GH-releasing Hormone mRNA Research Articles

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.

Growth Hormone Releasing Hormone Expression During Postnatal Development in Growth Hormone-Deficient Ames Dwarf Mice:mRNA in situ Hybridization

Several genetic mutations in mice and rats that produce lifelong growth hormone (GH) deficiency result in overexpression of GH-releasing hormone (GHRH) mRNA in hypothalamic arcuate nucleus neurons. In order to examine the development of this condition, GHRH mRNA expression was quantified in Ames dwarf (df/df) and normal (DF/?) mice at 1 (day of birth), 3, 7, 14, 21 and 60 postnatal days (d) following in situ hybridization. Total mRNA was assessed using computer-assisted densitometry after X-ray film autoradiography, and mRNA expression per neuron was quantified by counts of grains per cell after emulsion autoradiography. Total GHRH mRNA was the same in dwarf and normal mice at 1, 3 and 7d. GHRH mRNA in dwarfs increased at 14d to 240% of that in DF/? (p < 0.005); the percentage overexpression in dwarf mice remained ≧200% through 60d, although total GHRH mRNA increased in both dwarfs and normals during this period. GHRH mRNA per neuron was the same in normal and dwarf mice at 1d, then increased in dwarfs to 190% of that in normals at 3d (p < 0.05), and rose to 300% of normal levels by 7d and beyond (p < 0.005). There was no sexual dimorphism in expression by either measure in normal or dwarf mice. These results indicate that an increase in GHRH mRNA in Ames dwarf mice is first detectable at 3d, a period of approximately 7d after the failure to initiate GH production, which occurs normally at embryonic day 17.5. The onset of GHRH overexpression occurs earlier than the decline of either hypophysiotropic somatostatin or dopamine in Ames dwarf mice. This difference may be due to the stimulatory action of GHRH, as opposed to the inhibitory effects of factors examined previously.

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.

Central Administration of a Growth Hormone (GH) Receptor mRNA Antisense Increases GH Pulsatility and Decreases Hypothalamic Somatostatin Expression in Rats

To test the hypothesis of the involvement of centrally expressed rat growth hormone receptors (rGH-R) in the ultradian rhythmicity of pituitary GH secretion, adult male rats were submitted to a 60 hr intracerebroventricular infusion of an antisense (AS) oligodeoxynucleotide (ODN) complementary to the sequence of rGH-R mRNA. Eight hour (10 A.M.-6 P.M.) GH secretory profiles, obtained from freely moving male rats infused with 2.0 nmol/hr of rGH-R AS, revealed a marked increase in GH peak amplitude (150 +/- 12 vs 101 +/- 10 ng/ml), trough levels (16.2 +/- 3.0 vs 5.4 +/- 1.4 ng/ml), and number of peaks (2.9 +/- 0.3 vs 1.8 +/- 0.2). No change was observed in rats treated with an ODN complementary to the prolactin receptor mRNA sequence (2.0 nmol/hr). Infusion of increasing ODN concentrations resulted in a dose-dependent stimulation of GH release. In parallel, somatogenic binding sites in the choroid plexus were decreased by 40%, and levels of rGH-R mRNA were increased in the periventricular nucleus (PeV) but unchanged in the arcuate nucleus (ARC). Levels of somatostatin mRNA, in the PeV but not in the ARC, were lowered by the treatment. Levels of GH-releasing hormone mRNA in the ARC were not affected. These data suggest that GH negative feedback results from a direct effect on central GH receptors and a subsequent activation of hypophysiotropic somatostatin neurons located in the anterior periventricular hypothalamus.

The role of galanin and its receptor in the feedback regulation of growth hormone secretion.

GH controls its own secretion through a mechanism involving short-loop feedback regulation of the synthesis and release of GH-releasing hormone (GHRH). GHRH neurons coexpress the peptide galanin, but the functional significance of this coexpression is unknown. In this study, we tested the hypotheses that 1) galanin gene expression in GHRH neurons is regulated by GH and 2) somatostatin (SS) or GHRH neurons are a target for the action of galanin in the hypothalamus. First, we compared levels of galanin messenger RNA (mRNA) in GHRH neurons between normal male rats and Lewis dwarf rats, which have markedly reduced blood levels of GH. The brains of normal and dwarf animals were processed for detection of galanin mRNA and GHRH mRNA by double-label in situ hybridization. We observed that Lewis dwarf rats had significantly reduced levels of galanin mRNA in their GHRH neurons (P < 0.05). Next, we tested the hypothesis that GH regulates galanin gene expression in GHRH neurons by experimentally altering circulating levels of GH. Three groups of adult male rats were used: 1) intact rats (n = 7); 2) hypophysectomized (hypox) rats (n = 7); and 3) hypox rats treated with 1.5 mg of rat GH (rGH) over a 3-day period (n = 6). At the end of the treatment period, the animals were killed, and their brains were collected and processed for double-label in situ hybridization for GHRH mRNA and galanin mRNA. The signal level of galanin mRNA in GHRH neurons was reduced in hypox animals to less than 10% of that in intact controls (P < 0.0001); whereas, the levels of galanin mRNA signal in GHRH neurons did not differ significantly between the groups of intact and rGH-treated hypox rats. Finally, to determine whether SS or GHRH neurons are targets for galanin, we used double-label in situ hybridization to determine whether either of these populations of neurons express galanin receptor mRNA. A subset of SS neurons in the PeN appeared to express the galanin receptor mRNA, whereas few, if any, GHRH neurons appeared to do so. We conclude that galanin, like its cotransmitter GHRH, is a target for GH action, and we infer that galanin may play a role in the feedback control of GH secretion by exerting a direct effect on SS neurons.

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.

The role of galanin and its receptor in the feedback regulation of growth hormone secretion

GH controls its own secretion through a mechanism involving short-loop feedback regulation of the synthesis and release of GH-releasing hormone (GHRH). GHRH neurons coexpress the peptide galanin, but the functional significance of this coexpression is unknown. In this study, we tested the hypotheses that 1) galanin gene expression in GHRH neurons is regulated by GH and 2) somatostatin (SS) or GHRH neurons are a target for the action of galanin in the hypothalamus. First, we compared levels of galanin messenger RNA (mRNA) in GHRH neurons between normal male rats and Lewis dwarf rats, which have markedly reduced blood levels of GH. The brains of normal and dwarf animals were processed for detection of galanin mRNA and GHRH mRNA by double-label in situ hybridization. We observed that Lewis dwarf rats had significantly reduced levels of galanin mRNA in their GHRH neurons (P < 0.05). Next, we tested the hypothesis that GH regulates galanin gene expression in GHRH neurons by experimentally altering circulating levels of GH. Three groups of adult male rats were used: 1) intact rats (n = 7); 2) hypophysectomized (hypox) rats (n = 7); and 3) hypox rats treated with 1.5 mg of rat GH (rGH) over a 3-day period (n = 6). At the end of the treatment period, the animals were killed, and their brains were collected and processed for double-label in situ hybridization for GHRH mRNA and galanin mRNA. The signal level of galanin mRNA in GHRH neurons was reduced in hypox animals to less than 10% of that in intact controls (P < 0.0001); whereas, the levels of galanin mRNA signal in GHRH neurons did not differ significantly between the groups of intact and rGH-treated hypox rats. Finally, to determine whether SS or GHRH neurons are targets for galanin, we used double-label in situ hybridization to determine whether either of these populations of neurons express galanin receptor mRNA. A subset of SS neurons in the PeN appeared to express the galanin receptor mRNA, whereas few, if any, GHRH neurons appeared to do so. We conclude that galanin, like its cotransmitter GHRH, is a target for GH action, and we infer that galanin may play a role in the feedback control of GH secretion by exerting a direct effect on SS neurons.

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.

Modulation of growth axis gene expression by in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the weaning Holtzman rat

While thein utero and lactational effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on both male and female reproductive systems appear to be severe, little is known about its effects on the developing growth axis. The objective of this study was to describe changes in growth axis gene expression that accompany exposure to TCDD duringin utero and lactational development. Pregnant Holtzman rats were administered 1 μg TCDD/kg maternal body weight or vehicle control on gestational day 15 by gavage. Using ribonuclease protection assays, we compared mRNA levels measured in 21-d-old female pups exposed to TCDD with levels measured in control animals for the following genes: somatostatin, growth hormone-releasing hormone (GHRH), hypothalamic and pituitary galanin (GAL), growth hormone (GH), and insulin-like growth factor-I (IGF-I). Serum GH concentrations measured by radio-immunoassay were significantly increased, although GH mRNA levels were unchanged from controls by TCDD exposure. Hypothalamic GAL mRNA was decreased in TCDD-treated animals, whereas pituitary GAL mRNA in TCDD-treated animals was not altered. GHRH mRNA was increased in hypothalami from TCDD-exposed animals. IGF-I mRNA in the liver was decreased to 67% of controls. These data indicate that the growth axis is sensitive to the effects of TCDD delivered during critical periods of development. The alterations observed in growth axis gene expression with exposure to TCDD add to the body of data demonstrating a potent effect of this compound on the fetal and neonatal endocrine system.

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.

A novel peptide from the growth hormone releasing hormone gene stimulates Sertoli cell activity.

The growth hormone releasing hormone (GHRH) gene is believed to produce a single functional peptide, GHRH (1). Although GHRH mRNA and peptide have been identified in various tissues, the only accepted function of GHRH is the stimulation of pituitary growth hormone. We have discovered that the GHRH mRNA encodes a second peptide that is abundant in testicular germ cells and specifically activates Sertoli cell expression of stem cell factor (SCF), a factor crucial for the normal progression of spermatogenesis (2). These observations suggest that the GHRH gene produces two distinct peptides, each with tissue-specific activities.

A novel peptide from the growth hormone releasing hormone gene stimulates Sertoli cell activity

The growth hormone releasing hormone (GHRH) gene is believed to produce a single functional peptide, GHRH (1). Although GHRH mRNA and peptide have been identified in various tissues, the only accepted function of GHRH is the stimulation of pituitary growth hormone. We have discovered that the GHRH mRNA encodes a second peptide that is abundant in testicular germ cells and specifically activates Sertoli cell expression of stem cell factor (SCF), a factor crucial for the normal progression of spermatogenesis (2). These observations suggest that the GHRH gene produces two distinct peptides, each with tissue-specific activities.

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.

Peptides of the growth hormone-releasing hormone family

Growth hormone-releasing hormone (GHRH) belongs to a family of peptides expressed at high levels in the brain and digestive system of mammals. We have identified GHRH mRNA and peptide in rat and human germ cells, and detected a GHRH receptor mRNA in Sertoli cells. GHRH treatment of cultured Sertoli cells results in accumulation of cAMP and increased expression ofc-fos and stem cell factor (SCF), two factors critical for normal germ cell development. The current study was designed to localize within testis the transcription of other members of the GHRH family, and their receptors, and to determine if they also stimulate SCF. RNAs from separated testicular cells were amplified by comparative reverse transcription-polymerase chain reaction (RT-PCR). Southern blot analysis of the PCR products verified the presence of five GHRH family peptide and receptor transcripts in distinct testicular cell types. Transcripts encoding VIP and glucagon, and the receptors for pituitary adenylate cyclase activating peptide (PACAP) and glucagon, were detected predominantly in Leydig cells. In contrast, expression of GHRH, PACAP, secretin, and secretin receptor predominated in germ cells. Receptors for GHRH and VIP were expressed equally in all testicular cell types. To determine if, like GHRH, any of these other peptides activate Sertoli cell expression of SCF, primary Sertoli cell cultures were treated for 4-6 h with 10 or 100 nM of each individual factor. There was no consistent stimulation of SCF mRNA by VIP, PACAP, glucagon, or secretin. Differential expression of these peptides and their receptors suggests that they may each have unique paracrine functions within the testis.

Sex-related alterations in hypothalamic growth hormone-releasing hormone mRNA-but not somatostatin mRNA-expressing cells in genetically obese Zucker rats.

The possibility that the growth hormone (GH) suppression associated with obesity is due to alterations in hypothalamic GH-releasing hormone (GHRH) and/or somatostatin (SRIH) has been considered, but the data are not consistent. In the present study, we sought to clarify the roles of GHRH and SRIH in obesity by using in situ hybridization to localize and quantify the level of expression of GHRH mRNA- and SRIH mRNA-containing neurons in the hypothalamus of male and female lean and obese Zucker rats (12 weeks of age; n = 6 per group). In lean animals, the number of GHRH mRNA-expressing cells in the arcuate nucleus and SRIH mRNA-containing neurons in the periventricular nucleus was 2- to 3-fold higher in males compared to females. The obese phenotype in the male was associated with a striking reduction in arcuate GHRH mRNA expression, both in terms of number of cells (-71%; p < 0.01) and grains/cell (-44%; p < 0.05). In contrast, in obese females, there was a marked augmentation (+ 175%; p < 0.05) in the number of GHRH mRNA-containing cells in the arcuate nucleus compared to their lean littermates. The small population of GHRH mRNA-containing neurons of the ventromedial nucleus was not modified in male obese rats, while it was considerably increased (p < 0.05) in obese females. Neither the number of labeling density of SRIH mRNA-containing neurons in the periventricular and arcuate nuclei of obese rats of either sex was changed when compared to their sex-matched lean counterparts. These results demonstrate that: (1) the obese male Zucker rat exhibits a marked diminution in hypothalamic GHRH mRNA expression, while a reverse pattern is evident in the obese female; (2) hypothalamic SRIH mRNA-containing neurons are not significantly altered in obese rats of both sexes. Our findings suggest that the impaired GH secretion of the obese Zucker rat is due, at least in part, to alterations in hypothalamic GHRH gene expression and that SRIH does not play a major role.

Evidence for the involvement of non-androgenic testicular factors in the regulation of hypothalamic somatostatin and GHRH mRNA levels

The growth hormone (GH) secretory pattern is dependent on sex and developmental stage. It is generally accepted that in the male rat this pattern is markedly influenced by androgens secreted by the Leydig cells. Recent findings, however, point to the existence of other non-androgenic testicular factors produced by the Sertoli cells and which regulate in vivo the GH responses to growth hormone releasing hormone (GHRH). The aim of this work was to investigate the role played by non-androgenic testicular factors on hypothalamic somatostatin (SST) and GHRH mRNA levels. Seventy-day-old male Sprague-Dawley rats were used throughout the work. They were divided into five groups: (1) control rats; (2) gonadectomized rats; (3) gonadectomized rats supplemented with exogenous administration of dihydrotestosterone (DHT); (4) ethylene dimethane sulphonate (EDS)-treated rats; (5) EDS-treated rats supplemented with exogenous administration of DHT. EDS is a cytotoxic agent that specifically destroys the Leydig cells. The rats were killed after 15 days of treatment. Hypothalamic SST mRNA levels were determined by Northern blot and by in situ hybridization, and GHRH mRNA levels assessed by Northern blot. We found that selective removal of Leydig cells with EDS greatly reduced the SST mRNA content in the periventricular nucleus of the hypothalamus. These levels were significantly lower than those found in gonadectomized rats. Furthermore, replacement treatment with dihydrotesterone (DHT) did not completely restore SST mRNA levels in EDS-treated rats, contrasting with the complete recovery of SST mRNA levels in gonadectomized rats. On the other hand, gonadectomy and EDS treatment produced a significant reduction in GHRH mRNA levels. DHT administration reversed the action of gonadectomy, but did not restore GHRH mRNA content in EDS-treated rats. These data suggest that, in addition to testosterone, as yet unidentified non-androgenic testicular factors can significantly influence SST and GHRH mRNA levels. This may indicate that non-androgenic testicular factors acting at hypothalamic level may be important in the neuroregulation of GH secretion and in the maintenance of sexual dimorphism in GH secretory pattern.

Growth hormone-releasing peptides: clinical and basic aspects.

Growth hormone (GH)-releasing peptides (GHRPs), a family of synthetic oligopeptides which stimulate GH release, were identified more than a decade ago. The effects of these peptides on GH release have been described in vivo and in vitro, in both animals and humans, using various doses and administration routes. It is generally accepted that GHRPs stimulate the release of GH by acting at the level of the pituitary through a receptor different to that for the endogenous GH-releasing hormone (GHRH). In addition, it has been reported that there are specific binding sites for these peptides in the hypothalamus and that systemic administration of GHRPs increases the expression of the immediate early gene c-fos in a subpopulation of hypothalamic neurons. However, the identity of these hypothalamic neurons and the mechanism of action of GHRPs at both the hypothalamic and pituitary levels remain unknown. One interesting aspect of GHRPs is that they are orally active and this phenomenon has been demonstrated in both animals and humans. Furthermore, these drugs stimulate GH secretion in humans dose-dependently with the magnitude and duration of this response being comparable to that seen with an intravenous peptide bolus. We have studied the oral activity of GHRP-2 on GH release in normal children. In addition, we have analyzed the response to GHRP-2 of obese adolescents, as well as the effects of an intravenous bolus of GHRH alone and GHRH plus GHRP-2. Orally administered GHRP-2 stimulates GH secretion in normal children and, although it seems that this drug is more potent in girls, there were no statistical differences between the groups. Characteristically, GH levels started to increase by 15 min, peaked at 60 min and returned to basal concentrations by 180 min. The effect of GHRP-2 was synergistic with GHRH 1-29 NH2. In addition, obese subjects appeared to have a greater response to this peptide than did normal controls. To study the effects of GHRPs on hypothalamic GHRH and somatostatin neurons, female dwarf rats (dw/dw) were treated continuously with GHRP-6 (1 mg/kg per 24 h) for 14 days. In situ hybridization for GHRH and SS was performed. We found that GHRP-6 stimulated GHRH mRNA levels in the posterior arcuate nucleus (ARC), with no significant effect in the anterior ARC or ventromedial hypothalamic neurons. SS mRNA levels in the posterior periventricular nucleus (PeN) were decreased after GHRP-6 treatment, while no effect was seen in the anterior PeN, ARC, or lateral paraventricular nucleus. These results suggest that GHRP-6 treatment modulates hypothalamic neurons controlling GH secretion; however, whether this effect is direct or mediated through another factor remains to be elucidated.

Induction of c-fos mRNA in the arcuate nucleus of normal and mutant growth hormone-deficient mice by a synthetic non-peptidyl growth hormone secretagogue.

We have studied by in situ hybridization histochemistry the mRNA expression of the c-fos immediate early gene in the brains of wild type and dwarf(dw/dw) and little(lit/lit) mutant mice after systemic injections of the synthetic GH secretagogues GHRP-6 and L-163,191. Both GH secretagogues induced a marked c-fos mRNA expression in the arcuate-ventromedial hypothalamus (ARC-VMH) of both control and mutant mice indicating a possible action on growth hormone releasing hormone (GHRH) neurones in the ARC-VMH. Both dw/dw and lit/lit mice showed a 5-fold elevation in GHRH mRNA expression in the ARC-VMH compared with control animals under basal conditions. Since lit/lit mice have a reduced ability to secrete GH and lack a functional GHRH receptor while dw/dw mice lack both GH and presumably GHRH receptors, the GH-secretagogue-induced c-fos mRNA in the brain of these mutants are unlikely to be mediated by an indirect action of GH or a interaction of the synthetic GH-secretagogue with the GHRH receptor.