Improvement in clinical features of hypercortisolism during osilodrostat treatment: findings from the Phase III LINC 3 trial in Cushing's disease.

PURPOSE: Cushing's disease is associated with substantial morbidity and impaired quality of life (QoL) resulting from excess cortisol exposure. The current study explored improvements in clinical signs and additional specific manifestations of hypercortisolism during osilodrostat (potent oral 11ß-hydroxylase inhibitor) therapy by degree of control of mean urinary free cortisol (mUFC). METHODS: LINC 3 (NCT02180217) was a prospective, open-label, 48-week study of osilodrostat (starting dose: 2 mg bid; maximum: 30 mg bid) that enrolled 137 adults with Cushing's disease and mUFC > 1.5 times the upper limit of normal (ULN). mUFC (normal range 11‒138 nmol/24 h), cardiometabolic parameters (blood pressure, weight, waist circumference, body mass index, total cholesterol, fasting plasma glucose, glycated haemoglobin), physical manifestations of hypercortisolism (facial rubor, striae, fat distribution, bruising, hirsutism [females], muscle atrophy) and QoL were evaluated. mUFC was defined as controlled if ≤ ULN, partially controlled if > ULN but ≥ 50% reduction from baseline, and uncontrolled if > ULN and < 50% reduction from baseline. Concomitant medications were permitted throughout the study. RESULTS: At weeks 24 and 48, respectively, mUFC was controlled in 93 (67.9%) and 91 (66.4%) patients, partially controlled in 20 (14.6%) and 13 (9.5%), and uncontrolled in 24 (17.5%) and 33 (24.1%). Overall, mean improvements from baseline in cardiometabolic at week 24 were greater in patients with controlled or partially controlled versus uncontrolled mUFC; at week 48, improvements occurred irrespective of mUFC control. Generally, physical manifestations and QoL progressively improved from baseline irrespective of mUFC control. CONCLUSIONS: Improvements in clinical signs and additional specific manifestations of hypercortisolism associated with Cushing's disease occurred alongside decreases in mUFC. Trial registration NCT02180217 (first posted July 2014).

Safety and efficacy of growth hormone (GH) during extended treatment of adult Japanese patients with GH deficiency (GHD).

OBJECTIVES: To assess the effects of a growth hormone (GH) replacement therapy using a GH dose regimen based on serum insulin-like growth factor (IGF-I) concentrations in Japanese adults with GH deficiency (GHD). DESIGN: In this multicentre, uncontrolled, open-label study, Japanese adults with GHD who had received either GH replacement therapy (GH-GH group, n=35) or placebo (Placebo-GH group, n=36) in a previous randomised, double-blind, placebo-controlled trial were treated with GH replacement therapy for 48 weeks. GH treatment was started at a dose of 0.003 mg/kg/day administered by subcutaneous injection for the first 8 weeks, after which the dose was adjusted to maintain patients' serum IGF-I levels within the reference range adjusted for age and gender. Body composition, serum lipids, serum IGF-I and IGF binding protein-3 (IGFBP-3) levels were measured throughout study. Symptom and quality of life scores were also determined. RESULTS: Lean body mass (LBM) was increased compared with baseline (the end of the preceding double-blind trial) at 24 and 48 weeks, with a mean (+/-SD) increase of 1.3% (+/-4.2%) at week 48 in the GH-GH group (an increase of 6.6% [+/-6.0%] from the start of the preceding double-blind trial) and a larger increase of 4.7% (+/-5.9%) in the Placebo-GH group. Body fat mass (BFM) increased slightly from baseline in the GH-GH group with a mean increase of 2.9+/-10.6% at week 48 (a decrease from the start of the preceding double-blind trial at 48 weeks of 7.8% [+/-15.0%]) but decreased by 6.5% (+/-11.7%) at week 48 in the Placebo-GH group. Serum lipids were unchanged or slightly increased from baseline in the GH-GH group but patients' lipid profiles improved in the Placebo-GH group. In patients who received placebo during the double-blind study, individualised GH therapy in this open-label study increased mean LBM at 48 weeks by 6.2+/-6.8% in patients with CO GHD and by 3.0+/-4.4% in patients with AO GHD. Changes in mean LBM and mean BFM at week 48 were +4.1+/-4.5% and -2.4+/-10.5%, respectively, in females and +5.0+/-6.7% and -8.9+/-11.8%, respectively, in males. In patients who received GH treatment during the double-blind study, overall changes in LBM, BFM and IGF-I SD score after 24 weeks and 48 weeks were small, with no significant differences between subgroups. While the overall incidence of adverse events was broadly similar in the GH-GH and Placebo-GH groups (97% and 89%, respectively), the incidence of treatment-related events was higher in the GH-GH group (83% vs 42% in the Placebo-GH group). Most adverse events in both treatment groups were of mild or moderate severity and not clinically significant. The incidences of oedema and cases of high IGF-I during the IGF-I level-adjusted treatment regimen were lower than those during the preceding fixed dose titration. CONCLUSION: Long-term GH replacement therapy was well tolerated in Japanese adults with GHD. GH treatment maintained the improvements in body composition and lipid profiles in the patients previously treated in the double-blind study (GH-GH group) and improved these parameters in previously untreated patients (Placebo-GH group). Individualised GH administration based on IGF-I levels was well-tolerated and effective.

Chemokine (C-X-C motif) ligand 1 is a myokine induced by palmitate and is required for myogenesis in mouse satellite cells.

AIM: The functional significance of the myokines, cytokines and peptides produced and released by muscle cells has not been fully elucidated. The purpose of this study was to identify a myokine with increased secretion levels in muscle cells due to saturated fatty acids and to examine the role of the identified myokine in the regulation of myogenesis. METHODS: Human primary myotubes and mouse C2C12 myotubes were used to identify the myokine; its secretion was stimulated by palmitate loading. The role of the identified myokine in the regulation of the activation, proliferation, differentiation and self-renewal was examined in mouse satellite cells (skeletal muscle stem cells). RESULTS: Palmitate loading promoted the secretion of chemokine (C-X-C motif) ligand 1 (CXCL1) in human primary myotubes, and it also increased CXCL1 gene expression level in C2C12 myotubes in a dose- and time-dependent manner. Palmitate loading increased the production of reactive oxygen species along with the activation of nuclear factor-kappa B (NF-κB) signalling. Pharmacological inhibition of NF-κB signalling attenuated the increase in CXCL1 gene expression induced by palmitate and hydrogen peroxide. Palmitate loading significantly increased CXC receptor 2 gene expression in undifferentiated cells. CXCL1 knockdown attenuated proliferation and myotube formation by satellite cells, with reduced self-renewal. CXCL1 knockdown also significantly decreased the Notch intracellular domain protein level. CONCLUSION: These results suggest that secretion of the myokine CXCL1 is stimulated by saturated fatty acids and that CXCL1 promotes myogenesis from satellite cells to maintain skeletal muscle homeostasis.

Identification of somatostatin receptor subtypes and an implication for the efficacy of somatostatin analogue SMS 201-995 in treatment of human endocrine tumors.

The presence of somatostatin receptors has been demonstrated in various endocrine tumors as well as in normal tissues. We recently have cloned five human somatostatin receptor subtypes (SSTR1-SSTR5). These mRNAs are expressed in a tissue-specific manner. In this study, we have determined the somatostatin receptor subtypes expressed in various endocrine tumors using a reverse transcriptase polymerase chain reaction method. In two cases of glucagonoma and its metastatic lymph nodes in one case, all the SSTR subtype mRNAs except SSTR5 mRNA were expressed. In four cases of insulinoma, SSTR1 and SSTR4 mRNAs were detected, but SSTR2 mRNA was not detected in one case and SSTR3 mRNA was not detected in two cases, indicating a heterogeneous expression of SSTR subtypes in insulinomas. Interestingly, SSTR3 mRNA, which is highly expressed in rat pancreatic islets, is not expressed in normal human pancreatic islets, while SSTR1, SSTR2, and SSTR4 mRNAs are expressed. In three cases of pheochromocytoma, SSTR1 and SSTR2 mRNAs were detected, showing an expression pattern identical to that of normal adrenal gland. In a carcinoid, SSTR1 and SSTR4 mRNAs were detected. We have also found that human SSTR2 shows a high affinity for SMS 201-995, which has been used clinically for the treatment of endocrine tumors. Since SMS 201-995 was effective in the treatment of a patient with glucagonoma in which SSTR2 mRNA was present, but had no effect in a patient with carcinoid in which SSTR2 mRNA was not detected, this study suggests that the efficacy of SMS 201-995 may depend, at least in part, on the expression of SSTR2 in tumors.

Efficacy and safety of growth hormone (GH) in the treatment of adult Japanese patients with GH deficiency: a randomised, placebo-controlled study.

OBJECTIVE: The aim of this study was to assess the effect of growth hormone (GH) replacement therapy on lean body mass (LBM) and other variables including body fat mass, serum lipids and quality of life measures in GH-deficient Japanese adults. DESIGN: This was a multicentre, double-blind, placebo-controlled, parallel group study. Following initial screening, patients were randomly assigned to GH treatment (n=37) or placebo (n=36). GH treatment was started at an initial dose 0.003 mg/kg/day s.c. each day for the first 4 weeks after which the dose was increased to 0.006 mg/kg/day for 4 weeks and then to 0.012 mg/kg/day for the last 16 weeks (n=37). Body composition, serum lipids, serum IGF-I and IGFBP-3 levels were measured during the 24-week study. Short Form-36 and Quality of Life Assessment of GH Deficiency in Adults scores were also determined. RESULTS: LBM was significantly increased from baseline at 24 weeks in GH-treated patients, with a mean (+/-SD) increase of 4.7% (+/-5.3%) compared with an increase of 1.0% (+/-4.4%) in the placebo group (p<0.0001 versus baseline, p=0.0003 versus placebo). Percentage body fat decreased significantly from baseline in GH-treated patients (9.3%, p<0.0001), compared with a non-significant 0.2% increase in the placebo group (p<0.0004 for difference between treatment groups). In addition, significantly increased serum IGF-I and IGFBP-3 levels and improvements in the patients' serum lipid profiles were observed in patients who received GH therapy. Changes in quality of life measures did not differ between treatments, probably because of the small number of patients studied. GH therapy was well tolerated, with adverse events of any cause reported in 86.5% of the GH treatment group and 83.3% of the placebo group. CONCLUSION: GH treatment significantly improved body composition and serum lipid profiles in adult Japanese patients with GH deficiency compared with placebo and had no clinically relevant adverse effects.

Germline deletion and a somatic mutation of the PRKAR1A gene in a Carney complex-related pituitary adenoma.

OBJECTIVE: The objective was to assess involvement of loss of the PRKAR1A gene encoding a type 1α regulatory subunit of cAMP-dependent protein kinase A located on 17q24 in a Carney complex (CNC)-related pituitary adenoma. DESIGN: We investigated aberrations of the PRKAR1A gene in a CNC patient with a GH-producing pituitary adenoma, whose family has three other members with probable CNC. METHODS: A gene mutation was identified by a standard DNA sequencing method based on PCR. DNA copy number was measured to evaluate allelic loss on 17q24 by quantitative PCR. The breakpoints of deletion were determined by cloning a rearranged region in the deleted allele. RESULTS: A PRKAR1A mutation of c.751_758del8 (p.S251LfsX16) was found in genomic DNA obtained from a pituitary adenoma, but not leukocytes from the patient. Reduced DNA copy number at loci including the PRKAR1A gene on 17q24 was detected in both the tumor and leukocytes, suggesting a deletion at the loci at the germline level. The deletion size was determined to be ∼ 0.5 Mb and this large deletion was also found in two other family members. CONCLUSION: This is the first case showing a CNC-related pituitary adenoma with the combination of somatic mutation and a large inherited deletion of the PRKAR1A gene. Biallelic inactivation of PRKAR1A appears to be necessary for the development of CNC-related pituitary adenoma.

Possible involvement of gamma-aminobutyric acid in growth hormone release induced by a Met5-enkephalin analog in conscious rats.

The interaction between opioid peptides and gamma-aminobutyric acid (GABA) in regulating GH secretion was studied in unanesthetized male rats with chronically implanted intraatrial catheters. GH secretion was pulsatile with GH bursts (mean +/- SE, 259.1 +/- 70.3 ng/ml) occurring at regular intervals (mean +/- SE, 3.6 +/- 0.2 h) in control rats. When a potent met5-enkephalin analog, FK 33-824 ([D-Ala,MePhe4,Met(O)-ol]enkephalin; 10 micrograms/100 g BW), was injected iv in the interval between two anticipated spontaneous GH bursts, plasma GH abruptly increased to a peak value of 716.0 +/- 144.9 ng/ml 20 min after the injection, and the following spontaneous GH burst appeared at longer intervals than expected (5.0 +/- 0.4 h, P less than 0.025 vs. control). The plasma GH increase induced by FK 33-824 was blunted by a specific opiate antagonist, naloxone (125 micrograms/100 g BW, iv), and the following spontaneous GH bursts occurred at the same time as in controls. The plasma GH response to FK 33-824 was significantly inhibited by two GABA antagonists, picrotoxin (0.3 mg/100 g BW, iv) and bicuculline (60 micrograms/100 g BW, iv), with peak GH values of 24.6 +/- 6.4 and 136.4 +/- 35.2 ng/ml, respectively (P less than 0.01 vs. FK 33-824 alone). The following natural GH bursts were also inhibited by these GABA antagonists (50.1 +/- 21.3 and 35.3 +/- 9.6 ng/ml; P less than 0.01 vs. control). These findings suggest the possible involvement of GABA in GH release induced by opioid peptides in the rat.

Possible mechanisms involved in growth hormone secretion induced by galanin in the rat.

The mechanisms by which central or systemic administration of galanin stimulates GH secretion were investigated in either conscious or urethane-anesthetized male rats. Intracerebroventricular injection of synthetic porcine galanin, a 29-amino acid gut-brain peptide (0.12, 0.6, and 3 nmol/rat), resulted in a dose-related increase in plasma GH. The plasma GH level was increased by an N-terminal galanin fragment [galanin-(1-19)], but not by C-terminal fragments [galanin-(2-29) and -(21-29)]. Intravenous injection or infusion of galanin (0.6 and 3 nmol/100 g BW) also raised plasma GH. The plasma GH increase induced by galanin was inhibited by pretreatment with rabbit antiserum specific for rat GRF. Pretreatment with yohimbine or phenoxybenzamine, alpha-adrenergic blockers, or picrotoxin, a gamma-aminobutyric acid (GABA) antagonist, blunted the plasma GH increase induced by intracerebroventricular injection of galanin. On the other hand, the plasma GH increase induced by iv injection of galanin was suppressed by picrotoxin, but not by phenoxybenzamine. These findings suggest that 1) both central and systemic administration of galanin stimulate GH secretion in the rat; 2) the N-terminal structure of galanin is required to stimulate GH secretion; 3) the stimulating effect of galanin is mediated, at least in part, by hypothalamic GRF; and 4) central alpha-adrenergic and GABAergic mechanisms may be involved in GH release induced by central administration of galanin, whereas systemic injection of galanin stimulates GH release predominantly through GABAergic mechanisms in the rat.

Interrelation between somatostatin output from the mediobasal hypothalamus and plasma growth hormone levels in conscious rats: effects of glucagon administration.

The interrelationship between SRIF output from the mediobasal hypothalamus and plasma GH levels was studied in conscious male rats using the push-pull perfusion technique in combination with repeated blood samplings. The MBH was perfused with artificial cerebrospinal fluid at the rate of 30 microliter/min, and blood samples were collected every 20 min from 1000-1700 h. In control animals, which received injection of acidified saline at 1100 h into the lateral ventricle, two large episodes of spontaneous GH secretion occurred regularly at around 1200 and 1540 h, and troughs occurred around 1400 h. In contrast, SRIF levels from mediobasal hypothalamus perfusate fluctuated at random, ranging from 10-116 pg/ml, with a mean value of 39.2 pg/ml. Mean SRIF levels at 1200 and 1540 h (43.4 +/- 9.0 and 24.4 +/- 4.2 pg/ml, respectively; n = 8) were not different from those at 1400 h (39.9 +/- 12.2 pg/ml). When glucagon (10 micrograms/rat) was injected at 1100 h, plasma GH levels decreased and remained low until 1600 h, whereas perfusate SRIF levels were elevated and remained high for the period. In these animals, the mean plasma GH levels during 1120-1540 h were lower than those in control rats [17.2 +/- 2.4 ng/ml (n = 9) vs. 143.4 +/- 17.5 ng/ml (n = 8); P less than 0.01]. In contrast, the mean SRIF levels in glucagon-treated rats were higher than those in controls [112.5 +/- 15.9 pg/ml (n = 9) vs. control 40.1 +/- 4.3 pg/ml (n = 8); P less than 0.01]. These results suggest that SRIF plays a role in tonic inhibition of GH release in response to the intracerebroventricular injection of glucagon in conscious rats, although SRIF plays, if any, a minor role in regulating episodic GH secretion.

Inhibition by gastrin-releasing peptide of growth hormone (GH) secretion induced by human pancreatic GH-releasing factor in rats.

Intravenous injection of synthetic human pancreatic GH-releasing factor (1-44) [hpGRF(1-44)] (50 ng-1 micrograms/100 g BW) resulted in a dose-related increase in plasma GH levels in urethane-anesthetized male rats. Pretreatment with cysteamine (30 mg/100 g BW, sc, 4 h previously) or anti-SRIF rabbit serum (0.5 ml/rat, iv, 1 h previously) raised basal plasma GH levels and markedly exaggerated the plasma GH response to hpGRF (1-44) (80 ng/100 g BW, iv). The intraventricular injection of gastrin-releasing peptide (GRP) (1 micrograms/rat) completely inhibited the increase of plasma GH induced by hpGRF (80 ng/100 g BW, iv) in control rats. However, in the rats treated with cysteamine or anti-SRIF rabbit serum, the inhibitory effect of GRP on hpGRF-induced GH release was significantly attenuated. hpGRF (10(-11)-10(-8) M) stimulated in a dose-related manner GH release from rat anterior pituitary cells superfused in vitro. GRP (10(-5) M) did not affect pituitary GH release induced by hpGRF (10(-9) M) in vitro. These results indicate that hpGRF stimulates rat GH secretion by acting at the pituitary level and the hypothalamic SRIF interacts with the action of GRF, and that GRP inhibits GH secretion, at least in part, by stimulating SRIF release from the hypothalamus.

Effects of glucagon, Neurotensin, and vasoactive intestinal polypeptide on somatostatin release from perifused rat hypothalamus.

The effects of neuropeptides on the release of immunoreactive somatostatin (SRIF) from the rat hypothalamus were examined in vitro using a perifusion system. Twelve hypothalamic halves of male rats were placed on a Sephadex G-25 column and continuously eluted with Krebs-Ringer bicarbonate buffer, poH 7.4, at 37 C. A high potassium concentration (56 mM) stimulated SRIF release in a calcium-dependent manner. The infusion of glucagon (10(-7), 5 x 10(-7), and 10(-6) M) resulted in a dose-related increase in the release of SRIF. Neurotensin (10(-6) M) also stimulated SRIF release, whereas vasoactive intestinal polypeptide (10(-7) and 10(-6) M) inhibited SRIF release. SRIF release was not affected by cholecystokinin-octapeptide (10(-6) M), cholecystokinin-tetrapeptide (10(-6) M), or tRH (10(-6) M). These findings suggest that SRIF release from the rat hypothalamus is influenced by glucagon, neurotensin, and vasoactive intestinal polypeptide.

Effect of prostaglandin E1 on vasoactive intestinal polypeptide release from the hypothalamus and on prolactin secretion from the pituitary in rats.

In order to elucidate the mechanisms by which prostaglandin (PG) affects PRL secretion, the effect of PGE1 on vasoactive intestinal polypeptide (VIP) release from the rat hypothalamus was examined by determining plasma VIP levels in rat hypophysial portal blood in vivo and VIP release from the perifused hypothalamus in vitro. Intraventricular injection of PGE1 (1 and 5 micrograms/rat) caused a 2- to 3-fold increase in the concentration of plasma VIP in hypophysial portal blood in anesthetized rats. The flow rate of portal blood was slightly increased after the injection of PGE1. VIP release from the perifused rat hypothalamus was stimulated by high potassium levels (56 mM). The infusion of PGE1 (10 microM) resulted in a significant increase in VIP release from the hypothalamus in vitro. Both these responses were calcium dependent. The intraventricular injection of PGE1 (1 and 5 micrograms/rat) resulted in a dose-related increase in peripheral plasma PRL levels in the rat. These findings suggest that PGE1 plays a stimulatory role in regulating VIP release from the hypothalamus into hypophysial portal blood and causes PRL secretion from the pituitary in rats.

Stimulation by serotonin of vasoactive intestinal polypeptide release into rat hypophysial portal blood.

The effect of serotonin (5-HT) on plasma vasoactive intestinal polypeptide (VIP) levels in hypophysial portal blood was studied in urethane-anesthetized rats. Portal blood was collected by the parapharyngeal approach and plasma VIP was determined by radioimmunoassay. Mean (+/- SE) basal plasma VIP level was 1799 +/- 232 pg/ml, which was slightly decreased during the control experiments in which physiological saline was injected either intraventricularly or intravenously. Intraventricular injection of 5-HT (2 and 10 micrograms/rat) resulted in a significant increase in plasma VIP concentrations within 20 min. Intravenous injection of L-5-hydroxytryptophan (5-HTP, 1 mg/100 g BW), a precursor of 5-HT, also caused an increase in VIP concentrations in hypophysial portal plasma. The flow rate of hypophysial portal blood did not change throughout the experiments. These findings suggest that 5-HT stimulates VIP release from the median eminence into the hypophysial portal vessels in the rat.

Immunoreactive vasoactive intestinal polypeptide in rat hypophysial portal blood.

Vasoactive intestinal polypeptide (VIP) was measured by RIA in the hypophysial portal blood of rats after total hypophysectomy. The mean (+/- SE) VIP concentrations were 1332 +/- 171 pg/ml (range, 300-3868 pg/ml) under urethane anesthesia and 1735 +/- 707 pg/ml under pentobarbital anesthesia. The concentrations of VIP in peripheral plasma were, in most animals, less than 100 pg/ml. The secretion rate of VIP was not considerably changed during the blood collection. Immunoreactive VIP in hypophysial portal blood was identical to authentic VIP on gel chromatography. These findings suggest that VIP secreted from the hypothalamus may modulate pituitary function via the portal circulation. (Endocrinology 108: 395, 1981)

Stimulation by leumorphin of prolactin secretion from the pituitary in rats.

The effect of leumorphin (LM), one of big leu-enkephalins derived from preproenkephalin B, on PRL secretion was studied in the rat in vivo and in vitro. Intracerebroventricular injection of synthetic porcine LM (0.06-6 nmol/rat) caused a dose-related increase in plasma PRL levels in urethane-anesthetized male rats and in conscious freely moving rats. Intravenous injection of LM (3 nmol/100 g BW) also raised plasma PRL levels in these animals. The plasma PRL response to intracerebroventricular LM (0.6 nmol/rat) was blunted by naloxone (125 micrograms/100 g BW, iv). The stimulating effect of LM on PRL release was the most potent among the peptides derived from preproenkephalin B. In in vitro studies, PRL release from superfused anterior pituitary cells was stimulated in a dose-related manner by LM (10(-9)-10(-6) M), and the effect was blunted by naloxone (10(-5) M). These results suggest that LM has a potent stimulating effect on PRL secretion from the pituitary in the rat by acting, at least in part, directly at the pituitary through an opiate receptor.

Growth hormone (GH) secretory dynamics in a case of acromegalic gigantism associated with hyperprolactinemia: nonpulsatile secretion of GH may induce elevated insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 levels.

We describe a case of pituitary gigantism with low levels of growth hormone (GH), elevated insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 (IGF-BP-3). The patient had characteristic clinical features of gigantism and acromegaly. The basal serum GH levels ranged from 1.2-1.9 micrograms/L, which were considered to be within normal limits. Serum GH response to either insulin-induced hypoglycemia or GH-releasing hormone was blunted. Frequent blood samplings during daytime and at night showed nonpulsatile GH secretion. Serum prolactin, IGF-I and IGF-binding protein-3 levels were elevated. After unsuccessful surgery, bromocryptine treatment normalized serum prolactin without affecting serum GH and IGF-I levels. Combined administration of octreotide and bromocryptine reduced serum GH and IGF-I levels. GH bioactivity as measured by Nb2 cell proliferation assay was within reference range. In the present case, nonpulsatile GH secretion and enhanced tissue sensitivity to GH may induce hypersecretion of IGF-I and IGF-BP-3 and cause clinical acromegalic gigantism.

Stimulation of growth hormone release by vasoactive intestinal polypeptide from human pituitary adenomas in vitro.

The effects of vasoactive intestinal polypeptide (VIP), TRH, dopamine, and rat median eminence extract on GH release from GH-secreting pituitary adenomas were studied in vitro using a sensitive superfusion method. Dispersed pituitary tumor cells obtained from three patients with acromegaly were placed in a superfusion column, and the amounts of GH in the superfusate were determined. The addition of VIP (10(-6) M) to the perfusion system resulted in a marked increase in GH release in all three cases, and a dose-response relationship in VIP (10(-8) 10(-6) M) induced GH secretion was observed in one case studied. TRH (10(-7) M) and median eminence extract (1 equivalent/ml) also caused an abrupt and marked increase in GH release in all of the experiments. The infusion of either dopamine (10(-7) M) or bromocriptine (10(-7) M) inhibited GH secretion. These results suggest that VIP as well as TRH stimulate GH secretion by a direct action on GH-secreting pituitary tumor cells in at least some acromegalic patients.

Secretory dynamics of growth hormone in an acromegalic patient associated with Graves' disease.

The dynamics of GH secretion were investigated in an acromegalic patient with Graves' disease during treatment with antithyroid drugs and radioactive iodine. The mean (+/- SE) basal plasma GH levels were 16.5 +/- 0.9 and 7.8 +/- 0.7 micrograms/L during the hyperthyroid and euthyroid states, respectively. There was a positive correlation (r = 0.902) between basal GH and free T4 levels. Twenty-four-hour plasma GH and urinary GH excretion both increased during the thyrotoxic state. Intravenous administration of TRH induced a marked increase in plasma GH levels during euthyroidism and a modest increase when the patient was hyperthyroid, whereas GH release induced by GH-releasing hormone was not altered by thyroid status. These findings suggest that hyperthyroidism stimulated spontaneous GH secretion from the pituitary adenoma, but inhibited the stimulating effect of TRH.

Characterization of Prophet of Pit-1 gene expression in normal pituitary and pituitary adenomas in humans.

Prophet of Pit-1 (Prop-1), which is a paired-like homeodomain transcription factor, is capable of binding to sites in an early enhancer of the Pit-1 gene and regulating its expression. According to a previous report, Prop-1 messenger RNA (mRNA) is expressed in the developing pituitary gland before Pit-1 mRNA expression and maximum expression are observed at e 12.0. After e 14.5, Prop-1 mRNA expression rapidly decreases, and only trace amounts of mRNA are detectable in adult mouse pituitary. Human Pit-1 is expressed considerably, not only in normal adult pituitary but also in pituitary adenomas, so we studied human Prop-1 gene expression in adult pituitary and pituitary adenomas. We also cloned human Prop-1 complementary DNA (cDNA) and sequenced the Prop-1 cDNAs in pituitary adenomas. The amino acid sequence of human Prop-1 cDNA that we cloned was identical to that of the previously reported sequence, except Thr substituted at codon 142 instead of Ala. This amino acid substitution is considered to be a polymorphism because it did not alter transcriptional activity, and 7 of 28 alleles were Ala. Human Prop-1 transcript was detected in normal adult pituitary, by Northern blot analysis, and in all pituitary adenomas examined by RT-PCR analysis. The expression of human Prop-1 in pituitary adenomas was confirmed by in situ hybridization in one of the somatotroph adenomas. The sequence analysis of human Prop-1 cDNAs in these pituitary adenomas revealed that there were no mutations, except 5 silent nucleic acid substitutions, suggesting that mutations of Prop-1 gene do not represent a frequent mechanism of human pituitary tumorigenesis.

A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans.

The synergistic relationship between GH-releasing secretagogue (GHS) and GH-releasing hormone (GHRH) with respect to GH secretion is well known. In the present study, we report a similar relationship between GHRH and ghrelin, a recently identified endogenous ligand for the GHS receptor. In normal male adults, various doses of ghrelin were intravenously administered alone or together with 1.0 microg/kg GHRH. At small doses of 0.08 and 0.2 microg/kg ghrelin, combined administration of the two peptides significantly stimulated GH release in a synergistic manner; the mean GH response values of the two peptide combinations were more than the summed mean GH response values of each peptide alone (P < 0.05). In addition, at 1.0 microg/kg ghrelin, the tendency of the synergistic effect was observed, although the comparison was not statistically significant probably due to a submaximal dose ceiling effect. No synergistic effects with respect to ACTH or prolactin secretion were observed. In conclusion, the synergistic interaction between ghrelin and GHRH was clearly shown and might be useful for a provocation test to diagnose GH deficiency.