GH-releasing Hormone mRNA Levels Research Articles

Intrahypothalamic neurohormonal interactions in the control of growth hormone secretion.

The secretion of growth hormone (GH, somatotropin) is regulated by two neurohormones: one inhibitory, somatotropin release-inhibiting hormone (SRIH) or somatostatin, and one stimulatory, GH-releasing hormone (GHRH). There are several lines of evidence for reciprocal interactions between SRIH and GHRH neuronal networks. Anatomically, GHRH terminals contact SRIH-containing neurons in the periventricular nucleus and SRIH-containing fibres innervate GHRH-containing neurons in the arcuate nucleus. Physiologically, SRIH and GHRH are secreted into the portal blood in complementary oscillating patterns. Results from immunizations with anti-SRIH antisera suggest that endogenous SRIH blocks GHRH release from the median eminence. Intracerebroventricular injections of SRIH stimulate secretion of GHRH indirectly, probably via autoinhibition of SRIH neurons in the anterior periventricular region. High resolution autoradiography allowed us to visualize high affinity, specific [125]SRIH receptors on 30% of GHRH mRNA-containing cells in the ventrolateral portion of the arcuate nucleus. The functional importance of these receptors was demonstrated by blocking endogenous SRIH action with cysteamine, which resulted in an increase in GHRH mRNA levels and desaturation of SRIH receptors in the ventrolateral part of the arcuate nucleus. Alterations in GH production caused by hypophysectomy or acute and chronic GH hypersecretion have opposite effects on the synthesis of SRIH and GHRH, giving further evidence for reciprocal interactions between these two neurohormonal systems.

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.

Expression Analysis of Hypothalamic and Pituitary Components of the Growth Hormone Axis in Fasted and Streptozotocin-Treated Neuropeptide Y (NPY)-Intact (NPY+/+) and NPY-Knockout (NPY –/–) Mice

In the fasted and the streptozotocin (STZ)-induced diabetic male rat, hypothalamic growth hormone (GH)-releasing hormone (GHRH) mRNA levels, and pulsatile GH release are decreased. These changes are believed to be due to a rise in hypothalamic neuropeptide Y (NPY) that inhibits GHRH expression. To directly test if NPY is required for metabolic regulation of hypothalamic neuropeptides important in GH secretion, NPY, GHRH and somatostatin (SRIH) mRNA levels were determined in fasted (48 h) and STZ-treated wild-type (NPY^+/+) and NPY-knockout (NPY^–/–) mice by ribonuclease protection assay. In addition, pituitary receptor mRNA levels for GHRH (GHRH-R), ghrelin (GHS-R) and SRIH (sst2) were assessed by RT-PCR. Under fed conditions the GH axis of NPY^+/+ and NPY^–/– did not differ. In the NPY^+/+ mouse, fasting resulted in a 23% weight loss and >250% increase in NPY mRNA accompanied by a significant reduction in both GHRH and SRIH mRNA. These changes were associated with increases in pituitary expression of GHRH-R and GHS-R and a concomitant suppression of sst2. In the NPY^–/– mouse, fasting also resulted in a 23% weight loss and comparable changes in GHRH-R and sst2, but failed to alter GHRH, SRIH and GHS-R mRNA levels. Fasting resulted in an overall increase in circulating GH, which reached significance in the fasted NPY^–/– mouse. Induction of diabetes in NPY^+/+ mice, using a single, high-dose, STZ injection (150 mg/kg), resulted in modest weight loss (5%), and a 158% increase NPY expression which was associated with reciprocal changes in pituitary GHS-R and sst2 expression, similar to that observed in the fasted state, but no change in hypothalamic GHRH or SRIF expression was observed. Induction of diabetes in NPY^+/+ and NPY^–/– mice, using a multiple, low-dose, STZ paradigm (5 consecutive daily injections of 40 mg/kg), did not alter body weight, hypothalamic neuropeptide expression or pituitary receptor expression, with the exception that sst2 mRNA levels were suppressed and GH levels did rise in the NPY^–/– mouse. These observations demonstrate that NPY is not required for basal regulation of the GH axis, but is required for fasting-induced suppression of GHRH and SRIH expression, as well as fasting-induced augmentation of pituitary GHS-R mRNA. In contrast to the rat, fasting clearly did not suppress circulating GH levels in mice, but resulted in an overall rise in mean GH levels, similar to that observed in other mammalian species. The fact that many of the fasting-induced changes in the GH axis were observed in the high-dose STZ-treated mice, but were not observed in the multiple, low-dose paradigm, suggests STZ-mediated modulation of GH axis function is dependent on the severity of the catabolic state and not hyperglycemia.

GHRH and sleep.

A significant portion of the total daily growth hormone (GH) secretion is associated with deep non-REM sleep (NREMS). GH secretion is stimulated by the hypothalamic neurohormone, GH-releasing hormone (GHRH). Exogenous GHRH promotes NREMS in various species. Suppression of endogenous GHRH (competitive antagonist, antibodies, somatostatinergic stimulation, high doses of GH or insulin-like growth factor) results in simultaneous inhibition of NREMS. Mutant and transgenic animals with a defect in GHRHergic activity display permanently reduced NREMS which cannot be reversed by means of GH supplementation. GHRH contents and mRNA levels in the hypothalamus correlate with sleep-wake activity during the diurnal cycle and sleep deprivation and recovery sleep. Stimulation of NREMS by GHRH is a hypothalamic action. GABAergic neurons in the anterior hypothalamus/preoptic region are candidates for mediating promotion of NREMS by GHRH. In contrast to NREMS, stimulation of REMS by GHRH is mediated by GH. Simultaneous stimulation of NREMS and GH secretion by GHRH may promote adjustment of tissue anabolism to sleep.

Fasting-induced changes in the hypothalamic-pituitary-GH axis in the absence of GH expression: lessons from the spontaneous dwarf rat.

Fasting results in a reciprocal shift in hypothalamic neuropeptide Y (NPY) and GH-releasing hormone (GHRH) expression in the adult male rat. It is hypothesized that the fasting-induced rise in NPY is responsible for the GHRH decline and subsequent attenuation of pulsatile GH release. Fasting also leads to a decrease in circulating IGF-I, attributed to both reduced GH release and peripheral GH resistance. Although pituitary GH output is suppressed in the fasted rat, we report herein that pituitary GHRH receptor (GHRH-R) and GH secretagogue receptor (GHS-R) mRNA levels are increased, while pituitary expression of the somatostatin receptor subtype 2 (sst2) and 5 (sst5) is decreased, as determined by real-time reverse transcription (RT)-PCR. A shift in the expression of pituitary receptor subtypes to favor GH synthesis and release may be due, at least in part, to a decline in GH/IGF-I negative feedback. In order to test this hypothesis, we compared hypothalamic and pituitary response to fasting (72 h) in normal male rats and rats with isolated GH deficiency (spontaneous dwarf rats (SDR)). Circulating GH levels were undetectable in SDR, and IGF-I levels were less than 10% of normal controls. Fasting stimulated NPY mRNA levels in SDR; however, the rise in NPY mRNA levels was not accompanied by a fall in GHRH mRNA, as observed in fasted normal rats. In fact, GHRH mRNA levels paradoxically rose in the fasted SDR to 135% of fed controls. At the pituitary level, fasting did not alter sst2 and sst5 mRNA levels in SDR but did stimulate the expression of GHRH-R and GHS-R to 165% and 149% of fed controls, respectively. These results demonstrate that the fasting-induced changes in pituitary expression of sst2 and sst5, but not GHRH-R and GHS-R, are GH/IGF-I dependent. In addition, these results argue against the theory that the negative association of NPY and GHRH expression observed following fasting represents a simple cause-and-effect relationship and suggest that GH, either directly or indirectly, mediates the effects of fasting on hypothalamic GHRH expression.

Effects of caloric restriction on gene expression in the arcuate nucleus

Neuroendocrine alterations that repress energy-costly physiologic processes such as reproduction and growth and induce stress responses, might underlie the antiaging effect of caloric restriction (CR). Neurons in the arcuate nucleus of the hypothalamus (ARH) might have a pivotal role in these neuroendocrine alterations. We investigated the effects of CR on gene expression of neuropeptide Y (NPY), proopiomelanocortin (POMC), growth hormone-releasing hormone (GHRH), somatostatin (SRIH), and cyclophilin (CP) in the ARH in male F344 rats at 6 months of age. Rats were fed ad libitum or a 30% caloric restricted diet with a modified alternate-days feeding regimen from 6 weeks of age. Reverse transcription-polymerase chain reaction (RT-PCR) methods were used to quantify mRNA levels over multiple time points during the 12-h/12-h dark/light cycle over a 2-days feeding cycle. The present study demonstrated that CR increased NPY-mRNA levels, but decreased POMC, GHRH, and CP mRNA levels differentially over the feeding cycle. The SRIH level was not significantly affected by CR. The present results support the neuroendocrine hypothesis of CR.

Decreased galanin mRNA levels in growth hormone-releasing hormone neurons after perinatally induced growth retardation.

Intrauterine growth retardation (IUGR) is associated with persistent postnatal growth retardation accompanied by dysfunction of the hypothalamic components of the growth hormone (GH) axis. At the adult stage, this is reflected by increased somatostatin (SS) and decreased neuropeptide Y (NPY) mRNA levels, whereas the GH-releasing hormone (GHRH) mRNA levels are normal and the output of GH remains unchanged. To extend our insight into the hypothalamic control of GH secretion in growth retarded rats, we determined galanin (GAL) mRNA levels at the adult stage of perinatally malnourished (i.e. IUGR and early postnatally food restricted) rats. Analyses included comparison of GAL mRNA levels in GHRH neurons in perinatally malnourished adult rats using a semi-quantitative double labeling in situ hybridization technique. We report that IUGR is accompanied by a 60% decrease in GAL mRNA levels in all GHRH neurons in the male IUGR group whereas a tendency towards a decrease was observed in the male early postnatally food restricted (FR) group. These effects became more pronounced when the analysis was restricted to GHRH neurons coexpressing GAL mRNA i.e. decreased GAL mRNA levels were seen in both male and female IUGR rats and in FR males. These data show that GAL mRNA levels in GHRH neurons are persistently decreased after perinatal malnutrition. Taking these results together with our previous data on SS, NPY and GHRH mRNA levels, we can conclude that IUGR leads to a reprogramming of the hypothalamic regulation of GH secretion.

Chronic food-restriction alters the expression of somatostatin and growth hormone-releasing hormone in the ovariectomised ewe.

Changes in the secretion of GH induced by long-term alterations in nutritional status are thought to result from alterations in somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) at the level of the hypothalamus. To date however, the effect of nutrition on the gene expression of SRIF and GHRH in a species where GH secretion is increased by food restriction, as is the case for the sheep and human, remains unknown. We determined the effect of under-nutrition on the expression of SRIF and GHRH in the hypothalamus of sheep. Ovariectomised ewes were randomly divided into two groups and either fed an ad lib diet (n=6) or a restricted diet of 500 g lucerne chaff per day (food-restricted; n=5) for 7 months. In situ hybridisation was used to study hypothalamic gene expression for GHRH, SRIF and galanin (GAL). The food-restricted animals had elevated plasma concentrations of GH; this was associated with an increase in GHRH mRNA levels in the arcuate nucleus (ARC) and reduced SRIF in the rostral periventricular nucleus and ventromedial hypothalamic nucleus. The level of gene expression of GAL in the ARC and SRIF in the caudal periventricular nucleus was similar in ad lib and food-restricted animals. In conclusion, the effect of chronic food-restriction on the secretion of GH reflects increased GHRH and reduced SRIF synthesis in the hypothalamus.

The growth hormone (GH)-axis of GH receptor/binding protein gene-disrupted and metallothionein-human GH-releasing hormone transgenic mice: hypothalamic neuropeptide and pituitary receptor expression in the absence and presence of GH feedback.

Elevation of circulating GH acts to feed back at the level of the hypothalamus to decrease GH-releasing hormone (GHRH) and increase somatostatin (SRIF) production. In the rat, GH-induced changes in GHRH and SRIF expression are associated with changes in pituitary GHRH receptor (GHRH-R), GH secretagogue receptor (GHS-R), and SRIF receptor subtype messenger RNA (mRNA) levels. These observations suggest that GH regulates its own synthesis and release not only by altering expression of key hypothalamic neuropeptides but also by modulating the sensitivity of the pituitary to hypothalamic input, by regulating pituitary receptor synthesis. To further explore this possibility, we examined the relationship between the expression of hypothalamic neuropeptides [GHRH, SRIF, and neuropeptide Y (NPY)] and pituitary receptors [GHRH-R, GHS-R, and SRIF receptor subtypes (sst2 and sst5)] in two mouse strains with alterations in the GH-axis; the GH receptor/binding protein gene-disrupted mouse (GHR/BP-/-) and the metallothionein promoter driven human GHRH (MT-hGHRH) transgenic mouse. In GHR/BP-/- mice, serum insulin-like growth factor I levels are low, and circulating GH is elevated because of the lack of GH negative feedback. Hypothalamic GHRH mRNA levels in GHR/BP-/- mice were 232 +/- 20% of GHR/BP+/+ littermates (P < 0.01), whereas SRIF and NPY mRNA levels were reduced to 86 +/- 2% and 52 +/- 3% of controls, respectively (P < 0.05; ribonuclease protection assay). Pituitary GHRH-R and GHS-R mRNA levels of GHR/BP-/- mice were elevated to 275 +/- 55% and 319 +/- 68% of GHR/BP+/+ values (P < 0.05, respectively), whereas the sst2 and sst5 mRNA levels did not differ from GHR/BP intact controls as determined by multiplex RT-PCR. Therefore, in the absence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor stimulation of GH synthesis and release. In MT-hGHRH mice, ectopic hGHRH transgene expression elevates circulating GH and insulin-like growth factor I. In this model of GH excess, endogenous (mouse) hypothalamic GHRH mRNA levels were reduced to 69 +/- 6% of nontransgenic controls, whereas SRIF mRNA levels were increased to 128 +/- 6% (P < 0.01). NPY mRNA levels were not significantly affected by hGHRH transgene expression. Also, MT-hGHRH pituitary GHRH-R and GHS-R mRNA levels did not differ from controls. However, sst2 and sst5 mRNA levels in MT-hGHRH mice were increased to 147 +/- 18% and 143 +/- 16% of normal values, respectively (P < 0.05). Therefore, in the presence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor suppression of GH synthesis and release.

Persistent changes in somatostatin and neuropeptide Y mRNA levels but not in growth hormone-releasing hormone mRNA levels in adult rats after intrauterine growth retardation.

A reduction in the availability of oxygen and nutrients across the placenta in the last trimester of pregnancy may lead to intrauterine growth retardation (IUGR) which, in turn, may cause a persistent postnatal growth failure. However, it is unknown whether this persistent growth retardation is centrally mediated through alterations in the components of the growth hormone (GH)-axis. We tested the hypothesis that alterations in the development of the central components of the GH-axis contribute to the persistent growth failure observed after experimentally induced IUGR or early postnatal food restriction (FR) in the rat. Using semi-quantitative in situ hybridization, we compared somatostatin (SS), GH-releasing hormone (GHRH) and neuropeptide Y (NPY) mRNA levels in adult rats experimentally subjected to IUGR or FR. We report that IUGR increased the expression of SS mRNA in the periventricular nucleus (PeN) of adult male and female rats by 128% and 153% respectively, did not alter the expression of GHRH mRNA in the arcuate nucleus (ARC) and decreased the NPY mRNA expression in the ARC by 73% in males and 61% in females, whereas in the FR group no changes in the expression of these mRNAs were observed. These data show that the timing of malnutrition or the presence of the placenta is important for the long-term alterations since the effects only occurred in the prenatally induced growth retardation and not in the early postnatally induced growth retardation group.

Human uterine and ovarian expression of growth hormone–releasing hormone messenger RNA in benign and malignant gynecologic conditions

Objective: To determine uterine and ovarian expression of growth hormone–releasing hormone (GHRH) messenger RNA (mRNA) in benign and pathologic gynecologic states. Design: Case–control study. Setting: Tertiary-care academic department. Patient(s): Women undergoing hysterectomy for benign or malignant gynecologic conditions. Intervention(s): Ovarian and uterine tissue was obtained for measurement of GHRH mRNA levels by reverse transcription polymerase chain reaction. Main Outcome Measure(s): Levels of GHRH mRNA in normal tissues were compared with those in tissues with pathologic abnormalities. Result(s): Growth hormone–releasing hormone mRNA was detectable in the ovary, endometrium, myometrium, fallopian tubes, and placenta. Levels of GHRH mRNA were significantly increased in secretory endometrium compared with proliferative endometrium. Hormone replacement therapy did not affect endometrial GHRH mRNA levels. Uterine myomas expressed similar levels of GHRH mRNA as normal myometrium. No changes in endometrial GHRH mRNA were detected in endometrial cancers compared with normal endometrium or myometrium obtained from the same patient; however, these levels were higher than those in noncancerous myometrial tissue obtained from other patients with benign gynecologic disease. In ovarian tissue, no differences in GHRH mRNA were found between premenopausal and postmenopausal women. Ovarian GHRH mRNA was significantly decreased in endometriotic cysts, whereas significantly greater GHRH expression occurred in ovarian cancer compared with normal ovarian tissue. Conclusion(s): Endometrial and ovarian GHRH gene transcription are altered in selective physiologic and pathologic states and are influenced by such factors as ovarian hormones. Because it is a growth factor, GHRH may promote endometrial proliferation and may be involved in the pathogenesis of ovarian and endometrial cancer and endometriosis.

Sleep deprivation increases rat hypothalamic growth hormone-releasing hormone mRNA.

Much evidence indicates that growth hormone-releasing hormone (GHRH) is involved in sleep regulation. We hypothesized that GHRH mRNA would increase and somatostatin (SRIH) mRNA would decrease during sleep deprivation. With the use of RT-PCR and truncated internal standards, rat hypothalamic GHRH mRNA and SRIH mRNA levels were evaluated after sleep deprivation. After 8 or 12 h of sleep deprivation there was a significant increase in rat hypothalamic GHRH mRNA expression compared with time-matched control samples. Hypothalamic GHRH mRNA levels were not significantly different from control values after 1 or 2 h of recovery after 8 h of sleep deprivation or after 2 h of recovery after 12 h of sleep deprivation. In control animals, variations in hypothalamic GHRH mRNA levels were observed. GHRH mRNA expression was significantly higher in the afternoon than at dark onset or during the dark period. SRIH mRNA levels were significantly suppressed at the termination of an 8-h sleep deprivation period and were significantly higher after dark onset than in the morning. The alterations in GHRH and SRIH mRNA expressions after sleep deprivation and recovery support the notion that GHRH plays an important role in sleep homeostasis and suggest that these neuropeptides may interact reciprocally in modulating sleep as they do in the control of growth hormone secretion.

Experimentally induced lysosomal dysfunction disrupts processing of hypothalamic releasing factors

Previous studies have shown that experimentally induced lysosomal dysfunction elicits various features of aging in the cortical telencephalon. The present study used cultured slices to test if: (1) it causes similar changes in the hypothalamus, and/or (2) modifies the processing of two releasing factors important to aging. A 2-day exposure to N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), a selective inhibitor of cathepsins B and L, triggered a pronounced increase in the numbers of lysosomes in the ventromedial and dorsomedial nuclei, and in lateral hypothalamus. Continued incubation with the inhibitor for 3-12 days resulted in the spread of endosomes-lysosomes into dendrites and, in the lateral hypothalamus, the formation of massive, lysosome-filled expansions of neuronal processes (meganeurites). These effects did not occur in the arcuate nucleus, making it the first region so far examined in which lysosomal proliferation is not initiated by hydrolase inhibitors. Despite this, a dense plexus of axons and terminals in the median eminence was partially depleted of growth hormone releasing hormone (GHRH) within 48 hours after addition of ZPAD. Moreover, the inhibitor caused axonal GHRH to become collected into large puncta, an effect highly suggestive of a partial failure in axonal transport. GHRH mRNA levels were not greatly affected by 6 days of ZPAD exposure, indicating that reduced expression did not play a major role in the peptide changes seen at 48 hours. Similar but less pronounced immunocytochemical changes were recorded for the somatostatin system in the arcuate and periventricular nucleus. It is concluded that lysosome dysfunction: (1) has different consequences for the arcuate nucleus than other brain regions, and (2) disrupts transport of hypothalamic releasing factors. The potential significance of the results to endocrine senescence is discussed.

Experimentally induced lysosomal dysfunction disrupts processing of hypothalamic releasing factors

Previous studies have shown that experimentally induced lysosomal dysfunction elicits various features of aging in the cortical telencephalon. The present study used cultured slices to test if: (1) it causes similar changes in the hypothalamus, and/ or (2) modifies the processing of two releasing factors important to aging. A 2-day exposure to N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), a selective inhibitor of cathepsins B and L, triggered a pronounced increase in the numbers of lysosomes in the ventromedial and dorsomedial nuclei, and in lateral hypothalamus. Continued incubation with the inhibitor for 3–12 days resulted in the spread of endosomes-lysosomes into dendrites and, in the lateral hypothalamus, the formation of massive, lysosome-filled expansions of neuronal processes (meganeurites). These effects did not occur in the arcuate nucleus, making it the first region so far examined in which lysosomal proliferation is not initiated by hydrolase inhibitors. Despite this, a dense plexus of axons and terminals in the median eminence was partially depleted of growth hormone releasing hormone (GHRH) within 48 hours after addition of ZPAD. Moreover, the inhibitor caused axonal GHRH to become collected into large puncta, an effect highly suggestive of a partial failure in axonal transport. GHRH mRNA levels were not greatly affected by 6 days of ZPAD exposure, indicating that reduced expression did not play a major role in the peptide changes seen at 48 hours. Similar but less pronounced immunocytochemical changes were recorded for the somatostatin system in the arcuate and periventricular nucleus. It is concluded that lysosome dysfunction: (1) has different consequences for the arcuate nucleus than other brain regions, and (2) disrupts transport of hypothalamic releasing factors. The potential significance of the results to endocrine senescence is discussed. J. Comp. Neurol. 401:382–394, 1998. © 1998 Wiley-Liss, Inc.

Gene expression of hypothalamic somatostatin and growth hormone-releasing hormone in dexamethasone-treated rats.

Supraphysiological doses of glucocorticoids inhibit growth hormone (GH) secretion in man and experimental animals. We investigated whether glucocorticoids inhibit GH secretion through changes in the gene expression of GH, hypothalamic somatostatin (SS) and GH-releasing hormone (GHRH), and whether such changes vary with the dose and duration of glucocorticoid excess. Male rats, 6 weeks of age, were treated with injections of either saline or different doses of dexamethasone (40, 200, 500 or 1,000 micrograms/kg/day) intraperitoneally for 3 or 8 days. Total RNA extracted from the anterior pituitary and hypothalamus was analyzed by Northern blot hybridization. SS mRNA level was also assessed in smaller hypothalamic fragments containing predominantly the periventricular and paraventricular nuclei, and by in situ hybridization. A biphasic effect on SS mRNA levels was observed such that a significant increase (p < 0.001) was demonstrated in the periventricular nucleus after 3 days of dexamethasone 1,000 micrograms/kg/day, but a reduction in hypothalamic SS mRNA was seen after 8 days for all doses employed (p < 0.05 or p < 0.01). On the other hand, hypothalamic GHRH mRNA levels showed a reduction which appeared to increase with the dose and duration of treatment and became statistically significant after 8 days at doses > or = 200 micrograms/kg/day (p < 0.05). Pituitary GH mRNA levels were increased after 3 days at doses > or = 500 micrograms/kg/day (p < 0.05) but showed no significant change at all doses after 8 days. We conclude that glucocorticoid excess is associated with changes in the gene expression of GH, hypothalamic SS and GHRH, which vary with the dose and duration of glucocorticoid treatment. Glucocorticoids inhibit GH secretion in vivo through a reduction in hypothalamic GHRH gene expression and, in animals with shorter duration of glucocorticoid excess also through an increase in SS gene expression in the periventricular nucleus.

Regulation of hypothalamic somatostatin, growth hormone-releasing hormone, and growth hormone receptor messenger ribonucleic acid by glucocorticoids

Although it is well known that chronic treatment with glucocorticoids inhibits somatic growth, the mechanism of action of this inhibitory effect is not completely understood. It is likely that glucocorticoids act at various levels, including pituitary, hypothalamus, and peripheral organs modulating GH synthesis, secretion, and action. In this work, we evaluated the effect of dexamethasone on hypothalamic somatostatin and GH-releasing hormone (GHRH) messenger RNA (mRNA) levels by in situ hybridization. We found a significant decrease of somatostatin mRNA content in the periventricular nucleus of the hypothalamus after 3, 8, and 15 days of treatment with dexamethasone. Furthermore, we observed a reduction in GHRH mRNA levels in the arcuate nucleus after 8 and 15 days of treatment with this steroid. As it has been shown that GH feeds back to regulate somatostatin and GHRH expression at the hypothalamic level through high affinity GH receptors, we evaluated the possibility of a GH-mediated action in the inhibitory effect of glucocorticoids on somatostatin and GHRH mRNA levels. To address this issue, we first studied the GH receptor mRNA content in both the periventricular and arcuate nuclei of the hypothalamus after dexamethasone treatment. Secondly, the effect of dexamethasone on somatostatin and GHRH mRNA levels in hypophysectomized animals also was assessed. We found a significant decrease in GH receptor mRNA levels in the periventricular nucleus and in the arcuate nucleus after 1, 3, 8, and 15 days of glucocorticoid administration. Finally, in hypophysectomized rats, dexamethasone treatment for 15 days did not reduce somatostatin mRNA levels in the periventricular nucleus but significantly decreased GHRH mRNA content in the arcuate nucleus. In summary, our results suggest an inhibitory GH-mediated effect of dexamethasone on somatostatin mRNA levels in the periventricular nucleus and an inhibitory direct effect of dexamethasone on GHRH neurones in the arcuate nucleus.

Regulation of hypothalamic somatostatin, growth hormone-releasing hormone, and growth hormone receptor messenger ribonucleic acid by glucocorticoids.

Although it is well known that chronic treatment with glucocorticoids inhibits somatic growth, the mechanism of action of this inhibitory effect is not completely understood. It is likely that glucocorticoids act at various levels, including pituitary, hypothalamus, and peripheral organs modulating GH synthesis, secretion, and action. In this work, we evaluated the effect of dexamethasone on hypothalamic somatostatin and GH-releasing hormone (GHRH) messenger RNA (mRNA) levels by in situ hybridization. We found a significant decrease of somatostatin mRNA content in the periventricular nucleus of the hypothalamus after 3, 8, and 15 days of treatment with dexamethasone. Furthermore, we observed a reduction in GHRH mRNA levels in the arcuate nucleus after 8 and 15 days of treatment with this steroid. As it has been shown that GH feeds back to regulate somatostatin and GHRH expression at the hypothalamic level through high affinity GH receptors, we evaluated the possibility of a GH-mediated action in the inhibitory effect of glucocorticoids on somatostatin and GHRH mRNA levels. To address this issue, we first studied the GH receptor mRNA content in both the periventricular and arcuate nuclei of the hypothalamus after dexamethasone treatment. Secondly, the effect of dexamethasone on somatostatin and GHRH mRNA levels in hypophysectomized animals also was assessed. We found a significant decrease in GH receptor mRNA levels in the periventricular nucleus and in the arcuate nucleus after 1, 3, 8, and 15 days of glucocorticoid administration. Finally, in hypophysectomized rats, dexamethasone treatment for 15 days did not reduce somatostatin mRNA levels in the periventricular nucleus but significantly decreased GHRH mRNA content in the arcuate nucleus. In summary, our results suggest an inhibitory GH-mediated effect of dexamethasone on somatostatin mRNA levels in the periventricular nucleus and an inhibitory direct effect of dexamethasone on GHRH neurones in the arcuate nucleus.

Hypothalamic growth hormone-releasing hormone mRNA varies across the day in rats.

Experiments were performed to determine whether growth hormone-releasing hormone (GHRH) mRNA displays diurnal variations in the hypothalamus and cortex of the rat. Levels of GHRH and beta-actin mRNA were measured from hypothalamic and cortical extracts using the reverse transcriptase polymerase chain reaction method in rats sacrificed at 4 h intervals across a 12:12 h light:dark cycle. Hypothalamic GHRH mRNA peaked around the light onset, declined during the light period, and stayed low in the dark. Variations in hypothalamic beta-actin and cortical GHRH mRNA levels were not observed. beta-Actin mRNA expression in the cortex was higher in the dark than in the light period. The results demonstrate that hypothalamic GHRH mRNA displays diurnal variations.

Differential regulation of the growth hormone receptor gene: effects of dexamethasone and estradiol.

GH receptor (GHR) expression differs during development between central and peripheral tissues. Peripheral GHR expression is known to be sensitive to gonadal and adrenal steroids, but little is known about their effects on GHR in the central nervous system. We have now studied the effects of estradiol (E2) or dexamethasone on GHR expression in rat arcuate nucleus (ARC) and hippocampus, using quantitative in situ hybridization. Dexamethasone, which strongly down-regulates hepatic GHR expression, had no effect on central GHR transcript abundance, whereas E2 treatment, which stimulates hepatic GHR expression, significantly reduced ARC GHR messenger RNA (mRNA) levels. E2 also increased somatostatin (SS) expression significantly in both ARC and periventricular nuclei but did not reduce ARC GH-releasing hormone (GHRH) mRNA levels. Ovariectomy stimulated GHR and GHRH mRNA levels in the ARC, whereas it lowered ARC SS expression. E2 replacement in ovariectomized animals restored GHRH and SS mRNA levels to control values. Hippocampal GHR mRNA transcripts showed the same response to these endocrine manipulations as seen in the ARC. The induction of hepatic GHR expression by E2 is known to involve the transcription of an alternate 5' untranslated first exon, GHR1. This was readily detectable in the liver using a specific GHR1 probe but could not be detected in any CNS area. Our results show that GHR expression in the CNS is sensitive to regulation by peripheral steroids but that CNS and hepatic expression of GHR is differentially regulated by the same treatments.

Immunosuppressant agent FK506 stimulates growth hormone (GH) secretion and gene expression of hypothalamic GH-releasing hormone in the rat.

Immunosuppressant agent FK506 has been reported to stimulate ACTH release from pituitary cells. We examined the effects of FK506 on GH release from the rat anterior pituitary cells and the effects of FK506 on hypothalamic GH- releasing hormone (GRH) and somatostatin (SS) gene expression in conscious male rats. In vitro experiments, the monolayer pituitary culture and reverse hemolytic plaque assay were employed to examine the GH release from the rat anterior pituitary cells. In in vivo experiments, the FK506 was administered for 7 days and then sequential blood sampling was performed every 20 min during 6 h in conscious rats. The hypothalamus was removed, and total RNA was extracted for Northern blot analysis. The FK506 significantly stimulated GH release from the rat anterior pituitary cells in a dose-dependent manner in vitro. In in vivo experiments, the area under the curve of GH surges was significantly increased in FK506-treated rats, although the peak height and the trough level of GH surges were not altered. Pituitary GH messenger RNA (mRNA) levels were significantly increased by the FK506 treatment. Hypothalamic GRH mRNA levels were significantly increased in FK506- treated rats, whereas hypothalamic SS mRNA levels were not altered. These findings indicate that FK506 stimulates GH secretion and gene expression of hypothalamic GRH in the rat.