Cholinergic control of growth hormone (GH) responses to GH-releasing hormone in insulin dependent diabetics: evidence for attenuated hypothalamic somatostatinergic tone and decreased GH autofeedback

OBJECTIVE We have investigated the effects of cholinergic modulation with pirenzepine and pyridostigmine and of GH pretreatment on the subsequent GH response to a maximal stimulatory dose of GH-releasing hormone (GHRH) in patients with insulin dependent diabetes mellitus (IDDM). We have also investigated the relationship between the differences in metabolic control and other parameters of disease state with the differences in GH responses in IDDM. PATIENTS Thirteen male subjects with IDDM and no clinical evidence of complications were selected based on HbA, levels to provide a wide range of metabolic control. Seven normal subjects were also studied. DESIGN Twelve of the subjects with IDDM and six normal subjects received pirenzepine 200 mg and pyriostigmine 120 mg pretreatment 60 minutes and GH pretreatment 3 hours before an i.v. injection of GHRH (1–44) (80 μg) in random order. All subjects underwent a control study with GHRH alone. MEASUREMENTS Serum GH and plasma glucose were measured at regular intervals throughout the study. Fasting plasma glucose and HbA, were measured before each study to provide measures of metabolic control. RESULTS Subjects with IDDM demonstrated exaggerated GH responses to GHRH compared to normals. Pirenzepine significantly reduced GH responses in both normal and diabetic subjects. However, the GH response to GHRH after pirenzepine was higher in subjects with IDDM (mean GH: IDDM vs normals; 8.1 ± 1.3 vs 2.9 ± 0.7 mU/l, P < 0.05). Pyridostigmine 120 mg significantly augmented the GH response to GHRH in normal subjects. In diabetic subjects, pyridostigmine failed to increase GH response to GHRH compared to GHRH alone (mean GH: pyridostigmine vs control: 75.7 ± 12.6 vs 38.9 ± 5.4 mU/l, P= NS). GH responses to GHRH after pyridostigmine pretreatment in both normal and diabetic subjects did not differ and the GH response to GHRH after pyridostigmine in normal subjects did not differ from the GH response to GHRH alone in diabetic subjects. In normal subjects, GH pretreatment significantly reduced subsequent GH responsiveness to GHRH (Δpeak GH 26.4 ± 5.2 vs 7.7 ± 5.4 mU/I, P< 0.04). In contrast, GH pretreatment did not cause any significant reduction in GH responsiveness to GHRH In diabetics (Δpeak GH 53.6 ± 9.7 vs 33.4 ± 11 mU/I, P= NS). No significant correlation was demonstrated between measures of diabetic control and the responses to GHRH alone or after cholinergic modulation and GH pretreatment. CONCLUSION These data suggest that ambient hypothalamic cholinergic tone in diabetes is high, and of similar degree to the enhanced cholinergic tone in normal subjects pretreated with pyridostigmine. We suggest that in diabetic subjects, the reduced responsiveness to autofeedback may be secondary to the enhanced cholinergic tone demonstrated in these patients. The mechanisms linking the uncontrolled diabetic state to this abnormal neuroregulation of GH remains unknown at present.     

Sexual dimorphism of pyridostigmine potentiation of growth hormone (GH)-releasing hormone-induced GH release in humans

Sex differences in the neuroregulation of GH secretion are not now known in humans. To investigate whether activation of cholinergic tone by pyridostigmine could cause a sex-related difference in the pituitary responsiveness to GH-releasing hormone (GHRH), we have studied the GH response to GHRH in 16 normal subjects (8 men and 8 women) tested after oral placebo or different doses of pyridostigmine (30, 60, and 120 mg). Each subject presented a normal response after iv administration of 50 micrograms GHRH and placebo. In men each dose of pyridostigmine induced a significant increase in the GH response to GHRH, as assessed by both the maximal GH peak and the area under GH curve. In women, on the contrary, the GH response to GHRH was not potentiated by pretreatment with pyridostigmine at any given dose. Only five female subjects were tested with 120 mg pyridostigmine because of the severe side-effects of the drug at this dosage. Our present data strongly suggest that in humans there is a sex-related difference in the neuroregulation of GH secretion and this is probably expressed through a different cholinergic tone.     

Insulin treatment normalizes reduced free insulin-like growth factor-I concentrations in diabetic children

OBJECTIVE We have recently demonstrated multiple aberrations in the GH–IGF axis in the sera of children with untreated insulin-dependent diabetes mellitus (IDDM) which were restored after insulin replacement. However, the net result of these alterations in the IGF system on the concentrations of free/biologically available IGF-I in the serum have not been examined directly in diabetic children. In the present study, the effect of diabetes and subsequent insulin replacement on the circulating free IGF-I concentrations are assessed. DESIGN Fasting venous serum samples were obtained longitudinally, before and at various times after the initiation of insulin treatment in untreated diabetic subjects. SUBJECTS Ten prepubertal, aged (mean ± SEM) 6.3±1.0 years, and six adolescent, aged 12.7±1.1 years, subjects with newly diagnosed and untreated IDDM, and age and pubertal status-matched control children and adolescents were recruited. METHODS The serum samples were collected before initiating insulin treatment and 12–24h, 1 week, and 1 month thereafter in subjects with IDDM. Insulin doses ranged from 0.5 to 1.2 U/kg/day. MEASUREMENTS Free IGF-I concentration was assayed by a recently developed two-site immunoradiometric assay. Total IGF-I was measured by radioimmunoassay after acid–ethanol extraction of binding proteins. Differences in free and total IGF-I concentrations in IDDM subjects before and during insulin treatment were analysed by repeated measures analysis of variance followed by pairwise multiple comparisons test. In seven subjects with IDDM, where serum IGFBP-1 and IGFBP-3 concentrations, and IGFBP-3 protease activity had also been measured in a previous study, the relationship between these variables and circulating free IGF-I concentrations were examined by linear regression analysis. RESULTS Free IGF-I concentrations in prepubertal subjects with IDDM were 0.9±0.2, 1.5±0.3, 1.6±0.3 and 2.5±0.4μg/l before, 1 day, 1 week and 1 month after insulin treatment, respectively. Free IGF-I concentrations of control prepubertal children were 2.6±0.5μg/l. Pubertal subjects had higher free IGF-I concentrations than prepubertal subjects but demonstrated a similar type of pattern; before insulin 2.3±1.1, 1 day 3.8±1.3, 1 week 3.7±0.6, 1 month 6.5±1.5 vs pubertal controls 7.7±2.0μg/l. Total IGF-I concentrations were also reduced in untreated diabetic subjects and showed a slower pattern of normalization than free IGF-I concentrations. Free IGF-I concentrations correlated positively with total IGF-I and negatively with IGFBP-1 concentrations. There was no significant correlation between free IGF-I and either serum IGFBP-3 concentrations or IGFBP-3 protease activity. CONCLUSION Alterations in the IGF system during untreated IDDM lead to a reduction in circulating free IGF-I concentrations which is restored progressively during insulin treatment. An increase in free IGF-I precedes that of total IGF-I suggesting that the former is a more sensitive indicator of the metabolic status. An inverse correlation between free IGF-I and IGFBP-1 supports the hypothesis that IGFBP-1 plays an important role in the acute modulation of free IGF-I levels.     

Effect of pyridostigmine and pirenzepine on GH responses to GHRH in hyperthyroid patients

OBJECTIVE: We wished to investigate whether thyrotoxicosis can influence the cholinergic modulation of GH secretion. DESIGN: Pyridostigmine was given orally, then GHRH injected i.v., and levels were measured. In a separate study, pirenzepine was injected i.v., then GHRH, and growth hormone levels were measured. PATIENTS: Thyrotoxic patients were compared with normal subjects. MEASUREMENTS: GH was measured from -30 to +120 minutes at intervals of 15 minutes. RESULTS: Pyridostigmine markedly increased GH responses to GHRH in normal subjects, but not in thyrotoxic patients. Pirenzepine abolished the GH response to GHRH in thyrotoxic patients. CONCLUSIONS: GH responses to GHRH in hyperthyroid patients were suppressed by cholinergic muscarinic receptor blockade with pirenzepine. Activation of cholinergic pathways with pyridostigmine did not increase GH responses to GHRH in these patients. This may be a consequence of increased hypothalamic cholinergic function or reduced hypothalamic GHRH activity in hyperthyroidism. Our findings demonstrate a further mechanism by means of which thyroid status may affect the secretory activity of the somatotroph.     

Effects of exogenous growth hormone pretreatment on the pituitary growth hormone response to growth hormone-releasing hormone alone or in combination with pyridostigmine in type I diabetic patients.

We evaluated the effects of iv pretreatment with exogenous GH on the GH response to GHRH either alone or in combination with pyridostigmine in 14 Type I diabetic patients and 6 normal subjects. All the subjects received an iv bolus injection of biosynthetic human GH, 2 IU; 2 h later they received either a. pyridostigmine, 120 mg orally, or b. placebo, 2 tablets orally, followed 1 h later by iv injection of GHRH(1-29) NH2, 100 micrograms. In normal subjects the median GH peak after GH+ GHRH was 1.8, range 1.2-6.9 micrograms/l. Pyridostigmine enhanced the GH response to GHRH in all subjects. The median GH peak after pyridostigmine + GH + GHRH was 32.7, range 19.8-42.1 micrograms/l (p less than 0.001 vs GHRH alone). Seven diabetic subjects had median GH peaks after GH + GHRH greater than 6.9 micrograms/l (the maximum GH peak after GH + GHRH in normal subjects) (group A: median GH peak 35.7, range 21.7-55 micrograms/l). The other diabetic subjects had GH peak lower than 6.9 micrograms/l (group B: median GH peak 4.4, range 2.1-6.5 micrograms/l). Pyridostigmine significantly increased the GH response to GHRH in group B patients (median GH peak 29.3, range 15.7-93.4 micrograms/l, p less than 0.001 vs GH + GHRH alone), but not in group A patients (median GH peak 39.9, range 21.9-64.9 micrograms/l). Group A diabetic patients were younger and had higher HbA1c and blood glucose levels than group B patients. In those diabetic patients with an exaggerated GH response to GH + GHRH, pyridostigmine failed to cause the increase in GH secretion observed in diabetic and control subjects with no responses to GH + GHRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin stimulates growth hormone secretion through pathways other than the growth hormone-releasing hormone

OBJECTIVE There is evidence that melatonin plays a role in the regulation of GH secretion. The aim of this study was to investigate the neuroendocrine mechanisms by which melatonin modulates GH secretion. Thus we assessed the effect of oral melatonin on the GH responses to GHRH administration and compared the effects of melatonin with those of pyridostigmine, a cholinergic agonist drug which is likely to suppress hypothalamic somatostatin release. DESIGN The study consisted of four protocols carried out during the afternoon hours. Study 1: oral melatonin (10 mg) or placebo were administered 60 minutes prior to GHRH (100 μg i.v. bolus). Study 2: GHRH (100 μg i.v. bolus) or placebo were administered at 0 minutes; oral melatonin or placebo were given at 60 minutes and were followed by a second GHRH stimulus (100 μg i.v. bolus) at 120 minutes. Study 3: placebo; oral melatonin (10 mg); oral pyridostigmine (120 mg); melatonin (10 mg) plus pyridostigmine (120 mg) were administered on separate occasions. Study 4: placebo; oral melatonin (10 mg); oral pyridostigmine (120 mg); melatonin (10 mg) plus pyridostigmine (120 mg) were administered on separate occasions 60 minutes prior to a submaximal dose (3 μg i.v. bolus) of GHRH. SUBJECTS Four groups of eight normal male subjects, ages 22–35 years, were randomly assigned to each protocol. MEASUREMENTS Growth hormone was measured by RIA at 15-minute intervals. RESULTS Oral melatonin administration had a weak stimulatory effect on GH basal levels. Prior melatonin administration approximately doubled the GH release induced by supramaximal (100 μg) or submaximal (3 μg) doses of GHRH. Melatonin administration restored the GH response to a second GHRH challenge, given 120 minutes after a first GHRH i.v. bolus. The GH releasing effects of pyridostigmine, either alone or followed by GHRH, were greater than those of melatonin. However, the simultaneous administration of melatonin and pyridostigmine was not followed by any further enhancement of GH release, either in the absence or in the presence of exogenous GHRH. CONCLUSIONS Our data indicate that oral administration of melatonin to normal human males increases basal GH release and GH responsiveness to GHRH through the same pathways as pyridostigmine. Therefore it is likely that melatonin plays this facilitatory role at the hypothalamic level by inhibiting endogenous somatostatin release, although with a lower potency than pyridostigmine. The physiological role of melatonin in GH neuroregulation remains to be established.     

Impaired growth hormone (GH) response to pyridostigmine in type 1 diabetic patients with exaggerated GH-releasing hormone-stimulated GH secretion

In the present study we investigated the effects of the acetylcholinesterase inhibitor pyridostigmine (PD), which is hypothesized to decrease hypothalamic somatostatin tone, alone and in association with GH-releasing hormone (GHRH) on GH secretion in 18 type 1 diabetic patients and 12 normal subjects using a randomized double blind placebo-controlled protocol. All subjects received either 120 mg oral PD or placebo 60 min before iv injection of either human GHRH-(1-29) NH2 (100 micrograms) or sterile water (2 mL). In normal subjects both PD alone and GHRH alone caused a significant increase in GH. PD and GHRH acted in a synergistic fashion when combined. In diabetic patients the GH response to GHRH was variable. To segregate the responses, the ratio between the GH increase after GHRH plus PD and after GHRH alone was calculated for each subject. In 10 diabetic patients (group A) the ratio was lower than 2 SD (P less than 0.05) from the mean response of normal subjects. These patients showed an exaggerated GH increase after GHRH and a lower GH increase after PD with respect to normal subjects. Eight diabetic patients (group B) showed a ratio similar to that in normal subjects and similar GH responses to the stimuli. No significant differences were found between groups A and B with respect to age, body mass index, and blood glucose levels. Duration of diabetes was longer and basal GH levels were higher in group A. Hemoglobin-A1c was higher in group A, but of only borderline statistical significance (P = 0.052). Our data demonstrate that in diabetic patients with exaggerated GH responses to GHRH an increase in cholinergic tone does not affect GH secretion. These data suggest that in some type 1 diabetic patients an altered somatostatinergic control of GH secretion may contribute to their abnormal GH response to GHRH.     

GH FEEDBACK OCCURS THROUGH MODULATION OF HYPOTHALAMIC SOMATOSTATIN UNDER CHOLINERGIC CONTROL: STUDIES WITH PYRIDOSTIGMINE AND GHRH

We have studied the effect of increased cholinergic tone on the GH response to growth hormone-releasing hormone (GHRH) and on GH feedback, using pyridostigmine, an acetylcholinesterase inhibitor. In six healthy male adult volunteers 120 mg oral pyridostigmine increased basal GH secretion compared to placebo and augmented the GH response to 100 micrograms i.v. GHRH (1-29) NH2; the effect was more than the additive effect of pyridostigmine and GHRH when each was given alone. Pretreatment with 2 IU methionyl-hGH given i.v. abolished the serum GH response to GHRH given 3 h later, demonstrating a negative feedback loop of GH on the response to GHRH; this inhibited response to GHRH was restored in subjects given pyridostigmine as well as methionyl-hGH. The data demonstrate that enhanced cholinergic tone releases GH, augments the serum GH response to GHRH and unblocks the negative feedback effect of methionyl-hGH pretreatment on the GH response to GHRH. These results suggest that GH negative feedback effects on its own secretion occur predominantly through increased hypothalamic somatostatin secretion; this somatostatin secretion is under inhibitory cholinergic control.     

Effect of enhancement of endogenous cholinergic tone with pyridostigmine on the dose-response relationships of growth hormone (GH)-releasing hormone-induced GH secretion in normal subjects

It is well known that GH responses to GH-releasing hormone (GHRH) show marked interindividual variations in normal subjects, which have been attributed to a variable somatostatinergic tone. Recently, it has been shown that enhancement of cholinergic tone with the acetylcholinesterase inhibitor pyridostigmine (PD), which presumably acts by inhibiting somatostatin release, stimulates basal GH secretion and GH responses to a maximal dose of GHRH. In this study we have investigated the effects of PD on the dose-response relationships of GHRH-induced GH secretion in normal subjects. Our data showed that PD (120 mg, orally, at-60 min) induced a clear-cut increase in basal GH levels, significantly different from that after saline treatment, at 15, 30, 45, 60, 90, and 120 min. Moreover, PD administration markedly potentiated GH responses to GHRH at doses of 500, 100, 25, 10, and 3 micrograms/subject, as assessed by either area under the curve or maximal peak GH levels. In fact, GH responses to pyridostigmine plus 3 micrograms GHRH were similar to those to the administration of 500 and 100 micrograms GHRH alone. Our findings of marked increases in GH response to GHRH after pyridostigmine administration show that with enhancement of cholinergic tone, the dose of GHRH needed to induce a similar increase in GH is reduced 30 times.     

Cholinergic receptor activation by pyridostigmine restores growth hormone (GH) responsiveness to GH-releasing hormone administration in obese subjects: evidence for hypothalamic somatostatinergic participation in the blunted GH release of obesity

GH secretion in response to provocative stimuli is decreased in obese individuals. However, the precise mechanism causing this decrease is unknown. In an attempt to determine if reduced cholinergic stimulation accounts for the decreased GH secretion, we studied the effect of enhanced cholinergic tone induced by pyridostigmine on GHRH-stimulated GH secretion in a group of seven obese and seven normal subjects. When GHRH (100 micrograms, iv) was administered after placebo in the obese group, mean plasma GH rose from 0.5 +/- (0.1 (+/- SE) to 3.6 +/- 1.5 micrograms/L at 30 min. When the same obese subjects were given GHRH 60 min after pyridostigmine administration (120 mg, orally), the mean plasma GH level rose from 1.8 +/- 0.6 to 21.0 +/- 7.5 micrograms/L at 30 min. The responses to placebo and pyridostigmine were significantly different at 15, 30, 45, 60, and 90 min. In the normal subjects, a similar dose of GHRH induced a GH peak of 24.3 +/- 7.1 micrograms/L, and the GHRH-stimulated peak was significantly higher (56.2 +/- 16.8 micrograms/L) after pyridostigmine administration. To study the effect of pyridostigmine alone six other obese and six other normal subjects were tested with pyridostigmine or placebo on different days. In the normal subjects the mean peak plasma GH level after pyridostigmine was 12.5 +/- 3.1 micrograms/L, and in the obese subjects it was 4.6 +/- 1.3 micrograms/L. Thus, pyridostigmine potentiated the action of GHRH, rather than merely being additive. We conclude that pyridostigmine stimulates GH secretion in obese as well as normal subjects, although the response was less in the former group. Pyridostigmine potentiates the response to GHRH in both groups, but again, the response was less in the obese subjects. These results suggest that the impaired somatotroph responsiveness in obese subjects may be due to chronically decreased hypothalamic cholinergic tone, resulting in enhanced somatostatinergic tone.     

ROLE OF CHOLINERGIC MUSCARINIC PATHWAYS ON THE FREE FATTY ACID INHIBITION OF GH RESPONSES TO GHRH IN NORMAL MEN

In order to explore the mechanisms by which free fatty acids (FFA) inhibit GH secretion, we studied the effect of the acetylcholinesterase inhibitor pyridostigmine (120 mg p.o.) on the FFA blockade of GH responses to the administration of GHRH (100 micrograms i.v.) in seven normal subjects. GHRH-induced GH secretion was significantly reduced following elevation of circulating FFA levels by lipid-heparin infusion and significantly potentiated by previous pyridostigmine treatment. Peak GH levels following combined administration of pyridostigmine plus lipid-heparin plus GHRH were significantly higher (P less than 0.01) than after GHRH alone and significantly lower than after pyridostigmine plus GHRH (P less than 0.01). In conclusion, central cholinergic activation by pyridostigmine, with the presumed reduction in somatostatin discharge, reversed the blocking effect of FFA on GHRH-stimulated GH release. Conversely, FFA were able to reduce even a maximal GH stimulation by pyridostigmine plus GHRH.     

Effect of cholinergic muscarinic receptor blockade on human growth hormone (GH)-releasing hormone-(1-44)-induced GH secretion in acromegaly and type I diabetes mellitus

In 10 patients with type I diabetes mellitus, 9 acromegalic patients, and 8 normal subjects, serum GH levels after an iv injection of 1 microgram/kg BW human GH-releasing hormone-(1-44) (GHRH) with and without cholinergic muscarinic receptor blockade (1 mg atropine, im, 15 min before GHRH) were measured. The GH increase after GHRH treatment in type I diabetic patients did not differ significantly from that in normal subjects. Pretreatment with atropine almost completely blocked the GH response to GHRH in patients with type I diabetes and normal subjects, but did not suppress the GH response to GHRH in acromegalic patients. These data indicate that cholinergic muscarinic receptors modulate GH secretion in normal man as well as in patients with type I diabetes mellitus, whereas acromegalic patients have a defect of cholinergic control of GH secretion.     

Influence of hyperthyroidism on growth hormone secretion

OBJECTIVE Hyperthyroidism is associated with altered GH secretion. Whether this is due to changes of somatotroph responsiveness or reflects an alteration in negative feedback signals at the hypothalamic level is unknown. We therefore performed a series of studies to shed some light onto this issue. DESIGN Study 1: GHRH (1 μg/kg b.w.) was injected i.v. in 38 hyperthyroid patients and in 30 normal subjects; in 11 of the patients the GHRH test was repeated following methimazole-induced remission of hyperthyroidism. Study 2: hGH (2 U i.v.) or saline were administered 3 hours prior to GHRH; six hyperthyroid patients and six normal subjects were studied. Study 3: ten normal subjects and ten hyperthyroid patients were given 75 g oral glucose or water 30 minutes before GHRH. Study 4: 11 normal subjects and eight hyperthyroid patients were studied. TRH or vehicle were dissolved in 250 ml of saline solution and infused at a rate of 400 μg/h for 150 minutes. Thirty minutes after the beginning of the infusions, l -arginine (30 g infused over 45 min i.v.) was administered. PATIENTS Hyperthyroid patients were compared to normal subjects. MEASUREMENTS Growth hormone was measured by RIA at 15-minute intervals. RESULTS GH responses to GHRH were subnormal in hyperthyroid patients. Following antithyroid drug treatment with methimazole, GH responses to GHRH increased in these patients in comparison to pretreatment values. Serum IGF-I levels, which were elevated before treatment, decreased after methimazole administration. Exogenous GH administration induced a clear decrease of GH responses to GHRH in both control and hyperthyroid subjects. On the other hand, oral glucose load decreased the GH responses to GHRH in normal but not in hyperthyroid subjects. TRH administration did not modify the GH responses to arginine in either normal subjects or hyper-thyroid patients. CONCLUSIONS Hyperthyroidism is associated with increased serum IGF-I levels and marked alterations in the neuroregulation of GH secretion. These changes involve decreased GH responsiveness to GHRH at the pituitary level and, at the hypothalamic level, a lack of suppressive effect of an oral glucose load. The normal inhibitory effect of exogenous GH administration but not of an oral glucose load in hyperthyroid patients suggests that these two feedback signals act through different mechanisms. The lack of effect of a TRH infusion on GH responses to l -arginine in normal and hyperthyroid patients makes an inhibitory role for TRH in GH secretion unlikely, at least in Caucasian subjects.     

Growth hormone-binding protein related immunoreactivity is regulated by the degree of insulinopenia in diabetes mellitus

OBJECTIVE Derangements in the GH/IGF axis are common in patients with diabetes mellitus. In insulin-dependent diabetes mellitus (IDDM), these disturbances seem to be due to a partial defect in GH action on its own receptor or via a post-receptor defect. In non-insulin-dependent diabetes mellitus (NIDDM), data are limited, and the regulation of the GH receptor (GHR) remains unclear. However, animal studies with diabetic rats demonstrated that the GHR density may be influenced by insulin disposal at the hepatocyte. With respect to this hypothesis we studied the relation between peripheral insulin status and the serum GH-binding protein (GHBP), which reflects indirectly the GHR density in the tissues. Patients with IDDM were compared to a NIDDM group as well as to a group of healthy subjects. DESIGN AND PATIENTS Basal blood samples for the determination of serum GHBP, GH, and IGF-I were obtained from patients with IDDM (n = 27), subjects with NIDDM (n = 112) and healthy controls (n = 42). Insulin, proinsulin, C-peptide and IGF-binding protein 1 (IGFBP-1) serum levels were used to estimate the insulin status in diabetic patients. RESULTS GHBP serum levels were significantly lower in patients with IDDM than in either NIDDM or controls (P < 0.001). Conversely, the IGF-I levels were reduced in both groups of diabetics. A subgroup of hypoinsulinaemic NIDDM patients showed significantly decreased GHBP concentrations (P < 0.05) compared to the NIDDM subgroup with hyperinsulinaemia. Furthermore, GHBP levels were significantly decreased in insulin-treated patients with NIDDM compared to either non-insulin-requiring subjects or normal controls (P < 0.05). A significant direct relation was found between levels of GHBP and total insulin dose (P < 0.01) in patients with IDDM. In the NIDDM group, GHBP was correlated with proinsulin (P < 0.001), C-peptide (P < 0.01), insulin (P < 0.05) and inversely with IGFBP-1 (P < 0.001). Multiple linear regression analysis indicated a significant contribution of proinsulin and IGFBP-1 to the variation of GHBP. CONCLUSIONS Decreased GHBP levels in IDDM as well as in NIDDM correlate with insulinopenia. Since the degree of insulinopenia depends on the capability of the β-cells to secrete proinsulin, C-peptide and insulin, we hypothesize that these hormones at least partially influence the serum level of GHBP. Low GHBP levels may reflect a reduced GH receptor density and a concomitant GH insensitivity, which leads to an impaired IGF generation in insulin-deficient patients.     

IGF-I levels rise and GH responses to GHRH decrease during long-term prednisone treatment in man

Glucocorticoid excess is associated with a blunted GH response to GHRH. IGF-I levels in hypercortisolism are controversial and have been reported as low, normal or high. The aim of this study was to evaluate longitudinally time-dependent changes in the GH response to GHRH, IGF-I, IGFBP-3 and albumin values in patients during corticotherapy. Six patients received GHRH before and after one week and one month of prednisone administration (20-60 mg/d, orally). IGF-I, IGFBP-3 and albumin were determined in each test, at time 0. Ten normal controls were also evaluated in one occasion. There were no differences in basal GH values, GH response to GHRH, IGF-I and IGFBP-3 levels between controls and patients before starting corticotherapy. Albumin (g/l; mean+/-SE) values were lower in patients before treatment (31+/-4) than in controls (43+/-1). After one week of prednisone administration there was a significant decrease in peak GH (microg/l) levels (before: 18.8+/-7.4; 1 week: 5.0+/-1.3), which was maintained after one month (8.1+/-3.5). IGF-I (microg/l) levels increased significantly, from 145+/-23 to 205+/-52 after one week of therapy, reaching levels of 262+/-32 after one month. IGFBP-3 (mg/l) values did not increase significantly (before: 2.1+/-0.2; 1 week: 2.5+/-0.3; 1 month: 2.8+/-0.2). Albumin levels showed a significant rise both after one week (36+/-4) and one month (42+/-3) of corticotherapy. In summary, we observed a marked decrease in the GH response to GHRH after one week and one month of prednisone administration associated with an increase in circulating IGF-I and albumin values. The physiological implications of these findings are still uncertain. It is possible that glucocorticoids increase hepatic IGF-I and albumin synthesis, although other mechanisms may have a role.     

Growth hormone, IGF-I and its binding proteins (IGFBP-1 and -3) in adult uraemic patients undergoing peritoneal dialysis and haemodialysis

OBJECTIVE: The GH/IGF axis is altered in chronic renal failure (CRF). CRF patients usually show normal or high serum concentrations of GH and IGF-I, whereas all IGF binding proteins (IGFBP-1 to -6), except IGFBP-5, considerably increase with declining renal function. The aims of the present study were to quantify serum concentrations of GH, IGF-I, IGFBP-1 and IGFBP-3 in a group of patients with CRF, and determine whether there were differences according to the type of dialysis, that is, peritoneal dialysis (PD) and haemodialysis (HD). DESIGN: A cross-sectional study in the setting of a dialysis unit of a general hospital. PATIENTS AND MEASUREMENTS: We studied 108 dialysis patients treated by PD (n = 54, 32 males and 22 females, mean age 61.0 +/- 1.4 years) or HD (n = 54, 31 males and 23 females, age 62.6 +/- 1.5 years). A group of 42 healthy subjects of similar age, sex and body mass index (BMI) served as the control group. Baseline serum concentrations of GH, insulin, IGF-I, IGFBP-1 and IGFBP-3 were measured in all patients and control subjects. RESULTS: Fasting serum concentrations of IGF-I and its binding proteins (IGFBP-1 and IGFBP-3) were significantly higher in dialysis patients than in subjects with normal renal function. IGF-I (248.9 +/- 23.4 vs. 205.5 +/- 15.5 micro g/l, NS), IGFBP-3 (5.6 +/- 0.4 vs. 5.5 +/- 0.2 mg/l, NS) and IGFBP-1 (36.1 +/- 5.9 vs. 44.1 +/- 6.5 micro g/l, NS) concentrations were similar in both groups of dialysis (PD vs. HD) patients. However, GH (2.3 +/- 0.3 vs. 1.1 +/- 0.1 micro g/l, P < 0.001) and insulin (40.4 +/- 4.5 vs. 30.1 +/- 3.1 micro U/ml, P < 0.05) levels were significantly higher in the PD group than in the HD group. Both groups of dialysis patients showed significantly higher levels of insulin than healthy subjects (14.7 +/- 1.9 micro U/ml, P < 0.0001 and P < 0.01 for PD and HD, respectively). In both groups of dialysis patients, IGF-I correlated inversely with IGFBP-1 (PD group r = -0.46, P = 0.0006; HD group r = -0.57, P = 0.0001) and directly with IGFBP-3 (PD group r = 0.44, P = 0.001; HD group r = 0.73, P = 0.001). No correlation between insulin and IGFBP-1 was found in any of the groups studied. CONCLUSIONS: These findings demonstrate that adult dialysis patients have elevated IGF-I, IGFBP-1 and IGFBP-3 serum concentrations compared with subjects with normal renal function. Only GH and insulin show statistically significant differences in relation to type of dialysis. Finally, the negative correlation between IGF-I and IGFBP-1 and the positive correlation between IGF-I and IGFBP-3 are maintained in both groups of adult dialysis patients.     

Pyridostigmine enhances, but does not normalise, the GH response to GH-releasing hormone in obese subjects

Obese patients are characterised by several neuroendocrine abnormalities, including characteristically a decrease in growth hormone responsiveness to GH-releasing hormone. In normal subjects, the GH response to GHRH is enhanced by the acetylcholinesterase inhibitor, pyridostigmine. We have studied the effect of this drug on GH secretion in gross obesity. Twelve obese patients were studied (mean weight 156% of ideal) and compared with a group of 8 normal volunteers. Each subject was initially studied on two occasions, in random order, with GHRH (1-29) NH2 100 micrograms iv alone and following pretreatment with pyridostigmine 120 mg orally one hour prior to GHRH. In obese patients, the GH response to GHRH was significantly blunted when compared to controls (GH peak: 20 +/- 4 vs 44 +/- 16 micrograms/l; mean +/- SEM). After pyridostigmine, the response to GHRH was enhanced in the obese subjects, but remained significantly reduced compared to non-obese subjects treated with GHRH and pyridostigmine (GH peak: 30 +/- 5 vs 77 +/- 20 micrograms/l, respectively). In 6 subjects, higher doses of GHRH or pyridostigmine did not further increase GH responsiveness in obese patients. Our results suggest that obese patients have a disturbed cholinergic control of GH release, probably resulting from increased somatostatinergic tone. This disturbed regulation may be responsible, at least in part, for the blunted GH responses to provocative stimuli.     

Use of the ligand immunofunctional assay for human insulin-like growth factor ((IGF) binding protein-3 (IGFBP-3) to analyze IGFBP-3 proteolysis and igf-i bioavailability in healthy adults, GH-deficient and acromegalic patients, and diabetics

The ligand immunofunctional assay for plasma insulin-like growth factor (IGF) binding protein (IGFBP)-3 developed in our laboratory provides for specific measurement of intact, as opposed to proteolyzed, IGFBP-3. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration; thereafter, intact and proteolyzed IGFBP-3 are captured by a monoclonal antibody in a solid-phase assay and incubated with (125)I-IGF-I, which detects the intact protein but not its proteolytic fragments. This assay was combined with assays for IGF-I (RIA of the ultrafiltrate) and total IGFBP-3 (immunoradiometric assay) to quantify the percentage of proteolyzed IGFBP-3 (percent proteolyzed IGFBP-3) and to calculate the IGF-I/intact IGFBP-3 ratio as an index of the fraction of exchangeable IGF-I bound to IGFBP-3. This fraction represents most of the IGF-I that is bioavailable. Because GH and insulin control the hepatic production and plasma concentrations of IGF-I and IGFBP-3, we set out to determine whether variations in the secretion of the two hormones are involved in the regulation of IGFBP-3 proteolysis. The study included adult populations of 36 healthy subjects, 23 hypopituitary patients untreated with GH, 43 acromegalics (13 untreated), 42 insulin-treated type 1 diabetics [insulin-dependent diabetes mellitus (IDDM)] patients, and 50 type 2 diabetics [non-IDDM (NIDDM)] patients, 22 of whom were insulin-treated and the remaining 28 treated with sulfonylurea and/or metformin). Unlike IGF-I and (to a lesser extent) total IGFBP-3 levels, which decline with age, percent proteolyzed IGFBP-3 seemed relatively stable. In healthy adults, the mean +/- SEM was 29.4 +/- 1.9 for subjects less than 45 yr old and was slightly (but not significantly) lower, 25.7 +/- 3, for those of more than 45 yr. There was no difference between male and female subjects. In GH-deficient patients, despite severely depressed IGF-I levels, percent proteolyzed IGFBP-3 and IGF-I/intact IGFBP-3 ratios were within the normal range. Among acromegalics, percent proteolyzed IGFBP-3 was elevated: 36.6 +/- 3.3 for patients of less than 45 yr, 33.3 +/- 3.2 for patients of more than 45 yr (P = 0.02 vs. healthy subjects). Consequently, the effects of excessive IGF-I synthesis are exacerbated by the enlarged exchangeable fraction of IGFBP-3-bound IGF-I. There was no significant difference in percent proteolyzed IGFBP-3 between GH-deficient patients before and after GH treatment or between treated and untreated acromegalics. In IDDM patients, the means for percent proteolyzed IGFBP-3 were higher than those in healthy adults: 36.7 +/- 3.7 (P = 0.03) and 31.3 +/- 3.3 for subjects of less than 45 and more than 45 yr, respectively. In NIDDM patients, all of whom were more than 45 yr old, the means were 35.2 +/- 2.5 (P = 0.02) for insulin-treated patients and 33 +/- 2.5 for the group treated orally. Among the diabetics, increased IGFBP-3 proteolysis resulted in an IGF-I/intact IGFBP-3 ratio that was normal for IDDM patients of less than 45 yr and above normal (P = 0.01) for the others. Percentage proteolyzed IGFBP-3 and the IGF-I/intact IGFBP-3 ratio were inversely related to body mass index in IDDM patients (r = -0.42, P = 0.008; and r = -0.31, P = 0.05, respectively) and to percentage glycosylated hemoglobin in all insulin-treated diabetics (r = -0.25, P = 0.05; and r = -0.33, P = 0.008, respectively). There was also an inverse relationship between IGF-I/intact IGFBP-3 ratios and IGFBP-1 levels in healthy adults (r = -0.39, P = 0.03) and orally treated NIDDM patients (r = -0.37, P = 0.05). Percentage proteolyzed IGFBP-3 was positively correlated to total IGFBP-3 in healthy adults (r = 0.65, P = 0.0001) and in all the groups of patients. It was negatively correlated to IGF-I/total IGFBP-3 in healthy subjects (r = -0.40, P = 0.02) and diabetics (r = -0.30, P = 0.005). This suggests an autoregulatory mechanism controlling the bioavailability of IGFBP-3-bound IGF-I in the 140-kDa complexes. In the pathological conditions studied here, regulation of IGF-I bioavailability by limited proteolysis of IGFBP-3 contributes toward an appropriate adaptation to insulin deficiency and/or resistance but not to disturbances of GH secretion.     

Pyridostigmine partially restores the GH responsiveness to GHRH in normal aging

In 11 elderly normal subjects and in 17 young healthy subjects we studied the response of plasma growth hormone to GH-releasing hormone (GHRH(29), 1 microgram/kg iv) alone and preceded by pyridostigmine (120 mg orally 60 min before GHRH), a cholinesterase inhibitor likely able to suppress somatostatin release. The GH response to pyridostigmine alone was also examined. Basal plasma GH levels were similar in elderly and young subjects. In the elderly, GHRH induced a GH rise (AUC, median and range: 207.5, 43.5-444.0 micrograms.l-1.h-1) which was lower (p = 0.006) than that observed in young subjects (548.0, 112.5-2313.5 micrograms.l-1.h-1). The pyridostigmine-induced GH rise in the elderly was similar to that in young subjects (300.5, 163.0-470.0 vs 265.0, 33.0-514.5 micrograms.l-1.h-1). Pyridostigmine potentiated the GH responsiveness to GHRH in both elderly (437.5, 152.0-1815.5 micrograms.l-1.h-1; p = 0.01 vs GHRH alone) and young subjects (2140.0, 681.5-4429.5 micrograms.l-1.h-1; p = 0.0001 vs GHRH alone). However, the GH response to pyridostigmine + GHRH was significantly lower (p = 0.0001) in elderly than in young subjects. In conclusion, the cholinergic enhancement by pyridostigmine is able to potentiate the blunted GH response to GHRH in elderly subjects, inducing a GH increase similar to that observed after GHRH alone in young adults. This finding suggests that an alteration of somatostatinergic tone could be involved in the reduced GH secretion in normal aging. However, a decreased GH response to combined administration of pyridostigmine and GHRH in elderly subjects suggests that other abnormalities may coexist, leading to the secretory hypoactivity of somatotropes.     

The effect of anorexia nervosa and refeeding on growth hormone-binding protein, the insulin-like growth factors (IGFs), and the IGF-binding proteins.

We studied the relationship of serum insulin-like growth factor-I (IGF-I), IGF-II, the IGF-binding proteins IGFBP-1, IGFBP-2, and IGFBP-3, and GH-binding protein (GHBP; which is postulated to be derived from the extracellular portion of the GH receptor) in normal volunteers and patients with anorexia nervosa before and after a refeeding program. Serum GHBP, IGF-I, and IGFBP-3 were all significantly decreased in low weight patients with anorexia nervosa and returned to nearly normal levels with refeeding. Fasting serum GH and serum IGFBP-1 and IGFBP-2 were significantly increased in low weight patients with anorexia nervosa and also returned to nearly normal levels with refeeding. Serum IGF-II was 27% lower in the low weight group than in normal subjects, but this difference was not statistically significant. Both serum IGF-I and IGF-II were positively correlated with serum IGFBP-3 and negatively correlated with serum IGFBP-1 and IGFBP-2. These data are consistent with the hypothesis that nutritional deprivation alters the GH-IGF axis by down-regulation of the GH receptor or its postreceptor mechanisms, and that this effect is reversible with refeeding.