Stimulatory Effects of Growth Hormone and Thyroxine on the Concentration of Gangliosides in the Snell Dwarf Cerebrum

The concentration of gangliosides in the Snell dwarf mouse cerebrum was monitored from postnatal day 5 to day 40. In the dwarf cerebrum, the concentration of total gangliosides increased up to postnatal day 20 and then stopped, whereas in the control cerebrum, it continued to increase up to postnatal day 40. At postnatal day 40, the ganglioside level in the dwarf cerebrum was 70% of that in the control cerebrum. Among the ganglioside species, the concentrations of GM4, GM2, GM1, GD1a, GD3, GD1b, GT1b, and GQ1b were significantly lower in the dwarf cerebrum than in the controls at postnatal day 40. The reduced concentrations of ganglioside species GM2, GD1a, GD3, GD1b, and GQ1b were completely restored by administration of bovine growth hormone (GH) during the first 20 days of postnatal life. The reduced concentration of the GM1 and GM4 species were most efficiently restored by administration of bovine GH plus thyroxine (T4) during the second 20 days of postnatal life. These results indicate that the lower ganglioside concentrations in the dwarf cerebrum can be elevated by hormone therapy and that there exist distinct GH and T4 actions on the enzymes participating in ganglioside metabolism.     

Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.     

Delayed appearance of liver growth hormone binding sites and of growth hormone-induced somatomedin production during rat development

The present study was designed to determine whether the apparent paradox of high circulating growth hormone levels in the fetus and the minimal effect of this hormone on growth might reflect a diminished responsiveness of fetal target organs to GH. Specific uptake by rat liver of [125I] bGH was very low in fetuses as compared to suckling and adult rats. Also, liver uptake of the iodinated hormone decreased proportionally with the simultaneous injection of increasing amounts of growth hormone, but was not modified by the simultaneous injection of unlabelled chemically-related hormones. Since the water content is significantly greater in fetal than adult tissues, results were expressed by liver dry weight and again, [125I] bGH liver uptake continued to increase with age. After bovine growth hormone administration to adult rats, plasma somatomedin C concentrations increased significantly, while they had no effect in fetuses. These results suggest that reduced liver somatogenic binding sites in the fetus prevents growth hormone from inducing growth-promoting effects during intrauterine life.     

Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide present in the plasma of man and rat that is thought to mediate the actions of pituitary growth hormone on cartilage to promote skeletal elongation. In the rat, plasma levels of IGF-I show both developmental and hormonal regulation: levels are low at birth, increase with age, and are decreased in growth hormone-deficient adult animals. The present study demonstrates that these changes in plasma IGF-I reflect the abundance of IGF-I RNA in rat liver. A human IGF-I cDNA probe hybridized to multiple RNA species in adult rat liver with sizes 8.6, 4.6, 3.2, 2.1, and 1.0-1.4 kilobases. These RNA species were decreased by greater than 80% in neonatal (2- and 12-day-old) rat liver and by greater than 90% in liver from adult rats made growth hormone-deficient by hypophysectomy. Treatment of hypophysectomized rats with growth hormone increased the abundance of all species of IGF-I RNA. These results suggest that growth hormone regulates the expression of its physiological mediator by altering the synthesis, stability, or both of IGF-I RNA in rat liver.     

双抗体免疫沉淀法纯化牛生长激素poly(A)~+RNA的研究

我们用双抗体免疫沉淀法,从牛垂体多聚核糖体中分离出牛生长激素特异多聚核糖体,由此多聚核糖体纯化的牛生长激素Poly(A)~+RNA,可以在麦胚体外翻译系统中和兔网织红细胞体外翻译系统中促进~(14)C-亮氨酸的参入。合成的含~(14)-亮氨酸的翻译产物中有91％可以被牛生长激素抗体沉淀。用SDS-11％PAGE对翻译产物进行鉴定表明,翻译产物在25KD处呈一条放射自显影带,与报导的牛生长激素前体分子量相吻合。     

Presence of prolactin receptors in eel liver and carp kidney and growth hormone receptor in eel liver.

1. 125I-labelled ovine prolactin and bovine growth hormone were used to test for the presence of prolactin and growth hormone receptors in membrane prepared from tissues of the white eel Anguilla japonica, the carp Ctenopharynogodon idellus and the ricefield eel Monopterus albus. 2. High levels of specific 125I-labelled ovine prolactin binding were found in white eel liver membranes and carp kidney membranes. 3. High levels of specific 125I-labelled bovine growth hormone binding were detected in white eel liver membranes. 4. Tissues of the ricefield eel did not bind 125I-labelled ovine prolactin or bovine growth hormone. 5. The results suggest the presence of prolactin receptors in white eel liver and carp kidney membranes and growth hormone receptors in white eel liver membranes.     

Effect of bovine growth hormone on rat liver plasma membranes as studied by circular dichroism and fluorescence using the extrinsic probe 7,12-dimethylbenzanthracene

Conformational changes produced by in vitro bovine growth hormone addition to plasma membranes of hypophysectomized rat liver proteins and lipids have been studied by circular dichroism as well as intrinsic and extrinsic fluorescence. 7,12-Dimethylbenzanthracene has been used as a fluorescent probe of changes in membrane structure. The exposure of membranes to bovine growth hormone produced a change in membrane negative ellipticity. Dimethylbenzanthracene at concentrations similar to those employed in fluorescence studies had no effect on the membrane circular dichroism spectrum. Its presence did, however, prevent a response to growth hormone. There was a decrease in peak fluorescence intensity and a peak shift when bovine growth hormone (0.5 · 10^?12 M) was added to liver membranes. The addition of dimethylbenzanthracene (1.6 · 10^?6 M) to membranes resulted in a decrease in the intensity of the protein fluorescence peak at 335 nm and the appearance of two peaks at 430 and 407 nm, assignable to the probe. The addition of bovine growth hormone (0.5 · 10^?12 M) produced a decrease in fluorescence at 335 nm and also in the peaks at 407 and 430 nm. These data are consistent with the conclusion that bovine growth hormone produces a conformational change in rat liver plasma membrane proteins and lipids.     

Lack of ornithine decarboxylase activity in isolated rat liver parenchymal cells

Polyamines are associated with fundamental metabolic and functional steps in cell metabolism. The activity of ornithine decarboxylase, the key enzyme in polyamine metabolism, was followed during the preparation of rat liver parenchymal cells and in the isolated cells during incubation. In experiments in which ornithine decarboxylase was not induced in vivo, enzyme activity dropped to barely measurable values during the preparation. An even more drastic loss of enzyme activity was noted in livers in which ornithine decarboxylase activity was stimulated in vivo 20-40fold by previous injection of bovine growth hormone, or thioacetamide or elevated because of circadian rhythmical changes of the enzyme activity. Within the first 20 min of liver perfusion to disintegrate the tissue, ornithine decarboxylase activity decreased by up to 80%. The presence of bovine growth hormone during cell preparation cannot prevent the loss of enzyme activity. Incubation of the isolated cells for periods of up to 240 min did not restore the enzyme activity. Furthermore, incubation of the cells with bovine growth hormone did not induce ornithine decarboxylase, even though the medium was supplemented with amino acids in physiological concentrations. During normal liver perfusion and in contrast to the situation with isolated cells, there is no loss of enzyme activity but a small rise. Following pretreatment of the animals with bovine growth hormone or thioacetamide the highly stimulated activity of ornithine decarboxylase declined slowly during liver perfusion, but never dropped to values lower than normal for perfusion periods of up to 240 min. Moreover, in the intact perfused organ ornithine decarboxylase remains responsive to bovine growth hormone. The experiments demonstrate that enzymatic tissue dispersion by collagenase in particular or the preparation of isolated cells in general drastically alters the metabolic and functional state of rat liver parenchymal cells.     

Elevated serum growth hormone in protein-deprived rats, and decreased liver RNA after hypophysectomy.

Serum growth hormone was significantly higher in rats fed on a non-protein than on a 25% casein diet. In both dietary groups, hypophysectomy provoked a decrease in the RNA/DNA ratio of the liver, and, especially in the protein-deprived rats, a decrease in the RNA synthesis per liver cell, measured as the product of the RNA/DNA ratio, and the ratio of specific RNA activity to specific nucleotide activity, two hours after administration of [3H]orotic acid. In the protein-deprived rats, full reconstitution of these figures was obtained with a combination of growth hormone, hydrocortisone and triiodothyronine, but not quite with growth hormone alone. The possible role of growth hormone in protein deprivation is discussed.     

Alteration of liver plasma membrane protein conformation by bovine growth hormone in vitro

The response of liver plasma membranes prepared from hypophysectomized, bovine growth hormone-treated hypophysectomized, and normal rats to addition of bovine growth hormone in vitro were studied by circular dichroism and spectrofluorometry. Membranes of hypophysectomized but not normal or treated rats showed increased negative ellipticity in the presence of bovine growth hormone (10^?9) without any change in trough position; at higher concentrations (10^?7?10^?6, m) there was less negative ellipticity. Membranes of hypophysectomized, normal, and growth hormone-treated rats all showed decreased emission of fluorescence and a small shift of the emission peak from 333 to 338 nm in the presence of bovine growth hormone (10^?17?10^?7, m). The excitation of fluorescence was quenched by bovine growth hormone. Polarization of excitation of fluorescence was unchanged.     

Restoration of microcephalic cerebrum with hypomyelination in the growth hormone-deficient mouse (lit): Stimulatory effects of GH restricted to the first 20 days of postnatal life

We administered bovine growth hormone to the Little (lit), a promising model of isolated growth hormone deficiency, during the first and second 20 days after birth. Positive results were obtained only when bovine growth hormone was given during the first 20 days of postnatal life. We observed a distinct increase in cerebral weight, DNA content, and 2, 3-cyclic nucleotide 3-phosphohydrolase activity. The latter administration of bovine growth hormone was ineffective. These data prove that growth hormone has an independent action on cerebral development, apart from the complementary or synergistic action of thyroid hormones, and that the administration of exogenous growth hormone led to increased myelinogenesis through its stimulatory effects on glial proliferation, as evidenced by the increase in cerebral DNA content.     

Tissue-specific expression and dietary regulation of a chimeric phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice

A series of transgenic mice was produced by microinjection of a segment of DNA, containing 460 base pairs of the phosphoenolpyruvate (P-enolpyruvate) carboxykinase promoter-regulatory region ligated to the bovine growth hormone structural gene, into the male pronucleus of fertilized mouse eggs. Founder animals which contained the gene were selected for further analysis and for breeding. The concentration of bovine growth hormone in the serum of animals which were shown to contain the gene ranged from a low of 5 ng/ml serum to approximately 2300 ng/ml serum. Mice with high levels of bovine growth hormone had growth rates double that of their litter mates which did not contain the transgene. The transgene was expressed only in the liver and kidney of the animals studied, and the level of specific mRNA for bovine growth hormone in these tissues could be regulated by diet in a manner similar to the endogenous P-enolpyruvate carboxykinase gene. Feeding the animals a diet high in carbohydrate for 1 week caused a 90% decrease in the concentration of bovine growth hormone in the blood, suggesting that the expression of the chimeric P-enolpyruvate carboxykinase/bovine growth hormone gene is sensitive to insulin. When the same animals were then refed a diet high in protein, but devoid of carbohydrate, the concentration of bovine growth hormone in their blood was induced 30-fold within a week. The administration of dibutyryl cyclic AMP to the transgenic mice caused a 2-fold induction in the level of bovine growth hormone in the serum within 90 min. Thus the region between -460/+73 in the P-enolpyruvate carboxykinase promoter-regulatory region contains sequences which can direct the tissue-specific expression, as well as hormonal and dietary responsiveness, of a linked structural gene.     

Additive effects of thyroid hormone,growth hormone and testosterone on deoxyribonucleic acid-dependent ribonucleic acid polymerase in rat-liver nuclei

1. The stimulations of DNA-dependent RNA polymerase in isolated rat-liver nuclei by thyroid hormone, human growth hormone and testosterone are compared. 2. Single or multiple administrations of growth-promoting doses of tri-iodo-l-thyronine, human growth hormone and testosterone stimulate the Mg²⁺-activated RNA-polymerase reaction in nuclei from thyroidectomized, hypophysectomized and castrated rats respectively. The magnitude of stimulation was proportional to the degree of enhancement of liver growth by each hormone. After a single injection, the latent period preceding the stimulation was 1, 2 and 10hr. for growth hormone, testosterone and tri-iodothyronine respectively. The time-course of stimulation of enzyme activity and the synthesis of rapidly labelled nuclear RNA in vivo were also different for each hormone. 3. Growth hormone administration failed to stimulate the Mn²⁺/ammonium sulphate-activated RNA-polymerase reaction. Thyroid hormone and testosterone, however, stimulated it but the effect was less pronounced and occurred several hours later than that observed for the Mg²⁺-activated RNA-polymerase reaction. 4. In combination experiments, hypophysectomized or the thyroidectomized rats were given growth hormone or tri-iodothyronine in a single or repeated doses at levels that produced the maximum stimulation of Mg²⁺-activated RNA-polymerase activity. Taking into account the different latent period for each hormone, a single administration of the second hormone caused an additional stimulation of the enzyme activity. Similar additive effects were observed in thyroidectomized–castrated rats after treatment with tri-iodothyronine and testosterone. The magnitude of the additional stimulation caused by the administration of the second hormone was compatible with the capacity of that hormone to promote liver growth in rats deprived of it. 5. It is concluded that, although these hormones have some similar effects, the regulation of nuclear RNA synthesis may be mediated via different routes for each hormone.     

Application of recombinant DNA technologies to studies on chicken growth hormone

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.     

Specific modification of carboxyl-groups in bovine growth hormone by a soluble carbodiimide and glycinemethylester

The reactivity of the carboxyl groups in bovine growth hormone was studied by reaction with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide in the presence of an excess of glycinemethylester. Localization in the molecule of the various kinetically distinguishable carboxyl groups was achieved. Highest reactivity was found in carboxyl groups 30, 125, 127 and 128. These residues were followed, as to reactivity, by a set including carboxyl groups 65, 105, 151, 152, 185 and 190. Modification of approximately one fifth of the carboxyl groups in bovine growth hormone led to an important decrease in its growth promoting activity and capacity to compete with 125I-labeled growth hormone for rat liver binding sites. Demethoxylation restored most of the original biological activity.     

Pretranslational regulation of sex-dependent testosterone hydroxylases by growth hormone in mouse liver

The effects of growth hormone on the expression of sex-dependent testosterone 16 alpha- and 15 alpha-hydroxylases were studied in growth hormone-deficient Little (lit/lit) mice at the activity as well as at the mRNA levels. The male isozyme of testosterone 16 alpha-hydroxylase ("C"-P-450(16)alpha) was repressed in the liver of male lit/lit mice, and the injection of bovine growth hormone resulted in an increase of the isozyme at both activity and mRNA levels to those seen in control lit/+ male mice. On the other hand, the female isozymes of testosterone 16 alpha- ("I"-P-450(16)alpha) and 15 alpha-hydroxylase (P-450(15)alpha) were increased in livers of both male and female lit/lit mice. The increased I-P-450(16)alpha and P-450(15)alpha in lit/lit mice were suppressed by growth hormone but only when it was injected once every 12 h. Thus, the results indicate that growth hormone acts as a masculinizing factor for testosterone hydroxylase activity by activating and inhibiting the expression of male and female isozymes of testosterone hydroxylases in mice, respectively. When growth hormone was infused to simulate a continuous secretion pattern, it showed no significant effect on the expression of hydroxylases in lit/lit mice, suggesting that growth hormone may not be a feminizing factor for testosterone hydroxylase activity in female mice. The changes of specific hydroxylase activities modulated by growth hormone in the mice correlated well with those amounts of hydroxylase mRNAs. The action of exogenous growth hormone to regulate the hydroxylases was so slow that it took 2 days to show a significant effect.     

Effect of hyperoxia on erythropoietin biogenesis in the "endocrine" kidney model

The erythropoietin plasma level and RNA synthesis in both kidneys were studied in rats with the H. Selye "endocrine" kidney under 4-hour hyperoxia. It was shown that a short period of hyperoxia leads to a 2-fold decrease in erythropoietin plasma level and to the fall of RNA synthesis in the "endocrine" and intact kidneys. From the evidence obtained it is concluded that hyperoxia inhibits erythropoietin production in the kidneys. Changes in high-polymeric RNA synthesis suggest that DNA-dependent RNA synthesis is one of the mechanisms of the hormone biogenesis.     

Human chorionic gonadotropin increases chromatin solubility in isolated bovine and human luteal nuclei

We have previously demonstrated that bovine and human luteal nuclei contain human chorionic gonadotropin/luteinizing hormone (hCG/LH) receptors and that these gonadotropins can directly stimulate nuclear membrane enzyme activity (nucleoside triphosphatase) involved in messenger ribonucleic acid (mRNA) transport from the nucleus to the cytoplasm. The present studies were undertaken to investigate the effect or hCG on chromatin solubility, reflecting perhaps synthesis and transport of RNA, in isolated bovine and human luteal nuclei. hCG increased chromatin solubility in a concentration-dependent manner. This hCG effect is either blocked or substantially reduced by the addition of hCG antiserum; denatured hCG had no effect and cyclic adenosine 3',5'-monophosphate could not mimic the hCG response. hCG had no effect on chromatin solubility in bovine liver or kidney nuclei and hormones other than hCG, human LH, or the beta subunit of hCG had no effect on chromatin solubility in bovine luteal nuclei, demonstrating the tissue and hormone specificity of the response. These findings further strengthen the concept of direct gonadotropin regulation of nuclear functions of luteal cells.