Native Growth Hormone Research Articles

THU048 Determination Of Receptor Binding Affinity Of Growth Hormone Analogues To The Growth Hormone Receptor In A Drug Screening Context

Abstract Disclosure: S. Erlendsson: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. A. Kulakova: Employee; Self; Novo Nordisk. S. Poulsen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. P. Thygesen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. Growth hormone (GH) and GH analogues are characterized by their ability to bind and activate the GH receptor (GHR). Binding affinity and functional activity are pivotal parameters for development and characterization of new GH analogues for treatment of growth disorders. The GHR is a member of the class I cytokine family and obtains a tripartite structure consisting of two extracellular fibronectin type III domains (ECD), a single-pass transmembrane domain (TMD), and a disordered intracellular domain (ICD). These receptors lack intrinsic kinase activity but rely on recruitment of intracellular signalling kinases and regulatory proteins. Activation of the downstream JAK-STAT signalling cascade is initiated by formation of an asymmetric GHR dimer induced by binding of GH to the GHR ECDs via two partially overlapping binding sites designated site I and II. The structural rearrangement in the ECDs propagates through the TMD and results in a separation of the ICDs which leads to activation through cross-phosphorylation of the Janus kinases 2 (JAK2). Here we utilize isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and small angle x-ray scattering (SAXS) to characterize the binding of native GH and GH analogues including somapacitan and pegvisomant to the monomeric GHR as well as an artificial soluble stabilized dimeric GHR. The GH analogues with good binding properties were subsequently tested in vitro for biological activity using a STAT3-luciferase reporter gene assay (RGA) and a proliferation assay with BAF3 cells expressing the human GHR. Both assays were established in two formats enabling testing of both receptor agonists and antagonists. Using SPR we show that the binding of all tested compounds is preserved between the monomeric and dimeric GHR with affinities ranging from near-picomolar to lower micromolar and we are subsequently able to confirm the expected stoichiometry by utilizing a combination of ITC and SAXS. In the RGA and the proliferation assay we demonstrate that a 1:2 stoichiometry is critical for receptor activation regardless of the affinity and therefore we propose high throughput screening of receptor stoichiometry as key in discovery of new growth disorder therapeutics. Presentation: Thursday, June 15, 2023

High level expression and purification of recombinant flounder growth hormone in E. coli.

Recombinant flounder growth hormone was overproduced in E. coli by using codon optimized synthetic gene and optimized expression conditions for high level production. The gene was cloned into PET-28a expression vector and transformed into E. coli BL21 (DE3). Induction at lower temperature, lower IPTG concentrations and richer growth media during expression resulted in increased expression level. The protein expression profile was analyzed by SDS-PAGE, the authenticity was confirmed by western blotting and the concentration was determined by Bradford assay. In addition, several attempts were made to produce soluble product and all resulted in insoluble product. The overexpressed protein was efficiently purified from inclusion bodies by moderate speed centrifugation after cell lysis. Among the solubilization buffers examined, buffer with 1% N-lauroylsarcosine in the presence of reducing agent DTT at alkaline pH resulted in efficient solubilization and recovery. The denaturant was removed by filtration and dialysis. The amount of the growth hormone recovered was significantly higher than previous reports that expressed native growth hormone genes in E. coli. The methodology adapted in this study, can be used to produce flounder growth hormone at large scale level so that it can be used in aquaculture. This approach may also apply to other proteins if high level expression and efficient purification is sought in E. coli.

OR07-2 Simultaneous stimulation of human 20 kDa growth hormone and 22 kDa growth hormone secretion by ghrelin

The World Anti Doping Agency has banned the use of recombinant human growth hormone (rhGH). Regardless, rogue athletes may use rhGH in pursuit of improved performance. Current immunoassaybased detection of illicit use of rhGH is complicated by the short half-life of injected rhGH, by the chemical identity of rhGH and a native growth hormone (GH), and by the physiologically high peaks of GH that occur with the pulsatile secretion of GH by a normal pituitary. We hypothesized that injection of rhGH changes the expression patterns of GHmessenger RNA (mRNA) in circulating white blood cells and of microRNA (miRNA) in plasma. Objective: To measure circulating GH mRNA and miRNAs in humans. Methods: We used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) with nested primers to detect and quantify GHmRNA.We usedmiRNAmicroarrays to identify rhGH-dependent miRNA expression and then measured the concentrations of selected miRNAs by RT-qPCR. Following Institutional Review Board approval, we obtained whole-blood or plasma, from 9 healthy volunteers and from 36 patient volunteers. All patients had known or suspected disorders of growth hormone production including panhypopituitarism treated with physiologic doses of rhGH (0.2 to 0.8mg/day) and acromegaly. Results: GH mRNA was quantified in blood from all healthy volunteers and patients with disorders of GH at concentrations ranging from 36 to 4815 copies/100 mL blood. The intra-individual GH mRNA concentration varied less than 3-fold in 17 samples collected over the period of one year in a healthy, athletic volunteer; however, variability increased markedly in the group with pituitary disorders. Initially, 9 specimens from: 3 patients receiving therapeutic growth hormone replacement, 4 patients with acromegaly, and 2 normal controls, were subject to plasma-derived miRNA microarrays. Of 1105 miRNA’s measured in each microarray, 3 were present in plasma of all rhGH replacement users and undetectable in all patients with acromegaly and normal controls. Conclusions: The narrow intra-individual range of its concentration suggests that GH mRNA in blood is not present as a result of pulsatile secretion and thus is worthy of study as a marker of doping with supraphysiologic amounts of rhGH. Further investigation of miRNA in plasma should be performed on samples obtained before and after administration of supraphysiologic amounts of rhGH to confirm the preliminary results in patients receiving therapeutic replacement doses of rhGH.

OR07-1 Circulating RNAs as markers of growth hormone doping

The World Anti Doping Agency has banned the use of recombinant human growth hormone (rhGH). Regardless, rogue athletes may use rhGH in pursuit of improved performance. Current immunoassaybased detection of illicit use of rhGH is complicated by the short half-life of injected rhGH, by the chemical identity of rhGH and a native growth hormone (GH), and by the physiologically high peaks of GH that occur with the pulsatile secretion of GH by a normal pituitary. We hypothesized that injection of rhGH changes the expression patterns of GHmessenger RNA (mRNA) in circulating white blood cells and of microRNA (miRNA) in plasma. Objective: To measure circulating GH mRNA and miRNAs in humans. Methods: We used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) with nested primers to detect and quantify GHmRNA.We usedmiRNAmicroarrays to identify rhGH-dependent miRNA expression and then measured the concentrations of selected miRNAs by RT-qPCR. Following Institutional Review Board approval, we obtained whole-blood or plasma, from 9 healthy volunteers and from 36 patient volunteers. All patients had known or suspected disorders of growth hormone production including panhypopituitarism treated with physiologic doses of rhGH (0.2 to 0.8mg/day) and acromegaly. Results: GH mRNA was quantified in blood from all healthy volunteers and patients with disorders of GH at concentrations ranging from 36 to 4815 copies/100 mL blood. The intra-individual GH mRNA concentration varied less than 3-fold in 17 samples collected over the period of one year in a healthy, athletic volunteer; however, variability increased markedly in the group with pituitary disorders. Initially, 9 specimens from: 3 patients receiving therapeutic growth hormone replacement, 4 patients with acromegaly, and 2 normal controls, were subject to plasma-derived miRNA microarrays. Of 1105 miRNA’s measured in each microarray, 3 were present in plasma of all rhGH replacement users and undetectable in all patients with acromegaly and normal controls. Conclusions: The narrow intra-individual range of its concentration suggests that GH mRNA in blood is not present as a result of pulsatile secretion and thus is worthy of study as a marker of doping with supraphysiologic amounts of rhGH. Further investigation of miRNA in plasma should be performed on samples obtained before and after administration of supraphysiologic amounts of rhGH to confirm the preliminary results in patients receiving therapeutic replacement doses of rhGH.

Immunogenicity, toxicology, pharmacokinetics and pharmacodynamics of growth hormone ligand–receptor fusions

A fundamental concern for all new biological therapeutics is the possibility of inducing an immune response. We have recently demonstrated that an LR-fusion (ligand-receptor fusion) of growth hormone generates a potent long-acting agonist; however, the immunogenicity and toxicity of these molecules have not been tested. To address these issues, we have designed molecules with low potential as immunogens and undertaken immunogenicity and toxicology studies in Macaca fascicularis and pharmacokinetic and pharmacodynamic studies in rats. Two variants of the LR-fusion, one with a flexible linker (GH-LRv2) and the other without (GH-LRv3), were tested. Comparison was made with native human GH (growth hormone). GH-LRv2 and GH-LRv3 demonstrated similar pharmacokinetics in rats, showing reduced clearance compared with native GH and potent agonist activity with respect to body weight gain in a hypophysectomized rat model. In M. fascicularis, a low level of antibodies to GH-LRv2 was found in one sample, but there was no other evidence of any immunogenic response to the other fusion protein. There were no toxic effects and specifically no changes in histology at injection sites after two repeated administrations. The pharmacokinetic profiles in monkeys confirmed long half-lives for both GH-LRv2 and GH-LRv3 representing exceptionally delayed clearance over rhGH (recombinant human GH). The results suggest that repeated administration of a GH LR-fusion is safe, non-toxic, and the pharmacokinetic profile suggests that two to three weekly administrations is a potential therapeutic regimen for humans.

Growth enhanced brown trout show increased movement activity in the wild

Summary 1Rapid growth is often associated with several fitness-related benefits. However, in most organisms growth rates are rarely maximized, suggesting that trade-offs limit the benefits of rapid growth. To enable sustained high growth, behavioural modifications incurring costs may be required, and these may be responsible for evolution of sub-maximal growth rates. 2In a field experiment it was tested whether rapid growth in brown trout is achieved by increasing potentially costly movement activity. Causal relations were obtained by manipulating the growth rate using bovine growth hormone (GH) implants and monitoring movement activity of stream-dwelling brown trout for two months using radiotelemetry. 3GH-treated trout grew significantly faster than sham-treated trout both in terms of body mass and length. The GH-treated trout also had a significantly larger absolute movement during the experiment, moving about 68% more than the sham-treated trout. There were no GH effects on diel movement or home range between the two treatments. Plasma GH levels were lower in the GH-treated fish, indicating negative feedback inhibition of native GH secretion, and that the bovine GH implant was still functioning at the end of the study. 4This is the first study to show increased movement activity in GH-enhanced fish in the wild. Because increased movement is expected to lead to increased metabolic costs, the higher movement activity in the GH-treated trout was likely associated with increased foraging activity. The results demonstrate that increased movement activity is associated with rapid growth in brown trout. Such movement activity may be unfavourable under certain ecological conditions or life stages (i.e. high risk of predation or low resource levels), and may thus represent one component of the cost limiting evolution of maximum growth rates.

Growth enhanced brown trout show increased movement activity in the wild

1 Rapid growth is often associated with several fitness-related benefits. However, in most organisms growth rates are rarely maximized, suggesting that trade-offs limit the benefits of rapid growth. To enable sustained high growth, behavioural modifications incurring costs may be required, and these may be responsible for evolution of sub-maximal growth rates. 2 In a field experiment it was tested whether rapid growth in brown trout is achieved by increasing potentially costly movement activity. Causal relations were obtained by manipulating the growth rate using bovine growth hormone (GH) implants and monitoring movement activity of stream-dwelling brown trout for two months using radiotelemetry. 3 GH-treated trout grew significantly faster than sham-treated trout both in terms of body mass and length. The GH-treated trout also had a significantly larger absolute movement during the experiment, moving about 68% more than the sham-treated trout. There were no GH effects on diel movement or home range between the two treatments. Plasma GH levels were lower in the GH-treated fish, indicating negative feedback inhibition of native GH secretion, and that the bovine GH implant was still functioning at the end of the study. 4 This is the first study to show increased movement activity in GH-enhanced fish in the wild. Because increased movement is expected to lead to increased metabolic costs, the higher movement activity in the GH-treated trout was likely associated with increased foraging activity. The results demonstrate that increased movement activity is associated with rapid growth in brown trout. Such movement activity may be unfavourable under certain ecological conditions or life stages (i.e. high risk of predation or low resource levels), and may thus represent one component of the cost limiting evolution of maximum growth rates.

A ligand-receptor fusion of growth hormone forms a dimer and is a potent long-acting agonist

Cytokine hormones have a short plasma half-life and require frequent administration. For example, growth hormone replacement involves daily injections. In common with other cytokines, the extracellular domain of the growth hormone receptor circulates as a binding protein, which naturally prolongs the biological half-life of growth hormone. Here we have studied the biological actions of a ligand-receptor fusion of growth hormone and the extracellular domain of its receptor. The genetically engineered ligand-receptor fusion protein was purified from mammalian cell culture. In rats, the ligand-receptor fusion had a 300-times reduced clearance as compared to native growth hormone, and a single injection promoted growth for 10 d, far exceeding the growth seen after administration of native growth hormone. The ligand-receptor fusion forms a reciprocal, head-to-tail dimer that provides a reservoir of inactive hormone similar to the natural reservoir of growth hormone and its binding protein. In conclusion, a ligand-receptor fusion of cytokine to its extracellular receptor generates a potent, long-acting agonist with exceptionally slow absorption and elimination. This approach could be easily applied to other cytokines.

Production and characterization of recombinantly derived peptides and antibodies for accurate determinations of somatolactin, growth hormone and insulin-like growth factor-I in European sea bass ( Dicentrarchus labrax)

A specific radioimmunoassay (RIA) for European sea bass ( Dicentrarchus labrax) growth hormone (GH) was developed and validated. For this purpose, a stable source of GH was produced by means of recombinant DNA technology in a bacteria system. The identity of the purified protein (ion exchange chromatography) was demonstrated by Western blot and a specific GH antiserum was raised in rabbit. In Western blot and RIA system, this antiserum recognized specifically native and recombinant GH, and it did not cross-react with fish prolactin (PRL) and somatolactin (SL). In a similar way, a specific polyclonal antiserum against the now available recombinant European sea bass SL was raised and used in the RIA system to a sensitivity of 0.3 ng/ml (90% of binding of tracer). Further, European sea bass insulin-like growth factor-I (IGF-I) was cloned and sequenced, and its high degree of identity with IGF-I peptides of barramundi, tuna, and sparid fish allowed the use of a commercial IGF-I RIA based on barramundi IGF-I antiserum. These assay tools assisted for the first time accurate determinations of SL and GH–IGF–I axis activity in a fish species of the Moronidae family. Data values were compared to those found with gilthead sea bream ( Sparus aurata), which is currently used as a Mediterranean fish model for growth endocrinology studies. As a characteristic feature, the average concentration year round of circulating GH in growing mature males of European sea bass was higher than in gilthead sea bream. By contrast, the average concentration of circulating SL was lower. Concerning to circulating concentration of IGF-I, the measured plasma values for a given growth rate were also lower in European sea bass. These findings are discussed on the basis of a different energy status that might allowed a reduced but more continuous growth in European sea bass.

Cis and trans acting factors in the regulation of parathyroid hormone (PTH) mRNA stability by calcium and phosphate

Calcium and phosphate regulate parathyroid hormone (PTH) mRNA stability through differences in binding of parathyroid proteins to an element in its 3′-untranslated region. One of the proteins is AUF1 (A+U-rich element binding factor 1). An in vitro degradation assay showed that transcripts for PTH and chimeric growth hormone (GH)–PTH 63 nt, but not for native GH, were stabilized by PT proteins from rats on low calcium diets and destabilized by proteins from rats on low phosphate diets, correlating with PTH mRNA levels in vivo. In transfection experiments the 63 nt binding element destabilized mRNAs of reporter genes and this was prevented by over-expression of AUF1. Our results identified a functional cis element in PTH mRNA. Differences in protein binding to this element determine PTH mRNA stability and its regulation by calcium and phosphate.

An insight into the possible mechanism of working of two-cistronic gene expression systems and rational designing of newer systems.

The initial attempts at hyper-expressing buffalo/goat growth hormone (GH)-ORFs in Escherichia coli directly under various strong promoters were not successful despite the presence of a functional gene. High level expression of GH was achieved as a fusion protein with glutathione-S-transferase (GST). To produce native GH in an unfused state, we adapted an established strategy of two-cistronic approach in our system. In this strategy, utilizing one of the highly efficient reported sequences as the first cistron led to a nearly 1000-fold enhancement in the level of expression under an E. coli promoter (trc). In search of a newer first-cistron sequence as well as to see the generality of the two-cistronic approach, we explored the ability of different lengths of a highly expressing natural gene to act as an efficient first cistron. Surprisingly, GST, which is naturally highly expressible in E. coli, could not be fitted into a successful two-cistronic construct. In addition, placement of the entire two-cistronic expression cassette (which had earlier given high-level GH expression under trc promoter) under the T7 promoter in E. coli failed to hyper-express GH. These results suggest that the successful exploitation of the two-cistron arrangement for hyper-expression of eukaryotic ORFs in bacteria is not as straightforward as was previously thought. It appears probable that factors such as the sequence context, together with the length and codons used in the first cistron are important as well.

A Conserved cis-Acting Element in the Parathyroid Hormone 3′-Untranslated Region Is Sufficient for Regulation of RNA Stability by Calcium and Phosphate

Calcium and phosphate regulate parathyroid hormone (PTH) gene expression post-transcriptionally by changes in protein-PTH mRNA 3'-untranslated region (UTR) interactions, which determine PTH mRNA stability. We have identified the protein binding sequence in the PTH mRNA 3'-UTR and determined its functionality. The protein-binding element was identified by binding, competition, and antisense oligonucleotide interference. The sequence was preserved among species suggesting its importance. To study its functionality in the context of another RNA, a 63-base pair cDNA PTH sequence was fused to the growth hormone (GH) gene. There is no parathyroid (PT) cell line and therefore an in vitro degradation assay was used to determine the stability of transcripts for PTH, GH, and a chimeric GH-PTH 63 nucleotides with PT cytosolic proteins. The full-length PTH transcript was stabilized by PT proteins from rats fed a low calcium diet and destabilized by proteins from rats fed a low phosphate diet, correlating with PTH mRNA levels in vivo. These PT proteins did not affect the native GH transcript. However, the chimeric GH transcript was stabilized by low calcium PT proteins and destabilized by low phosphate PT proteins, similar to the PTH full-length transcript. Therefore, we have identified a PTH RNA-protein binding region and shown that it is sufficient to confer responsiveness to calcium and phosphate in a reporter gene. This defined element in the PTH mRNA 3'-UTR is necessary and sufficient for the regulation of PTH mRNA stability by calcium and phosphate.

Antibodies for Growth Hormone and Prolactin Using Multiple Antigen Peptide Immunogens

: Antibodies elicited by novel synthetic peptide antigens derived from a highly conserved domain of the growth hormone (GH) and prolactin (PRL) of vertebrates were developed using the multiple antigen peptide approach. The sequence of the antigens is located near the carboxy-terminus in the D domain of the GH and PRL in a cluster of 11 and 10 conserved amino acids, respectively, within a sequence of 18 residues. The synthetic peptides were manually synthesized, purified by high-performance liquid chromatography, and the corresponding antibodies, elicited in rabbits, were cross-reacted with the GH and PRL of a variety of mammalian (human, bovine, ovine, pig, and equine) and nonmammalian (chicken, coho salmon, chum salmon, rainbow trout, catfish and striped bass) vertebrates. The cross-reactivity between the immunogen and its corresponding antigen was tested by immunobloting using either GH or PRL. The GH and PRL of the organisms tested cross-reacted specifically with the corresponding antibody. Chicken and fish GH and PRL showed stronger antibody cross-reactivity than that observed in mammalian sources. These results demonstrate the utility of peptide-derived polyclonal antibodies in the detection of native and recombinant GH and PRL of a variety of vertebrates.

Studies with a Growth Hormone Antagonist and Dual-fluorescent Confocal Microscopy Demonstrate that the Full-length Human Growth Hormone Receptor, but Not the Truncated Isoform, Is Very Rapidly Internalized Independent of Jak2-Stat5 Signaling

We have investigated trafficking of two negative regulators of growth hormone receptor (GHR) signaling: a human, truncated receptor, GHR1-279, and a GH antagonist, B2036. Fluorescent-labeled growth hormone (GH) was rapidly internalized by the full-length GHR, with >80% of the hormone internalized within 5 min of exposure to GH. In contrast, <5% of labeled GH was internalized by cells expressing truncated GHR1-279. Using another truncated receptor, GHR1-317 fused to enhanced green fluorescent protein (EGFP), we have exploited fluorescence energy transfer to monitor the trafficking of ligand-receptor complexes. The data confirmed that internalization of this truncated receptor is very inefficient. It was possible to visualize the truncated GHR1-317-EGFP packaged in the endoplasmic reticulum, its rapid movement in membrane bound vesicles to the Golgi apparatus, and subsequent transport to the cell membrane. The GH antagonist, B2036, blocked Jak2-Stat5-mediated GHR signaling but was internalized with a similar time course to native GH. 1) demonstrate the rapid internalization of GH when studied under physiological conditions; 2) confirm the hypothesis that internalization of cytoplasmic domain truncated human GHRs is very inefficient, which explains their dominant negative action; and 3) show that the antagonist action of B2036 is independent of receptor internalization.

Growth hormone (GH) receptors, binding proteins and IGF-I concentrations in the serum of transgenic mice expressing bovine GH agonist or antagonist.

We have examined the regulation of hepatic growth hormone receptors (GH-R) and serum GH binding proteins (GHBP) in transgenic mice expressing an antagonist of bovine growth hormone (bGH), G119K-bGH, and consequently exhibiting a growth suppressed dwarf phenotype. Specific GHBP could be measured in transgenic dwarf mouse serum only by immunological methods (RIA), because these mice have a very high concentration of mutated bGH in circulation (> 1 microgram/ml) and, therefore, almost all GHBP is bound to G119K-bGH and cannot be quantitated in binding assays. The concentrations of GHBP were 0.6 +/- 0.4 nM and 1.7 +/- 0.4 nM for normal and dwarf mice respectively. The concentrations of free GHBP in normal mice and in transgenic mice expressing wild-type GH can be calculated using chromatographic techniques as the dissociation constant (Kd) and the ratio of bound 125I-GH to free 125I-GH in the serum ([GHBP]free = B/F.Kd). In agreement with the assumption that GHBP reflects GH-R status, liver uptake of injected labeled bGH was greatly reduced in transgenic dwarfs in comparison with normal mice or with transgenic mice expressing wild-type bGH (liver/blood ratio of 0.48 +/- 0.21, 2.7 +/- 0.2, and 1.3 +/- 0.3 respectively) indicating that the high concentration of the mutated bGH (G119K-bGH) prevents labeled bGH uptake, as was expected from the dwarf phenotype. 125I-bGH taken up by the liver of transgenic dwarf mice was found in a smaller molecular species than in normal mice, compatible with the presence of 1:1 [(GH-R):GH] complexes instead of the 2:1 [(GH-R)2:GH] or 2:2 [(GHBP)2:(GH)2] complexes found in normal mice. The concentration of IGF-I, the principal mediator of GH activity, in the G119K-bGH transgenic mice was correlated with the concentration of free GHBP. This allowed us to use free GHBP concentration as a marker of the effects of the active endogenous hormone (mGH) on liver receptors in the presence of different concentrations of the antagonist of GH. The levels of GHBP in serum, as well as the concentration of GH-R in liver microsomes from mice expressing the bGH antagonist, are up-regulated by the high concentration of G119K-bGH (85%), but significantly less so than that which could be expected for the same concentration of native GH (220-275%). This up-regulation suggests that the G119K-bGH antagonist is internalized and induces synthesis of the receptor and of the binding protein.

Optimization of the solubilization and renaturation of fish growth hormone produced by Escherichia coli.

Growth hormone (GH) enhances the growth rate of aquacultured fish and shellfish, but it is difficult to extract native GH from fish pituitary glands. However, fish recombinant GH (rGH) can be efficiently synthesized by Escherichia coli cells, although it exists in denatured form in inclusion bodies (IB). We studied the solubilization of IB and the renaturation of rGH to help facilitate the production of a large amount of biologically active rGH. A 100-ml sample of rGH-producing E. coli produced 73.43 +/- 5.47 mg IB (dry weight, n = 3) after 20 h induction by 1 mM isopropyl beta-o-thiogalactopyranoside. Interestingly, if the bacteria were induced by 0.1 mM beta-lactose, 95.3 +/- 3.43 mg of IB was obtained. The optimal conditions for denaturation and renaturation of rGH were when IB were solubilized in 6 M guanidine hydrochloride and then dialysed against pH 10 dialysis buffer (50 mM ammonium bicarbonate and 2 mM EDTA) containing 100 mM L-arginine, 2 mM oxidized glutathione and 2 mM reduced glutathione for 24 h at 4 degrees C in a volume ratio of 3 to 500. At least 20% of the denaturated rGH in IB was renatured. Juvenile black sea bream injected with 0.05 microgram/g resultant rGH once every 2 weeks exhibited significant increases (P < 0.05) in weight gain (84%) relative to fish in the control group over a 16-week period. This process is an economical and effective way to obtain an active form of rGH biosynthesized by a prokaryotic system.

Angiogenic activity of anterior pituitary tissue and growth hormone on the chick embryo chorio-allantoic membrane : A novel action of GH

A useful system to evaluate the angiogenic activity of hormones and growth factors is the chorioallantoic membrane (CAM) of chick embryos. The present studies examined the angiogenic activity of chicken anterior pituitary glands and both fibroblast growth factor (FGF) and growth hormone (GH). Grafts of anterior pituitary gland evoked an angiogenic response on the CAM which was lost if the adenohypophyseal tissue was first boiled. The magnitude of the angiogenic response to anterior pituitary glands increased with the age of the donor (from a minimum 15 days of embryonic development to a maximum between 2 and 6 weeks old). In view of the similarity of the profile of the angiogenic response and the reported changes in GH secretion, the angiogenic activity of GH was then examined. Considerable angiogenic responses were observed with GH; there being increases (P < 0.05) in number of new blood vessels on the CAM of chick embryos on which native chicken GH or native bovine GH or recombinant bovine GH were added. These data support GH having an angiogenic action.

Secretory Synthesis of Active Recombinant Fish Growth Hormone by Insect Cells Using a Baculovirus Vector

A growth hormone cDNA of yellowfin porgy (Acanthopagrus latus) was inserted into baculovirus vectors, pAcT7, under the control of the polyhedrin promoter. A 22-kilodalton (kDa) protein, similar to that of native growth hormone, was detected in the supernatant of insect cells infected with the recombinant virus at the level of 2–8% of total proteins. This protein was immunoprecipitated with rabbit polyclonal antibodies raised against native chum salmon (Oncorhynchus keta) growth hormone. Furthermore, a radiolabeled protein band of 22 kDa was also seen in the recombinant-virus-infected cell culture supernatant after labeling with [35S]methionine. Injection of individual tilapia with 0.1 μg of crude supernatant protein containing recombinant growth hormone once a week resulted in significant increases (p &lt; 0.01) in weight (54%), weight gain (260%), length (21%), and feed efficiency rate (210%) relative to a similarly treated control group and an untreated control group at week 5 onwards.

Interaction of Thyroid Hormone and Retinoic Acid Receptors on the Regulation of the Rat Growth Hormone Gene Promoter

Retinoic acid transcriptionally regulate growth hormone (GH) gene expression through sequences located in the 5′ -flanking region of the gene. A partial induction by retinoic acid was obtained with the −181 bp of the rat GH promoter, and sequences up to −209 were required for a full response. These sequences contain the previously identified thyroid hormone responsive element. The retinoid X receptor RXR increased transactivation by T3 and RA. The retinoid was relatively more effective in stimulating the native GH promoter than an heterologous promoter which contains the response element, thus showing the importance of the promoter context on transactivation by the nuclear receptors.

Chronic fasting reduces the response of the thyroid to growth hormone and TSH, and alters the growth hormone-related changes in hepatic 5′-monodeiodinase activity in rainbow trout, Oncorhynchus mykiss

Chronically fasted rainbow trout ( Oncorhynchus mykiss) had significantly lower plasma l-thyroxine (T 4) and triiodo- l-thyronine (T 3), and higher plasma growth hormone (GH) concentrations than fed animals. Fasted and fed trout were administered bovine thyrotropic hormone (bTSH), native ovine GH (oGH), or recombinant human GH (rhGH) alone, or GH in combination with bTSH to further study the effects of food deprivation on the activity of the pituitary-thyroid axis and on the control of hepatic T 3 production. Although the fasted rainbow trout retained the ability to respond to bTSH challenge, the resultant elevation in plasma T 4 concentration was significantly lower than that of fed animals; there was no plasma T 3 response to bTSH challenge in either fed or fasted trout, except for a significant elevation in fed bTSH-injected fish and a significant depression in fed saline-injected fish sampled 2.5 hr after the injection. GH when administered alone had no significant effect on plasma T 4 concentrations of either fed or fasted animals, and stimulated an increase in plasma T 3 concentration and an increased hepatic T 3 content only in the fed fish, despite a significant stimulation by both oGH and rhGH of in vitro hepatic 5′-monodeiodinase activity (MDA) in both fed and fasted groups. bTSH appeared to suppress rhGH- and oGH-stimulated MDA in fasted groups, and rhGH-stimulated MDA in fed trout. The data suggest that chronic fasting induced a down-regulation of the response of thyroid tissue to bTSH challenge, and of the GH-stimulation of T 3 production, in vivo, although in vitro hepatic MDA was elevated following GH administration to both fed and fasted rainbow trout.