Activity Of Human Growth Hormone Research Articles

The Preservation of Lyophilized Human Growth Hormone Activity: how Do Buffers and Sugars Interact?

One of the most common classes of excipients used in protein formulations are buffers. The aim of this work is to investigate the effect of buffers on protein stabilization given by sugars during freeze drying. Molecular Dynamics simulations of human growth hormone (hGH) in the presence of sucrose and trehalose were performed, and the impact of phosphate and citrate buffers on their protective action was analyzed. We found that buffers broke the hydrogen bonding network formed by excipients, and the consequences of this disruption of structure ordering were different in sucrose-based or trehalose-based formulations. More specifically, we observed that buffers increased protein recovery in the presence of excipients, such as sucrose, that exert their action mainly by preferential exclusion. By contrast, the opposite effect was sometimes noted in the case of excipients, such as trehalose, whose protective action is related to the formation of a highly structured matrix. We found that buffers have important properties, other than the control of pH, that can contribute to the overall stability of proteins. Some of these properties are related to their interaction with the other components of the formulation.

Intergenic GWAS SNPs are key components of the spatial and regulatory network for human growth.

Meta-analysis of genome-wide association studies has resulted in the identification of hundreds of genetic variants associated with growth and stature. Determining how these genetic variants influence growth is important, but most are non-coding, and there is little understanding of how these variants contribute to adult height. To determine the mechanisms by which human variation contributes to growth, we combined spatial genomic connectivity (high-throughput conformation capture) with functional (gene expression, expression Quantitative Trait Loci) data to determine how non-genic loci associated with infant length, pubertal and adult height and contribute to gene regulatory networks. This approach identified intergenic single-nucleotide polymorphisms (SNPs) ∼85 kb upstream of FBXW11 that spatially connect with distant loci. These regulatory connections are reinforced by evidence of SNP-enhancer effects and altered expression in genes influencing the action of human growth hormone. Functional assays provided evidence for enhancer activity of the intergenic region near FBXW11 that harbors SNP rs12153391, which is associated with an expression Quantitative Trait Loci. Our results suggest that variants in this locus have genome-wide effects as key modifiers of growth (both overgrowth and short stature) acting through a regulatory network. We believe that the genes and pathways connected with this regulatory network are potential targets that could be investigated for diagnostic, prenatal and carrier testing for growth disorders. Finally, the regulatory networks we generated illustrate the power of using existing datasets to interrogate the contribution of intergenic SNPs to common syndromes/diseases.

Synthetic growth hormone releasers detected in seized drugs: new trends in the use of drugs for performance enhancement

Gone are the days when gym users in search of drugs to boost performance and muscular appearance resorted to drug regimes of just oral and injectable versions of anabolic steroids. Today, information gleaned from seizures made by law enforcement agencies, personal anecdotes and hearsay shared both in gyms and on the internet, drug analysis reports, articles in newspapers and muscle genre magazines and observation of illicit suppliers' websites reveals that a host of synthetic peptide hormones are now available on the ‘life-style’ drug market 1. These include growth hormone releasers such as CJC-1295, GHRP-2 and GHRP-6 2-7. These drugs have similar effects on the body, mainly triggering the pulsatile releasing activity of human growth hormone (hGH) from the anterior pituitary gland 8. This hormone, in turn, is involved in the regulation of body composition, due to its anabolic and lipolytic actions, as well as physical performance and wellbeing 9. Custom officers at Copenhagen Airport (Denmark) intercepted a package from China—shipped via FedEx®—containing five identical cardboard boxes, each holding 10 clear glass vials (Fig. 1). The vials were handed over to the medicine regulatory authority in Denmark (the Danish Health and Medicines Authority) and given to us to examine for research purposes. None of the vials were labelled, nothing was printed on the cardboard boxes and there were no accompanying package leaflets, giving the name of the drugs, strength and route of administration. All vials in four of the boxes had orange-coloured plastic caps; the vials in the remaining box had blue caps. Otherwise, there was no way to tell the vials apart. A closer inspection revealed that the vials contained a white lyophilized powder, presumably for the preparation of solutions for injection. To identify the active substance(s), we analysed the contents of one vial from each cardboard box using high-performance tandem mass spectrometry. Unlabelled vials containing the growth hormone releasers CJC-1295 (orange caps) and GHRP-2 (blue caps) seized at Copenhagen Airport (Denmark) Our analysis showed that four vials (with orange-coloured caps) contained CJC-1295 and one vial (with a blue cap) contained GHRP-2. We also found mannitol in the CJC-1295 preparation—a substance previously detected in illicit drugs, where it is mixed in to add bulk 10. These findings add to reports of growth hormone releasers found in illicit preparations in Europe 2-7 and Australia 11, and suggest new drug-using trends. The potential harms relating to the consumption of growth hormone releasers for performance enhancement are unknown. However, users are exposed to risks from a broad range of defects discovered in illicit market products, including unknowing exposure to undeclared active substances, unknown amounts of active substance and/or contamination 12-14. The marketing of growth hormone releasers as improved ways to increase performance and appearance, compared with ‘old’ drugs such as anabolic steroids, can be seen as a sign of the increasing commodification of ‘life-style’ drugs 15. Further, the existence of global shipment options (including well-established delivery services) that are cheap and fast allows transnational distribution of illicit drugs, as highlighted by this study. Systematic intelligence-gathering concerning new trends in substance use for performance enhancement purposes and the harms it may cause is needed urgently, especially as tailored public health responses are limited. Health-care professionals encountering users seeking treatment or health-related advice relating to ‘performance-enhancing drugs’ should be aware of the variety of new drugs on the market. None. The authors thank the Danish Health and Medicines Authority for providing the vials. The authors also thank the Audio Visual Services at Vendsyssel Hospital for providing pictures. This study was supported through the Obelske Family Foundation grants.

Potential Mechanism of Action of Human Growth Hormone on Isolated Human Penile Erectile Tissue

Potential Mechanism of Action of Human Growth Hormone on Isolated Human Penile Erectile Tissue

Potential mechanism of action of human growth hormone on isolated human penile erectile tissue

Potential mechanism of action of human growth hormone on isolated human penile erectile tissue

Potential Mechanism of Action of Human Growth Hormone on Isolated Human Penile Erectile Tissue

To evaluate the mechanisms of growth hormone (GH) action on isolated human penile erectile tissue. Human GH (hGH) has been suggested to play a role in male reproductive function, including penile erection. Nevertheless, it still remains unclear which intracellular pathways mediate the physiological effects of GH on the human corpus cavernosum (HCC). Using the organ bath technique, the effects of GH were investigated on electrical field stimulation (EFS)-induced relaxation of isolated HCC in the absence and presence of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine (l-NOARG, 10 microm). Effects of GH on the production of tissue cyclic guanosine monophosphate (cGMP) in the absence and presence of ODQ and l-NOARG were also elucidated using radioimmunoassay. ODQ and l-NOARG abolished the relaxation of the tissue induced by EFS, whereas amplitudes were increased by physiological concentrations of GH (1-100 nm). The attenuation of EFS-induced amplitudes by l-NOARG but not ODQ was, in part, reversed by GH. The production of cGMP (pmol cGMP/mg protein) induced by 10 nm GH was abolished in the presence of 10 microm ODQ. In contrast, the combination of GH (10 nm) and l-NOARG (10 microm) maintained cGMP production significantly greater than baseline (0.68 +/- 0.36 vs 1.07 +/- 0.48 pmol cGMP/mg protein). Our data provide evidence that GH may act on human HCC by an NO-independent effect on guanylyl cyclase activity and may thus explain how growth factors, such as hGH, regulate male erectile function.

MP-12.05: Mechanisms of action of human growth hormone on isolated human penile erectile tissue

MP-12.05: Mechanisms of action of human growth hormone on isolated human penile erectile tissue

1072: Mechanisms of Action of Human Growth Hormone(GH) on Isolated Human Penile Erectile Tissue

1072: Mechanisms of Action of Human Growth Hormone(GH) on Isolated Human Penile Erectile Tissue

Thymic activity of human growth hormone on murine thymocytes: Potential role in promoting thymopoiesis following bone marrow transplant

Thus far, very few agents have been demonstrated to positively affect thymic function. Many cytokines have been shown to promote peripheral T cell function but only a select few (i.e. IL-7 and SCF) have been shown to promote thymopoiesis or T progenitor cell survival. Most cytokines were meant to exert local effects and it is often difficult to generate the levels needed after systemic administration to yield sustained biological effects. Hormones, by their very nature, are well suited for systemic administration.

Identification of Residues Outside the Two Binding Sites That Are Critical for Activation of the Lactogenic Activity of Human Growth Hormone

Human growth hormone (hGH) binds lactogenic or somatotrophic receptors, creating active heterotrimeric complexes. Comparison of hGH structures, either free or bound to a single lactogenic or somatotrophic receptor, shows binding is associated with structural changes. Changes in hGH structure are unique when binding either lactogenic or somatotrophic receptors and they influence the spatial arrangement of residues constituting the second receptor-binding site. Using site-directed mutagenesis, we identified a contiguous set of largely hydrophobic residues that forms a motif communicating between the two receptor-binding sites of hGH. The residues are external to the receptor-binding epitopes and were identified when their mutation reduced site 2 function without changing site 1 function. The motif includes Phe44, Leu93, Tyr160, Leu163, and Tyr164, located in two hydrophobic clusters between the receptor-binding sites. Their mutation to Glu disrupts hydrophobic interactions and reduces lactogenic activity between 4.7- and 85-fold with little effect on somatotrophic activity or spectroscopic properties. These differential effects indicate that loss of lactogenic activity is not a result of global mis-folding. We propose the loss of lactogenic activity results from disruption of specific hydrophobic clusters that disables the site 1 binding-induced structuring of the second receptor-binding site.

IFN-γ Increases the hGH Gene Promoter Activity in Rat GH3 Cells

Aim: To study the effect(s) of interferon gamma (IFN-γ) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the molecular mechanism underlying the effect(s). Methods: Cell transfection and luciferase reporter gene were used. Results: IFN-γ (10² and 10³ U/ml) increased the activity of hGH in GH3 cells. The addition of the mitogen-activated protein kinase inhibitor PD98059 (40 µmol/l) to the cells blocked the stimulatory effect of IFN-γ. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IFN-γ induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IFN-γ, four deletion constructs of hGH gene promoter were created. The stimulatory effect of IFN-γ was abolished following deletion of the –250 to –132 fragment. Conclusions: IFN-γ increases the activity of hGH gene promoter in rat pituitary GH3 cells. This stimulatory effect of IFN-γ appears to require the intracellular mitogen-activated protein kinase-dependent signaling pathway. The effect of IFN-γ requires the promoter sequence that spans the –250 to –132 fragment of the gene, but is unrelated to Pit-1 protein.

Actions of monoclonal antibodies on the activity of human growth hormone (GH) in an in vitro bioassay

An in vitro bioassay for GH was established, based on the response of the 3T3-F442A mouse preadipocyte cell line, together with a parallel receptor-binding assay using the same cells. The effects of monoclonal antibodies on the biological activity of human GH in vitro were then explored. Antibodies that did not bind GH had no effect on the bioassay or on receptor binding. Antibodies EB1 and EB2, which strongly enhance growth-promoting actions in vivo, inhibited the actions of human GH in the in vitro bioassay, and blocked binding of human GH to receptors. Antibody NA71, which weakly enhances growth promotion by human GH in vivo, enhanced biological activity in vitro but did not affect receptor binding. Thus, enhancement of the biological activity of human GH has been shown in this in vitro system, but the effect does not correlate completely with the established enhancement effects in vivo. Of the various mechanisms that have been proposed to explain the enhancement effect these results support the ‘restriction hypothesis’ — the idea that monoclonal antibodies may enhance GH action in vivo by preventing binding of GH to receptors/binding sites that are not involved in growth promotion.

Human growth hormone site 2 lactogenic activity requires a distant tyrosine164

Comparison of crystallographic structures of human growth hormone, either bound to the prolactin receptor or free of receptors, reveals that human growth hormone binding to the prolactin receptor at site 1 is associated with a structural change in human growth hormone that influences the organization of residues which constitute site 2. We have identified Tyr 164 as a residue that is critical for the propagation of this structural rearrangement. Tyr 164 is a structural epitope for site 1 and is distal to site 2. Mutation of Tyr 164 to glutamic acid (Y164E) does not affect the somatotrophic activity, absorption or fluorescence spectra or binding to the human prolactin receptor when compared to wild-type human growth hormone, indicating the subtle effects of the mutation. Lactogenic assays using extended concentrations of Y164E human growth hormone produce dose-response curves that are characterized by a right-shifted agonist phase and an unchanged antagonist phase when compared to wild-type human growth hormone. These results indicate that Tyr 164 is required for the lactogenic activity of human growth hormone and that mutation to glutamic acid disrupts the lactogenic function of site 2.

Monoclonal antibody enhanced stimulation of human growth hormone action in vitro using ovine costal cartilage growth plate chondrocytes.

Investigations into the mechanism underlying antibody-mediated enhancement of growth hormone action have been hampered by the lack of an in vitro assay system. In this work, we have used isolated ovine costal cartilage growth plate chondrocytes to demonstrate, for the first time, that monoclonal antibody EB1 can enhance the proliferative actions of human growth hormone on this cell type. Chondrocytes were cultured for 14 days prior to exposure to GH+/-monoclonal antibody EB1 for a 4-day treatment period. Human growth hormone alone promoted a significant dose-dependent increase in chondrocyte proliferation; maximal stimulation was achieved at about 3.3-10 ng/ml growth hormone, and at higher doses of growth hormone, the response declined. Monoclonal antibody EB1 was shown to enhance the proliferative activity of 10 ng/ml human growth hormone in a significant dose dependent fashion. In conclusion, our results demonstrate that an antibody capable of enhancing GH activity in vivo also has the capacity to potentiate GH activity in vitro. This system may provide an important tool for investigations into the mechanism of GH action, and how this is modified by GH enhancing MAbs.

Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands.

We have previously suggested that although salivary glands function in an exocrine manner they might none the less offer a useful way to deliver therapeutic proteins systemically. As a direct functional test of this hypothesis, we constructed a recombinant adenovirus (AdCMVhGH) encoding human growth hormone (hGH) and then studied the biological action of hGH produced following transfer of the hGH gene to rat submandibular glands. At 48 h following infusion of AdCMVhGH into these glands via cannulation of the main excretory duct, serum levels of hGH were approximately 16 ng/ml and rat insulin-like growth factor-1 was elevated approximately 25%. Moreover, serum chemistry profiles of rats subjected to in vivo gene transfer displayed alterations in the BUN:creatinine ratio and triglyceride levels presumably reflecting the anabolic actions of the hGH. These results provide the first demonstration of systemic biological action from a transgene product secreted in an endocrine fashion from the salivary glands.

Identification of a Motif Associated with the Lactogenic Actions of Human Growth Hormone

Human growth hormone (hGH) stimulates somatogenic and lactogenic actions through the GH and prolactin (PRL) receptors, respectively. In contrast, non-primate GHs stimulate only somatotropic action. Phe44, of the human GH sequence is present in all hormones stimulating lactogenic action and absent in all hormones stimulating only somatotropic action. We speculate that the presence of Phe44 is a feature necessary for specifying lactogenic activity. In this report, the role of Phe44 was investigated by its deletion or substitution with alanine or leucine. Deletion of Phe44 or substitution with leucine did not significantly change the structure of hGH as determined by circular dichroism, absorbance, and fluorescence spectroscopies. In contrast, substitution of alanine perturbed the structure. Deletion of Phe44 reduced binding affinity for the lactogenic receptor, resulting in a reduced activation. Substitution with either alanine or leucine partially restored lactogenic receptor binding affinity, which correlated with the hormones' activity in the Nb2 rat lymphoma cells. All the recombinant hGHs had similar somatotropic activities in FDC-P1 cells transfected with the hGH receptor. These data indicate that the hydrophobic side chain of Phe44 is required for lactogenic receptor binding and activation but is unnecessary for somatotropic action.

Mitogenic and receptor activities of human growth hormone 108ndash;129

We have selectively synthesized a number of peptides encompassing the region of helix 3 of growth hormone (GH). These peptides and native human (h) GH have been evaluated for mitogenic and receptor activities in 3T3-F442A preadipocytes. In this system, wild type hGH is anti-mitogenic. In contrast, hGH 108-129 stimulated DNA synthesis while other GH-derived peptides were ineffective. hGH (L) 108-129 had an EC50 of about 0.2 nM and was maximally effective at about 0.5 nM in stimulating [3H]thymidine incorporation in 3T3-F442A cells. hGH (L) 108-129 was mitogenically as active as insulin-like growth factor-I and more active than insulin. It was less effective than transforming growth factor-beta. By cell cycle analysis, hGH (L) 108-129 increased the proportion of cells in S/G2/M phases to 28%. hGH, when coincubated with hGH (L) 108-129, blocked the mitogenic response of the peptide. A monoclonal antibody to the GH receptor significantly reduced binding of 125I-hGH to its receptor but had no effect on binding of 125I-hGH (L) 108-129. Affinity cross-linking of 125I-hGH to its receptor was not duplicated with 125I-hGH (L) 108-129. No other GH peptides or insulin competed for binding of 125I-hGH 108-129. Scatchard analysis indicated a Kd of 5.2 nM with 5.6 x 10(5) binding sites/cell for hGH (L) 108-129. These studies indicate that hGH (L) 108-129, a sequence encompassing helix 3 of hGH, acts by binding to a site other than the GH receptor and evokes high mitogenic responses.

Selective modification of human growth hormone by site-directed mutagenesis: implications for the diversity of biological actions.

Human growth hormone (GH) is an anabolic hormone required for normal growth. In addition, human GH affects the metabolism of proteins, carbohydrates and fats and possesses lactogenic effects. Although the GH receptor has recently been cloned, it is not clear whether the diverse biological activities of human GH are transduced through a single or through several types of related receptors. To address this question, 15 recombinant analogues of human GH were prepared and tested in bioassays in vitro and in vivo, in which the hormone action is mediated through lactogen or somatogen receptors. The results clearly suggest that recombinant analogues of human GH that recognize either somatogen or lactogen receptors, or both, but have selectively modified post-receptor effects, are helpful in elucidating the diverse biological activities of GH. These differences are most probably due to minor structural variability in GH and lactogen receptors in different organs and/or species. Genetic engineering of human GH may lead to production of modified analogues with changed and narrower specificities. One of the possible applications would be for a human GH analogue devoid of diabetogenic activity.

Mitogenic and receptor activities of human growth hormone 108-129.

We have selectively synthesized a number of peptides encompassing the region of helix 3 of growth hormone (GH). These peptides and native human (h) GH have been evaluated for mitogenic and receptor activities in 3T3-F442A preadipocytes. In this system, wild type hGH is anti-mitogenic. In contrast, hGH 108-129 stimulated DNA synthesis while other GH-derived peptides were ineffective. hGH (L) 108-129 had an EC50 of about 0.2 nM and was maximally effective at about 0.5 nM in stimulating [3H]thymidine incorporation in 3T3-F442A cells. hGH (L) 108-129 was mitogenically as active as insulin-like growth factor-I and more active than insulin. It was less effective than transforming growth factor-beta. By cell cycle analysis, hGH (L) 108-129 increased the proportion of cells in S/G2/M phases to 28%. hGH, when coincubated with hGH (L) 108-129, blocked the mitogenic response of the peptide. A monoclonal antibody to the GH receptor significantly reduced binding of 125I-hGH to its receptor but had no effect on binding of 125I-hGH (L) 108-129. Affinity cross-linking of 125I-hGH to its receptor was not duplicated with 125I-hGH (L) 108-129. No other GH peptides or insulin competed for binding of 125I-hGH 108-129. Scatchard analysis indicated a Kd of 5.2 nM with 5.6 x 10(5) binding sites/cell for hGH (L) 108-129. These studies indicate that hGH (L) 108-129, a sequence encompassing helix 3 of hGH, acts by binding to a site other than the GH receptor and evokes high mitogenic responses.

Insulin-Like Growth Factor Binding Proteins: Structural and Molecular Relationships

The insulin-like growth factors (IGFs) are important metabolic and mitogenic factors involved in cell growth and metabolism. IGF-I and -II belong to a family of peptide hormones that include relaxin and insulin, and they share a high degree of structural similarity with proinsulin. They are produced in the liver and multiple other tissues, and are partially growth hormone dependent. These peptides interact with specific high affinity receptors, designated as type I and type II IGF receptors, as well as with the insulin receptor. IGFs have direct effects on somatic growth and on the proliferation of many tissues and cell types, both in vivo and in vitro. IGF-I is currently thought to be the main mediator of the growth promoting actions of human growth hormone and IGFs also have a profound effect on carbohydrate and lipid metabolism, distinct from that of insulin. A recently recognized class of proteins which have high affinity and specificity for the IGFs, the IGF binding proteins (designated as IGFBPs; Ballard et al., 1989; Rosenfeld et al., 1990), have been shown to be involved in modulation of the proliferative and mitogenic effects of IGFs on cells. The molecular mechanisms involved in the interaction of the IGFBPs with the IGFs and their receptors remain unclear, but it is certain that a full understanding of the actions of IGF in vivo will require the characterization of these binding proteins.