Twenty-kilodalton human growth hormone (20K hGH) secretion from growth hormone-secreting pituitary adenoma cells in vitro

Circulating human growth hormone (GH) consists of several molecular isoforms. Increased proportion of circulating non-22K hGH and 20K hGH was reported in active acromegaly. In this study, we studied the release of 20K and 22K hGH from cultured GH-producing human pituitary adenoma cells in vitro. Pituitary adenoma cells obtained from 6 acromegalic patients were cultured and submitted to perifusion experiments. Concentrations of 20K and 22K hGH in the serum and the perifusion effluent were determined by specific enzyme-linked immunosorbent assays recently developed. The %20K value varied in a wide range from 3.58 to 8.72% in vitro and was lower than in the serum (mean+/-SD: 6.57+/-1.88% vs 9.08+/-2.12%, P<0.05). There was no correlation between the %20K values in vitro and in vivo (r=0.31, P>0.05). The in vitro secretions of 20K and 22K hGH were in parallel and strongly correlated (r=0.953, P<0.001). These findings suggest that different GH-producing pituitary adenoma cells secrete 20K hGH in variable amounts and that the proportion of 20K hGH in the serum might be affected by metabolic clearance of hGH isoforms. It was also suggested that 20K and 22K hGH might be secreted in toto from GH-producing human pituitary adenoma cells.     

Isolated human growth hormone deficiency due to the hGH-I gene deletion with (type IA) and without (the Israeli-type) hGH antibody formation during hGH therapy

Three Japanese patients with isolated growth hormone deficiency from two different families were shown to be homozygous for deletion of the structural gene for human growth hormone (hGH-I gene). These three patients had the same restriction fragment length polymorphism haplotypes. In patient No. 1, the growth rate initially responded well to pituitary human growth hormone, but growth rapidly ceased concomitantly with the development of high levels of anti-hGH antibodies. He again responded well to recombinant methionyl hGH and recombinant hGH without the methionine residue, even though having high hGH antibodies. Two siblings (Patients No. 2 and 3) showed a rather good response to pituitary hGH treatment without hGH antibodies ever being detected (the Israeli-type). hGH-I gene deletions may not necessarily result in hGH antibody formation. Heterogeneity has been observed in isolated hGH deficiency due to hGH-I gene deletion. hGH-I gene analysis should not be limited to patients with hGH antibody formation and subnormal growth responses to hGH therapy.     

Thyrotropin-releasing hormone (TRH)-induced growth hormone (hGH) responses in cirrhotic men.

The plasma growth hormone (hGH) responses to an intravenous challenge of 400 micrograms of thyrotropin-releasing hormone (TRH) were evaluated in 14 normal controls and in 29 chronic alcoholic men. The normal controls had either a minimal or no hGH response to TRH, having basal hGH levels of 0.9 +/- 0.2 ng per ml and peak hGH levels of 2.0 +/- 0.5 ng per ml. In contrast, the chronic alcoholic men had a basal hGH level of 2.8 +/- 0.4 ng per ml, 3 times the basal level of the normal controls (P less than 0.01). The peak hGH response of the alcoholic men was 7.4 +/- 1.5 ng per ml (P less than 0.01). The 29 alcoholic men could be divided into two groups based upon the presence or absence of cirrhosis as determined by liver biopsy. The 16 alcoholic men with cirrhosis had greater basal hGH levels (3.5 +/- 0.6 ng per ml) and peak hGH levels (9.5 +/- 2.3 ng per ml) than did the 13 alcoholic men without cirrhosis (basal hGH 2.1 +/- 0.6 ng per ml, peak hGH 4.9 +/- 1.5 ng/ml). Plasma estradiol levels were similar in the normal controls and in the alcoholic men. In contrast, plasma estrone was greater in the alcoholic men (32.2 +/- 3.5 pg per ml) than in the normal controls (18.9 +/- 1.8 pg per ml) (P less than 0.05). However, when the plasma estrone levels of alcoholic men with cirrhosis were compared to those of the alcoholic men without cirrhosis no difference existed. Thus it is difficult to ascribe the increased hGH responses of the cirrhotic alcoholic men when compared to those of the noncirrhotic alcoholic men as being a result of increased basal estrogen levels.     

Arginine induced growth hormone (hGH) response and paradoxical hGH secretion stimulated by TRH in diabetes mellitus

Summary Growth hormone (hGH) reserve following arginine administration and the paradoxical hGH response to thyrotropin-releasing hormone (TRH) were studied in 30 diabetics without evidence of vascular complications. The diabetics were divided into 4 groups according to the type of their disease and to the metabolic condition within the IDDM group (insulin-dependent: IDDM, in acceptable response and in poor metabolic control; non-insulin-dependent: NIDDM, and juvenile diabetics not requiring insulin at least for two years after diagnosing their disease: NIDDY). The results were compared with controls of identical age and normal weight. A paradoxical hGH response to TRH stimulation was found only in IDDM patients in poor metabolic control. In this group the hGH reserve revealed by arginine was significantly larger than in the others. It was shown that the induced hGH release was independent of the sex distribution of the groups and of the basal hGH values. Magnitude of the hGH reserve and appearance of the paradoxical hGH response were not necessarily correlated but the substantial reserve was frequently associated with a paradoxical response. It can be assumed that the unfavorable metabolic condition is of decisive importance in giving rise to these anomalies. Our observations seem to confirm the need for good metabolic control if the pathological hGH secretion in diabetics is to be prevented.     

Effect of growth hormone deficiency and recombinant hGH (rhGH) replacement on the hypothalamic-pituitary-adrenal axis in children with idiopathic isolated GH deficiency

Objective Recombinant hGH (rhGH) therapy may unmask central hypoadrenalism in adults with organic GH deficiency (GHD), likely by normalizing 11β‐hydroxysteroid dehydrogenase type 1 isoenzyme (11βHSD1) activity and reducing cortisone to cortisol conversion. The aim of the present study was to evaluate the hypothalamic–pituitary–adrenal (HPA) axis in children with idiopathic isolated GHD and normal pituitary magnetic resonance imaging (MRI) both before and during rhGH therapy. Design and patients This was a single‐centre study of 10 consecutive children [five males and five females, mean age: 12·2 ± 1·0 year]. Evaluation was performed at baseline and on rhGH (mean duration: 10·9 ± 2·9 months, mean dose: 0·030 ± 0·002 mg/kg bw/day). Measurements HPA function was assessed by serum cortisol levels before and after appropriate provocative stimuli, that is, 1 µg ACTH test (N = 5 patients) or insulin tolerance test (ITT, N = 5 patients), evaluating all children with the same stimulation test both before and during rhGH therapy. Central hypoadrenalism was excluded by the presence of either a peak of > 500 nmol/l or a rise in cortisol levels of > 200 nmol/l, after both tests. Results On rhGH therapy, serum IGF‐I levels normalized, while serum cortisol and ACTH levels did not significantly differ from those recorded at baseline. The mean serum cortisol peak after both provocative tests was not significantly different on rhGH therapy and at baseline (498 ± 41 vs. 580 ± 35 nmol/l, respectively, P = 0·06), the mean cortisol rise being 280 ± 45 and 270 ± 36 nmol/l on rhGH and at baseline, respectively. Conclusions According to the diagnostic criteria, no child became hypoadrenal on rhGH, contrary to what observed in patients with organic GHD, further supporting the view that only in patients with organic multiple pituitary hormone deficiency GHD masks the presence of a hidden central hypoadrenalism.     

Long term pulsatile growth hormone (GH)-releasing hormone therapy in children with GH deficiency

We treated seven GH-deficient children with 3-hourly 1 microgram/kg sc pulses of GHRH-(1-44) for 6 months and 2 micrograms/kg.pulse for another 6 months. Four patients had a serum GH response to iv GHRH before treatment, and an additional patient responded to iv GHRH after 1 month of pulsatile sc GHRH administration. The mean cumulative growth velocity increased from a pretreatment mean of 2.7 +/- 0.2 (+/- SE) to 8.4 +/- 2.5 and 5.4 +/- 0.7 cm/yr after 2 months and 1 yr of treatment, respectively. Low dose pulsatile GHRH therapy was effective in promoting growth in five of seven children, with height gain ranging from 4.4-7.5 cm at the end of 1 yr's therapy. Only one of the two patients who did not respond to GHRH had an improvement in linear growth when they were subsequently treated with synthetic GH. The other patient, a 16.5-yr-old pubertal girl who had both satisfactory GH and somatomedin-C responses during GHRH therapy, did not respond to either GHRH or, later, synthetic GH. The pretreatment serum GH response to iv GHRH, the serum somatomedin-C concentrations, and the peak serum GH response during sc GHRH therapy were not reliable predictors of clinical response.     

Long-term therapy with recombinant human growth hormone (Saizen) in children with idiopathic and organic growth hormone deficiency

In an open-label study, 69 children with organic or idiopathic growth hormone deficiency (GHD) were treated with recombinant human growth hormone (Saizen) for an average of 64.4 mo, with treatment periods as long as 140.9 mo. Auxologic measurements, including height velocity, height standard deviation score, and bone age, were made on a regular basis. The data suggest that long-term treatment with Saizen in children with GHD results in a positive catch-up growth response and proportionate changes in bone age vs height age during treatment. In addition, long-term Saizen therapy was well tolerated, with the majority of adverse events related to common childhood disorders or existing baseline medical conditions and not to study treatment. There were no significant changes in laboratory safety data or vital signs, and no positive antibody tests for Saizen.     

Human growth hormone (hGH) secretion in milk of goats after direct transfer of the hGH gene into the mammary gland by using replication-defective retrovirus vectors.

Mammary-specific promoters have been used in transgenic animals to limit transgene expression to the mammary gland. Gene therapy techniques to target just one organ for introduction of a foreign gene have also been demonstrated. We have directly infused replication-defective retroviruses encoding hGH into the mammary gland of goats via the teat canal during a period of hormone-induced mammogenesis. This resulted in the secretion of hGH into the milk when lactation commenced on day 14 of the regime. Levels of hGH in the milk were highest on the first day of lactation, averaging approximately 60 ng/ml, and declined to a plateau of 12 ng/ml from day 9 to day 15 of lactation. Thus we report targeting of replication-defective retroviruses to the mammary secretory epithelial cells to produce foreign proteins in the milk of ruminants.     

Human pituitary growth hormone cells in old age.

Pituitaries obtained at autopsy from 18 men and 17 women, over 80 years of age, who died of various acute illness, were investigated histologically by various staining procedures, including the immunoperoxidase technique. Compared with pituitaries of 10 male and 10 female subjects who died of short-lasting diseases between 20 and 56 years of age, no involution of growth hormone cells was found in pituitaries of the older group. Incidence, distribution, granulation and immunoreactivity of growth hormone cells showed no apparent differences related to age and sex. Lack of regression of growth hormone cells with advancing age indicates that the pituitary can produce growth hormone in old subjects.     

Evaluation of adult idiopathic growth hormone deficiency with other pituitary hormones deficiency

The type and percentage of multiple pituitary hormone deficiency (MPHD) were studied in 42 patients with idiopathic growth hormone deficiency (IGHD). It was found that the development of secondary sexual characteristics was poor or absent in 39 patients (93%) with gonadotropin deficiency (GnD). Mean serum testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in the 39 patients were significantly less than those of normal adult males (P less than 0.01). Mean testicular volume in 36 patients with GnD was significantly less than that in 3 with normal T level. We also found that 24-hour urinary free cortisol level (24 hour UFC) was low in 24 (57.1%) of 42 patients, but it is important that none had obvious symptoms of hypoadrenocorticism such as hypoglycemia, hypotension etc and received adrenal-corticosteroid treatment. 22 (52.4%) of the 42 patients suffered from hypothyroidism, with serum thyroxine (T4) level lower than normal but thyrotrophin (TSH) within normal range. 6 patients with hypothyroidism had moderate symptoms such as cold intolerance, constipation, rough and dry skin, slowing down both mentally and physically. 17 patients have treated with thyroxine. From the results mentioned above, 14 of the 41 patients with MPHD had pan-pituitary hormones (LH, FSH, TSH, ACTH) deficiency, only one had isolated growth hormone deficiency. Among all the patients, 23 underwent breech delivery and 11 patients had birth asphyxia. We therefore conclude that: (1) most of the IGHD cases are complicated with other pituitary hormone deficiency; (2) most of the IGHD cases have with MPHD; (3) Breech delivery and birth asphyxia were important etiological factors of IGHD.     

Growth hormone therapy for adult growth hormone deficiency.

GH deficiency in adult life is associated with a number of adverse biological changes including osteopenia, reduced exercise capacity, altered body composition, deleterious alterations in the lipid profile and insulin status, and reduced quality of life. Potentially, most of these changes can be reversed by GH replacement therapy. In an era of health rationing, however, GH replacement is unlikely to be offered to every GH-deficient adult. Therefore, we have proposed a strategy aimed at delineating which adults with GH deficiency might benefit most from GH therapy.     

Reversible dimerization of 20 kilodalton human growth hormone (hGH).

A noncovalent dimer of the 22 kilodalton human growth hormone (22 K-hGH) is known to have diminished somatogenic activity compared with monomeric 22 K-hGH. In the present study, we examined the biological activity and physicochemical behaviour of a noncovalent dimer of the 20 kilodalton human growth hormone (20 K-hGH), an isoform of 22 K-hGH. Analysis of the equilibrium between monomeric and associated forms revealed that the associated 20 K-hGH was present in the dimeric form in aqueous solution. The kinetics of dimerization in rat plasma followed the theory of dissociation-association equilibrium, and more than 99% of 20 K-hGH molecules existed as a monomer in the equilibrium state at the physiological hGH concentration. Analysis of the pharmacokinetics showed that the ratio of the administrated dimer in rat circulation decreased from 43% to less than 4% in 2 h. A preparation of noncovalent dimeric 20 K-hGH had essentially the same degree of biological potency as that of a monomer in both in vitro and in vivo bioassays. In conclusion, dimerization of 20 K-hGH is reversible both in vitro and in vivo and a noncovalent dimer can function as a pharmaceutically active component of a 20 K-hGH preparation, in contrast to a 22 K-hGH preparation.     

The 20-kilodalton (kDa) human growth hormone (hGH) differs from the 22-kDa hGH in the effect on the human prolactin receptor.

Previously we have demonstrated that 20-kDa human GH (20K-hGH) is a full agonist for hGH receptor (hGHR) even though its complex formation with hGHR and hGH-binding protein differs from that of 22-kDa human GH (22K-hGH). In this study, we focused on the effect of 20K-hGH on human PRL receptor (hPRLR). To elucidate the effects of 20K-hGH on hPRLR and compare them with those of 22K-hGH, we prepared two cells stably expressing full-length hPRLR, Ba/F3-hPRLR and CHO-hPRLR. In the proliferation of Ba/F3-hPRLR cells, which can grow in a dose-response to lactogenic hormones, both 20K- and 22K-hGH exhibited bell-shaped curves in the absence of exogenous zinc ion (Zn2+); however, the curve of 20K-hGH was shifted to a 10-fold higher concentration than that of 22K-hGH in view of EC50 value (the EC50 of 20K- and 22K-hGH were 15 nM and 1.5 nM, respectively). Addition of Zn2+ up to 25 microM increased the activities of both 20K- and 22K-hGH; however, the enhancement by Zn2+ was greater in 20K-hGH than in 22K-hGH, thereby the activities of both hGH isoforms reached the same level at 25 microM Zn2+. Nevertheless, in the presence of 0.25-1 microM free Zn2+, which is equal in human serum, the activity of 20K-hGH was still lower than that of 22K-hGH. The modest effect of 20K-hGH on activating hPRLR in the absence of Zn2+ was confirmed in the rat serine protease inhibitor 2.1 (Spi2.1) gene promoter activation and JAK2/Stat5 tyrosine phosphorylation in CHO-hPRLR. In addition, in human breast cancer cell T-47D, 20K-hGH was proved to stimulate Stat5 tyrosine phosphorylation to much lower degree than 22K-hGH via not hGHR but hPRLR. Taken together, our data suggest that 20K-hGH may be a weaker agonist for hPRLR than 22K-hGH in the human body; therefore 20K-hGH may alleviate the hPRLR-mediated side-effects such as breast cancer when administered to human body.     

Childhood-onset growth hormone (GH) deficiency in adult life

Over the last decade GH replacement therapy for adults has progressed in status from research study to a mainstream clinical indication. An area ripe for further research, however, is the difference between adults who developed GHD before and after completion of growth and puberty. That differences exist, not only in aetiology, but also in phenotype and response to GH therapy is clear. However, whether these differences are intrinsic to the timing of onset of GHD, or related to secondary factors including the method of assessment or dose of GH employed is uncertain. This chapter discusses the current state of knowledge in this area and poses further questions, not only for the researcher attempting to understand the mechanisms underlying these differences, but also for the physician seeking to ameliorate the impact of GHD in patients who acquired GHD in childhood.     

Proteolytic cleavage of human growth hormone (hGH) by rat tissues in vitro: influence on the kinetics of exogenously administered hGH

The presence of several endogenous molecular forms of human GH (hGH), including proteolytically cleaved two-chain forms, has been proposed to be related to the diverse biological activity of hGH. The present study characterized hGH degradation in the rat to determine how peripheral metabolism may influence the kinetics and pharmacology of exogenously administered hGH. In vitro studies indicated that hGH was proteolytically degraded by thyroid gland and skeletal muscle, but not liver and kidney homogenates. The proteolytic activity, localized to the 9000 x g pellet fraction, was characterized as a chymotrypsin-like serine protease using class-specific inhibitors. N-Terminal sequencing of hGH peptides formed by the thyroid gland and skeletal muscle indicated that cleavage sites were almost exclusively at Tyr/Phe-Xaa bonds, with similar points of cleavage observed in the two tissues. Immunoreactive two-chain forms of hGH were also formed. The two-chain molecules had similar cleavage sites, but differed in apparent mol wt when analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To understand the potential significance of two-chain product formation, we compared the kinetics and degradation of hGH with those of a synthetic two-chain derivative of hGH (Des-1-8,135-145; 2-CAP). The in vitro tissue distribution of 2-CAP proteolysis was the same as that for hGH. The fragmentation pattern of 2-CAP was less complex when analyzed by reverse phase HPLC. The major peptide fragments formed from 2-CAP were chromatographically similar to those formed from hGH. The plasma kinetics of 2-CAP were compared to those of hGH with a RIA using polyclonal antiserum to hGH. After im and sc administration of 2-CAP (125 micrograms/kg), the area under the plasma concentration curve was 3.2- and 4.5-fold greater, respectively, than after administration of hGH (125 micrograms/kg). Both compounds had a greater area under the curve by the im than the sc route. 2-CAP had 2- to 3-fold greater bioavailability than hGH by the im and sc routes. Plasma from rats treated 30 min earlier with hGH im was immunoextracted and analyzed by Western blotting. A circulating immunoreactive fragment was detected which had similar electrophoretic mobility as a two-chain hGH product formed during the in vitro incubations of hGH with skeletal muscle and thyroid gland homogenates. The results indicate that hGH is proteolytically processed in peripheral tissue homogenates, with the formation of two-chain products. The greater bioavailability of 2-CAP suggests that metabolism of hGH to two-chain forms may influence the in vivo kinetics of hGH.