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Original Article |
Division of Endocrinology and Metabolism (E.R., A.D.R., D.B., O.L.V., W.C., J.D.V.), Department of Pediatrics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; and Division of Endocrinology (C.Y.B.), Department of Medicine, Tulane University Medical Center, New Orleans, Louisiana 70112-2699
Address all correspondence and requests for reprints to: J.D. Veldhuis, M.D., Endocrine Division, Mayo Clinic and Medical School, 200 First Street SW, Rochester, Minnesota 55905.
Abstract
A single injected pulse of GH inhibits the time-delayed secretion of GH in the adult by way of central mechanisms that drive somatostatin and repress GHRH outflow. The marked amplification of spontaneous GH pulse amplitude in puberty poses an autoregulatory paradox. We postulated that this disparity might reflect unique relief of GH-induced autonegative feedback during this window of development. The present study contrasts GH autonegative feedback in: 1) normal prepubertal boys (PP) (n = 6; Tanner genital stage I, chronologically aged 8 yr, 9 months to 10 yr, 1 month; median bone age 8.5 yr); 2) longitudinally identified midpubertal boys (MP) (n = 6; Tanner genital stages III/IV, aged 12 yr, 6 months to 15 yr, 6 months; median bone age 15 yr); and 3) healthy young men (YM) (n = 6, aged 18–24 yr; bone age >18 yr). Subjects each underwent four randomly ordered tandem peptide infusions on separate mornings while fasting: i.e. 1) saline/saline infused iv bolus at 0830 h and 1030 h; 2) saline/GHRH (0.3 µg/kg iv bolus) at the foregoing times; 3) recombinant human (rh) GH (3 µg/kg as a 6-min square-wave iv pulse)/saline; and 4) rhGH and GHRH. To monitor GH autofeedback effects, blood samples were obtained every 10 min for 5.5 h beginning at 0800 h (30 min before GH or saline infusion). Serum GH concentrations were quantitated by ultrasensitive chemiluminometry (threshold 0.005 µg/liter). On the day of successive saline/saline infusion, MP boys maintained higher serum concentrations of: 1) GH (µg/liter), 2.2 ± 0.25, compared with PP (0.61 ± 0.10) or YM (0.88 ± 0.36) (P = 0.011); 2) IGF-I (µg/liter), 493 ± 49 vs. PP (134 ± 16) and YM (242 ± 22) (P < 0.001); 3) T (ng/dl), 524 ± 58 vs. PP (<20) (P < 0.001); and 4) E2 (pg/ml),19 ± 3 vs. PP (< 10) (P = 0.030) (mean ± SEM). Consecutive saline/GHRH infusion elicited comparable peak (absolute maximal) serum GH concentrations (micrograms per liter) in the three study groups, i.e. 18 ± 5.0 (PP), 9.6 ± 1.7 (MP), and 14 ± 5.3 (YM) (each P < 0.01 vs. saline; P = NS cohort effect). Injection of rhGH attenuated subsequent GHRH-stimulated peak serum GH concentrations (micrograms per liter) to 7.8 ± 1.9 (PP), 5.8 ± 1.2 (MP), and 4.8 ± 1.1 (YM) (each P < 0.01 vs. saline; P = NS pubertal effect). GH autofeedback reduced non-GHRH-stimulated (basal) serum GH concentrations by 0.74 ± 0.28 (PP), 5.7 ± 1.7 (MP) and 1.4 ± 0.27 (YM) fold, compared with saline (P = 0.016 for MP vs. PP or YM). In addition to greater fractional autoinhibition, MP boys exhibited markedly accentuated postnadir escape (4.6-fold steeper slope) of suppressed GH concentrations (P < 0.001 vs. PP or YM). Linear regression analysis of data from all 18 subjects revealed that the fasting IGF-I concentration negatively predicted fold-autoinhibition of GHRH-stimulated peak GH release (r = -0.847, P = 0.006) and positively forecast fold-autoinhibition of basal GH release (r = +0.869, P < 0.001). In contrast, the kinetics of rhGH did not differ among the three study cohorts. In summary, boys in midpuberty manifest equivalent responsiveness to exogenous GHRH-stimulated GH secretion; heightened susceptibility to rhGH-induced fractional inhibition of endogenous secretagogue-driven GH release, compared with the prepubertal or adult male; and accelerated recovery of GH output after acute autonegative feedback. This novel tripartite mechanism could engender recurrent high-amplitude GH secretory bursts that mark sex hormone-dependent activation of the human somatotropic axis.
AUTONEGATIVE FEEDBACK WITHIN a neuroendocrine axis denotes the ability of a secreted hormone to inhibit its own release reversibly after a time delay (1, 2, 3, 4). For example, a single pulse of GH rapidly suppresses GHRH-stimulated GH secretion in healthy men and women (3, 5). Autorepressive mechanisms serve to maintain pulsatile GH secretion within the physiological range (1, 6, 7, 8, 9, 10, 11, 12).
GH autoinhibition is mediated by way of cognate receptors in the hypothalamus. Distal central nervous system effector pathways drive somatostatin release and inhibit GHRH outflow (7, 13, 14, 15). Thus, pretreatment with presumptive somatostatin antagonists relieves GH autofeedback (7, 16). GH represses its own secretion more prominently in the adult male than female rodent (4, 13, 14, 15, 17). Whether GH autofeedback is modulated by sexual maturation and/or gender in the human is not known.
The precise hypothalamo-pituitary mechanisms that mediate amplification of pulsatile GH secretion in normal puberty have not been elucidated (1). We reasoned that among other considerations temporary relief of GH autonegative feedback could sustain high-amplitude GH pulses at this time. To test this clinical hypothesis, we compared the magnitude and time course of recombinant human (rh) GH-induced autoinhibition in the healthy pre-, mid-, and postpubertal male and correlated autoinhibition with concurrent serum GH, IGF-I, and sex-steroid hormone concentrations.
Subjects and Methods
Study subjects
The protocol was approved by the Human Investigation Committee of the University of Virginia School of Medicine. The reason for the investigation and the potential risks were discussed. Volunteers were informed that they would not receive personal benefit. All subjects under the age of 18 yr gave individual assent, and a parent provided written consent.
The study groups comprised 18 healthy males: i.e. prepubertal boys (PP, n = 6), midpubertal boys (MP, n = 6), and young men (YM, n = 6). MP subjects were recruited during a 7-yr longitudinal study to document attainment of midpuberty (18). The height and weight of each subject fell within 95% normative limits for chronological age based on clinical standards of the U.S. National Center for Health Statistics (Atlanta, GA). Prepuberty was defined as Tanner genital stage I and midpuberty as Tanner genital stage III or IV. Young men had completed puberty at least 2 yr earlier.
Clinical protocol
Volunteers had an unremarkable medical history, physical examination, and screening biochemical tests of hematological, renal, metabolic, endocrine, and hepatic function (19, 20). None reported any acute illness, chronic disease, psychiatric disorder, recent transmeridian travel (three or more time zones traversed within 10 d), or significant weight change (1 kg or more within 6 wk).
To allow overnight adaptation to the study unit, volunteers were admitted to the General Clinical Research Center (GCRC) on the evening before blood sampling. Admissions were assigned in randomized order and scheduled at least 3 d apart (below). A standardized snack of 12 kcal/kg (55% carbohydrate, 15% fat, and 30% protein) was provided at 1800 h. Subjects remained fasting overnight and during the sampling and infusion protocol the next morning. Lights were put out at 2000 h, and an iv catheter was inserted into a forearm vein at 0700 h. Blood samples (1.0 ml) were withdrawn at 10-min intervals for 5.5 h beginning at 0800 h (30 min before the first saline or GH infusion). Sera were frozen for later assay of GH by chemiluminometry (below).
The infusion protocol comprised an iv injection of saline or rhGH (3.0 µg/kg, administered as a 6-min square-wave pulse at 0830 h followed by bolus iv infusion of saline or a submaximally effective dose of rhGHRH-1, 44-amide (0.33 µg/kg) 2 h later. A time delay of 120 min permits demonstrable autonegative feedback and allows six half-lives of decay of injected GH. Sampling was continued for an additional 3 h. At 1330 h, lunch was provided before discharge from the GCRC.
Assays
Serum GH concentrations were measured in each sample in duplicate by an automated ultrasensitive chemiluminescence-based assay (modified Luma Tag hGH assay, Nichols Diagnostic Institute, San Juan Capistrano, CA). The modified assay achieves a sensitivity of 0.005 µg/liter at 5 SD above the zero-dose tube (21, 22). The rhGH (22 kDa) is the assay standard. The median intraassay coefficient of variation was 8.5%, 6.5%, and 4.8% at serum GH concentrations spanning the ranges of 0.02 to 1.0, 0.2 to 1.0, and 1.0 to 8.5 µg/liter, respectively. Samples from the four study sessions in any given subject were assayed together. GH concentrations were detectable in all samples. Unknown values were interpolated from the standard curve via a four-parameter monotonic (sigmoidal) function (23).
Fasting (morning) serum concentrations of T, E2, LH, FSH, PRL, total IGF-I, T4, and TSH were quantitated by RIA or immunoradiometric assay, as reported earlier (19, 24, 25). Estimates at or below sensitivity of the T and E2 RIAs were arbitrarily assigned the corresponding detection limits of 20 ng/dl (0.70 nmol/liter) and 10 pg/ml (36.7 pmol/liter), respectively.
Analytical methods
Exogenous GHRH stimulation and rhGH-induced feedback inhibition. Absolute peak (simple maximum), fractional (fold) increase (peak value divided by the prepeak nadir serum GH concentration), and incremental (peak minus nadir) GH release were used as model-free measures of GHRH-stimulated GH secretion.
The magnitude of GH autofeedback was quantitated as ratio of the serum GH concentration (mean or peak) observed after saline infusion to that attained after rhGH injection. Thus, a ratio of unity denotes no detectable autoinhibition, whereas a higher ratio signifies relative GH-induced suppression over saline. Post-rhGH-infused serum GH concentrations declined gradually (see Kinetic estimates, below). In the absence of GHRH stimulation, the de facto minimum (nadir) emerged during the 1-h interval encompassing 210–270 min after the injection of GH. This interval was defined as post-rhGH basal GH release. The rate of recovery of serum GH concentrations from the absolute nadir was quantitated as the slope of the linear regression of rising GH concentrations over time.
Kinetic estimates. The GH distribution volume (liters per kilogram) was computed as the quotient of the dose of rhGH injected (3.0 µg/kg) and the subsequent peak serum GH concentration (µg/liter). The metabolic clearance rate (liters per kilogram per day) was calculated as the dose of rhGH injected divided by the time-integrated serum GH concentration over the subsequent 120 min (micrograms per liter x minutes). The rate of elimination of rhGH was defined by the relationship: half-life = [ln 2/metabolic clearance rate] x GH distribution volume.
Statistical analysis. ANOVA was applied to compare measured and derived GH responses among the three study groups. Ratio data were logarithmically transformed to reduce heterogeneity of variance. Post hoc contrasts were evaluated by Tukey’s honestly significantly different criterion at an overall type I error rate (P value) of 0.05. Data are presented as the mean ± SEM. Linear regression analysis was applied to data in the combined group of 18 males to relate autofeedback measures to mean serum GH, IGF-I, E2, or T concentrations.
Results
Table 1
summarizes median values (and absolute ranges) of chronological and bone ages and morning mean (±SEM) serum concentrations of GH, IGF-I, and sex steroids. ANOVA revealed higher serum IGF-I concentrations in MP than either PP or YM (< 0.001). T and E2 concentrations were comparably elevated in both MP and YM (P < 0.001 over PP). Serum T4, TSH, and PRL concentrations were normal in all subjects and invariant of pubertal status (data not shown).
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illustrates serum GH concentration profiles for one subject in each developmental cohort (PP, MP, and YM).
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plots individual GHRH-stimulated peak (maximal) serum GH concentrations in relation to the fasting total serum IGF-I concentration. Values are shown following the randomly ordered infusion of saline (top) and rhGH (bottom). Figure 2B presents the corresponding fractional (fold) GHRH-stimulated peak GH responses. The stimulatory effect of GHRH was significant in each study group (P < 0.001) and did not differ by pubertal status. Comparable statistical inferences applied to simple mean and incremental GH release (peak minus nadir) (data not shown).
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). Injection of rhGH also caused a significantly greater absolute decrement (micrograms per liter) in the serum GH concentration in MP than in YM (but not than in PP) (P = 0.046) (Fig. 3B). In fact, autoinhibition of basal (over GHRH-stimulated) GH release was 2.5-fold more prominent in MP than in YM (but not than in PP) (P = 0.028).
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depicts the time course of postnadir recovery of basal (non-GHRH-stimulated) serum GH concentrations. Nadir GH values occurred at statistically comparable median times after the rhGH injection, i.e. 190 min (PP), 200 min (MP), and 210 min (YM) (± 32 min pooled SEM; P = NS by study group). Absolute nadir serum GH concentrations (micrograms per liter) were lower in YM at 0.09 ± 0.02 than MP (0.24 ± 0.08) or PP (0.39 ± 0.13) (P < 0.01). The rate of rise (slope of the linear regression) of postnadir serum GH concentrations was 4.6-fold greater in MP than in PP and YM (P < 0.001).
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, top) and positively predicted fold-reduction of basal (non-GHRH-stimulated) GH concentrations (r = +0.869, P < 0.001) (Fig. 5, bottom).
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).
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The present clinical investigation tests the hypothesis that pubertal boys are resistant to rhGH-induced autonegative feedback, compared with pre- or postpubertal individuals. Statistical comparisons of feedback responses in a controlled autofeedback paradigm revealed several important new insights. First, contrary to our a priori hypothesis, a single pulse of rhGH enforced comparable autoinhibition of exogenous GHRH action before, during, and after sexual maturation. Accordingly, we postulate that GH-induced somatostatin outflow does not differ remarkably among the three developmental strata studied. This notion is based on the primary role of GH-induced somatostatin release in suppressing the response to exogenous GHRH in this paradigm, (see Introduction). Second, a single pulse of rhGH reduced basal (non-GnRH-stimulated) GH concentrations by 4.1- and 7.7-fold more in midpubertal than pre- or postpubertal males, respectively. This unexpected outcome signifies greater rather than less autonegative feedback on spontaneous (endogenous GHRH-driven) GH secretion in pubertal boys. And third, the rate of recovery of GH secretion following the nadir was 4.6-fold more rapid in midpuberty than in prepuberty or adulthood. Accelerated escape from GH autoinhibition could denote heightened hypothalamic secretagogue activity and/or abbreviated hypothalamic somatostatin release in adolescence (below). The foregoing feedback distinctions in pubertal boys were specific mechanistically because sexual maturational status did not influence the kinetics of infused rhGH (Table 2
).
Hypothalamic somatostatin content increases by several-fold in the adult, compared with juvenile animal (26). Orchidectomy represses and androgen repletion restores somatostatin gene expression in the periventricular nucleus (27, 28, 29). On the other hand, whereas puberty also increases somatostatin mRNA in the female rodent, ovariectomy and estrogen repletion do not affect this response consistently (28, 30). Whether this gender distinction applies to the sex steroid-dependent control of somatostatin secretion in the human is not known. In fact, unlike the primary regulatory role of androgen in murine species, estrogen appears to mediate the stimulatory actions of testosterone in the human (1, 31, 32, 33). In this context, the present data are unique in showing developmentally unchanged GH autofeedback on an exogenous GHRH stimulus. This finding would suggest that feedback-induced somatostatin outflow does not increase greatly in pubertal boys.
Infusion of rhGH suppressed basal more than GHRH-stimulated GH secretion in each of the three developmental strata. This distinction presumably reflects the greater stimulatory efficacy of exogenous rh GHRH-1, 44-amide than endogenous GHRH, and/or ghrelin signal in opposing GH feedback-induced somatostatin outflow. The foregoing difference was significantly (2.5-fold) greater in midpubertal boys than young men. Unexpectedly, young men given a pulse of rhGH exhibited 2.4- to 4.2-fold lower absolute nadir GH concentrations than pre- or midpubertal boys, respectively. Lower nadir GH concentrations induced by a potent exogenous GH autofeedback signal (if equally applicable to endogenous GH autofeedback) in young men could contribute to the known decline in GH concentrations in young adulthood, compared with puberty. Indeed, the mechanisms underlying the latter transition are otherwise largely unknown.
The present analysis quantitates autofeedback on basal GH release using an ultrasensitive assay system (19, 21, 25, 34, 35, 36, 37). Thus, our observations will be important to affirm in other assays as well as in additional larger study cohorts. In addition, investigations will be needed to assess the developmental regulation of GH autofeedback in girls.
Boys in midpuberty achieved more rapid postnadir recovery of GH release following rh GH-imposed inhibition. Accelerated escape of GH from suppression could reflect sex steroid-dependent augmentation of feed-forward signaling by hypothalamic secretagogues (e.g. GHRH or ghrelin) and/or abbreviation of the duration of GH-induced somatostatinergic outflow (33). Indeed, both mechanisms would be expected to unleash high-amplitude GH pulses (Fig. 6).
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Linear regression analyses revealed, first, that serum GH concentrations predicted IGF-I concentrations in the ensemble cohort, consistent with expected stimulation of hepatic IGF-I production by systemic GH (47). Second, increased IGF-I concentrations forecasted lesser autofeedback on GHRH-stimulated GH release (Fig. 5, top). The latter association would support but not prove the conjecture that hypothalamic GHRH and/or GHRP drive is elevated in midpuberty, thereby opposing feedback repression but elevating IGF-I output. Third, serum IGF-I concentrations positively determined the magnitude of rhGH-induced inhibition of basal GH release (Fig. 5, bottom). This finding would be consistent with in vivo negative feedback of systemic IGF-I concentrations on basal GH secretion. In this regard, short-term blockade of tissue GH receptors with pegvisomant lowers systemic IGF-I concentrations by 30% and stimulates pulsatile GH secretion by 2-fold in young adults (48). Conversely, iv infusion of rhIGF-I suppresses GH release (33, 40, 41, 49).
In summary, rhGH-induced autonegative feedback on an exogenous GHRH stimulus is comparable in healthy males in pre-, mid-, and postpuberty. Such data indicate that inducible somatostatin outflow does not change greatly with sexual maturation. In contrast, pubertal boys manifest marked fractional autofeedback on endogenous GHRH-driven GH release and more rapid recovery of GH secretion after the nadir. The foregoing tripartite mechanisms could contribute to augmentation of GH pulse amplitude in puberty in boys.
Acknowledgments
Footnotes
Present address for A.D.R.: INSMED, Inc., P.O. Box 2400, Glen Allen, Virginia 23058.
Abbreviations: rhGH, Recombinant human GH.
Received October 29, 2001.
Accepted May 9, 2002.
References
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), and six young men (
), each given a 6-min iv pulse of saline (control, top) or rhGH 3.0 µg/kg (GH, bottom) 120 min earlier (see Fig. 1
