Abstract
Growth hormone (GH) consists of several isoforms. We have studied the proportion, expressed as percentage of total GH concentration, of non-22 kDa (non-22K) GH isoforms and 20K GH during 8-week oral treatment with MK-677 25 mg daily in 12 obese males.
The proportion of non-22K GH isoforms in peak total GH samples after the initial MK-677 administration was higher than that after 2 and 8 weeks (p<0.01 and p<0.05, respectively). In selected non-peak total GH samples after the initial MK-677 administration, however, the proportion of non-22K GH isoforms was similar to that in the peak total GH samples after 2 and 8 weeks. The proportion of 20K GH in 2-h samples after the initial MK-677 administration was lower than that after 2 and 8 weeks (p<0.01 and p<0.05, respectively).
We concluded that the proportion of non-22K GH isoforms was higher in peak, but not in non-peak, total GH samples after the initial MK-677 administration than that observed after multiple doses. The proportion of 20K GH in 2-h samples after the initial MK-677 administration was lower than that after 2 and 8 weeks. These moderate changes in the proportion non-22K GH isoforms are likely of small importance for the clinical response to MK-677 treatment.
Introduction
The work by Bowers [1], [2], [3] resulted in the discovery of growth hormone-releasing peptides (GHRPs) including the hexapeptide GHRP-6. Later research has led to the development of the GHRPs GHRP-1, GHRP-2, and hexarelin [4], [5], and non-peptidyl GH secretagogues (GHSs) such as the orally active spiroindoline sulfonamide MK-677 [6]. A cell membrane G-protein coupled receptor has been cloned and shown to be a target for the GHSs [7]. An endogenous ligand for the GHS receptor, ghrelin, has been isolated [8].
The main GH isoform produced by the GH-N gene in the pituitary is 22K GH [9]. Another isoform, the 20K GH, which lacks residues 32–46 of 22K GH, is produced in the pituitary by alternative splicing of 22K GH messenger ribonucleic acid [10], [11], [12]. Studies show that 20K GH enhances linear growth in hypophysectomised rats [13] and exerts lipolytic activity in vitro [14]. 20K GH binds to GH receptors similarly to 22K GH [15], but differs in acute insulin-like activity [16] and binding to lactogenic receptors [17], [18]. Furthermore, exogenous 20K GH administration suppresses endogenous 22K GH secretion in normal men [19].
In addition to 22K and 20K GH, several other GH molecular isoforms exist [9], [13], [14], [15]. These can arise in the pituitary through pre-transcriptional and posttranslational mechanisms, or in peripheral tissues, where 22K GH may be degraded to various recirculating GH fragments [20], [21], [22], [23]. The physiological role of these various GH isoforms is not fully known. However, these non-22K GH isoforms may differ from 22K GH regarding biological activity, binding properties, and metabolic clearance [9], [23]. 22K GH binds to one GH receptor by a first binding site, and then to another GH receptor by a second binding site, resulting in receptor homodimerisation and activation [24], [25]. Thus, GH isoforms, partially or completely lacking one of these two binding sites, may act as weak agonists or antagonists of the GH receptor [26], [27]. It has also been suggested that some non-22K GH isoforms and fragments may bind to specific receptors [26], [28].
Little is known about the regulation of non-22K isoforms. In a study by Baumann et al. [29], the relative proportion of circulating GH isoforms was not changed after administration of GH-releasing hormone (GHRH) or l-dopa in healthy subjects, or after administration of thyroid stimulating hormone in acromegalic patients [29]. However, in acromegaly, several other studies have shown an increased proportion of various non-22K GH isoforms [30], [31], [32], [33]. In a study by Tsushima et al. [34], the ratio between 20K and 22K GH was unchanged after administration of insulin or GHRH in healthy subjects, whereas it was increased in acromegaly and anorexia nervosa [34].
We have previously reported that 8-week MK-677 treatment elicited a sustained increase in serum peak total GH levels, although the initial serum peak total GH response to MK-677 was higher than the peak GH responses to MK-677 at 2 and 8 weeks of treatment [35]. The aim of the present study was to determine the released proportion of non-22K GH isoforms and 20K GH after MK-677 administration in healthy obese males using the 22K GH exclusion assay (GHEA) [36] and a 20K-specific enzyme-linked immunosorbent assay (ELISA) [34].
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Subjects
Twelve males, 19–49 years of age, with body mass index (BMI) >30 kg/m2 and waist:hip ratio >0.95 were studied. At study start, mean age was 36.8 (2.7) years and mean BMI was 32.0 (0.4) kg/m2. They were recruited using advertisements in local newspapers. Exclusion criteria included fasting blood glucose >6.4 mmol/L and blood pressure >165/95 mm Hg. Except for obesity, all subjects were in good general health and none used concomitant medication.
One subject was discontinued from the study after
Serum total GH concentrations
The geometric mean serum peak total GH concentration, analysed concomitantly with the GHEA, was 23.5 (4.6) μg/L after the first MK-677 administration, 6.2 (1.5) μg/L at 2 weeks, and 5.1 (1.4) μg/L at 8 weeks. The mean time from MK-677 administration to peak total GH concentration was 0.96 (range 0.5–1.5) hours after the initial MK-677 administration, and 1.58 (range 1–4) hours and 1.21 (range 0.5–2) hours, respectively, after MK-677 administration at 2 and 8 weeks. In the selected non-peak GH
Discussion
This study revealed that the proportion of non-22K GH isoforms in peak, but not in non-peak, total GH samples was higher after the initial MK-677 administration than that after MK-677 administration at 2 and 8 weeks of treatment. The proportion of 20K GH in 2-h samples was lower after the initial MK-677 administration than that after MK-677 administration at 2 and 8 weeks. Serum non-22K GH concentration was correlated with serum total GH concentration at all timepoints. Serum concentrations of
Acknowledgments
This study was supported by grants from the Swedish Medical Research Council (Nos. 11285, 11331, 11502, 11576, and 1162), and from Merck Research Laboratories, Rahway, NJ, USA. We are indebted to Lena Wirén, Anne Rosén, Ingrid Hansson, Sigrid Lindstrand, and Annika Reibring at the Research Centre for Endocrinology and Metabolism for their skillful technical support. We are grateful to Dr. Noboru Satozawa at Nihon Schering K.K., Chiba, Japan, for the gift of 20K antibodies.
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