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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 7 3326-3331
Copyright © 2004 by The Endocrine Society

Augmentation of Growth Hormone Secretion after Testosterone Treatment in Boys with Constitutional Delay of Growth and Adolescence: Evidence against an Increase in Hypothalamic Secretion of Growth Hormone-Releasing Hormone

Michael S. Racine, Kathy V. Symons, Carol M. Foster and Ariel L. Barkan

Department of Internal Medicine, Division of Endocrinology and Metabolism (M.S.R., A.L.B.), and Department of Pediatrics and Communicable Diseases, Division of Endocrinology (M.S.R., C.M.F.), University of Michigan Medical Center; and Department of Veterans Affairs Medical Center (K.V.S.), Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Ariel L. Barkan, M.D., Division of Endocrinology and Metabolism, 3920 Taubman Center, Box 0354, University of Michigan Medical Center, Ann Arbor, Michigan 48109. E-mail: abarkan{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The increase in pituitary GH secretion that occurs during mid-late puberty in boys follows an increase in circulating testosterone (T) concentration; the direct mechanism by which this occurs is unknown. We hypothesized that T increases GH secretion during puberty by augmenting hypothalamic output of GHRH. Using constant infusions of a GHRH antagonist, we tested this hypothesis in six early pubertal boys with constitutional delay of growth and adolescence who had a mean chronological age of 14.0 ± 0.3 yr and mean bone age of 11.4 ± 0.2 yr. Blood samples were obtained from subjects every 15 min for 24 h during the overnight infusion of normal saline (2000–0600 h) and again during the overnight infusion of GHRH antagonist (0.33 µg/kg/h) the following night. Subjects then received transdermal T (5-mg patch) for 12 h nightly and were studied again after 4 wk of treatment. Serum samples were assayed for GH and total ghrelin; the percent suppression of GH during GHRH antagonist infusion was calculated. Morning serum T rose from 0.44 ± 0.09 to 4.43 ± 0.74 µg/liter (P = 0.005). T treatment was associated with a 92.6% increase in mean nocturnal GH secretion area under the curve (830 ± 177 to 1599 ± 340 µg/24 h·liter). Infusion of GHRH-antagonist suppressed mean nocturnal GH area under the curve by 29.1% before T treatment (830 ± 177 to 621 ± 168 µg/24 h·liter), and by 29.4% after T treatment (1599 ± 340 to 1182 ± 249 µg/24 h·liter; P = 0.99). Somatotroph sensitivity to GHRH was tested with 0.1- and 1.0-µg/kg doses of GHRH-44 iv; GH response did not change with regard to T treatment. The mean 24-h concentration of total ghrelin was unchanged with regard to T treatment. In summary, nightly transdermal T administration in six boys with constitutional delay of growth and adolescence increased GH output almost 2-fold, whereas the degree of GH suppressibility by GHRH antagonist remained unchanged. We conclude that the T-associated augmentation of GH secretion during early puberty in boys is unlikely to involve an absolute increase in hypothalamic GHRH output.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE RAPID ACCELERATION in linear growth during puberty in boys is associated with a significant transitory increase in pituitary GH secretion (1, 2) with augmentation of GH secretory pulse amplitude and duration, whereas pulse frequency remains unchanged (3, 4, 5, 6). Maximal 24-h GH secretion occurs during mid-late puberty in boys, concurrent with maximal growth velocity that may reach 12 cm/yr (2). Total GH production and concentrations of circulating IGF-I during this time of accelerated growth are similar to those in adults with acromegaly.

The neuroregulatory mechanisms proximate to the pubertal enhancement of GH release are unclear, and the potential relative involvement of augmented hypothalamic GHRH secretion, decreased hypothalamic somatostatin (SRIH) secretion, or augmented gastric ghrelin secretion is uncertain (6). The increased concentration of circulating gonadal steroids that follows the activation of the hypothalamic-pituitary-gonadal axis at the onset of puberty is a prerequisite for normal augmentation of GH secretion and acceleration of growth velocity. Delayed hypothalamic-pituitary-gonadal activation defers the pubertal acceleration of growth in boys with constitutional delay of growth and adolescence (CDGA); administration of the aromatizable androgen testosterone (T) enhances GH pulse amplitude in boys with CDGA (7, 8) and in boys with hypogonadotropic hypogonadism (9) and accelerates growth velocity. In contrast, the nonaromatizable androgens dihydrotestosterone (10, 11, 12) and oxandrolone (7, 8) do not augment GH pulse amplitude. GH secretion during mid-puberty is dampened by the estrogen receptor blocker tamoxifen (13) but is not affected by the androgen receptor blocker flutamide (14). These findings have been widely interpreted as demonstrating that the pubertal enhancement of GH secretion in boys is dependent upon the conversion of T to estradiol (E2) by aromatase.

An increase in hypothalamic GHRH secretion has been proposed as a proximate mechanism accounting for the increase in GH secretion during puberty (6, 9, 10). However, the confirmation of this hypothesis presents a substantial investigational problem, as direct sampling of the pituitary-portal circulation and selective immunoneutralization of GHRH and SRIH are impractical in human subjects. Our group has validated a model for the semiquantification of hypothalamic GHRH secretion in vivo in humans, employing the principle that the suppressibility of GH secretion during the infusion of a reversible, competitive antagonist of the GHRH receptor [(N-Ac-Tyr1, D-Arg2)GHRH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)NH2] is inversely proportional to the magnitude of endogenous hypothalamic GHRH output (15, 16). We have employed this model in examining the effect of transdermal T administration upon GHRH secretion in six boys with CDGA, using a dose of GHRH antagonist (0.33 µg/kg·h) that produced submaximal suppression of GH in young and elderly men (15).

We also measured the serum concentration of total ghrelin in all blood samples before and after T administration and assessed the 24-h ghrelin profiles.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Six boys undergoing an evaluation for short stature were enrolled from the Pediatric Endocrinology Clinic at the University of Michigan. Subjects 2–6 had a family history of CDGA. Subject 5 had a history of infectious colitis. He had been off antibacterial therapy for 1 yr before the study and had normal nutritional status and no gastrointestinal symptoms. A year after the study, his symptoms recurred, and he is under investigation for possible ulcerative colitis. No other child had any significant prior illness. All had normal values for free T4, TSH, IGF-I, IGF binding protein 3, complete blood count, comprehensive metabolic panel, and erythrocyte sedimentation rate. No child had intrauterine growth retardation. Birth weights ranged from 3.1–4.1 kg. All parents were of normal height, within 10–90 percentiles for normal adults.

All subjects had delayed adolescence with absent pubic hair growth but testicular volumes (6–8 cm3), and bone ages were appropriate for their degree of pubertal development. In the absence of another diagnosis, all children were thought to have CDGA at the time of the study. Subject characteristics are shown in Table 1Go. The mean height age was 11.5 ± 0.2 yr, consistent with the mean bone age of 11.4 ± 0.2 yr.


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  		TABLE 1. Subjects characteristics

 
Upon completion of the study the patients remained were followed in the Pediatric Endocrine Clinic at the University of Michigan Medical Center. All of them subsequently entered spontaneous puberty and had marked acceleration of statural growth. Currently, all of them are within the mean ± 2 SD of the normal height distribution.

Procedures

The study was approved by the Institutional Review Board of the University of Michigan Medical School. All subjects participated as paid volunteers. Assent was obtained from all subjects, and written informed consent was provided by a parent or guardian. Each subject was admitted to the University of Michigan General Clinical Research Center on two occasions, before and after 4 wk of T treatment. During each admission, meals were standardized. Lights were turned off at 2200 h and on at 0600 h daily, and sleep was not allowed during daytime hours.

On d 1, subjects were admitted in the evening and had an iv catheter placed in each arm. On d 2, intermittent blood sampling every 15 min for GH and ghrelin began at 0600 h and continued for a 28-h period. At 2000 h, a continuous iv infusion of saline was started and continued through the night. On d 3, GHRH, 0.1 µg/kg and 1.0 µg/kg, was administered iv at 0600 h and 0800 h, respectively. Saline infusion and blood sampling were suspended at 1000 h, 2 h after the second GHRH bolus. Blood sampling every 15 min restarted at 2000 h, concurrent with the start of an iv infusion of GHRH antagonist at 0.33 µg/kg·h. Blood sampling continued overnight until 0800 h, and subjects were discharged the morning of d 4. After the first admission, subjects were prescribed transdermal T (Testoderm 5-mg patch) to be worn nightly for 12 h. Subjects returned after 4 wk for a repeat study, in which the protocol was followed exactly as during the first admission. One of the subjects (number 3) did not start on T after the baseline study as instructed. He started using T patches only after 16 wk and was studied 4 wk later. The T patches were worn overnight during the second admission. The dose of GHRH antagonist (0.33 µg/kg·h) was chosen based on our previous studies in which this dose produced submaximal suppression of nocturnal GH secretion in young and elderly men (15). Thus, the use of this dose optimized our chances to detect any shift in GH suppressibility during T treatment. A limitation of this method is its limited sensitivity, whereby a minimum 3- to 10-fold difference in the GHRH milieu is needed for a reliable performance of the model (15, 16). The interpretation of the data was performed with this limitation in mind.

Hormone assays

Serum GH concentrations were measured in duplicate by chemiluminescent immunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA) with a lower limit of sensitivity of 0.01 µg/liter, and intra- and interassay coefficients of variation of 5.4 and 8.6%, respectively. Total T was measured from pooled serum (collected at each General Clinical Research Center admission from 0500–0800 h during the morning of d 2) by fluoroimmunoassay (Delfia Wallac, Turku, Finland) with a sensitivity limit of 0.035 µg/liter and intra- and interassay coefficients of variation of 4.5 and 7.5%, respectively. Serum IGF-I was measured by RIA (Diagnostic Systems Laboratory, Webster, TX) with a sensitivity of 0.80 µg/liter and intra- and interassay coefficients of variation of 3.4 and 8.2%, respectively. Serum E2 was measured by RIA (Diagnostic Products Corporation, Los Angeles, CA) with a sensitivity limit of 5 pg/ml and intra- and interassay coefficients of variation of 4.7 and 7.6%, respectively. Total ghrelin was measured using an in-house sandwich ELISA with a lower limit of sensitivity of 0.01 µg/liter and intra- and interassay coefficients of variation of 3% each (17).

Statistical analyses

GH secretion was analyzed as area under the curve (AUC) for hormone concentration vs. time for 24-h and nocturnal (2000–0600 h) profiles. Ghrelin data were also calculated as 24-h AUC. The suppressibility of nocturnal GH output by GHRH antagonist was calculated as a percentage of nocturnal GH AUC during the antagonist infusion as compared with GH AUC during saline infusion in the same individual, both before and after T treatment. The percent suppression of GH secretion was calculated as 100% (GH secretion during saline infusion) minus percent residual secretion (GH secretion during GHRH antagonist infusion). GH responses to GHRH-44 were calculated as the differences between the maximal GH concentration within 2 h after GHRH-44 bolus and the GH concentration at time 0, immediately before the GHRH-44 bolus. GH pulsatility was studied by Cluster Analysis version 7.0 with a cluster size of 2 x 2 and t-statistic of 3 and 2 to detect significant increases and decreases in GH, respectively. The program was generously provided by Dr. M. Johnson from the University of Virginia and used in our previous studies (18).

All data are presented as mean ± SE of the mean. Statistical comparisons were conducted using paired Student’s t tests. P values <0.05 were regarded as indicating statistical significance.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
T, E2, and IGF-I

Mean serum concentrations of total T, E2, and IGF-I before and during T administration are shown in Table 2Go. The mean baseline morning T concentration was low and increased during treatment to within the expected range for mid-late puberty males. Concentrations of serum E2 were below the limit of detectability (5 pg/ml) in all subjects before T and remained undetectable in all subjects during T treatment except in subject number 1, in whom it was reported at 5.11 pg/ml (P = 0.36). Baseline mean serum IGF-I was 168 ± 31 µg/liter and increased to 258 ± 55 µg/liter during T treatment (P = 0.048).


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  		TABLE 2. Hormonal parameters before and after T administration

 
Twenty four-hour and nocturnal GH secretion

The compiled 24-h and nocturnal GH AUC data are shown in Table 2Go. The nocturnal GH secretion profiles of a representative subject during saline and GHRH antagonist infusions before and after T are shown in Fig. 1Go.



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  		FIG. 1. Nocturnal GH profiles in a representative patient (no. 1) at baseline (left panel) and after 4 wk of T treatment (right panel). Data are shown for both saline infusion (•) and GHRH antagonist infusion ({circ}). During T treatment, mean nocturnal GH increased by 19%; the percent inhibition of nocturnal GH by GHRH antagonist remained the same (57.1 and 59.1%, respectively).

 
In the six subjects as a group, mean 24-h GH AUC during T increased by 115.7% (P = 0.014; Table 2Go). Mean overnight (2000–0800 h) GH AUC also increased by 92.6% (P = 0.022; Table 2Go).

Data for GH pulsatility as assessed by Cluster Analysis are shown in Table 2Go. After T, mean pulse amplitude increased by 112.3%, from 2.44–5.18 µg/liter (P = 0.007), and mean maximal pulse amplitude increased by 85.8%, from 6.35–11.80 µg/liter (P = 0.006). Pulse frequency was not affected (P = 0.6), and the data are not shown. The mean trough GH concentration (defined as the mean of the five lowest GH concentrations per subject per 24 h) was 0.04 µg/liter before T and 0.09 µg/liter after T (P = 0.005), an increase of 125%.

GHRH antagonist infusion

Before T treatment, the infusion of GHRH antagonist suppressed nocturnal GH secretion in all subjects except number 5, in whom GH secretion was 52% greater during the night of the GHRH antagonist than during saline infusion the previous night. In the other five subjects, GHRH antagonist infusion suppressed mean nocturnal GH AUC by 35.0 ± 9.1% (range, 13.2–57.1%) before T.

During T, nocturnal secretion of GH during the infusion of GHRH antagonist was suppressed in all subjects except number 6, in whom GH secretion increased by 96% during GHRH antagonist. In the remaining five subjects, GH AUC was suppressed by a mean of 35.2 ± 11.7% (range, 13.6–68.3%) after T.

Using the data from the four subjects (numbers 1–4) whose GH secretion was suppressed during GHRH antagonist relative to saline both before and after T, mean GH AUC was decreased by 31.7 ± 9.5% before T and by 39.7 ± 12.1% after T (P = 0.68). Including all six subjects in the analysis, mean GH AUC was suppressed by 29.1 ± 9.5% before T and by 29.4 ± 11.2% after T (P = 0.99; Fig. 2Go).



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  		FIG. 2. Suppression of nocturnal GH output by GHRH antagonist before and during T treatment.

 
Pituitary response to GHRH-44

Pituitary sensitivity to GHRH was tested using consecutive iv boluses of GHRH-44, 0.1 µg/kg and 1.0 µg/kg, during the infusion of saline and before and after T treatment. Response to GHRH-44 was recorded as the peak GH concentration during the 2 h after the bolus minus the GH concentration at the time of the bolus (peak minus baseline), giving a value for {delta}-GH.

GHRH-44 0.1 µg/kg, pre- and post-T

Mean {delta}-GH in response to GHRH-44 0.1 µg/kg before T was 0.6 ± 0.3 µg/liter (range, 0–1.9); the {delta}-GH of subject number 3 was 30.0 µg/liter but was not included in analysis as his GH profile revealed an endogenous GH pulse immediately preceding the administration of GHRH-44. The vigorous GH response in this subject was assumed to have been due to the compounded effect of endogenous and exogenous GHRH. This is a known problem of assessing GH responses to exogenous GHRH (19). After T exposure, mean {delta}-GH in response to GHRH 0.1 µg/kg (again excluding subject number 3) was 2.1 ± 0.8 µg/liter (range, 0.0–5.4), similar to the response before T (P = 0.2).

GHRH-44 1.0 µg/kg, pre- and post-T

Mean {delta}-GH in response to GHRH-44 1.0 µg/kg before T was 4.3 ± 1.0 µg/liter (range, 1.1–7.0; P = 0.03 compared with GHRH 0.1 µg/kg before T). After T, mean {delta}-GH in response to GHRH-44 1.0 µg/kg was 5.3 ± 1.3 µg/liter (range, 1.2–9.6; P = 0.56 compared with {delta}-GH in response to the same dose of GHRH-44 before T).

Plasma ghrelin

Plasma ghrelin was measured at 3-h intervals. The mean plasma total ghrelin concentration in 24 h did not change during T treatment (Table 2Go), and there was no detectable circadian rhythm of ghrelin concentrations before or after T (Fig. 3Go).



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  		FIG. 3. Daily ghrelin profiles (3-h sampling) before and during T treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
GH secretion (4, 5) and serum IGF-I concentrations (20, 21, 22) are highest during the adolescent growth spurt, after physiological priming of the somatotropic axis by gonadal steroid. In boys, T may affect GH secretion via direct pituitary or hypothalamic effects (23, 24), and it has been suggested that GHRH secretion is increased during the pubertal growth spurt (6, 9, 10).

Although the mechanisms that produce the pubertal augmentation of GH secretion are unclear, the decline in GH secretion during the somatopause is associated with decreased hypothalamic GHRH secretion (15, 25). In a previous study using graded GHRH antagonist infusions, we have shown increased GH suppressibility in healthy elderly men as compared with young men with similar levels of T, indicating decreased hypothalamic GHRH output with aging in men (15). When elderly women were compared with young women under the same model, however, we were unable to show similar evidence for decreased hypothalamic GHRH with aging (16). This may be due to a mechanism other than decreased GHRH secretion in the somatopause of women, or could reflect the limited resolution of our experimental paradigm, where a 3- to 10-fold difference in GHRH milieu is necessary to reliably measure a difference in the degree of GH suppression by GHRH antagonist (15). In view of the decrease in GHRH secretion in aged female primates vs. their young counterparts (26), the latter possibility seems more likely.

In the current experiment we hypothesized that the major mechanism that produces the augmented GH secretion of puberty is increased secretion of hypothalamic GHRH, representing a mechanism opposite to that responsible for the somatopause in men. As a model of early puberty in males, we used normal prepubertal volunteers with constitutional delay of growth and adolescence, in whom the controlled administration of sex steroid would allow for observation of the effects of T upon the somatotropic axis. The use of transdermal T patches worn only overnight was designed to reproduce the physiological pattern of increased nocturnal T in normal early-pubertal males.

Ideally, the sensitivity of this subject population to GH suppression by GHRH antagonist would be tested with several doses of the antagonist to calculate the potential shift at the half-maximal inhibition dose. The impracticality of multiple blood sampling episodes in children obliged the use of only a single GHRH antagonist dose, 0.33 µg/kg·h. This dose was chosen from our previous studies where it produced a submaximal suppression of overnight GH output in young and elderly men (15) and women (16), being on the linear part of the dose-response curve. This dose thus provided the best chance to detect a potential shift in the degree of GH suppressibility by a single dose of GHRH antagonist. The doses of GHRH-44 (0.1 and 1.0 µg/kg iv) were also chosen based upon our previous work (15, 16) where these were the minimally and maximally effective doses of this neurohormone to produce an acute rise in serum GH.

The increase in GH pulsatility seen in our subjects is similar to previously published T-induced changes in pubertal boys (4, 5, 6, 7, 8) and is characterized mainly by increased GH pulse amplitude. The mean trough GH concentration, which is taken to reflect interpulse GH secretion, increased significantly by more than 2-fold during T administration, from 0.042 µg/liter to 0.090 µg/liter, suggesting either an increased interpulse (basal) GH secretion rate or lengthened serum half-life of GH. In a previous study, using infusions of GHRH antagonist, we demonstrated that basal GH secretion in men was not affected by GHRH antagonist infusion (18). Our current finding of a doubling of trough GH concentration may suggest a decrease in hypothalamic SRIH secretion during early puberty in males, in that the interpulse GH secretion rate in males may inversely reflect hypothalamic SRIH secretion (27).

In most studies of pubertal GH secretion in boys, the aromatization of T to E2 appears to be necessary for augmentation of somatotrope output (10, 11, 12, 13, 14), and serum E2 concentration was correlated in a positive, linear fashion with the basal GH secretion rate in prepubertal children and late adolescents as reported by Veldhuis et al. (6). In previous work, however, we found that an infusion of E2 in pubertal boys failed to augment bioactive or immunoreactive GH concentrations (28). E2 may oppose the GH-suppressive actions of SRIH in postmenopausal women (29), although whether that finding applies to boys during puberty is unknown. In the present study, serum E2 levels remained low during the administration of T, a finding that mirrors previously published results (2, 9) and that fails to directly support a role of increased circulating E2 in the enhancement of GH secretion during puberty. A role for intrapituitary or hypothalamic aromatization of T to E2 or for biologically relevant changes in serum E2 concentrations below the detectability limit of the assay remain a possibility.

The increased GH secretion of puberty is not apparently due to enhanced somatotrope sensitivity to native GHRH stimulation. In our subjects, GH response to low-dose and maximal physiological GHRH stimulation was similar before and after T exposure, a finding that supports data reported previously (9). The variability of GH responses to single GHRH boluses complicated the analysis, as was noted previously by Cho et al. (30). The variability can be theoretically minimized by short-term pretreatment with somatostatin (30), but this complicates the study and introduces yet another variable; hence, we decided against using this approach.

Previous studies in humans and animals (31, 32, 33) reported conflicting data on plasma and stomach ghrelin during sexual maturation and after administration of gonadal steroids. Our ghrelin assay measured total serum ghrelin, which might have missed meaningful changes in bioactive ghrelin during puberty. We could detect no circadian rhythm of total ghrelin concentration either before or after T, and mean ghrelin concentrations were not different after vs. before T replacement. In two previous cross-sectional reports of serum ghrelin concentrations in normal children, fasting ghrelin decreased from early childhood through adolescence (34, 35). To ascertain the role of ghrelin in the neuroregulation of GH secretion during puberty, if any role exists, it will be necessary to explore any potential alterations in the concentrations of active ghrelin by sex steroids and during puberty.

Although we could detect no change in GHRH secretion in our subjects, the limitations of our method allow for the possibility that small changes in hypothalamic GHRH secretion, undetectable by our experimental paradigm, may result from exposure to T in early puberty. It is also possible that a measurable increase in GHRH secretion occurs with further pubertal maturity in males, as the current protocol examined the effects of T upon GH secretion in only the earliest stage of artificial pubarche. It is of interest that in our group of early pubertal boys the degree of GH suppressibility by GHRH antagonist, 0.33 µg/kg·h (~29%), was clearly higher than in young healthy men (~5–7%) and similar to that observed in elderly men during the somatopause (~30–50%) (15, 36). This suggests that GHRH output in our subjects might have been relatively low and could presumably increase with the progression of puberty. Whether this would be due to a longer exposure to T or to an activation of another, T-independent, mechanism is unknown. However, Giustina et al. (9) have demonstrated doubling of GH output after 4 wk of im T therapy in men with hypogonadotropic hypogonadism, and Metzger and Kerrigan (13, 14) have shown marked changes in GH secretion in normal boys exposed to flutamide and tamoxifen for only 3–4 d. Thus, the duration of our experimental paradigm was sufficiently long, and one should postulate either insufficient sensitivity of our method to detect changes in GHRH output or a true absence of such changes as an explanation of our data.

In conclusion, we could detect no change in hypothalamic GHRH secretion in a model of early puberty in boys, despite a doubling of 24-h GH secretion due to increased pulse amplitude after T exposure. The mean trough interpulse GH concentration was also doubled by T, suggesting that basal interpulse GH secretion is increased during puberty. The concentration of circulating total ghrelin was unaffected by T administration and demonstrated no discernible diurnal variation. We conclude that the augmentation of GH secretion in early puberty in boys may occur via a mechanism distinct from the sole augmentation of hypothalamic GHRH secretion. The combination of increased GH pulse amplitudes and interpulse GH levels under the influence of T may reflect diminution of hypothalamic somatostatin secretion.


   Acknowledgments
 
We thank all the participants in this study for their cooperation, the staff of the University of Michigan General Clinical Research Center for their excellent care of the subjects, Ms. Pamela Olton and Ms. Roberta DeMott-Friberg for their invaluable technical help, and Dr. Morton Brown for his assistance with the statistical analysis.


   Footnotes
 
This work was supported by USPHS Grants RO-1-DK-38449 (A.L.B.) and MO-1-RR00042 (University of Michigan General Clinical Research Center) and the Department of Veterans Affairs Medical Research Service (A.L.B.).

Abbreviations: AUC, Area under the curve; E2, estradiol; SRIH, somatostatin; T, testosterone.

Received November 10, 2003.

Accepted March 19, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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