory pattern present in males.     

Impaired ghrelin signaling is associated with gastrointestinal dysmotility in rats with gastroesophageal reflux disease

Gastroesophageal reflux disease (GERD) is often associated with decreased upper gastrointestinal motility, and ghrelin is an appetite-stimulating hormone known to increase gastrointestinal motility. We investigated whether ghrelin signaling is impaired in rats with GERD and studied its involvement in upper gastrointestinal motility. GERD was induced surgically in Wistar rats. Rats were injected intravenously with ghrelin (3 nmol/rat), after which gastric emptying, food intake, gastroduodenal motility, and growth hormone (GH) release were investigated. Furthermore, plasma ghrelin levels and the expression of ghrelin-related genes in the stomach and hypothalamus were examined. In addition, we administered ghrelin to GERD rats treated with rikkunshito, a Kampo medicine, and examined its effects on gastroduodenal motility. GERD rats showed a considerable decrease in gastric emptying, food intake, and antral motility. Ghrelin administration significantly increased gastric emptying, food intake, and antral and duodenal motility in sham-operated rats, but not in GERD rats. The effect of ghrelin on GH release was also attenuated in GERD rats, which had significantly increased plasma ghrelin levels and expression of orexigenic neuropeptide Y/agouti-related peptide mRNA in the hypothalamus. The number of ghrelin-positive cells in the gastric body decreased in GERD rats, but the expression of gastric preproghrelin and GH secretagogue receptor mRNA was not affected. However, when ghrelin was exogenously administered to GERD rats treated with rikkunshito, a significant increase in antral motility was observed. These results suggest that gastrointestinal dysmotility is associated with impaired ghrelin signaling in GERD rats and that rikkunshito restores gastrointestinal motility by improving the ghrelin response.