Journal Article A failed assay opened a new door in growth hormone research. Get access W H Daughaday, W H Daughaday Search for other works by this author on: Oxford Academic Google Scholar I E Karl, I E Karl Search for other works by this author on: Oxford Academic Google Scholar M M Karl M M Karl Search for other works by this author on: Oxford Academic Google Scholar Endocrinology, Volume 130, Issue 2, 1 February 1992, Pages 565–566, https://doi.org/10.1210/endo.130.2.1733709 Published: 01 February 1992

Complex, biologically active proteins (eg, enzymes and hormones) can be manufactured safely and cost-effectively through applications of biotechnology. Some of these proteins (eg, human insulin, human somatotropin, rennet for cheese manufacture) are currently approved for medical or food processing applications. Bovine somatotropin (bST) for lactating dairy cattle is another product that can be produced via biotechnology and may allow dairy farmers to produce milk at a lower cost. In 1985, based on an evaluation of toxicological data, the Food and Drug Administration concluded that milk and meat from bST-supplemented cows was safe and wholesome. The Food and Drug Administration has authorized the use of milk and meat from bST-supplemented cows in the commercial food supply. Its evaluation of the impact of bST supplementation on the long-term health of dairy cattle is near completion, and bST may be approved for commercial use in early 1991.

Pretreatment of confluent human fibroblast cultures for two days in dexamethasone, serum free medium increased 10-20 fold the sensitivity of the cells to insulin-like growth factor I (IGF I) stimulation of amino acid uptake using the amino acid analog alpha-aminoisobutyric acid (AIB). This increased sensitivity resulted both from the use of serum free medium and the addition of dexamethasone to the serum free media. Pretreatment of the cells for 1, 2, or 3 days before assay showed that the maximum increase in sensitivity was obtained after a two day pretreatment. Pretreatment of the cells also increased their sensitivity to insulin and bovine insulin-like growth factor II stimulation of AIB uptake similar to that seen for IGF I. No consistent effect of the pretreatment was observed on either the basal level of AIB uptake or the maximal hormonal stimulation of AIB uptake. Nor was any change noted in the shape of the dose response curves. Addition of IGF I to the pretreatment medium greatly reduced the sensitivity of pretreated cells. [125I]IGF I binding studies done on suspended fibroblasts indicated that there was up to a two fold increase in the number of receptors with no increase in their affinity for IGF I. Thus, pretreatment of fibroblasts with dexamethasone and serum free medium greatly enhances their sensitivity to IGF I stimulation of AIB uptake and makes this an excellent in vitro bioassay system for IGF I.

A supranormal rate of growth in intact, prepubertal, 26-day-old female rats was evoked by administration of large doses of highly purified rat GH (rGH). In response to daily doses of 1 and 5 mg/rat (13.6 mg/kg BW and 68 mg/kg BW), sc, for 20 days, body weight (BW) gain increased 51% and 73%, and skeletal growth increased 27% and 40%, respectively. Serum rGH in treated rats rose as much as 69-fold greater than that of controls. Feed efficiency, the ratio of weight gained to feed consumed, increased from 19.8% to 32.0%. This rGH treatment depleted pituitary GH content as much as 58% and caused hepatomegaly. These effects, as well as the accelerated growth rate, reverted to normal after cessation of rGH treatment. Onset of puberty in rGH-treated rats was delayed an average of 2.7 days. A similar stimulatory effect on BW gain, but not skeletal growth, as well as depletion of pituitary GH content, and hepatomegaly, was elicited by rGH treatment in adult, plateaued female rats. These effects in plateaued rats reverted to normal after cessation of GH treatment, and 50% of the body weight gain was rapidly lost. The largest dose of rGH used, 5 mg/day, was apparently toxic, resulting in a 20% higher mortality rate in treated prepubertal and plateaued female rats. Antibody formation was not the cause of the toxicity, since antibodies against rGH were undetectable at the lowest dilutions of serum tested. Serum rat insulin-like growth factor I (RIA), 3.5 U/ml in untreated intact prepubertal rats, increased to 4.7 and 5.0 U/ml, respectively, after 20 days of rGH treatment. In hypophysectomized rats, serum rat insulin-like growth factor I (RIA), undetectable in controls, was increased to 1.63 U/ml after 14 days treatment with 1 mg rGH/day. This study demonstrates that greater than normal growth can be stimulated in normal female rats by administration of large doses of homologous GH, but at the risk of serious adverse effects. Possible implications for the administration of GH to non-GH-deficient children, to promote taller stature, are clear.

Somatomedin-C (Sm-C) and growth hormone (GH) levels were determined before, during and after human growth hormone (hGH) treatment in 18 children with small-for-date short stature ( SDSS ), 7 children with significant idiopathic short stature ( SISS ) and 14 children with hypopituitarism. Data on the acute effects of hGH on Sm-C were compared to growth responses after 6 to 9 months therapy. Eleven of the 25 non-hypopituitary patients with normal basal and stimulated serum GH levels and normal basal Sm-C levels increased their rates of growth more than 3.0 cm/year. This compared with 11 of the 14 children with hypopituitarism who increased their rates of growth by at least 3.0 cm/year when treated with GH. Neither the basal somatomedin levels nor the GH-stimulated somatomedin levels correlated well with subsequent growth in the non-hypopituitary patients. These studies indicate that GH therapy may be effective in treating short stature in children without demonstrable GH deficiency.

It is known that the somatomedins exist in human serum complexed to specific binding proteins. The existence of unbound somatomedins in serum has never unequivocally been demonstrated. We have characterized the distribution of insulin-like growth factor (IGF) I in different fractions after gel filtration of serum through Sephadex G-200 in neutral buffer. IGF-I was measured by RIA after acid extraction. Seventy-two percent of serum IGF-I was associated with large complexes with an estimated size of about 150,000 daltons and 25% was associated with smaller complexes of about 50,000 daltons. No unbound IGF-I was detected. Ultracentrifugation of 10 ml fresh serum was carried out at 106,000 X g for 17 h, after which the tube was aspirated in 1-ml fractions beginning at the top. IGF-I by RIA in fractions 2 and 3 sedimented with albumin; in fractions 4 to 7, the sedimentation pattern approached that of immunoglobulin G. This shift is consistent with the size distribution of IGF-I complexes demonstrated by gel filtration. The failure to find any significant increase in the concentration of IGF-I relative to albumin in the top 30% of the tube (fractions 1-3) after centrifugation argues against the presence of measurable free IGF-I in these fractions. The ability of upper fractions to bind added [125I]IGF-II proved to closely approximate the binding of the initial serum, indicating little sedimentation of the accessible binding protein. The relative binding of [125I]IGF-II by serum aliquots proved to be markedly concentration dependent. At concentrations above 5% serum, the incremental increase of binding as a function of serum concentration was much reduced. We interpret this to indicate that with dilution there is a dissociation of complexes and an increase in accessible binding sites. This phenomenon may modify tissue delivery of somatomedins in interstitial fluid. The data suggest that in undiluted serum there is no significant concentration of free somatomedins but at the dilution of serum that exists in the interstitial fluid, dissociation of bound somatomedins may be facilitated.

We determined the cross-sectional natural history of retinopathy by prospective study of 461 insulin-dependent juvenile-onset diabetics. In so doing, we compared the sensitivity of ophthalmoscopy, photography, and fluorescein angiography in detecting retinopathy. Photography was far more reliable than ophthalmoscopy in detecting early retinopathy and equivalent to angiography. Retinopathy was not present at diagnosis of diabetes. After a lag period, the prevalence of retinopathy rose in sigmoidal fashion, reaching 50% at just over seven years duration, and asymptotically approaching 90% at 17--50 years. Proliferative retinopathy was first seen at 13 years duration, and its prevalence rose to 26% at 26--50 years. From the natural history we computed the dimensions of a proposed clinical trial to test the effect of tight metabolic control in prevention of retinopathy.

Plasma GH levels of fed male gentled rats (52+6 ng/ml) were significantlygreater than those of nongentled animals (24 + 5 ng/ml). Plasma corticosterone levels showed the opposite relationship—gentled, 4.6 ± 0.8 μg/100 ml, vs. nongentled, 8.4 ± 1.4 μg/100 ml. Pentobarbital anesthesia caused a significant rise in plasma GH in both gentled and nongentled rats and a corresponding decrease in plasma corticosterone. A statistically significant diurnal variation in plasma GH could not be demonstrated, although the lowest GH levels were generally observed at 5 PM and corresponded to peak values for plasma corticosterone. Ether anesthesia, hypertonic glucose, 2-deoxyglucose, insulin-induced hypoglycemia and epinephrine all resulted in a marked suppression of plasma GH, and an increase in plasma corticosterone, effects which were either partially or completely blocked by pentobarbital anesthesia. The effects of insulin-induced hypoglycemia and 2-deoxyglucose were not mediated by catecholamines since they were noted in adrenalectomized animals and the response was not abolished by depletion of endogenous catecholamines with reserpine. Femoral venous catheterization consistently and rapidly suppressed the plasma GH and this inhibitory effect was not blocked by pentobarbital anesthesia. (Endocrinology88: 909, 1971)

A sensitive radioimmunoassay for rat growth hormone (GH) has been modified and used to study pituitary content of GH in the Sprague-Dawley rat under varying conditions. Growth hormone does not appear in the rat fetal pituitary until the 19th day of gestation. Day-old rats have low GH content and concentration but GH rapidly accumulates with age. By puberty the male rat has more pituitary GH than the female rat. This difference increases as the rats grow older. Estrogen treatment of the male rat lowers GH concentration and causes an increase in pituitary size. Testosterone treatment of the female rat leads to an elevation of GH content and concentration. Castration of male rats results in levels of GH that are indistinguishable from those of female rats. These studies indicate that factors of age, sex and gonadal hormones interact to affect pituitary content of growth hormone in the rat. (Endocrinology81: 195,1967)

The secretion of growth hormone in response to hypoglycemia and the administration of arginine in man has been shown to be greatly influenced by certain non-pituitary hormones. Corticosteroids decrease growth hormone response to these stimuli in man (1,2) while estrogens and the synthetic estrogenic compound diethylstilbestrol facilitate the release of growth hormone(3,4). The mechanism of action of these modifying effects has not been established. Corticosteroids have little effect on the concentration of growth hormone in the pituitary of the normal rat(5). If corticosteroids are administered during the induction of hypoglycemia, the expected drop in growth hormone content is inhibited(5). Estrogens on the other hand decrease the concentration of growth hormone even when compared to pair fed control rats(6). The modifying effects of estrogens and corticosteroids on growth hormone secretion could be exerted either at the level of the hypothalamus by affecting the secretion of the somatotropin releasing factor or these hormones could affect the secretion of growth hormone by acting directly on the somatotropic cells themselves. The observations of Pecile and Müeller (5) have suggested that cortisol may deplete the hypothalamus of somatotropin releasing factor. In their experiments hypothalamic extracts from cortisol treated animals had a much reduced capacity to cause a depletion of bioassayable pituitary growth hormone in recipient animals when compared to hypothalamic extracts from normal rats. Cortisol-treated recipient rats retained their ability to respond with a depletion of bioassayable growth hormone when injected with hypothalamic extracts from normal rats. Although one can not have complete confidence in the specificity of bioassay of crude pituitary extracts, these observations suggest that cortisol affects growth hormone release at the level of the hypothalamus. Despite these observations we felt that it was important to study possible influences of corticosteroids and estrogens directly on the pituitary in the absence of hypothalamic tissue.