Growth Hormone Preparations Research Articles

P-5-1 Preparation of rat pituitary growth hormone and direct effects of growth hormone on insulin secretion

P-5-1 Preparation of rat pituitary growth hormone and direct effects of growth hormone on insulin secretion

Influence of triiodothyronine and growth hormone on growth of dwarf and normal chickens: Interactions of hormones and genotype

1. 1. The effects of dietary triiodothyronine (T 3), injections of a preparation of growth hormone (GH) (purified from chicken pituitary tissue) and their combination on growth were investigated in three lines of chickens. The three lines were the Cornell K strain (K) (a Single Comb White Leghorn strain), the Cornell K strain hemizygous for the sex-linked dwarfing gene (SLD), and the Cornell K strain homozygous recessive for the autosomal dwarfing gene (ADW). 2. 2. A dietary T 3 treatment by genotype interaction was observed. Dietary T 3 (0.1 ppm) decreased growth in the K line, tended to decrease growth in the ADW line while it tended to increase growth in the SLD line. 3. 3. Chicken growth hormone (100 μg/kg body wt) alone did not affect growth in any of the lines studied. There was, however, a GH treatment by T 3 treatment interaction. Chicken GH overcame the growth-depressing effects of T 3 in the K and ADW lines while it tended to promote growth in T 3 treated SLD birds. 4. 4. Dwarf (SLD) chickens had higher basal circulating GH concentrations, lower circulating immunoreactive somatomedin C concentrations, and lower circulating T 3 concentrations than the K or ADW chickens.

COMPARISON OF THE LIPOLYTIC POTENCY OF PITUITARY-DERIVED AND BIOSYNTHETIC GROWTH HORMONE IN HYPOPITUITARY CHILDREN

Whether lipolysis is an intrinsic property of the growth hormone (GH) molecule or is due to contaminants of pituitary origin remains controversial. We therefore compared the rise in plasma free fatty acid (FFA) levels measured with the Nefa Quick “BMY” kit, Boehringer) induced by either pituitary GH (pit-GH, Nanormon, Nordisk) or biosynthetic GH (syn-GH, Somatonorm, Kabi) in 34 hypopituitary children (age 5-18 years, peak plasma GH < 5 ng/ml after insulin and glucagon tolerance tests). The patients were admitted after an overnight fast and fasted until 1230 hr on both study days. On day 1, no injection was given. On day 2, 0.2 U/kg of either pit-GH (N=5, group A) or syn-GH (N=29, group B) was given IM at 0830 hr. The FFA levels at 1230 hr are expressed as % of the 0830 hr value. In group A, mean (± SEM) plasma FFA levels at 1230 hr were 138.7 ± 9.8 % of the 0830 hr value on day 1 and 233.1 ± 20.0 % on day 2. In group B, they were 130.3 ± 10.9 % and 267 ± 22.6 %. on days 1 and 2, respectively. Thus, both GH preparations induced a rise in plasma FFA levels greater than that induced by fasting alone (p < 0.001) but the two GH preparations were not significantly different from each other (p > 0.2). We conclude that the acute lipolytic effect probably represents an intrinsic property of the GH molecule.

Similarities and Differences Among Prolactins and Growth Hormones and Their Receptors

SYNOPSIS. Data from the literature and from our own studies on the receptors for prolactin (PRL) and growth hormone (GH) are reviewed and analyzed. Receptors for PRL have been studied in a wider range of species and in a greater diversity of target organs than have the binding sites for GH. Although GHs are structurally more highly conserved among the vertebrates than are PRLs, the available data indicate that there is greater diversity among GH receptors than there is among PRL receptors. In general, GH receptors show greater species specificity but less hormone specificity than do PRL receptors. The reason for the greater diversity among GH receptors as compared to PRL receptors is unknown; it bears no relationship to phylogeny. Data on the binding of purified preparations of mammalian PRL, GH and placental lactogen (PL) to renal and hepatic receptors for PRL and GH, respectively, of several vertebrate species are reviewed. The species and hormone specificity of the binding of the hormones to the two typesof receptors showed no consistent pattern. To explain this disarray, we propose that the receptor binding domains of PRL and GH were present in their common ancestral gene and that they havebeen retained to variable degrees by all of the descendant members of the PRL-GH family. We further propose that hormone and species specificity of binding is determined by hindering features on the hormones and on the receptors, rather than by merely the presence or absence of the appropriate binding determinants.

Development and validation of a salmon growth hormone radioimmunoassay

A highly specific and sensitive radioimmunoassay (RIA) for the measurement of plasma and pituitary growth hormone (GH) levels in salmonid fishes was developed using an antiserum raised in rabbit against chum salmon ( Oncorhynchus keta) GH (sGH). Pituitary extracts and plasma from chum, coho, masu, and amago salmon, and from rainbow trout and Japanese charr, all exhibited displacement curves parallel to the sGH standard. Samples from the eel, carp, goldfish, and tilapia, as well as plasma from hypophysectomized chum salmon and rainbow trout, all showed negligible cross-reactivity. None of the mammalian or teleostean GH or prolactin preparations tested cross-reacted with the antibody in the assay system. RIA sensitivity was 0.6 ng sGH/ml of plasma when 100 μl of plasma was employed. Intra- and interassay coefficients of variation were 3.9 and 4.1%, respectively. Plasma GH levels of the mature chum salmon caught in Otsuchi Bay were highly variable, especially in females (20.2 ± 8.2 ng/ml) as compared with males (16.0 ± 1.1 ng/ml), and there was no significant change after transfer to fresh water. Whereas there was no change in plasma GH levels in males kept in seawater, the levels in females increased with time in close correlation with the increase in plasma chloride.

Comparison of reversed-phase and anion-exchange high-performance liquid chromatography for the analysis of human growth hormones

Separation of human growth hormones (hGH) has been studied by three chromatographic methods: gel filtration chromatography, reversed-phase high-performance liquid chromatography (HPLC) and anion-exchange HPLC, Six growth hormone preparations were used to characterize the systems: two pituitary extracts (an old freeze-dried purified extract A ¯ , and a freshly extracted pituitary B ¯ ; two purified forms (hGH 20K and dimer); and two chemically modified monomeric forms (reduced and deamidated). Gel filtration chromatography (pH 8) separated the two pituitary extracts A ¯ and B ¯ into four components (monomeric, dimeric, aggregate and void material), the relative compositions of which were very similar in both extracts. Reversed-phase HPLC under acid dissociating conditions (pH 2) separated the extracts into four peaks (M 1, M 2, D and A). The first two components are both monomers: M 1 contains all those forms where no major conformaitonal change has occured; M 2 comprises forms with substantial conformational alteration ( e.g. disulphide bridge cleavage). Component D includes the interchain disulphide dimer, whilst A is an uncharacterized oligomeric form. Anion-exchange HPLC (pH 8) separated extract A ¯ into four regions of immunoreactivity. Region 1 contains hGH 20K separated from hGH 22K; region 2 includes other monomeric charge variants; region 3 is a broad peak which includes true dimers of hGH 20K and hGH 22K, as well as loosely aggregated monomer. Region 4 is another broad peak, presumably containilng a higher-molecular-weight hGH form or forms. All the hGH forms (identified and unidentified) can be separated by sequential use of two out of three chromatographic methods. No two forms found so far coeluted in all three systems.

A rapid method for the preparation of 1251-labelled human growth hormone for receptor studies, using reverse-phase high performance liquid chromatography

Human growth hormone was labelled with 125 Iodine by the stoichiometric modification of the chloramine-T method to a specific activity of 50-80 microCi/microgram, and the iodinated mixture was purified by reverse-phase high performance liquid chromatography using a C18 column (SynChropak RP-P) and a linear gradient. Compared with the usual Sephadex G-100 chromatography, HPLC gave a much better separation, with a higher yield and a considerably reduced analysis time (30 min vs 5 h). The [125I]-labelled preparation had normal binding to IM-9 lymphocyte receptors. The maximum bindability of the HPLC-purified preparation approximated 90%, which is the best value so far reported for human growth hormone. It is concluded that HPLC is a fast, convenient and reproducible method for obtaining an improved [125I]-labelled human growth hormone for receptor studies.

Detection of host-derived contaminants in products of recombinant DNA technology in E. coli: a comparison of silver-staining and immunoblotting

Contamination of medicines produced in E. coli by recombinant DNA methodology with host-cell proteins is considered a potential problem with this type of method. In this report techniques for the detection of trace quantities of host-cell proteins in SDS-gel electrophoretograms were examined. Detection of E. coli proteins by immunoblotting, using antisera raised in rabbits to lysates of E. coli, was compared with detection using the ultrasensitive silver stain. Silver staining detected a larger number of E. coli proteins in a one-dimensional electrophoresis system than did immunoblotting. Proteins that were markedly antigenic in the rabbit were detected at a greater sensitivity by the immunoblotting approach. Both techniques detected contaminant proteins in a preparation of methionyl human growth hormone produced in E. coli known to be contaminated with host-cell proteins. No contaminating proteins were seen by either technique in more rigorously purified preparations of growth hormone. A combination of these two approaches would provide useful evidence of purity of medicines produced by recombinant DNA technology, and is potentially applicable to a wide range of host-vector systems.

The International Standard for Human Growth Hormone for Bioassay: calibration and characterization by international collaborative study

Two preparations of human growth hormone (hGH) were prepared as candidates for the International Standard for Human Growth Hormone for Bioassay and were studied by 22 laboratories in 10 countries in an international collaborative study. The 2 candidate preparations, freeze-dried in ampoules coded 80 505 and 80 521 , were assayed against the International Standard for Growth Hormone, bovine, for Bioassay (IS bGH), by in vivo assays; against the International Reference Preparation of Growth Hormone, human, for Immunoassay (IRP hGH), by receptor-, immunoassays and other in vitro methods; and against each other by various methods. Both preparations contained the 2 recognized main growth hormone components (22 kDa and 20 kDa forms) and other components, but that in ampoules coded 80 505 had less deamidated hGH and contaminant hormones and pyrogen than that in ampoules coded 80 521 . The estimates by in vivo bioassays, using immature hypophysectomized rats, of the potency of 80 505 in terms of the IS bGH were heterogeneous (1–6 IU/ampoule), probably because of the dissimilarity of the preparations of bovine and human GH. Estimates with receptor- and immunoassays of 80 505 against the IRP hGH were also heterogeneous (3–9 IU/ampoule). Nevertheless, the majority of estimates from all assays tended to be between 3 and 6 IU/ampoule. Although 80 521 and 80 505 differed in relatively minor respects, assays of one directly against the other gave relative potency estimates with in vivo assays which were significantly different from estimates with in vitro methods. With the agreement of the participants in the study, the WHO Expert Committee on Biological Standardization established the preparation in ampoules coded 80 505 as the First International Standard for Human Growth Hormone for Bioassay, with the defined potency of 4.4 IU/ampoule. This corresponds to an approximate 2.5 IU/mg of hGH extract and maintains reasonable continuity of the unit.

Development and validation of a salmon prolactin radioimmunoassay

A highly specific radioimmunoassay (RIA) for the measurement of prolactin (PRL) in the plasma and pituitary of salmonid fishes was developed using a rabbit antiserum to chinook salmon ( Oncorhynchus tschawytscha) PRL. The PRLs purified from chinook salmon and chum salmon ( O. keta) pituitaries showed exactly the same competitive inhibition curves in the RIA, regardless of iodination of either hormone. The displacement curves for pituitary extracts and plasma from several salmonids, including chum, coho, and amago salmon, rainbow trout, and Japanese charr, were parallel to the salmon PRL standard, whereas those from the eel, goldfish, carp, and tilapia showed negligible cross-reactivity. Negligible cross-reactivity was also seen with plasma from hypophysectomized rainbow trout or coho salmon. None of the mammalian PRL or growth hormone (GH) preparations, bullfrog PRL, or presumptive chum salmon “gonadotropin” and eel “PRL” cross-reacted in the PRL RIA. Presumptive chum salmon GH showed less than 0.05% cross-reactivity. The RIA sensitivity was less than 0.1 ng of the salmon PRL standard per milliliter. The immunoreactive reactive plasma PRL levels in mature chum salmon were below 1 ng/ml in seawater. The plasma PRL in females increased to about 8 ng/ml 1 day after transfer to fresh water, and high levels (2–4 ng/ml) were maintained during 3–7 days after the transfer. In contrast, when males were transferred to fresh water, an increase in plasma PRL was seen only 1 day after the transfer. A significant decrease in plasma osmolality was observed in both males and females after transfer to fresh water. No change was observed either in plasma PRL or osmolality, when fish were transferred from seawater to seawater.

Highly purified human growth hormone fails to stimulate lipolysis in rabbit adipocytes in vitro or in rabbits in vivo

To determine whether the rapid lipolytic effect observed with human growth hormone (hGH) preparations in rabbits and rabbit adipose tissue is an intrinsic property of the hormone, we examined the lipolytic effects in vivo and in vitro of clinical grade preparations, hGH purified by DEAE cellulose chromatography, and hGH prepared by recombinant DNA techniques. Using isolated rabbit perirenal adipocytes, ACTH and clinical-grade hGH preparations both stimulated glycerol release to the same maximal rate with half-maximal hGH effects observed between 8 and 50 μg/mL. Purification of the most potent lipolytic preparation of clinical grade hGH by DEAE cellulose chromatography yielded a preparation (2 IU/mg of growth activity that retained insulinlike effects on rat fat pad [U- 14C] glucose metabolism) that, at concentrations up to 0.2 mg/mL, failed to stimulate lipolysis by adipocytes incubated for one or four hours in the presence or absence of dexamethasone or trypsin inhibitor or when preincubated for three hours prior to addition of hGH. Recombinant DNA-derived hGH did not stimulate glycerol release at 0.1 mg/mL. While antiserum to purified hGH blocked the increase in glucose oxidation in rat fat pads produced by clinical grade hGH, it did not inhibit its lipolytic effect using rabbit adipocytes. Purified hGH (0.1 mg/kg IV) was also unable to elicit a rise serum free fatty acid (FFA) levels of conscious rabbits while, at the same dose, clinical grade hGH increased FFA levels to 900 μEq/L over basal. Rapid lipolytic stimulation in rabbit adipocytes by hGH preparations could not be attributed to the 20,000 molecular weight variant of hGH (hGH20K) or the peptide corresponding to positions 32 to 46 of hGH (deletion peptide). Glycerol release was accelerated by a < 5000 molecular weight fraction obtained during the preparation of purified hGH. In conclusion, the lipolytic effect produced by clinical-grade hGH preparations in rabbits in vivo and in vitro could not be attributed to intact hGH, hGH20K, or deletion peptide, but is elicited by a contaminant separable from hGH on further purification.

Induction of sensitivity of fibroblast cultures to pituitary growth hormone by a thermostable serum factor

This paper presents data to show that highly purified pituitary growth hormone (GH) preparations, themselves unable to stimulate DNA biosynthesis in cultures of adult human skin fibroblasts, acquire this ability if the cells are treated simultaneously with a factor present in a thermostable and acid-resistant fraction of rat blood serum. Activity of this factor in rat blood serum has been shown to depend on the pituitary, and to increase after hypophysectomy. Human GH, while not exhibiting activity itself, if added to the medium simultaneously with serum fraction from hypophysectomized rats, stimulated DNA biosynthesis by fibroblasts significantly. The increase in tritium-thymidine incorporation under the influence of the hormone together with the serum fraction amounted to 233%. It is important to not that serum fraction of intact rats of the same age in a concentration of 1% was unable to induce sensitivity of the fibroblasts to human GH.

Reevaluation of lipolytic activity of growth hormone in rabbit adipocytes

The lipolytic activities of porcine pituitary fractions and purified growth hormone (GH) from human (h), porcine (p), ovine (o) and rabbit (Rb) origin as well as ovine placental lactogen (oPL), were compared to that of ACTH on rabbit adipocytes. All the GH preparations and oPL were equivalent in inhibiting the binding of labelled oGH to liver plasma membranes from pregnant rabbits. ACTH, and to a lesser extent porcine pituitary fractions and hGH, stimulated free fatty acid production by isolated adipocytes. The sensitivity of the adipocytes to these factors was increased when adenosine deaminase was added to the incubation medium. But, RbGH, pGH, oGH and oPL had no effect. We conclude that GH is not directly involved in the control of lipolysis in rabbit adipocytes and that the effect of hGH is rather due to a contamination of this preparation by other pituitary factors.

Diabetogenic activity of native and biosynthetic human growth hormone in obese (ob/ob) mouse.

It has been repeatedly suggested that diabetogenic activity is not an intrinsic property of native pituitary growth hormone (GH) and that the diabetogenic effects produced by GH preparations are due to low-molecular-weight contaminants or degradation products of the hormone. This possibility was evaluated in this study by assessing the ability of purified native human GH (hGH) and biosynthetic methionyl-hGH to exacerbate fasting hyperglycemia and glucose intolerance in the obese (ob/ob) mouse. Native hGH that had been purified by DEAE-cellulose chromatography (A-type; 1.8 IU/mg) produced fasting hyperglycemia and glucose intolerance in the ob/ob mouse when injected subcutaneously at doses of 50 micrograms/day or greater for 3 days. It had no effect when a single subcutaneous dose of 200 micrograms was administered 24 h previously. To eliminate possible contamination with smaller peptides, the hGH was gel-filtered on a column of Sephacryl S-200 in 6 M guanidine-HCl. When injected subcutaneously into ob/ob mice at a dose of 50 micrograms/day or greater for 3 days, the guanidine-treated hGH produced glucose intolerance. Also biosynthetic methionyl-hGH produced marked fasting hyperglycemia and glucose intolerance when injected subcutaneously at doses of 50 or 100 micrograms/day for 3 days. These results support the conclusion that hGH itself is indeed diabetogenic but that chronic exposure of the organism to the hormone is required for its effects on glucose metabolism to become clearly manifest.

Preparative electrophoresis for large scale preparation of highly purified bovine growth hormone

A method allowing large scale preparation of bovine growth hormone from pituitary glands in relatively few steps is described. In comparison to conventional purification techniques previously reported, the use of preparative polyacrylamide gel electrophoresis reduces the number of steps and the amount of time needed for the isolation of the hormone. In addition, the yield is several times greater, the hormone is purer and it has greater bioactivity (1.83 IU/mg).

Influences of Growth Hormone on Glucose Uptake by Avian Adipose Tissue

The uptake of glucose by chicken adipose tissue was determined in vitro using adipocytes or adipose explants using 14C orthomethyl glucose (OMG). The net uptake of 14C-OMG was enhanced by preparations of bovine and chicken GH and by insulin. Insulin and growth hormone (GH) (chicken or bovine) stimulated the uptake of 14C-OMG occurring in 1 hr by adipose tissue (both adipocytes and explants) from chicks hypophysectomized for 5 days but not from sham-operated birds. Tissue preincubated with GH became refractory to GH stimulation of 14C-OMG uptake. The short-term (2 to 15 min) uptake of 14C-OMG by explants was, however, stimulated by the bovine GH and insulin in tissue from sham-operated but not hypophysectomized chicks. Surprisingly, net decreases in 14C-OMG uptake, in the presence of bovine GH, were observed with adipose tissue (adipocytes and explants) from hypophysectomized birds, when incubated in the presence of epinephrine, and from hypophysectomized birds immediately following surgery.

Growth hormone acts at a pretranslational level in hepatocyte cultures

We have examined the effects of ovine growth hormone and recombinant DNA synthesized human growth hormone on hepatocytes maintained in serum free cultures. Both growth hormone preparations augmented or attenuated 3 specific mRNA sequences as revealed by two-dimensional gel electrophoresis of [35S] methionine labeled products synthesized in vitro in an mRNA dependent rabbit reticulocyte lysate system. The results clearly indicate that growth hormone, free of potential pituitary contaminants, acts directly on hepatocytes at a pretranslational level.

Development and validation of a carp growth hormone radioimmunoassay

Purified growth hormone (GH), isolated from pituitary glands of the common carp ( Cyprinus carpio), was shown to have biochemical and immunological properties in common with other teleost GH preparations. Intraperitoneal injections of the carp GH, at a dose of 1 μg/g body wt, resulted in significant increases in body weight in goldfish. In addition, the carp GH was used to prepare an antiserum for the development of a radioimmunoassay (RIA). The displacement curves for serum from goldfish with an intact pituitary gland were parallel to that of the purified cGH in this RIA. In contrasts serial dilutions of serum from hypophysectomized goldfish, and purified goldfish prolactin and carp gonadotropin did not have significant cross reaction. The present study strongly suggests that this RIA is suitable for the measurement of circulating GH levels in the goldfish.

Absence of lipolytic activity from purified human growth hormone in cultured 3T3-L1 adipocytes.

Although standard preparations of growth hormone activated hormone-sensitive lipase in differentiated 3T3-L1 adipocytes, more highly purified preparations of human and bovine hormones were not lipolytic in this system. We found that the standard preparations were contaminated with a lipolytic substance which was removed during purification of the growth hormone. The purified growth hormone likewise did not stimulate lipolysis in adipose tissue from fasted rats. Dexamethasone had no potentiating activity in either assay.

Fragments of human growth hormone produced by digestion with bromelain: Chemistry and biological properties

In an effort to produce small discrete fragments of human growth hormone (GH), we examine the action of the proteolytic enzyme, bromelain, on this molecule. Purified human GH incubated for 40 min at 22°C with crude bromelain and gel-filtered on Sephadex G-100 resulted in a major digestion product, peak 2. SDS-urea gel electrophoresis in the presence of β-mercaptoethanol suggested that peak 2 was composed of two polypeptide chains. Two polypeptide fractions were isolated by the reduction and S-alkylation of peak 2 in 6 M guanidine-HCl and subsequent chromatography on Sephacryl S-200 in 6 M guanidine-HCl. These two fractions, A and B, had the same mobilities as the two components of peak 2 on SDS-urea gels. Amino-terminal analysis, tryptic peptide mapping, carboxypeptidase digestion, cyanogen bromide cleavage, and amino acid analysis of fraction A indicated that it was peptide 1–135. Amino-terminal analysis and tryptic peptide mapping of fraction B suggested the presence of a mixture of peptides 143–191, 145–191 and 146–191. Thus, peak 2 is heterogeneous and appears to be a mixture consisting of peptide 1–135 + peptide 143–191, peptide 1–135 + peptide 145–191 and peptide 1–135 + peptide 146–191, in each case the N-terminal peptide being joined to the C-terminal peptide by the disulfide bridge between residues 53 and 165. In the weight-gain test in hypophysectomized rats, two preparations of peak 2 appeared to be somewhat less active than the native human GH preparations from which they were derived. Several preparations of peak 2 showed equivalent potency in stimulating [ 14C]glucose oxidation to 14CO 2 by isolated epididymal adipose tissue of hypophysectomized rats. Also, most of the peak 2 preparations were somewhat less active than native human GH in displacing 125I-labeled human GH bound to antibodies to human GH.