Further evaluation of IGF-I responsiveness to ACTH in children affected with IGHD.

In our previous studies we had demonstrated that, in children affected with isolated GH deficiency (IGHD), a short-term recombinant growth hormone (rGH) therapy increases the 11-deoxycortisol (S) secretion and induces an IGF-I responsiveness to the ACTH challenge. The aim of the present study was to further investigate the mechanisms by which IGF-I is secreted after ACTH challenge in children affected with IGHD by correlating IGF-I versus cortisol (F) time courses after ACTH administration. Ten children affected with IGHD were subjected to rGH therapy (4 IU/day subcutaneously) for 10 days. The responsiveness of IGF-I, F and S to the ACTH 1-17 test were evaluated before and at the end of the therapy. No IGF-I response to the ACTH test was recorded in the patients before the rGH treatment, whereas after rGH administration ACTH induced a significant IGF-I release (p < 0.001) which started at the 1st hour, reached a peak value between the 5th and 6th hours and disappeared at the 10th hour. In conclusion, our study confirms that a short-term rGH therapy induces an IGF-I responsiveness to ACTH and helps to better define the kinetics and the mechanism of this IGF-I response to ACTH.     

Localization of Carboxyl Ester Lipase in Human Pituitary Gland and Pituitary Adenomas

Carboxyl ester lipase (CEL) is an enzyme that hydrolyzes a wide variety of lipid substrates, including ceramides, which are known to show inhibitory regulation of pituitary hormone secretion in experimental models. Because no studies on CEL expression in human pituitary and pituitary adenomas have been reported in the literature, we investigated CEL expression in 10 normal pituitary glands and 86 well-characterized pituitary adenomas [12 FSH/LH cell, 17 α-subunit/null cell, 6 TSH cell, 21 ACTH cell, 11 prolactin (PRL) cell, and 19 GH cell adenomas] using IHC, immunoelectron microscopy, Western blotting, and quantitative RT-PCR. In normal adenohypophysis, CEL was localized in GH, ACTH, and TSH cells. In adenomas, it was mainly found in functioning GH, ACTH, and TSH tumors, whereas its expression was poor in the corresponding silent adenomas and was lacking in FSH/LH cell, null cell, and PRL cell adenomas. Ultrastructurally, CEL was localized in secretory granules close to their membranes. This is the first study demonstrating CEL expression in normal human pituitary glands and in functioning GH, ACTH, and TSH adenomas. Considering that CEL hydrolyzes ceramides, inactivating their inhibitory function on pituitary hormone secretion, our findings suggest a possible role of CEL in the regulation of hormone secretion in both normal and adenomatous pituitary cells. (J Histochem Cytochem 58:881–889, 2010)     

Dynamic of the GRF-induced GH response in genetically obese Zucker rats: influence of central and peripheral factors

To determine the time onset of the growth hormone (GH) alteration in the genetically obese rat, we studied the in vivo and in vitro rat growth hormone releasing factor (rGRF(1-29)NH2)-induced GH secretion in 6- and 8-week-old lean and obese male Zucker rats. Under sodium pentobarbital anesthesia, rGRF(1-29)NH2 (GRF) was injected intravenously at two doses: 0.8 and 4.0 micrograms/kg b.w. Basal serum GH concentrations were similar in lean and obese age-matched animals. The GH response to both GRF doses tested was unchanged in 6-week-old obese rats as compared to their lean litter mates. In contrast, a significant decrease of the GH secretion in response to 4.0 micrograms/kg b.w. GRF was observed in the 8-week-old obese rats. The effect of GRF (1.56, 6.25 and 12.5 pM) was further studied in vitro, in a perifusion system of freshly dispersed anterior pituitary cells of lean and obese Zucker rats. Basal GH release was similar in the 6-week-old animal group. In contrast, it was significantly decreased in 8-week-old obese rats as compared to their lean litter mates. Stimulated GH response to 1.56 and 6.25 pM GRF was significantly greater in the 6-week-old obese group than in the age-matched control group. In contrast, the GH response to all GRF concentrations tested was significantly decreased in the 8-week-old obese rats as compared to their respective lean siblings. In 8-week-old obese rats, a decrease of GH pituitary content and an increase of hypothalamic somatostatin (SRIF) concentration were observed. Insulin and free fatty acid serum were significantly increased in 8-week-old obese rats. In contrast, lower insulin-like growth factor I serum levels were observed in the obese animals as compared to their lean litter mates. Finally, to further clarify the role of the periphery in the inhibition of GH secretion observed in the 8-week-old fatty rats, we exposed cultured pituitary cells of 8-week-old lean animals to 17% serum of their obese litter mates. A significant decrease of GRF-stimulated GH secretion of lean rat pituitary cells exposed to the obese serum was noted (P less than 0.05). This study demonstrates that, in the obese Zucker rat, an alteration of the GH response to GRF is evident by the 8th week of life. This defective GH secretion could be related to peripheral and central abnormalities.     

Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3',5'-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids

We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.     

Melanin-concentrating hormone stimulates human growth hormone secretion: a novel effect of MCH on the hypothalamic-pituitary axis

Melanin-concentrating hormone (MCH), a 19-amino acid orexigenic (appetite-stimulating) hypothalamic peptide, is an important regulator of energy homeostasis. It is cleaved from its precursor prepro-MCH (ppMCH) along with several other neuropeptides whose roles are not fully defined. Because pituitary hormones such as growth hormone (GH), ACTH, and thyroid-stimulating hormone affect body weight and composition, appetite, insulin sensitivity, and lipoprotein metabolism, we investigated whether MCH exerts direct effects on the human pituitary to regulate energy balance using dispersed human fetal pituitaries (21-22 wk gestation) and cultured GH-secreting adenomas. We found that MCH receptor-1 (MCH-R1), but not MCH receptor-2, is expressed in both normal (fetal and adult) human pituitary tissues and in GH cell adenomas. MCH (10 nM) stimulated GH release from human fetal pituitary cultures by up to 62% during a 4-h incubation (P < 0.05). Interestingly, neuropeptide EI (10 nM), which is also cleaved from ppMCH, increased human GH secretion by up to 124% in fetal pituitaries. A milder, albeit significant, induction of GH secretion by MCH (20%) was seen in cultured GH-secreting pituitary adenomas. A comparable stimulation of GH secretion was seen when cultured mouse pituitary cells were treated with MCH. Treatment of cultured GH adenoma cells with MCH (100 nM) induced extracellular signal-regulated kinases 1 and 2 phosphorylation, suggesting activation of MCH-R1. In aggregate, these data suggest that MCH may regulate pituitary GH secretion and imply a potential cross-talk mechanism between appetite-regulating neuropeptides and pituitary hormones.     

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Effects of a leucine analog on growth hormone processing and secretion by cultured cells

Bovine and rat growth hormones (bGH and rGH, respectively) possess signal peptides that direct the hormone to the secretory pathway and are proteolytically cleaved prior to secretion. Previous in vitro translation studies indicated that incorporation of the polar leucine analog beta-hydroxyleucine into de novo synthesized polypeptides inhibits signal peptide function. To test the effects of this analog on GH secretion by cultured animal cells, transfections of mouse L-cells with a bGH expression plasmid or metabolic labeling of endogenous rGH in anterior pituitary cells was performed in the absence or presence of beta-hydroxyleucine. Transient expression of bGH in mouse L-cells or endogenous expression of rGH in anterior pituitary cells resulted in an accumulation of GH in the culture medium. Treatment with beta-hydroxyleucine resulted in a block in secretion as evidenced by an accumulation of GHs within these cells. Amino-terminal sequencing of the intracellular form of the analog-substituted GHs demonstrated accurate signal peptide cleavage. In contrast, in vitro translations of bGH RNA performed in the presence of beta-hydroxyleucine and microsomal membranes resulted in the inhibition of signal peptide cleavage. The results suggest that beta-hydroxyleucine can uncouple signal peptide processing and protein secretion in cultured cells.     

Effect of cortisol or adrenocorticotropic hormone on luteinizing hormone secretion by pig pituitary cells in vitro

The effect of cortisol or adrenocorticotropic hormone (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) was studied in vitro using dispersed pig pituitary cells. Pig pituitary cells were dispersed with collagenase and DNAase and then grown in McCoy's 5a medium containing 10% dextran charcoal-pretreated horse serum and 2.5% fetal calf serum for 3 days. Cells were preincubated with cortisol or ACTH before GnRH was added. When pituitary cells were incubated with 400 micrograms cortisol/ml medium for 6 h or longer, increase basal secretion of LH was observed. However, GnRH-induced LH release was reduced by cortisol. The degree of this reduction was dependent on cortisol, and a concentration of cortisol higher than 100 micrograms/ml was needed. Cortisol also inhibited the 17 beta-estradiol-induced increase in GnRH response. ACTH-(1-24), ACTH-(1-39), or porcine ACTH had no influence on GnRH-induced LH secretion. Our results show that cortisol can act directly on pig pituitary to inhibit both normal and estradiol-sensitized LH responsiveness to GnRH.     

The effects of 5-azacytidine on the expression of the rat growth hormone gene. Methylation modulates but does not control growth hormone gene activity

The role of DNA methylation in the expression of the rat growth hormone (rGH) gene was assessed by using a hypomethylating agent, 5-azacytidine, and the iso-schizomeric restriction enzymes MspI and HpaII. 5-Azacytidine increased rGH mRNA 3-8-fold in GH3D6 cells, a subclone of rat pituitary tumor cell lines that expresses one-tenth to one-fifteenth the GH expressed by two other clones, GH3 and GC. The effect was also detected at the level of pre-mRNA. The effect was independent of glucocorticoids and thyroid hormones and was found to be inheritable. The DNA methylation pattern generated by the isoschizomeric restriction enzymes indicated that the HpaII sites in the rGH gene were mostly methylated in GH3D6 cells but mostly unmethylated in GC cells. After treatment with 5-azacytidine, about 22% of these HpaII sites in GH3D6 cells became unmethylated. Thus, DNA methylation correlates inversely with the expression of the rGH gene in these cell lines. However, three other observations indicate that factors in addition to DNA methylation control rGH expression. First, in GC cells, even though most of the HpaII sites are unmethylated, the gene is not fully expressed. Second, in rat hepatoma cells, which do not express GH at all, the GH gene is less methylated than that in GH3D6 cells. Third, within the sensitivities of the assay methods, 5-azacytidine has no effect on the GH gene when it is completely silent. Taken together, the findings indicate that DNA methylation modulates but does not control GH gene expression. It is tempting to speculate that DNA methylation can influence expression only when the gene is committed to express.     

Induction of growth hormone release by dioscin from Dioscorea batatas DECNE

In this study, dioscin was isolated from Dioscoreae Rhizoma (DR), which is the rhizome of Dioscorea batatas D(ECNE). that inhabits broad areas of Korea and Japan. To determine whether dioscin induced growth hormone (GH) release, we evaluated its induction effects on GH release both in vitro and in vivo. The 70% methanol extract of DR, and its n-hexane and n-BuOH fractions, induced rat GH (rGH) release in rat pituitary cells 10-fold, 8-fold, and 5- fold higher than the control (0.36 +/- 0.02 nM), respectively (p < 0.05 each). The dioscin-induced rGH release of the cells was concentration-dependent and its ED(50) was 1.14 x 10(-5) M. Within 90 minutes after intravenous administration of 10 microg/kg (p < 0.05 at t(max)), dioscin caused the greatest increase in rGH concentration (C(max)) in the rat plasma (34.16 +/- 14.10 ng/ml) (n = 4), which was twice as high as the control group (12.88 +/- 3.29 ng/ml) (n = 27).     

Inhibitory effects of cysteamine on neuroendocrine function

The action of cysteamine on anterior pituitary hormone secretion was studied in vivo using conscious, freely moving male rats and in vitro using anterior pituitary cells in monolayer culture. Administration of 500 micrograms cysteamine into the lateral cerebral ventricles of normal rats caused the complete inhibition of pulsatile GH secretion for a minimum of 6 h. This treatment also significantly decreased plasma concentrations of LH for at least 6 h in orchiectomized rat, TSH in short-term (0.5 month) thyroidectomized rats, and PRL in long-term (6 months) thyroidectomized rats. The in vivo stimulation of GH, LH, TSH and PRL with their respective releasing hormones 60 min after administration of cysteamine was not different from the response observed in rats pretreated with saline except for PRL where cysteamine pretreatment significantly inhibited the expected PRL increase. In vitro, 1 mM cysteamine decreased basal and TRH stimulated PRL release while not affecting basal or stimulated GH, LH, TSH and ACTH secretion. These data demonstrate the dramatic and wide-ranging effects of cysteamine on anterior pituitary hormone secretion. This action appears to be mediated through hypothalamic pathways for GH, LH and TSH and through a pituitary pathway for PRL.     

Dexamethasone increases potassium channel messenger RNA and activity in clonal pituitary cells.

Glucocorticoid hormones are released as part of the stress response and regulate secretion by the pituitary. Since the activity of ion channels also influences secretion, we examined the effect of the glucocorticoid agonist dexamethasone on ion channel expression. K+ channel mRNA was detected in rat hypothalamus and anterior pituitary, with probes derived from the rat Kv1 gene, a member of the mammalian voltage-gated K+ channel superfamily. High levels were also detected in PRL-secreting clonal (GH3 and GH4C1) rat pituitary cells. Dexamethasone rapidly increased the steady state concentration of Kv1 mRNA in GH3 cells in a dose-dependent manner. This change in gene expression was accompanied by an increase in whole cell voltage-gated K+ current [lk(i)] with similar pharmacology to the Kv1 gene product. Our findings indicate that hormones may act directly on excitable cells to produce long term effects on electrical activity and secretion by regulating K+ channel expression.     

Effects of varying the position of thyroid hormone response elements within the rat growth hormone promoter: implications for positive and negative regulation by 3,5,3'-triiodothyronine

The thyroid hormone response element (T3RE) of the rat GH (rGH) promoter is located at -188 to -165 relative to the mRNA start site (TSS). Similar sites have been identified in other genes regulated by T3. We have investigated some of these T3REs in positions within the rGH promoter to assess the relative influences of DNA-binding site and position on positive and negative regulation by T3. Synthetic oligonucleotides were used with sequences from the rGH T3RE and proposed negative T3REs (nT3RE) from the rat and human alpha-subunit and rat beta TSH genes. The nT3REs were placed in the background of the wild-type rGH promoter in two positions, at -55 and down-stream of the TSS, with up- and down-mutations of the rGH T3RE. Rat GH T3RE elements were placed 700 basepairs up-stream of a basal rGH promoter and some also at the -55 and TSS positions. Constructions were tested in a transient transfection assay in rat pituitary tumor cells. Two copies of the rGHPAL (palindromic T3RE) placed 700 basepairs up-stream of the rGH promoter conferred 10-fold T3 induction. In the -55 position, the rGHPAL increased T3 induction compared to that in controls, whereas a fragment from the rat and human alpha-subunit gene in the same position reduced induction. Negative T3REs from rat beta TSH and human alpha-subunit reduced T3 induction 50% when placed at the TSS position of a rGH promoter containing an up-mutant T3RE. The T3REPAL placed at the same site increased T3 induction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thymosin fraction 5 (TF5) stimulates secretion of adrenocorticotropic hormone (ACTH) from cultured rat pituitaries

In vivo administration of a partially purified thymic hormone-containing extract of the thymus gland, TF5, causes an increase in serum glucocorticoids. The lack of a direct effect of TF5 on adrenal corticosterone secretion suggests that it is mediated at the level of the pituitary. Cultured rat pituitary monolayers were used to determine if the effect is mediated by stimulation of ACTH secretion from the pituitary. Two lots of TF5, BPP100 and C114080-01, caused a dose dependent secretion of ACTH from cultured pituitary monolayers. There was a synergistic effect when the cells were treated with both TF5 and corticotropin-releasing factor (CRF). Immunoneutralization studies were done in which the cells were treated with TF5 or CRF and an antibody to CRF. The antibody completely blocked CRF induced ACTH release, but had no effect on TF5 stimulated ACTH release, suggesting that the activity is not due to a CRF-like peptide in TF5. A number of peptides isolated from TF5, and certain other peptides produced by the immune system were evaluated for their ability to stimulate ACTH secretion. These included thymosin (TSN) alpha 1, alpha 11, and beta 4, prothymosin alpha (PT alpha, thymopoeitin 5 (TP5), factuer thymique serique (FTS), interferon alpha (INF alpha), INF gamma, interleukin 1 (IL-1), and interleukin 2 (IL-2). None of these factors had any effect on pituitary ACTH secretion. These results demonstrate that some peptide component of TF5 causes an increase in serum corticosteroids by stimulating pituitary ACTH release.     

Effect of dexamethasone on the release of pituitary hormones in monolayer cultures of rat pituitary cells

The effect of dexamethasone on the release of ACTH, GH, PRL, LH and TSH was studied in monolayer cultures of rat pituitary cells in 4-hour incubation. With or without the addition of rat hypothalamic extract, the release of GH was significantly inhibited by dexamethasone at concentrations higher than 10(-9) M, although less remarkably than that of ACTH. Intracellular ACTH and GH were unchanged. PRL, LH and TSH were not affected. These results indicate that dexamethasone, when exerted for 4 hours, suppressed the release of GH as well as ACTH, at least in part, at the pituitary level.     

The inhibitory action of corticotropin-releasing hormone on gonadotropin secretion in the ovariectomized rhesus monkey is not mediated by adrenocorticotropic hormone

Earlier observations in our laboratory indicated that i.v. infusion of human/rat corticotropin-releasing hormone (hCRH) suppresses pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in ovariectomized rhesus monkeys. Since cortisol secretion increased significantly as well, it was not possible to exclude the possibility that this inhibitory effect of hCRH on gonadotropins was related to the activation of the pituitary/adrenal axis. The purpose of the present study was to determine the role of pituitary/adrenal activation in the effect of hCRH on LH and FSH secretion. We compared the effects of 5-h i.v. infusions of hCRH (100 micrograms/h, n = 7) and of human adrenocorticotropic hormone (ACTH) (1-24) (5 micrograms/h, n = 3; 10 micrograms/h, n = 3, 20 micrograms/h, n = 3) to ovariectomized monkeys on LH, FSH, and cortisol secretion. As expected, during the 5-h ACTH infusions, cortisol levels increased by 176-215% of baseline control, an increase similar to that observed after CRH infusion (184%). However, in contrast to the inhibitory effect observed during the CRH infusion, LH and FSH continued to be released in a pulsatile fashion during the ACTH infusions, and no decreases in gonadotropin secretion were observed. The results indicated that increases in ACTH and cortisol did not affect LH and FSH secretion and allowed us to conclude that the rapid inhibitory effect of CRH on LH and FSH pulsatile release was not mediated by activation of the pituitary/adrenal axis.     

Galanin stimulates rat pituitary growth hormone secretion in vitro

The effect of galanin on growth hormone (GH) secretion was investigated in monolayer cultures of rat anterior pituitary cells. Galanin caused a gradual increase in GH concentrations into the culture medium that was maximal at 90 minutes and sustained after 180 minutes. The ED50 for galanin-stimulated GH secretion was approximately 200 nM compared to an ED50 for rat GH-releasing factor (rGRF)-stimulated GH secretion of 10pM. Galanin and rGRF were additive in increasing GH release into the incubation medium. These data indicate that porcine-derived galanin has a direct effect on pituitary GH secretion in vitro.