Abstract
Objective
To identify biomarkers of growth hormone (GH) and insulin-like growth factor 1 (IGF-1) action in human serum.
Background
The search for new markers of GH activity has received extensive attention given that the current biomarkers (IGF-1, IGFBP-3 and collagen peptides) show substantial variability in the population, and are not reliably predictive of either the physiologic effects of GH therapy or the detection of GH abuse by athletes. GH releasing hormone (GHRH) is a polypeptide synthesized in the hypothalamus that binds to receptors on pituitary somatotropes to promote the synthesis and release of GH. Serum GH and IGF-1 levels have been shown to increase with administration of GHRH or CJC-1295, a long-acting GHRH analog.
Design
Sera from 11 healthy young adult men before and one week after CJC-1295 injection were analyzed by two-dimensional gel electrophoresis for proteomic changes. Serum proteins displaying significant changes before and after treatment were subsequently identified using mass spectrometry. In addition, correlations between these proteins and GH or IGF-1 levels were evaluated.
Results
Two protein spots that displayed decreased intensities after treatment were identified as an apolipoprotein A1 isoform and a transthyretin isoform. Three protein spots upregulated by CJC-1295 treatment included beta-hemoglobin, a C-terminal fragment of albumin, and a mix of an immunoglobulin fragment and another C-terminal albumin fragment. A linear relationship was found between the spot containing immunoglobulin and albumin fragments and IGF-1 levels.
Conclusions
Although the molecular mechanisms linking the identified proteins to GH and IGF-1 biological activity remain to be clarified, the results suggest that they represent potential biomarkers of GH and/or IGF-1 action.
Introduction
Growth hormone releasing hormone (GHRH) is a 44-amino acid peptide synthesized in the hypothalamus. At the pituitary gland, GHRH binds to receptors on somatotropes to promote synthesis and release of GH. Together with somatostatin and ghrelin, GHRH is a key participant in the regulation of pulsatile GH secretion.
The administration of recombinant human GH (rhGH) has been approved by the FDA and various international regulatory agencies for several indications both in children (GH deficiency (GHD), Turner syndrome, children born short for gestational age, Prader–Willi syndrome, idiopathic short stature, etc.) and in adults (GHD, HIV-associated lipodystrophy, etc.) [1]. Treatment with rhGH is routinely performed as one subcutaneous daily injection, thus it does not resemble the physiological pulsatile pattern of GH release. On the other hand, the administration of GHRH or its analogs results in increased levels of GH in serum and more importantly, maintenance of the episodic pattern of GH secretion. In addition, contrary to rhGH administration which contains only the 22 kDa isoform of GH, therapy with GHRH-type molecules should result in the secretion of all forms of GH that are produced in the pituitary somatotropes (including post-translationally modified isoforms whose functions remain unknown [2]).
A major obstacle in assessing the efficacy of GH therapy is the lack of a consistent biological serum marker. Levels of IGF-1 and IGFBP-3 are widely used to monitor the efficacy of GH therapy, but unfortunately, there is considerable variability in responses (growth velocity, final stature, glucose tolerance, insulin levels, etc.) with a lack of consistent correlation between the measured response and the serum levels of GH, IGF-1, IGFBP-3, etc. Collagen peptides are also used as biomarkers of GH activity, but again their reliability as predictors of treatment efficacy is variable [3]. In addition, the abuse of rhGH by athletes to improve performance is a major concern, and underscores the need for new and more specific biomarkers of GH action [3], [4], [5], [6], [7]. Moreover, the finding of new biomarkers will expand our understanding of the molecular mechanisms and physiologic effects of GH action.
In a previous study, the long-acting GHRH analog CJC-1295 was shown to promote an increase in GH and IGF-1 levels in sera of healthy young adult men one week after administration, without affecting the pulsatility of GH secretion [8]. The extended half-life of the compound (8–10 days in humans) is due to its ability to bind to endogenous serum albumin through a free thiol group forming a covalent disulfide bond [9]. The present study was designed to identify biomarkers of GH and/or IGF-1 action in serum samples obtained before and one week after administration of CJC-1295 to healthy subjects. The samples were analyzed using a proteomic approach to search for serum proteins and protein isoforms that respond to increases in GH or IGF-1. Five protein spots displaying significant changes after treatment were identified and their serum levels evaluated for correlations to GH and IGF-1 serum concentrations.
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Section snippets
Materials and methods
The subjects and procedures followed for drug administration and GH and IGF-1 measurements have been previously reported [8].
Individual serum proteome patterns
The subjects’ serum proteomes studied displayed marked heterogeneity in the low molecular weight range (<25 kDa) when resolved by 2DE. The patterns observed were conserved in each subject at the two timepoints studied and consisted mainly of the presence or absence of two of the spot “trains” usually observed in that region (Fig. 1). The term spot “train” refers to a group of protein spots that have approximately the same molecular weight and varying isoelectric points. These characteristics
Discussion
The analysis of the serum proteome of 11 healthy male subjects revealed marked heterogeneity among gel images from different subjects, which might be partly explained by the alleles of haptoglobin present in each individual [16], [17]. Given that this study was aimed at finding clinically relevant protein biomarkers of GH and/or IGF-1, the criteria used for selection of spots were not only statistical significance between before and after drug administration, but also that the spots were
Acknowledgements
This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by grants from WADA, the Diabetes Research Initiative at Ohio University, and ConjuChem Inc. J.J.K. is also supported in part by NIH (AG019899-06, DK075436-01).
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However, the two proteins are high abundant proteins in human serum and plasma (vide supra). Sackmann-Sala et al. used a similar strategy for the discovery of potential biomarkers for the application of a long-acting GHRH analog (CJC-1295) [105]. Serum samples were albumin-depleted, then separated by 2D-PAGE, and differentially expressed proteins identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. View all citing articles on ScopusView full text
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