| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Michigan Medical Center (J.J.O., E.D., A.L.B.), and Department of Veterans Affairs Medical Center (K.S., A.L.B.), 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, Room 0354, University of Michigan Medical Center, Ann Arbor, Michigan 48109. E-mail: abarkan{at}umich.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The close interdependence between T and GH has been demonstrated in prepubertal and pubertal boys and hypogonadal men. The administration of exogenous T or stimulation of endogenous T production in hypogonadal or prepubertal models increases circulating GH and IGF-I concentrations (6, 7, 8, 9, 10).
Recently, Eakman et al. (11) suggested that the administration of T to a group of boys with constitutional delay in growth and adolescence increased GH pulse amplitude via augmented GHRH pulse amplitude. This effect appeared to be mediated via an estrogen-dependent mechanism, a finding previously reported by several other investigators (12, 13, 14, 15). However, little is known about the impact of T repletion on the somatotropic axis in older men.
Direct experimental approaches for the measurement of GHRH in humans, such as pituitary-portal or peripheral blood sampling, are either impractical or do not reflect its hypothalamic output, respectively. We have recently developed a model for semiquantification of hypothalamic GHRH output in vivo (16, 17). This model followed the approach proposed and validated by Hall et al. (18) for semiquantification of hypothalamic GnRH secretion during the different stages of the menstrual cycle and by Ferin et al. (19) and Cicero et al. (20) for assessment of endogenous opioid tone in rats. This model is based on well established principles of pharmacodynamics (21). By giving graded doses of a specific competitive GHRH receptor antagonist (N-Ac-Tyr1,D-Arg2)GHRH-(1–29)-NH2, we found a left shift of the dose-inhibition curve for a group of elderly men. Although other physiological mechanisms may account for this finding, as extensively discussed in our previous studies (16, 17), the simplest explanation is an age-dependent decrease in the hypothalamic GHRH output contributing to the somatopause in men (16).
We used the same experimental approach (16, 17) to examine whether the decline in GHRH output in elderly men is consequent upon their lower plasma T concentrations. We found that physiological concentrations of serum T created by short-term (5–6 wk) transdermal T administration in elderly men did not augment pituitary GH output and did not change the suppressibility of nocturnal GH secretion by the submaximal dose of GHRH antagonist.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The protocol was approved by the institutional review board of the University of Michigan Medical School and the Department of Veterans Affairs Medical Research Service. Each man signed a consent form before his participation in the study. Six young (20–23 yr old) and seven elderly (61–76 yr old) healthy men were recruited from the community. None was taking any medication known to modify GH or gonadotropin/T secretion. All were night sleepers. Elderly men were physically active and engaged in recreational exercise. The body mass index (BMI) was 27 kg/m2 or less in all participants. Elderly subjects were excluded if they had a history of prostate cancer, symptomatic benign prostatic hyperplasia, or other significant medical problems. The demographic data are summarized in Table 1
.
|
Young and elderly men were admitted to the General Clinical Research Center of University of Michigan on two and four different occasions, respectively. They were admitted at 2000 h on d 1 and were discharged at 0800 h on d 3. A heparin-filled venous cannula was placed in each arm, one for infusions and boluses, and another for blood sampling. Lights were turned off at 2300 h and on at 0700 h. Standard hospital meals were served at 0800, 1300, and 1900 h. Blood sampling was performed every 20 min from 0600 h on d 2 to 0800 h on d 3. Continuous iv infusions of normal saline or GHRH antagonist (0.33 µg/kg·h) were administered during each visit, from 0400 h on d 2 to 0800 h on d 3. The order of the infusions was randomized, and a minimum of 3 d elapsed between admissions. Sleeping was not allowed before 2300 h or after 0700 h. An iv bolus of GHRH-44 (Peninsula Laboratories, Inc., Belmont, CA; 0.33 µg/kg) was administered at 0600 h on d 3.
Upon completion of the first two visits, elderly patients started applying Testoderm TTS (Alza Corp., Palo Alto, CA; 5 mg every morning) for 35–40 d (until the end of the study), and the above studies were repeated. The T patches were rotated every 24 h as recommended by the manufacturer. Prostate-specific antigen was measured at the beginning and end of the study.
GHRH antagonist [(N-Ac-Tyr1,D-Arg2)GHRH-(1–29)] was manufactured by Bachem (King or Prussia, PA) according to good medical practice conditions. It was formulated by the investigational pharmacy of University of Michigan Hospital as 5 mg/ml solution in normal saline. The stock solution was stored at –20 C, thawed immediately before administration, and diluted in normal saline containing human serum albumin (1%) to prevent adherence of the peptide to the plastic.
Body composition
Total fat mass, lean body mass, and fat percentage were assessed in all elderly men and in five of the young men using a total body dual energy x-ray absorptiometry scan (IQ analysis software version 4.1, Lunar Corp., Madison, WI).
Assays
Blood samples were centrifuged, and plasma was stored at –20 C until assayed. All assays were run in duplicate. Plasma GH was measured in a chemiluminometric assay (Nichols Institute, Inc., San Juan Capistrano, CA), and the assay sensitivity was 10 ng/liter. The mean intraassay coefficient of variation (CV) was 4.7%, and the mean interaasay CV was 8.6%. Total IGF-I was measured after acid-ethanol extraction by a two-site immunoradiometric assay (Diagnostic Systems Laboratories, Inc., Webster, TX) with a sensitivity of 10 ng/liter. The mean intraassay CV was 6.5%, and the mean interassay CV was 5.4%. Free T was measured by a solid phase 125I RIA (Diagnostic Products Corp.) with a sensitivity of 0.15 ng/liter. The mean intraassay CV was 7.8%, and the mean interassay CV was 11.8%. Total T was measured by a solid phase 125I RIA (Diagnostic Products Corp.) with a sensitivity of 40 ng/liter. The mean intraassay CV was 5%, and the mean interassay CV was 7.9%. Dihydrotestosterone (DHT) was measured by RIA after extraction chromatography (Esoterix Laboratories, Calabasas Hills, CA) with a sensitivity of 20 ng/liter. The mean intraassay CV was 7.4%, and the mean interassay CV was 10.5%. Estradiol was measured by a no extraction, solid phase 125I RIA (Diagnostic Products Corp.) with a sensitivity of 8 ng/liter. The mean intraassay CV was 5.3%, and the mean interassay CV was 6.4%.
Calculations
The integrated GH concentration (IGHC; micrograms per liter per minute) was calculated by the trapezoidal rule. Maximal GH (micrograms per liter) was defined as the highest GH concentration measured during the specific time interval. In each subject, GH output (spontaneous or GHRH induced) during GHRH antagonist infusion was calculated as a percentage of the corresponding GH measurement during baseline saline infusion (percent residual output). The percent suppression of total 24-h GH by GHRH antagonist was calculated as 100% – the percent residual output. The responses to GHRH stimulation were defined as IGHC over the 2-h period after the GHRH bolus injection.
Discrete parameters of GH pulsatility were assessed using Cluster analysis of individual GH profiles (22). A power function fit of local variance, a 2 x 1 cluster size, and a t statistic of 2 were used. Cluster-identified GH pulses with an amplitude less than 0.03 µg/liter were excluded from the analysis because they could not be distinguished from the assay noise (23). Nadir GH was defined as the mean of the three lowest GH concentrations during the 24-h sampling, i.e. values belonging to the lowest 5% of the data array.
Statistical analysis
As the degree of GH suppression by GHRH antagonist was the primary outcome parameter for this study, we have planned it using the power analysis based on data obtained by us previously (16) in identical subjects receiving the same dose of GHRH antagonist. With six subjects per group we had 80% power to detect the difference of 1.7 SD between young and elderly subjects when using a two-tailed, two-sample t test at a 5% level of significance. The study of data from seven elderly subjects using a paired two-tailed t test at a 5% level of significance provided 80% power to detect a true difference of 1.3 SD in the degree of GH suppressibility by GHRH antagonist. Thus, the size of the groups employed in this study provided sufficient power to detect relevant changes in the points of interest based on our earlier use of this model. Data are presented as the mean ± SE. Data were analyzed by paired or nonpaired two-tailed t tests as appropriate. Statistical significance was assumed for P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Despite similar BMI, elderly men had higher fat mass, percentage of body fat, and waist to hip ratio (WHR) and lower lean mass than their younger counterparts (Table 1
). The only measured sex hormone that proved to be significantly lower in elderly subjects was free T (Table 2). Indeed, five of the elderly men had levels of free T in the hypogonadal range (three of them had also lower than normal total T levels).
|
Elderly men had lower IGHC, maximum GH, and IGF-I compared with young subjects (Table 2). Administration of 0.33 µg/kg GHRH-44 during the saline infusion (non-T) day elicited similar GH responses in both groups (204 ± 35 vs. 367 ± 84 µg/min·liter; P = 0.09).
Effects of GHRH antagonist
A GHRH antagonist dose of 0.33 µg/kg·h was ineffective in suppressing GH output in the young, but lowered it markedly in the elderly subjects (5.5 ± 9.9% vs. 35.8 ± 2.6%; P = 0.009). The percent suppression of 24-h GH output by GHRH antagonist in elderly men did not change after T delivery (35.8 ± 2.6 vs. 27.7 ± 6.5%, P = 0.24) during the GHRH antagonist infusion (Fig. 1).
|
Delivery of Testoderm TTS to elderly men did not significantly change their weight or WHR compared with their pretreatment values. Likewise, prostate-specific antigen levels were unchanged (data not shown).
Effect of exogenous T on sex hormones
Total T, free T, and DHT increased significantly after T administration [521.5 ± 56.3 vs. 395.4 ± 57.2 ng/dl (P = 0.021), 13.8 ± 1.3 vs. 10.1 ± 1.7 pg/ml (P = 0.017), and 71.4 ± 8.9 vs. 41 ± 8.1 ng/dl (P = 0.004), respectively]. Estradiol concentrations remained unchanged (Table 2). Free T normalized in all but one elderly man after T delivery. Total T became normal in all three subjects with previously low values. Actual dynamics of T and free T during T replenishment are shown in Fig. 2.
|
)There was no appreciable suppression by GHRH antagonist of the GH response to GHRH in the young men or the elderly men before and after T administration (P > 0.5 for all comparisons).
|
Discrete parameters of GH pulsatility are shown in Table 3. GH pulse frequency was similar in young and elderly men at baseline and did not change in the elderly during T replenishment. The same was true with regard to the nadir GH. The only parameter that distinguished between the young and the elderly subjects was mean GH pulse amplitude (P < 0.01), but it did not change in the elderly subjects during T replenishment.
|
Effect of exogenous T on GH parameters
IGHC decreased significantly after T supplementation (872 ± 115 vs. 1027 ± 121 µg/min·liter; P = 0.026), but maximum GH did not change significantly (5.3 ± 0.4 vs. 5.7 ± 0.9 µg/liter; P = 0.74). Plasma IGF-I did increase mildly, but in a statistically significant fashion (139.1 ± 10.4 vs. 129.1 ± 10.8 ng/ml; P = 0.02; Table 2). The GH response to 0.33 µg/kg GHRH-44 during the saline infusion day was not affected by T replenishment in the elderly men (226 ± 46 vs. 278 ± 84 µg/min·liter; P = 0.47).
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Similar to previous studies (24, 25, 26, 27, 28), elderly men had lower baseline IGHC, GH pulse amplitude, and IGF-I levels than younger men. Likewise, although both populations had similar BMI values, the elderly had higher fat mass, percentage of body fat, and WHR. It is known that age and relative adiposity are specific negative determinants of the frequency and amplitude of GH secretory pulses of GH in healthy men (29). Relative adiposity, particularly in the abdominal region (and even more so in the intraabdominal compartment), is a major negative determinant of stimulated GH secretion (30, 31). Therefore, the possibility of the increased adiposity in our older subjects causing lower parameters of GH secretion cannot be excluded. Indeed, in our earlier study (16) nocturnal GH output became identical in young and elderly subjects matched for degree of adiposity. Importantly, however, the difference in the ability of GHRH antagonist to suppress nocturnal GH output was still preserved between the young and elderly subjects despite perfectly matched body composition parameters (16). This further strengthened our conclusions of the inherently diminished endogenous GHRH milieu in elderly men.
It was also not surprising that five of the seven elderly men had levels of free T below the normal range (three of them had also low total T). These values normalized in all but one patient after short-term T supplementation. In contrast to free T, the baseline mean total T was not significantly different between young and elderly subjects, although three of the seven elderly subjects had subnormal total T concentrations. Thus, overall, the elderly subjects studied here presented a typical degree of aging-associated hypogonadism observed by other investigators (1, 2, 3). However, both mean final free and total T concentrations increased significantly after T administration into the young normal range. Estradiol was unchanged, but DHT was significantly increased with exogenous T supplementation, probably as a result of T exposure to high levels of 5
-reductase in the skin. Therefore, with our intervention we were able to achieve our goal of increasing the circulating androgen milieu without changing estradiol levels, thus isolating the androgen effect mediated through the androgen receptor.
It is clear that restoration of plasma T concentrations in prepubertal boys (6, 7, 8) or in hypogonadal men (9, 10) increases IGHC and IGF-I. However, little is known about the impact of T repletion on the somatotropic axis in older men. A recent study (32) found that only high doses of im T (200 mg weekly), resulting in supraphysiological androgen concentrations, significantly stimulated basal, pulsatile, and total daily GH secretion and normalized serum IGF-I concentrations in the elderly. In the present study transdermal T administered to elderly men did not normalize the overall GH output or discrete parameters of GH pulsatility. These observations contradict the hypothesis that physiological hypogonadism in elderly men is responsible for the somatopause. Whether more prolonged administration of T would be more effective is not known, but is unlikely, taking into account that even a shorter duration of T exposure was sufficient to augment GH output in boys with constitutional growth delay or isolated gonadotropin deficiency (10, 15). Interestingly, during transdermal T replacement that selectively increased the circulating androgen (but not the estrogen) milieu, plasma IGF-I levels increased slightly, but significantly, in the elderly men in parallel with a concomitant decline in the GH daily output. This is in complete agreement with our earlier data in rats (33), where DHT suppressed plasma GH and selectively increased both circulating IGF-I concentrations and hepatic IGF-I mRNA levels. Whether in our current study the rise in IGF-I was the actual cause of GH suppression is uncertain, taking into account that plasma IGF-I remained grossly subnormal. In any case, it suggests that the previously observed augmentation of GH secretion in the elderly men treated with supraphysiological doses of im T (32) was an artifact of high estrogen levels that were probably achieved by the peripheral conversion of T within the increased fat mass.
Our findings here contrast with those of previous experiments performed in rats. In that research (34, 35), castrated adult male rats had less GHRH mRNA in their hypothalami than intact rats or T-replete castrated rats. Exogenous administration of DHT, but not estradiol, prevented this deficiency from occurring. To assess the potential effects of androgens on GHRH secretion, we used a paradigm for semiquantification of GHRH output, as we previously demonstrated in men (16) and women (17), that was based on the original model by Hall et al. (18). We previously found that GHRH antagonist (0.33 µg/kg·h) suppressed IGHC in young men by 7 ± 13% compared with 59 ± 6% in the elderly (16). We here fully confirm these results in separate groups of subjects and, in addition, demonstrate that administration of transdermal T to elderly men, with subsequent normalization of their serum androgen concentrations, does not close the gap between young and elderly men.
It has been shown that exogenous administration of GHRH can rejuvenate the somatotropic axis, with normalization of GH and IGF-I (36). We found in this study that a bolus of GHRH-44 given during the saline infusion day elicited similar GH responses in young and elderly men. These data confirm the fact that pituitary insensitivity to GHRH is not the explanation for the somatopause. Even though there appeared to be a statistical trend toward lower GH responsiveness in the elderly, this was probably due to relatively small numbers of subjects per group, as we have previously shown (16, 17) in larger groups of similar subjects that pituitary sensitivity to GHRH is equal in young and elderly subjects with similar BMI values. We could not detect any significant suppression of the GH responses to bolus GHRH (0.33 µg/kg) by submaximal doses of GHRH antagonist in young or elderly men (both before and after T replenishment). Coupled with the observation that this dose of GHRH antagonist did not noticeably suppress endogenous GH output in the young, but was very effective in this regard in the elderly, this suggests that the magnitude of endogenous GHRH pulses in the latter group was markedly lower than that produced by the GHRH bolus employed here. This strengthens our original conclusion of the diminution of GHRH output in the elderly. Perhaps using a lower dose of exogenous GHRH might have been more informative (16). The variability of serial nocturnal GH outputs and of GH responses to GHRH had been noted in our previous study (16) and might have affected the calculated percent GH suppression by GHRH antagonist in this group. However, the sufficient number of subjects studied (as determined by power analysis) and the similarity of the pre- and post-T values argue strongly against the possibility that a real difference might have been missed. Most importantly, administration of T significantly suppressed the 24-h GH output in the elderly. This serves as additional evidence that augmentation of GHRH secretion by T replenishment is highly unlikely.
In summary, with the administration of transdermal T to a group of elderly men we were able to bring their total and free T concentrations into the range seen in young men. With the new T milieu, we expected to augment the hypothalamic GHRH output in elderly men with a subsequent increment in the parameters of GH secretion. However, the discrete parameters of pulsatile GH secretion did not apparently increase, and the ability of GHRH antagonist to suppress endogenous GH output did not change. In fact, selective normalization of androgen concentrations coupled with the supraphysiological DHT milieu and unchanged E2 concentrations actually decreased the 24-h GH output. We conclude that the androgen deficiency of old age is unlikely to be the proximate cause of the somatopause.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: BMI, Body mass index; CV, coefficient of variation; DHT, dihydrotestosterone; IGHC, integrated GH concentration; T, testosterone; WHR, waist to hip ratio.
Received September 3, 2003.
Accepted March 28, 2004.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  R. Muniyappa, J. D. Sorkin, J. D. Veldhuis, S. M. Harman, T. Munzer, S. Bhasin, and M. R. Blackman Long-term testosterone supplementation augments overnight growth hormone secretion in healthy older men Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E769 - E775. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Racine, K. V. Symons, C. M. Foster, and A. L. Barkan 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 J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3326 - 3331. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
