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Original Studies |
Metabolic Research Unit, Department of Medicine, and Statistics Section, Department of Social and Preventative Medicine, University of Queensland, Princess Alexandra Hospital (J.D.W., R.C.C., J.H.), 4102 Brisbane, Australia; Neuroendocrine Unit, Department of Medicine, Innenstadt University Hospital (M.B., C.J.S.), 80336 Munich, Germany; SEMPR, Serviço do Endocrinologia e Metabologia do Hospital de Clínicas da Universidade Federal do Paraná (C.L.B.), 80060-240 Curitiba, Brazil; Research Center for Endocrinology and Metabolism, Sahlgrenska Hospital (P.A.L., L.C., T.R.), S-413 45 Gothenberg, Sweden; Department of Endocrinology, St. Thomas’s Hospital (M.-L.H.), London, United Kingdom SE1 7EH; Department of Internal Medicine and Cardiovascular Sciences, Frederico II University (R.N.), 80131 Naples, Italy; and Department of Medicine M (Endocrinology and Diabetes), Aarhus University Hospital (R.D.), 8000 Aarhus, Denmark
Address all correspondence and requests for reprints to: Dr. Jennifer D. Wallace, Metabolic Research Unit, University of Queensland, Department of Medicine, Princess Alexandra Hospital, 4102 Brisbane, Australia. E-mail: jwallace{at}medicine.pa.uq.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Signal transduction conventionally follows two-site binding of the GH ligand and membrane-bound receptor dimerization. Intact 22-kDa GH possesses both receptor-binding sites and has been shown to exert growth-promoting, anabolic, lipolytic, and multiple effects on carbohydrate metabolism in humans (6, 8). Twenty-kilodalton GH, which has altered receptor binding site 1 conformation, was initially thought to be biologically inert (9), but recent studies have shown receptor binding and agonistic actions comparable to those of 22-kDa GH (10, 11). A variety of actions have been shown with other non-22-kDa GH isoforms (2, 12, 13, 14), raising the possibility of signaling mechanisms other than via the GH receptor (15).
Serum GH concentrations increase dramatically in response to acute exercise, with the response depending on the nature, duration, and relative workload of the exercise and the age, gender, and nutritional and training status of the individual (16). There are limited data on the response of isoforms other than 22-kDa GH to acute exercise (17, 18, 19, 20, 21, 22). GH may be important in homeostasis during or after exercise (16), but until the response of the major molecular isoforms to exercise has been fully characterized, the contribution of differing isoforms to this process cannot be further explored.
Recombinant DNA technology has provided ample supplies of synthetic GH, permitting athletes to illegally administer GH in attempts to enhance sporting performance. Detection of exogenous GH administration may be possible by assessing alterations in the ratios of endogenous GH isoforms (23). To investigate this strategy it is first necessary to characterize the response of GH isoforms to acute exercise. To explore both the question of metabolic homeostasis and GH dope detection, we aimed to characterize the GH isoform responses to acute exercise in trained athletic males using a variety of established and novel assays.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Selection criteria included: male gender, age between 18–40 yr , high level of habitual aerobic activity defined as at least four 30-min sessions of continuous aerobic type exercise per week, high aerobic fitness defined as maximal oxygen uptake (VO2max) greater than 45 mL/kg·min, and no illnesses or medications known to impair exercise or to alter endocrine function.
Screening
A full physical examination was performed, and blood was taken for routine biochemistry, hematology, and serum testosterone, T4, and T3 determinations. Urine samples were tested to exclude glycosuria. Skinfold thicknesses were measured with a Harpenden caliper at standard sites (biceps, triceps, subscapular, abdominal, and suprailiac), and the percent body fat was calculated (24). VO2max was measured by cycle ergometry (Lode Excalibur Sport, Grunningen, Holland) and respiratory gas analysis (Medical Graphics CPX-D Cardiopulmonary Exercise Testing System, Medical Graphics, Birmingham, UK) as previously described (25, 26).
Study design
All subjects provided informed consent for studies approved by
Guy’s and St. Thomas’s Hospital’s ethics committee (London, UK).
Subjects underwent both rest (Rest) and exercise (Ex) studies,
separated by 48 h, with random assignment of the sequence (Fig. 1
). Subjects were semirecumbent from -60
until just before 0 min, were upright from 0–30 min (chair sitting for
Rest study and upright cycle ergometer for Ex study), and were
semirecumbent from 30–120 min. Exercise on a cycle ergometer included
two 5-min warm-up stages at 1 and 2 watts/kg and 20 min at 65% of the
workload achieved at VO2max (corresponding to
80% VO2max). Blood samples were taken at
intervals from -30 to 120 min. Subjects drank 250 mL water immediately
after exercise and again at 60 min to replace predicted sweat loss.
During the Rest study, chair-sitting subjects were given 50 mL water at
these two times to ensure uniformity of fluid ingestion.
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Total serum GH (total GH) was assayed using a polyclonal immunoradiometric assay (Pharmacia & Upjohn, Inc., Uppsala, Sweden). The pituitary-derived GH standards provided in the immunoradiometric assay were calibrated against the First International Reference Preparation from WHO (IRP 80/505), in which 1 mg = 2.6 IU GH. The manufacturer specified that the assay had 100% cross-reactivity with 22-kDa, deamidated 22-kDa, dimeric 22-kDa, and 20-kDa GH isoforms. The within-assay coefficients of variation (CVs) were 10%, less than 5.0%, and less than 5.0%, and the between-assay CVs were 9.0%, less than 5.0%, and 7.0% at 10, 23, and 43 mU/L, respectively. The detection limit was 0.4 mU/L.
The 22-kDa GH was measured by a two-site, monoclonal antibody, time-resolved fluroimmunoassay (Delphia hGH assay, Wallac, Inc. Oy, Turku, Finland). The standard was biosynthetic rhGH calibrated against IRP 80/505. Cross-reactivity with 20-kDa GH was less than 0.001%. The intraassay CVs were 5.0%, 3.9%, 3.4%, 2.1%, and 4.1% at 0.6, 12.2, 21.5, 34.1, and 89.3 mU/L, respectively. Interassay CVs were 6.3%, 3.7%, 5.3%, 5.4%, and 4.2% at 0.05, 0.34, 1.14, 2.26, and 3.78 mU/L, respectively. The detection limit was 0.03 mU/L.
Non-22-kDa GH was measured with the 22-kDa GH exclusion assay (22-kDa GHEA) as previously described (27). The 22-kDa GH monomers and dimers were removed from serum with a specific monoclonal antibody (MCB) and magnetic beads. Non-22-kDa isoforms, including mostly 20-kDa GH and a small fraction of other monomers and fragments of GH, were detected by assaying the stripped serum with a polyclonal antibody GH immunoradiometric assay (identical to the total GH assay). The intra- and interassay CVs were less than 9% at concentrations between 82.2–94.6 mU/L, and 17% and 21.6% at concentrations of 19.2 and 20.8 mU/L, respectively. The detection limit was 0.05 mU/L.
"Pituitary" GH (Pit-GH) and "recombinant" or 22-kDa GH (rhGH) were assayed simultaneously in two sandwich-type immunoassays. The Pit-GH assay was based on mAb 1H6 and biotinylated mAb 10A7 and was permissive in recognizing preferentially pituitary GH consisting of a wide variety of molecular isoforms, including 22-kDa, 20-kDa, acidic, fragmented, and modified forms of GH. The rhGH assay used mAb 5D7 and biotinylated mAb 10A7 and preferentially recognized recombinant 22-kDa GH (23). These assays were developed to optimally recognize pituitary (IRP 80/505) and recombinant (IRP 88/624) GH, respectively. The standard for each assay was IRP 80/505. The assays were highly reproducible (intraassay CV, <5%; interassay CV, <6%). The intraassay CV for the ratio of rhGH/Pit-GH was 4.8%. The detection limits were 0.13 and 0.05 mU/L, respectively.
The 20-kDa GH was assayed with antibodies provided by Mitsui Pharmaceuticals (Tokyo, Japan) (28), with modifications. The method involved monoclonal antibody D05 and biotinylated mAb 7D5 in a solid phase fluorometric sandwich assay. The within- and between-assay CVs were 4.2% and 6.1%, respectively. The cross-reactivity with 22-kDa GH was less than 0.1%. The lower detection limit was 0.13 mU/L.
The immunofunctional GH assay (GH-IFA) detects GH isoforms possessing both receptor-binding sites, i.e. those capable of biological signaling via GH receptor dimerization (29). The method used an anti-hGH monoclonal antibody directed against binding site 2 of hGH to capture and immobilize GH from the serum. Biotin-labeled recombinant human GH-binding protein in a second incubation step formed a complex with those hGH molecular isoforms also possessing binding site 1. The signal was detected after a short third incubation step with labeled streptavidin. Cross-reactivity with 20-kDa GH was approximately 11% (Mitsui preparation). The within-assay CVs were 8.5%, 7.3%, and 6.1%, and the between-assay CVs were 12.8%, 9.4%, and 7.9%, at 1.1, 13.8, and 49.9 mU/L, respectively. The detection limit was 0.3 mU/L.
Statistics
Effects of exercise were assessed by split-plot, repeat measures ANOVA using a general linear model (SPSS 7.5 for Windows, SPSS, Inc., Chicago, IL), with within-subject factors being condition (Rest vs. Ex) and time point, and the between-subject factor being study order (whether subjects were studied under the Rest or Ex condition first). The relative proportions of GH isoforms were calculated at each time point, providing both analytes were above (not at) the assay detection limit; spurious ratios were deleted where values were statistical outliers. Differences between Rest and Ex conditions for 22-kDa GH/total GH, rhGH/Pit-GH, and 20-kDa GH/total GH ratios were analyzed using ANOVA, treating each time point as a discrete variable, as repeat measures ANOVA was not possible under these conditions of incomplete datasets due to the number of individual assays at or below the detection limit of the assay. Disappearance half-times were calculated from exponential curve fitting to the individual data after peak values were achieved. A one-way ANOVA was used to assess differences in disappearance half-times, with post-hoc t testing to ascribe individual distinctions. Simple linear regression analysis was used to assess relationships between variables. Results were reported as the mean ± SEM. Assays measured in mass units were converted to Systeme International units (1 mg = 2.6 U).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In response to acute exercise, the absolute concentrations of GH
measured by each assay increased (Fig. 2
and Table 1). Concentrations were low and
stable on the Rest day. Statistically, the condition x time point
interaction showed a difference from Rest for all analytes
(P < 0.0001 for each assay), but no order effect. Peak
concentrations occurred at the end of the 30-min exercise bout for all
analytes. The 20-kDa GH showed a broader peak, which extended to 15 min
after the end of exercise.
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and
Table 2. Each ratio was stable during the Rest study. On
cessation of exercise, the ratio of non-22-kDa GH/total GH continued to
increase, peaking 15 min after exercise and declining slowly thereafter
(P < 0.0001 for condition x time point
interaction; difference between end of exercise and +15 min,
P < 0.002). A similar postexercise increase in the
ratio of 20-kDa GH/total GH was observed. The ratio of rhGH/Pit-GH
increased at the end of exercise (i.e. an increase in the
relative proportion of 22 kDa), followed by a progressive fall to
levels below that recorded before exercise (P = 0.024,
Rest vs. Ex at 120 min). No change was observed in the ratio
of 22-kDa GH/total GH.
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shows the relationship between
total GH and non-22-kDa GH as a percentage of total GH for all paired
samples in both Rest and Ex studies. Considerable variability in the
proportion of non-22-kDa GH/total GH is seen at low total GH
concentrations, changing to a more uniform pattern of approximately 8%
at higher total GH concentrations.
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), with total GH, 22-kDa GH, rhGH, and GH-IFA
having mean disappearance half-times of approximately 19–20 min,
whereas Pit-GH and 20-kDa GH were significantly different from the
other isoforms, averaging approximately 25 min (P <
0.0001). There were significant differences in the degree of conformity
to an exponential decay between analytes (by one-way ANOVA for
r2, P < 0.0001). Non-22-kDa GH
did not disappear in a strictly exponential fashion, being markedly
different from total GH, 22-kDa GH, Pit-GH, rhGH, and GH-IFA
(P < 0.001 for each). The correlation coefficient for
20-kDa GH was good (r2 = 0.91 ± 0.03), but
was inferior to the exponential curve fit for GH-IFA (P
= 0.04), rhGH (P = 0.02), and Pit-GH (P
= 0.03).
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Correlations between different assays are shown in Table 4. These relationships held when Rest and Ex data were
compared separately (data not shown) or together.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study demonstrated that 22-kDa GH was quantitatively the major molecular isoform to appear in serum in response to acute exercise. Twenty-two-kilodalton GH represented 73% of total GH at peak concentrations, as assessed by comparing assays with calibrated standards (Pharmacia IRMA and Delphia 22-kDa GH assay). The former assay is known to detect, in addition to 22-kDa GH, deaminated 22-kDa, dimeric 22-kDa, 20-kDa GH, and 17-kDa GH. In comparing the two new assays for total and 22-kDa GH (Pit-GH and rhGH, respectively) with these commercial assays, very close agreement was observed in terms of quantitation of peak concentrations.
Exercise-induced responses in 20-kDa GH, non-22-kDa GH (measured by the 22-kDa GH exclusion assay), and GH-IFA have also been specifically demonstrated. As 20-kDa GH is produced as a messenger ribonucleic acid splice variant of 22-kDa GH, parallel pituitary secretion of 22- and 20-kDa GH during exercise is implied. Earlier reports of the response of 20-kDa GH to exercise either used crude subtraction assays or lacked resting control studies (17, 18, 19, 20, 21, 22). Non-22-kDa GH, measured by the 22-kDa GH exclusion assay, is predominantly composed of 20- and 17-kDa GH, representing approximately 5% and 3%, respectively, of total GH at the end of exercise (by subtraction). These findings conflict with prior reports that 17-kDa GH circulated in concentrations double that of 22-kDa GH (14, 30). The immunofunctional GH assay detected isoforms with the two binding sites necessary for receptor dimerization and signal transduction. The mean peak GH-IFA response was only 43% and 59% of total and 22-kDa peak concentrations, respectively, potentially relating to greater detection by the total and 22-kDa GH assays of dimerized, chemically altered, protein-bound, or smaller isoforms not immediately available for two-site receptor binding and the relative insensitivity of the GH-IFA to 20-kDa GH.
The magnitude of the GH response to exercise is, in general, proportional to the relative intensity of endurance-type exercise (16), given the parallel activation of sympatho-adrenal and other pituitary homeostatic responses (31). Nevertheless, the interindividual variability of peak GH responses, whether total GH or other isoforms, varied up to 10-fold between subjects despite completion of identical relative intensity, submaximal, nonexhaustive exercise. We concluded that the interindividual variability of GH responses was not caused by variations in isoforms of GH, as the correlations between total GH and other isoforms was high (r2 = 0.93–0.96).
A novel finding from this study was that the proportion of GH isoforms changed across acute exercise and into recovery. Although 22-kDa GH was the predominant isoform detected at peak concentrations (end of Ex), isoforms of GH other than 22-kDa increased during the postexercise period. This conclusion is derived from the peak in non-22-kDa GH/total GH occurring after the end of exercise, the progressive fall in rhGH/Pit-GH after a peak at the end of exercise (implying less 22-kDa GH), and the increase in 20-kDa GH/total GH after exercise. Taken together, these data suggest that the proportion of 20-kDa, 17-kDa, and possibly other non-22-kDa isoforms (dimers, oligomers, and GH bound to serum proteins) increased after exercise. The predicted change in the ratio of 22-kDa GH/total GH did not parallel those of the rhGH/Pit-GH ratio, possibly because the latter assay pair was optimized to distinguish between related isoforms.
The increase in the proportion of isoforms other than 22-kDa GH after exercise may be caused by 1) different pituitary isoform secretion; 2) appearance of isoforms from nonpituitary sources (3); 3) generation of fragments, dimers, and oligomers in the circulation (32); and 4) differences in clearance of different isoforms. Our data clearly show that there were differences in disappearance half-times, with slower disappearance of 20-kDa GH and Pit-GH, the latter having high affinity for dimeric forms (but limited sensitivity for 20-kDa GH). The 20-kDa GH has been shown to form both hetero- and homodimers with 20- and 22-kDa GH and to bind to large circulating binding proteins that have reduced clearance rates compared with 22-kDa GH bound to high affinity GH-binding protein (5, 33). We speculate that dimers and/or other more slowly cleared isoforms (22) contributed to the delayed disappearance of Pit-GH, because 20-kDa GH appeared unable to explain the difference quantitatively.
Our data support the contention that heterogeneity of isoforms is reduced during times of peak GH concentration (and presumed high pituitary secretory rate) and is increased at other times. Baumann also showed (34, 35) that the GH isoform mix was different between the basal and stimulated states. Using immunoadsorbent chromatography and gel electrophoresis to accurately measure GH isoforms at low concentrations, basal conditions revealed high variability of isoforms and immunoreactive fragments, with 30-, 16-, and 12-kDa isoforms being consistently identified. During states of high spontaneous secretion or pharmacological stimulation, the plasma GH pattern was predominantly composed of 22-kDa GH, 20-kDa GH, acidic GH, and dimers. The relative contributions of pituitary secretion and peripheral fragment generation to these processes remain to be determined.
The biological consequences of alterations of GH isoforms during and after exercise remain to be determined. Potentially, enhanced diabetogenic effects of smaller GH isoforms may prevent postexercise hypoglycemia (12, 15). In addition, stimulated GH concentrations are criteria for the diagnosis and treatment of GH deficiency in children and adults. The marked differences reported here between assays reinforces the need for assay standardization (36). Finally, our data may assist in the development of a test for exogenous recombinant human 22-kDa GH administration by athletes, which would rely on alterations in GH isoform ratios (23).
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Acknowledgments |
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Footnotes |
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Received May 30, 2000.
Revised September 6, 2000.
Accepted September 30, 2000.
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References |
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2-macroglobulin as a low-affinity
growth hormone-binding protein. Acta Padiatr. 417(Suppl):108–110.
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