Strength and its relationship to changes in fat-free mass, total body potassium, total body water and IGF-1 in adults with growth hormone deficiency: effect of treatment with growth hormone.

The present investigation examined changes in strength in growth hormone deficient (GHD) adults following treatment with recombinant human growth hormone (rhGH), and assessed their relationship to changes in fat-free mass (FFM), total body potassium (TBK), total body water (TBW), the concentration of TBK and TBW per kg FFM, and insulin like growth factor-1 (IGF-1). The investigation was double-blind and placebo-controlled for a period of 6 months; this was followed by a period of open treatment for a further 6 months. Patients were assigned randomly to experimental (E) and control (C) groups. In the first 6 months group E received rhGH and group C placebo; in the second 6 months both groups received rhGH. Serial data were analysed for 23 males (11 group E, 12 group C) and 20 females (10 group E, 10 group C). Body composition was assessed by dual energy X-ray absorptiometry, TBK and TBW. Muscle strength was recorded for arm flexion, leg extension and hand grip. Significant increases in FFM occurred in the first 6 months in group E (2.3 kg males, 1.4 kg females) and in the second 6 months in group C (2.4 kg males, 1.4 kg females). There was a modest increase in absolute strength with time, although only three increments were significant (knee extension in group E males and arm flexion in groups E and C females), all of which occurred during the 6-12 month period. Allometric scaling did not improve the identification of significant increments of strength. The mean concentrations of TBK (males 57.0-58.6, females 51.4-53.9 mmol) and TBW (males 0.65-0.69, females 0.65-0.68 l) per kg FFM, were significantly smaller at all stages of the trial than the reference values, suggesting that treatment had not fully normalized these variables. Likewise, the relationship between most of the increments of regional and total strength, and the corresponding increments of FFM, were generally poor and not significant. It was concluded that the reduced concentrations of TBK and TBW per kg FFM, which may be the effect of an inappropriate dose regime or mode of delivery, may, in part, contribute to the anomaly between increases in strength and FFM.

Hypokalaemia during insulin-induced hypoglycaemia in hypopituitary adults with and without growth hormone deficiency.

OBJECTIVE: We have investigated plasma potassium changes during insulin-induced hypoglycaemia (IIH) in adult patients with growth hormone deficiency (GHD) who have low total body potassium and may also have a vulnerable myocardium due to an increased prevalence of atherosclerosis. DESIGN: Hypoglycaemia was induced through the administration of intravenous soluble insulin (0.1 U/Kg, i.v.). SUBJECTS: 23 consecutive adult patients undergoing routine biochemical evaluation for hypopituitarism. MEASUREMENTS: GH, cortisol glucose, sodium and potassium levels were measured at 0, 30, 60, 90 and 120 min after insulin administration. Pituitary function was also assessed through measurement of TSH, thyroid hormones, LH/FSH, testosterone and 17-oestradiol. IGF-I concentrations were also analysed. RESULTS: All patients achieved adequate hypoglycaemia (nadir glucose < 2 mmol/l). 15 patients had GHD (peak GH < 10 mU/l) either in isolation or as part of a spectrum of pituitary hormone deficiencies. The remaining 8 patients had normal pituitary function. Plasma potassium concentrations (mean +/- SEM) fell from 3.8 +/- 0.3 mmol/l (normals) and 3.8 +/- 0.2 mmol/l (GHDs) to nadir concentrations of 3.0 +/- 0.2 mmol/l and 3.1 +/- 0.3 mmol/l, respectively (P < 0.005). Overt hypokalaemia (< 3.5 mmol/l) occurred in 13/15 GHDs and all normals. There were no significant differences between the groups. CONCLUSIONS: Insulin-induced hypoglycaemia causes similar degrees of significant hypokalaemia in patients with normal pituitary function and in those with GH deficiency, either alone or in combination with other pituitary hormone deficiencies. Therefore, insulin-induced hypoglycaemia does not appear to be associated with any greater risk of hypokalaemia in hypopituitary adults with GHD compared to those with normal anterior pituitary function.

Glutamate pathways mediate somatostatin responses to glucose in normal and diabetic rat hypothalamus.

We investigated the role of hypothalamic glutamate receptors in mediating the stimulatory effect of low glucose (< 5 mM) on somatostatin release. We also studied whether alteration in glutamate release might contribute to the reduced hypothalamic somatostatin response to low glucose observed in diabetic (Goto-Kakizaki) rat hypothalami. Hypothalamic somatostatin release in response to incubation with 1 mM D-glucose was inhibited by the ionotropic glutamate receptor antagonists MK801, D-AP5 and DNQX but not by the metabotropic antagonists L-AP3 or MCPG. The release of somatostatin was increased by the ionotropic agonists NMDA, AMPA and kainate but not by metabotropic agonists t-ACPD or L-AP4. Basal and peak glutamate release in response to incubation with 1 mM glucose, were significantly lower from GK hypothalami There were no significant differences in the basal or stimulated release of serine and GABA. These data indicate that ionotropic NMDA/AMPA/kainate receptors and not metabotropic receptors mediate the effects of glucose on rat hypothalamic somatostatin release. Reduced hypothalamic somatostatin release in response to low glucose in diabetic (Goto-Kakizaki) rats may well be secondary, at least in part, to reduced glutamate release.

Desensitisation of somatostatin, TRH and GHRH responses to glucose in the diabetic (Goto-Kakizaki) rat hypothalamus.

We have studied the effects of glucose on the release of somatostatin (SS), TRH and GHRH from incubated hypothalami of normal and genetically diabetic, Goto-Kakizaki (GK) rats. The active isomer D-glucose caused a dose-related inhibition of SS, TRH and GHRH from normal rat hypothalami over a 20-min incubation period in vitro. In contrast, in GK rats the effects of glucose on TRH and SS were significantly reduced and the effects on GHRH were abolished. These data indicate that the sensitivity of SS-, TRH- and GHRH-producing hypothalamic neurones is reduced in diabetic rats. The effect is most pronounced for GHRH release as there was no change in the release of this peptide with increasing glucose concentrations. In conclusion, it appears that the diabetic state in GK rats causes differential desensitisation (GHRH > TRH and SS) of neuronal responses to subsequent changes in glucose concentrations in vitro. This may be due to alterations in the neurotransmitter control and/or a reduction in number, affinity or function of glucose transporters on these peptidergic neurones or other intermediary neuronal pathways.

Effects of free fatty acids on somatostatin secretion, content and mRNA levels in cortical and hypothalamic fetal rat neurones in monolayer culture.

The effects of free fatty acids on somatostatin secretion, content and mRNA levels in fetal rat hypothalamic and cortical cell cultures were investigated. Somatostatin secretion and content were quantified by radioimmunoassay. Somatostatin mRNA levels were measured by Northern blot hybridization using a cRNA probe. Treatment with either caprylic acid (5 x 10(-3) M) or oleic acid (5 x 10(-5) M) for 90 min inhibited basal somatostatin secretion in both hypothalamic and cortical cell cultures. In addition, the increase in somatostatin secretion induced by incubation with veratridine (10(-4) M) or carbachol (10(-4) M) for 90 min was significantly reduced by the addition of caprylic acid, but somatostatin release stimulated by 5.6 x 10(-2) M KCl was not affected. Furthermore, treatment with these free fatty acids for 90 min markedly decreased somatostatin mRNA levels in both types of neurone culture. These inhibitory effects were transient, being observed after 90 min, but not after 5 h. These results support the probability that there is a role for free fatty acids in the regulation of somatostatin mRNA levels and somatostatin secretion in both hypothalamic and cortical cell cultures.

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 HbA1 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 pyridostigmine 120 mg pretreatment 60 minutes and GH pretreatment 3 hours before an i.v. injection of GHRH (1-44) (80 micrograms) 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 HbA1 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 (delta peak GH 26.4 +/- 5.2 vs 7.7 +/- 5.4 mU/l, P < 0.04). In contrast, GH pretreatment did not cause any significant reduction in GH responsiveness to GHRH in diabetics (delta peak GH 53.6 +/- 9.7 vs 33.4 +/- 11 mU/l, 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.

Immunochemiluminometric assays (ICMA) specific for growth hormone releasing hormone 1-44 NH2 and 1-40 OH.

We describe specific two-site immunochemiluminometric assays able to directly measure human growth hormone-releasing hormone 1-44 NH2 and 1-40 OH concentrations in unextracted plasma. A common N-terminal antibody was purified from polyclonal rabbit antisera to growth hormone-releasing hormone 1-44 NH2 on a growth hormone-releasing hormone 1-29 NH2 linked affinity column and labelled with chemiluminescent acridinium ester. C-terminal specific monoclonal antibodies to growth hormone-releasing hormone 1-44 NH2 and 1-40 OH were raised in Balb/C mice and used as solid phase antibodies. Assay of fasting specimens from normal individuals gave medians (and ranges) of 23 pg/ml (2-200) and 30 pg/ml (3-134) for growth hormone-releasing hormone 1-44 NH2 and 1-40 OH, respectively. Samples from a series of acromegalics showed that most have values in the normal range though median values were higher, 56 pg/ml for growth hormone-releasing hormone 1-44 NH2 (P < 0.001) and 52 pg/ml for 1-40 OH (P < 0.001). Using these assays it will be possible for the first time to directly study the physiology and pathophysiology of these two peptides.

Stimulatory effect of free fatty acids on growth hormone releasing hormone secretion by fetal rat neurons in monolayer culture.

The effects of free fatty acids (FFAs) on growth hormone releasing hormone (GHRH) secretion by fetal rat cortical and hypothalamic neurons in monolayer culture have been investigated. Treatment with caprylic or oleic acids for 90 min, induced a dose-dependent increase in GHRH secretion. The stimulatory effect of caprylic acid was time-dependent, being present after 90 min and 5 h but not after 24 h. Finally, GHRH release induced by 56 mM KCl or by the calcium ionophore A23187 was not modified by 5 x 10(-3) M caprylic acid. These data support a role for FFAs on GHRH secretion.

Effects of ambient glycaemic state on growth hormone responsiveness in insulin-dependent diabetes.

OBJECTIVE: We wished to examine the effects of ambient glycaemia on hypothalamic somatostatinergic tone in insulin-dependent diabetes mellitus. DESIGN: After 6-hour periods of either euglycaemia (5 mmol/l) or hyperglycaemia (15 mmol/l), exercise-induced GH secretion was measured. PATIENTS: Seven insulin dependent diabetics with no evidence of complications were recruited. RESULTS: There was no significant difference between basal GH levels during euglycaemia and hyperglycaemia (5.50 vs 5.53 mU/l). Nor was there a significant difference when mean delta GH levels (33.9 vs 27.4 mU/l) or mean area under GH curve (2331 vs 4038 mU min/l) during euglycaemia or hyperglycaemia were compared. CONCLUSIONS: We find no evidence therefore to support short-term effects of ambient glucose concentration on GH responsiveness, and by inference no direct effect of short-term changes in glycaemic control on hypothalamic release of somatostatin in the aetiology of GH hypersecretion in insulin-dependent diabetes mellitus.

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.

Occurrence of galanin-like immunoreactivity in growth hormone-releasing factor (GRF)-containing neurons of the monkey (Macaca fascicularis) infundibular nucleus and median eminence.

The distribution of growth hormone-releasing factor (GRF)- and galanin (GAL)-immunoreactive (-IR) neurons in the mediobasal hypothalamus of the monkey (Macaca fascicularis) was studied with immunohistochemistry using a direct double-labelling method. GRF- and GAL-IR cell bodies were demonstrated in the ventral part of the infundibular nucleus and dense aggregations of GRF- and GAL-IR fibers were seen in the external layer of the median eminence, closely surrounding portal vessels. Double-staining revealed that GRF and GAL were colocalized in cell bodies of the infundibular nucleus and in nerve fiber varicosities in the external layer of the median eminence. GAL has been reported to stimulate the secretion of growth hormone in both rats and humans, most likely via hypothalamic mechanism(s).

A clinical update on hypothalamic-pituitary control.

There is at present an accumulation of data which indicate the importance of stimulatory and inhibitory growth factors in the paracrine, and perhaps autocrine, regulation of anterior pituitary cell growth and function. There may well be several more specific pituitary growth factors which will be identified in the near future. Preliminary data are also emerging concerning the pattern of oncogene expression in human pituitary tumours. It is now necessary to relate growth factor production, oncogene expression and hypothalamic regulation to specific anterior pituitary cell populations and human pituitary adenomas. In the midst of these studies it should not be forgotten that coordinated vascular and supporting tissue growth is also crucial to the normal development and maintenance of anterior pituitary structure and function.

Dopamine stimulates release of thyrotrophin-releasing hormone from perfused intact rat hypothalamus via hypothalamic D2-receptors.

We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 mumol/l (increase over basal, 118 +/- 16.5 (S.E.M.) fmol TRH; P less than 0.001 vs basal). This effect was mimicked by the specific D2-agonist drugs bromocriptine (0.1 mumol/l) and LY 171555 (0.1 mumol/l) (increase over basal values, 137.5 +/- 13.75 fmol and 158.6 +/- 10.7 fmol respectively; P less than 0.001 vs basal), but not by the D1-agonist SKF 38393A. The stimulatory effect of dopamine (1 mumol/l) was blocked in a stereospecific manner by the active (D) but not by the inactive (L) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D2-antagonist domperidone (0.01 mumol/l) whereas the D1-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0.01 mumol/l) of this D1-antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D2-receptors.

The role of calcium and calmodulin in mediating release of thyrotrophin-releasing hormone by cultured hypothalamic cells.

We have developed a fetal rat hypothalamic cell culture system for the study of factors controlling the acute release of TRH. Release of TRH by the cells has been characterized by reversed-phase high pressure liquid chromatography and about 86% of the total immunoreactivity in the medium co-eluted with synthetic TRH. Release of TRH by the cells in response to 56 mmol K+/l increased between days 5 and 9 of culture but reached a plateau thereafter. Cell contents of TRH did not change significantly between days 5 and 14 of culture. Release of TRH from the cells was stimulated by K+ (56 mmol/l), veratridine (100 mumol/l) and ouabain (100 mumol/l) to 550, 480 and 335% of basal release respectively over a 1-h period. Release of TRH was dependent upon calcium in that it was absent when calcium-free medium was used and could be blocked by verapamil (20 mumol/l); however it could not be blocked by nifedipine (50 mumol/l). The calcium ionophore blocked by nifedipine (50 mumol/l). The calcium ionophore A23187 (1 mumol/l) stimulated TRH release to 340% of basal release. Tetrodotoxin (1 mumol/l) completely abolished the release in response to veratridine but had no effect on the release stimulated by K+ (56 mmol/l). The calmodulin antagonists trifluoperazine and triflupromazine (50 mumol/l) inhibited veratridine-stimulated TRH release. This was at a site after calcium influx as they also inhibited A23187-stimulated TRH release. The highly specific calmodulin antagonist W7 (10 mumol/l) also inhibited both veratridine and A23187-stimulated TRH release whereas, at the same concentration, its inactive analogue W5 did not significantly inhibit TRH release in response to either stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of thyroid status on brain catecholamine biosynthesis in adult rats: assessment by a steady-state method.

Effects of thyroid status on brain catecholamine turnover in adult rats were investigated using a steady-state method. Rats were treated for 3 weeks with s.c. injections of L-thyroxine (0.4 mg/kg), aminotriazole in drinking water (0.1%, w/v) or vehicle. After 2 weeks of treatment rats were implanted chronically with lateral intracerebroventricular (i.c.v.) cannulae. They were injected i.c.v. with [3H]tyrosine 1 week later. Catecholamine and tyrosine content and specific activity were measured in mediobasal hypothalamus, anterior hypothalamus and striatum, using high-performance liquid chromatography with electrochemical detection. Thyroxine treatment resulted in a significant increase in noradrenaline and dopamine synthesis localized to the mediobasal hypothalamus. Conversely, aminotriazole treatment resulted in a significant decrease in noradrenaline synthesis localized to the mediobasal hypothalamus. The localization of these changes in catecholamine turnover to the mediobasal hypothalamus suggests that they may be specific functional effects which are of importance in the overall integrated control of thyroid function.

Hypothalamic D2 receptors mediate the preferential release of somatostatin-28 in response to dopaminergic stimulation.

We have studied the effect of dopamine (DA) together with agonist and antagonist drugs of varying specificity on the release of immunoreactive forms of somatostatin (SS) from the perfused, adult rat hypothalamus in vitro. Levels of SS increased from 14.7 +/- 3.7 pg (mean +/- SE) under basal conditions to 137 +/- 23.0 pg after exposure to 10(-6) M DA. This dopaminergic effect was mimicked by the specific D2 agonists bromocriptine (10(-7) M) and LY 171555 (10(-6) M) but not by the D1 agonist SKF 38393A (10(-6) M). The stimulatory action of DA (10(-6) M) was blocked by the active (d) but not the inactive (l) isomer of butaclamol (10(-7) M). Similar blockade was achieved with the specific D2 antagonists metoclopramide (10(-8) M) and domperidone (10(-8) M), whereas the D1 antagonist SCH 23390 partially blocked the stimulation of DA but only when used at X100 greater concentration (10(-6) M). SCH 23390 (10(-8) M) did not affect the dopaminergic stimulation of SS release. HPLC characterization of the immunoreactive forms of SS yielded two peaks which corresponded to SS-28 and SS-14. The ratio of these forms varied significantly under different conditions. In the basal state the ratio of SS-28 to SS-14 was 1:4.4; in response to stimulation with DA, the ratio was 1:1.7 and in response to depolarization with 60 mM K+ the ratio was 1:3.1. In conclusion, the stimulatory action of DA on SS release is mediated via hypothalamic D2 receptors. Furthermore dopaminergic stimulation increases the molar ratio of SS-28 to SS-14 in the total immunoreactive SS which is released.

Growth hormone responses to growth hormone-releasing factor (1-29) in euthyroid, hypothyroid and hyperthyroid rats.

In order to investigate whether the impaired GH secretion associated with hypothyroidism and hyperthyroidism is due to a hypothalamic or a pituitary disorder, we have studied plasma GH responses to GH-releasing factor (1-29) (GRF) in euthyroid, hypothyroid and hyperthyroid rats. Hypothyroid rats showed a significant (P less than 0.001) reduction in GH responses to GRF (5 micrograms/kg) at 5 min (350 +/- 35 vs 1950 +/- 260 micrograms/l), 10 min (366 +/- 66 vs 2320 +/- 270 micrograms/l) and 15 min after GRF injection (395 +/- 72 vs 1420 +/- 183 micrograms/l; mean +/- S.E.M.) compared with euthyroid rats. Hyperthyroid rats showed a significant (P less than 0.05) decrease in GH responses to 5 micrograms GRF/kg after 30 min (200 +/- 14 vs 325 +/- 35 micrograms/l) but not at other time-points, or after the administration of 1 microgram GRF/kg. These data indicate that in hypothyroidism and perhaps hyperthyroidism there is an alteration in the responsiveness of the somatotroph to GRF administration.