Effect of oral glucose administration on plasma growth hormone-releasing hormone (GHRH)-like immunoreactivity levels in normal subjects and patients with idiopathic GH deficiency: evidence that GHRH is released not only from the hypothalamus but also from extrahypothalamic tissue

Using a specific and sensitive RIA for GH-releasing hormone (GHRH), we examined the effect of oral administration of 75 g glucose on peripheral plasma GHRH-like immunoreactivity (GHRH-LI) in normal subjects (n = 12) and patients with idiopathic GH deficiency (IGHD) (n = 6). The normal subjects had two peaks of plasma GHRH-LI after oral glucose administration. The initial peak GHRH-LI levels occurred 30-150 min after glucose ingestion and corresponded to an increase in blood glucose. The increment in plasma GHRH-LI levels 30 min after glucose ingestion [7.4 +/- 2.4 (+/- SEM) pg/ml] was significantly higher (P less than 0.05) than that during a control study. Second peaks in plasma GHRH-LI occurred 3.5-6 h after glucose ingestion, and the mean increment 5 h after glucose ingestion was 9.4 +/- 2.4 pg/ml. This second rise of plasma GHRH-LI coincided with a significant increase in plasma GH after reactive hypoglycemia. This second GHRH-LI peak and the rise of plasma GH after hypoglycemia were absent in patients with IGHD, whereas the first peak of plasma GHRH-LI appeared shortly after glucose ingestion in these patients as well as in normal subjects. In addition, hypoglycemia produced by iv injection of regular insulin (0.1 U/kg) was not accompanied by increases in plasma GHRH-LI and GH levels in patients with IGHD, whereas insulin-induced hypoglycemia resulted in significant elevations of both plasma GHRH-LI and GH levels in normal subjects. These findings suggest that peripheral plasma GHRH-LI is derived from the hypothalamus as well as from an extrahypothalamic source(s); extrahypothalamic GHRH is released shortly after glucose ingestion; and secretion of GHRH from the hypothalamus is stimulated by hypoglycemia.     

Studies on plasma insulin-like growth factor (IGF)-I levels in normal subjects and in patients with various endocrine and metabolic diseases using radioimmunoassay for synthetic IGF-I (26-46)

Determination of plasma IGF-I concentrations is not easily accessible to clinical use at present because of extremely limited supply of purified natural IGF-I essential for its assay system. Thus, an alternative method has recently been introduced by the development of a specific radioimmunoassay (RIA) for IGF-I (26-46). We examined the specificity and sensitivity of this assay system, and then investigated the changes in plasma concentrations of IGF-I in normal children, adults and in patients with various endocrine and metabolic diseases. Each plasma sample was subjected to acid-ethanol treatment before assay to separate IGF-I from its binding protein. The recovery rate of known amount of IGF-I (26-46) added to untreated plasma sample was more than 90%. The coefficients of variation of intra- and interassay were 9.0% and 13.6%, respectively. This assay system was able to detect IGF-I as low as 10 pg/tube. When plasma sample of a patient with active acromegaly was applied to Sephadex G-75 column, immunoreactive IGF-I was eluted at the position of 7,000 molecular weight. An inhibition curve of plasma extract from an acromegalic patient was parallel to that of IGF-I (26-46), indicating that the RIA could detect IGF-I. There was no remarkable difference between IGF-I values of plasma and serum from the same individual. The value of IGF-I concentration of cord plasma was considerably low (144 +/- 6.7 pg/ml, M +/- SEM) as compared with that of sera of 49 normal children aged 7-12 12 years (320 +/- 14.3 pg/ml). The highest value (460 +/- 54 pg/ml) was attained at the age of 13 years, followed by gradual decrease toward adult age. Plasma IGF-I concentration of normal adults between 20 and 69 years of age was 290 +/- 10 pg/ml. When plasma IGF-I values of adult males and females were separately plotted against age group of each decade, the value declined gradually with age in males while in females there was a remarkable increase in plasma IGF-I concentration at 4th and 5th decades, suggesting the effect of hormonal change at menopause on plasma IGF-I levels. There was a good correlation between disorders of GH secretion and plasma IGF-I concentrations. In 10 cases of active acromegaly the level was 506 +/- 67 pg/ml (285-970 pg/ml). On the other hand in 20 patients with pituitary dwarfism it was only 180 +/- 15 pg/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of digoxin on the somatotroph responsiveness to growth hormone-releasing hormone (GHRH) alone or combined with arginine in normal young volunteers

BACKGROUND: The activity of the GH/IGF-I axis, known to play a major role in myocardial structure and function, has been reported to be altered in patients with chronic heart failure. AIM AND DESIGN OF THE STUDY: In order to evaluate the possibility that clinically used cardioactive drugs may exert neuroendocrine influences on somatotroph secretion, we studied the effects of pretreatment with enalapril (20 mg/day orally for 3 days), furosemide (20 mg i.v. as a bolus at -5 minutes) or digoxin (0.25 mg orally 4x/day for 3 days) on the GH response to growth hormone-releasing hormone (GHRH) (1.0 microg/ kg i.v. as a bolus at 0 minutes) in 12 healthy male adults (age [mean +/- SEM] 30.2 +/- 1.4 years; BMI 22.7 +/- 0.7 kg/ m2). In a subgroup of 8 subjects the same study was performed testing the GH response to GHRH + arginine (ARG; 0.5 g/kg i.v. from 0 to + 30 minutes). RESULTS The GH response to GHRH (1,304.1 +/- 248-5 microg/l/h) was not modified by enalapril (1,368.7 +/- 171.2 microg/l/h) or by furosemide (1,269.3 +/- 185.2 microg/l/h) but was significantly blunted by digoxin (613.6 +/- 73.2 microg/l/h, P < 0.05). On the other hand digoxin, enalapril and furosemide did not modify the GH response to GHRH +ARG. CONCLUSIONS: Digoxin, but not enalapril or furosemide, inhibits the GH response to GHRH in normal subjects. The blunting effect of digoxin on the GHRH-induced GH response is counteracted by arginine. These findings show that digoxin possesses an inhibitory effect on somatotroph secretion that may be mediated at the hypothalamic level.     

Calcitonin suppresses growth hormone (GH) response to growth hormone-releasing hormone (GHRH) in man

Calcitonin (CT) receptors have been found in the hypothalamus, suggesting a neuroendocrine role for this peptide. We have recently shown that, in the rat, central administration of salmon calcitonin (sCT) suppresses basal and GHRH-stimulated GH secretion. To further investigate how sCT alters GH secretion, we studied the effects of sCT (100U MRC, im) or placebo on basal and GHRH (50 micrograms, iv)-stimulated GH secretion in 6 normal men. GHRH was administered 1 h after sCT injection. Basal GH levels were not altered by sCT administration. However, GH response to GHRH was markedly suppressed by sCT (area under the curve - sCT: 574.6 +/- 69.7 vs placebo: 1057.2 +/- 284.8 micrograms. min/L; p less than 0.02). Cortisol levels were higher in sCT-treated subjects compared to controls, from 45 to 105 min after sCT injection (p less than 0.05). However, no correlation was found between GH response to GHRH and cortisol levels. No changes in glucose, calcium and PTH levels were seen. These results demonstrate that sCT inhibits GHRH-induced GH secretion in man by a mechanism apparently independent of changes in peripheral cortisol, glucose, calcium and PTH levels.     

Effect of thyrotropin-releasing hormone on growth hormone release in normal subjects pretreated with human pancreatic growth hormone-releasing factor 1-44 pulsatile administration

Growth hormone (GH) increase after thyrotropin-releasing hormone (TRH) has been documented in many pathological conditions. In order to evaluate whether exposure to growth hormone-releasing factor (GRF) might contribute to this effect in normal subjects, we studied GH responses to placebo, TRH, GRF and GRF plus TRH either in basal condition or after GRF administration. Ten subjects received placebo, TRH, GRF and GRF plus TRH on four separate occasions. GRF induced a clear rise in plasma GH, statistically different from those obtained after placebo or TRH (p less than 0.01). TRH was completely ineffective in both stimulating GH release and amplifying the secretory GH response to GRF. Twenty subjects, subdivided in four groups, received 3 consecutive intravenous GRF boli at two-hour intervals. Two hours later they were given a fourth stimulus: 5 had another 25 micrograms GRF i.v., 5 had 200 micrograms TRH i.v., 5 were tested with simultaneous 25 micrograms GRF and 200 micrograms TRH i.v. injection, and 5 with 1 ml saline. GH secretory responses were quantitated by determining the net incremental area under the curve (nAUC) over 60 min after the administration of each stimulus. The pattern of GH secretion after 1-3 GRF boli was not statistically different among the four groups. Plasma GH nAUC was higher after the first GRF injection than after the following ones (p less than 0.01). The administration of a fourth GRF bolus also caused a GH increase which was significantly smaller than that after the first one (p less than 0.01), but greater than that after placebo (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of growth hormone-releasing hormone on pentagastrin-induced growth hormone release in normal subjects

In order to investigate the mechanisms by which gastrin cause GH release in humans we measured the GH response to pentagastrin alone (1.5 micrograms/kg/hour from 120 to 210 min) and following pretreatment with GHRH (GHRH 1-29,250 micrograms, iv at 0 min) in normal male subjects. Prior GHRH administration abolished the GH response to the second bolus of GHRH (1 micrograms/kg) administered two hours later. Pentagastrin infusion induced a rise in GH levels maximal at 60 min (9.1 + 0.6 ng/ml, mean + SE), but this rise was abolished by pretreatment with GHRH. Finally, we found that gastrin did not modify basal GH release or GH responses to GHRH by rat anterior pituitary cells in monolayer culture. Taken together, these data suggest that gastrin regulates GH secretion by acting at hypothalamic level.     

Inhibition of L-dopa induced growth hormone release in normal and diabetic subjects by glucose administration

Administration of L-dopa 1 g induced an increase of plasma growth hormone (GH) levels in seven of ten healthy volunteers and in six of ten hyperglycemic insulin-dependent diabetic subjects; the maximal GH response was higher in normal subjects. Addition of 100 g glucose orally to the L-dopa completely abolished the GH response of both groups. The difference between the effect of endogenous hyperglycemia and the effect of a sudden increase of blood sugar after glucose administration on L-dopa induced GH release in diabetic subjects may be explain by the resetting of the hypothalamic control for pituitary GH release to higher levels of blood glucose.     

Behavioural phenotyping of male growth hormone-releasing hormone (GHRH) knockout mice

ObjectiveGH-releasing hormone (GHRH) is a key regulator of GH secretion. The role of GH in anxiety is somewhat contradictory. The aim of this study is to elucidate the consequences of lack of GHRH on emotional behaviour in a mouse model of GH deficiency due to removal of the GHRH gene (GHRH knock out, GHRHKO).DesignHomozygous GHRHKO and wild type male mice were utilized for this study. The emotional behaviour was measured through a battery of behavioural tests (locomotor activity/open field, light–dark exploration, elevated plus maze, forced swim test, tail suspension test). To correlate the emotional behaviour with brain neurochemistry, we evaluated thyrotropin-releasing hormone (TRH) gene expression in hypothalamic tissue by real-time PCR, and the levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) in prefrontal cortex by HPLC analysis.ResultsGHRHKO mice showed increased exploratory activity. In the open field test (P < 0.005), light–dark box (P < 0.005) and elevated plus maze (P < 0.05), GHRHKO mice demonstrated a decrease in anxiety-related behaviour. In addition, GHRHKO mice showed reduced immobility time with respect to control in forced swim test and tail suspension test (P < 0.0001). The gene expression of hypothalamic TRH (P < 0.05) was increased, while NE levels in prefrontal cortex were decreased compared to control (P < 0.05).ConclusionThese results suggest that in male mice GHRH deficiency brings about an increased physical activity and decreased anxiety- and depression-related behaviour, possibly related to increased TRH and decreased NE levels in the brain.     

The mutant growth hormone-releasing hormone (GHRH) receptor of the little mouse does not bind GHRH.

The little mouse is a dwarf strain characterized by low levels of GH, pituitary hypoplasia, and an unresponsiveness to treatment with exogenous GHRH. The defect has been mapped to a missense mutation in the GHRH receptor gene that abolishes the function of the receptor, but the mechanism of this inactivation is unknown. Receptor function might be affected at the level of protein expression, maturation and processing, localization to the cell surface, ligand binding, or signaling. In this study, Western blots, using antiserum raised against the GHRH receptor and immunoprecipitation analysis of epitope-tagged receptors, demonstrate that both wild-type and mutant receptor proteins are expressed at equivalent levels in transfected cells. Immunofluorescence analysis of intact and permeabilized cells expressing the epitope-tagged receptors suggests that wild-type and little mouse receptors are similarly localized to the cell surface. A species homologous binding assay was developed and used to show that 125I-mouse GHRH binds with high affinity to the wild-type mouse receptor but not to the little mutant receptor. Consistent with this, the mutant receptor fails to stimulate intracellular cAMP accumulation. Our results demonstrate that the little mutation does not dramatically affect the expression level, glycosylation, or cellular localization of the receptor protein but that it blocks specific GHRH binding, and therefore, signaling does not take place.     

Effect of continuous somatostatin and growth hormone-releasing hormone (GHRH) infusions on the subsequent growth hormone (GH) response to GHRH. Evidence for somatotroph desensitization independent of GH pool depletion

Continuous infusions of growth hormone-releasing hormone (GHRH) attenuate the subsequent growth hormone (GH) response to GHRH. To test whether this phenomenon can occur in the absence of GH pool depletion, we examined the effects of continuous infusions of 10 nM GHRH and of 10 nM somatostatin (SRIH), separately or in combination, on dispersed, perifused rat anterior pituitary cells. Columns of these cells were given either GHRH alone for 5 h, GHRH and SRIH together for 3 h followed by GHRH alone, or SRIH alone for 3 h followed by GHRH or medium. SRIH blunted both basal GH release and the GH response to GHRH, without affecting the subsequent GH responses to GHRH. The GHRH infusions attenuated the subsequent GH response to GHRH, even when GH release was initially prevented by the concurrent infusion of SRIH. Furthermore, the degree of attenuation was similar in the presence or absence of SRIH, suggesting that pool depletion plays little role in the desensitization process under these experimental conditions. The results are consistent with the hypothesis that a short-term infusion of GHRH leads to attenuation of the GH response in rat anterior pituitary cells primarily through receptor effects rather than through GH pool depletion.     

Effect of subacute administration of human growth hormone on various serum lipid and hormone levels of hypercholesterolemic and normocholesterolemic subjects

Six hypercholesterolemic and five normocholesterolemic subjects were injected twice daily with human growth hormone for 7 days. During HGH administration, the average serum cholesterol of both types of subjects fell significantly, and their serum triglyceride levels increased. Peak insulin responses to glucose also increased. However, no change occurred in the serum phospholipid, FFA, cortisol, or thyroxine concentrations during HGH treatment.     

Repetitive growth hormone-releasing hormone administration restores the attenuated growth hormone (GH) response to GH-releasing hormone testing in normal aging

The plasma GH response to human pituitary GH (hpGH)-releasing hormone-40 (hpGHRH-40; 1 microgram/kg BW) was significantly lower in seven healthy aged men (age range, 65-78 yr) than in seven healthy young men (age range, 18-31 yr) 30, 60, and 90 min after acute hpGHRH-40 administration (P less than 0.0001, by Student's unpaired t test). To verify whether a priming regimen might be able to reverse the reduced GH response to GHRH, elderly subjects underwent repetitive administration of hpGHRH-40 and placebo in a double blind design (100 micrograms hpGHRH-40 or volume-matched saline iv as a single morning dose, every 2 days for 12 days). After the hpGHRH-40-priming regimen, plasma GH values 30, 60, and 90 min after the acute GHRH test were significantly higher than values at the corresponding time points after placebo treatment. These findings suggest that somatotroph cells become less sensitive to GHRH with normal aging and demonstrate that repetitive administration of GHRH restores the attenuated response.     

Octreotide administration,under particular temporal conditions,enhances the responses of growth hormone to growth hormone-releasing hormone in normal subjects

OBJECTIVE Somatostatin not only inhibits basal and GHRH-stimulated GH secretion but might also enhance pituitary GH responsivity to GHRH under different temporal conditions. We investigated whether octreotide, a long-acting somatostatin analogue, has any positive actions on GHRH-induced GH release in normal human subjects. DESIGN The study consisted of three protocols. At 0800 hours, after fasting overnight, all subjects received 1 μg/kg GHRH l.v. bolus at 0 minutes. In each protocol, either octreotide (200 μg s.c.) or placebo were given respectively 8, 12, or 16 hours prior to GHRH challenge. SUBJECTS Three groups of eight normal volunteers (four female and four male in each group), aged 18–35 years, were randomly assigned to each protocol. MEASUREMENTS Growth hormone was measured by IRMA. Samples for GH assay were taken at ?30 and 0 minutes and then at 15-minute intervals up to 120 minutes. RESULTS When placebo or octreotide were administered 8 hours before GHRH, peak GH levels were respectively (mean ± SE, mU/l) 56·2 ± 16·6 and 60·8 ± 11·4 (NS). Also, when placebo or the somatostatin analogue were administered 16 hours prior to GHRH, peak GH levels were comparable (61·0 ± 7·4 vs 58·8 ± 7·4, NS). However, in the group receiving placebo or octreotide 12 hours prior to GHRH, the GH responses to GHRH were clearly enhanced by octreotide administration (peak GH levels, mU/l, 55·6 ± 21·6 vs 104·0 ± 17·4, P < 0·02). This enhancement of GH responses was observed in all subjects. CONCLUSIONS Octreotide administration did not affect GH responses to GHRH when given either 8 or 16 hours prior to GHRH. However, octreotide enhanced GHRH-induced GH release when administered 12 hours prior to GHRH. It thus appears that, under particular temporal conditions, octreotide may act positively on GH secretion in man.     

Sex-related naloxone influence on growth hormone-releasing hormone-induced growth hormone secretion in normal subjects

The effect of opiate-receptor antagonist naloxone on growth hormone (GH) release after growth hormone-releasing hormone (GHRH) 1-44 administration was investigated in ten normal men and 18 normal women during different phases of their menstrual cycle. Naloxone was infused at a rate of 1.6 mg/h in women and 1.6- and 3.2 mg/h in men, starting one hour before GHRH administration (50 micrograms iv as a bolus). On different day sessions, naloxone, GHRH, or saline were administered as controls. Naloxone infusion reduced the GHRH-induced GH release in normal women. The mean % inhibition of peak GH response was 83% during follicular phase, 46.5% during periovulatory phase, and 77.6% during luteal phase. On the contrary, in normal men, both doses of naloxone infusion were ineffective in blunting the GH response to GHRH. Our studies indicate that naloxone infusion was capable of inhibiting GH release induced by direct stimulation with GHRH in normal women, suggesting an opiate-antagonist action at the anterior pituitary level. The absence of such an effect in normal men strongly indicates a sex dependence of naloxone effects and suggests a role of the sexual steroid environment in opioid modulation of pituitary hormone secretion.     

Effect of growth hormone-releasing hormone on human peripheral blood leukocyte chemotaxis and migration in normal subjects

The effect of synthetic human growth hormone-releasing hormone (GHRH) on chemotactic response and migration inhibition of human peripheral blood leukocytes has been studied. In the assay performed by using modified Nelson methods, significant inhibition of chemotactic response was observed at 10(-6)M-10(-8) M concentrations of GHRH. There is a strong negative correlation (r = -0.519; p less than 0.001) between the chemotactic response of peripheral blood leukocytes and the concentration of GHRH. In contrast, GHRH tested at the same concentration range was not active in the migration inhibition assay. This finding provides additional evidence for the neuroimmunomodulatory action of GHRH.     

Age-related decrease in plasma growth hormone: response to growth hormone-releasing hormone, arginine, and L-dopa in obesity

Aging is associated with a reduction in plasma growth hormone (GH) secretion in non-obese subjects. To determine whether or not age-related changes in plasma GH secretion exist in obese subjects, we measured (a) plasma GH response to growth hormone-releasing hormone (GRH; 1 microgram/kg body wt), arginine (0.5 g/kg body wt), L-dopa (500 mg), and (b) plasma glucose, insulin, and free fatty acids (FFAs) in 26 fasted obese subjects of various ages ranging from 16 to 71 years. Only subjects with a body mass index (BMI; kg/m2) between 30.0 and 39.0 were studied. Six subjects were adolescents, 9 were in their 20s, and 11 were 30 years or older. The mean peak levels of plasma GH in response to GRH, arginine, and L-dopa in obese subjects were 11.3 +/- 2.1, 21.9 +/- 4.4, and 5.2 +/- 0.3 ng/mL in adolescents, 8.2 +/- 1.6, 9.1 +/- 1.5, and 3.1 +/- 0.6 ng/mL in those in their 20s, and 4.5 +/- 0.4, 7.3 +/- 1.4, and 2.8 +/- 0.3 ng/mL in those 30 years or older, respectively, showing a significant decrease in peak GH level with advancing age (P less than .05 to P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)