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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2007-0043
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 6 2336-2345
Copyright © 2007 by The Endocrine Society

Tripartite Control of Growth Hormone Secretion in Women during Controlled Estradiol Repletion

Johannes D. Veldhuis, Mihaela Cosma, Dana Erickson, Remberto Paulo, Kristi Mielke, Leon S. Farhy and Cyril Y. Bowers

Endocrine Research Unit 9 (J.D.V., M.C., D.E., R.P., K.M.), General Clinical Research Center, Mayo Medical and Graduate Schools of Medicine, Mayo Clinic, Rochester, Minnesota 55905; Division of Endocrinology and Metabolism (L.S.F.), Department of Internal Medicine, School of Medicine, University of Virginia, Charlottesville, Virginia 22908; and Endocrine Division, Department of Medicine (C.Y.B.), Tulane University Health Sciences Center, New Orleans, Louisiana 70112

Address all correspondence and requests for reprints to: Johannes D. Veldhuis, Endocrine Research Unit 9, General Clinical Research Center, Mayo Medical and Graduate Schools of Medicine, Mayo Clinic, Rochester, Minnesota 55905. E-mail: veldhuis.johannes{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Studies of how aging attenuates GH secretion are confounded by differences in sex-steroid milieus, abdominal visceral fat mass (AVF), and IGF-I concentrations and limited in interpretability by the use of pharmacological doses of secretagogues.

Hypothesis: In a controlled estrogenic milieu, near-physiological secretagogue drive will unmask distinct influences of age, AVF, and IGF-I on GH secretion.

Location: The study was conducted at an academic medical center.

Subjects: Subjects included 10 healthy pre- (PRE) and 10 postmenopausal (POST) women.

Procedure: In a defined estradiol (E2) milieu, we compared GH secretion after submaximal stimulation with GH-releasing peptide (GHRP)-2 (ghrelin analog), GHRH, and L-arginine (an inhibitor of somatostatin outflow).

Analysis: We related GH responses to age stratum (dichotomous variable) and AVF and IGF-I concentrations (continuous variables).

Results: In the face of comparable concentrations of E2, testosterone, and SHBG: 1) age (P < 0.001) and secretagogue type (P < 0.001) independently determined GH secretion; 2) GH responses in POST subjects were only 26–33% of those in PRE (P ≤ 0.002) across all secretagogues; 3) POST women lost the PRE order of secretagogue potency (GHRP-2 > GHRH = L-arginine); and 4) in the combined cohorts, higher AVF predicted reduced L-arginine-stimulated GH secretion (R2 = 0.46, P = 0.0013), whereas higher IGF-I concentrations forecast increased GHRP-2 and GHRH drive (R2 ≥ 0.52, P ≤ 0.013).

Conclusion: A paradigm of near-physiological secretagogue drive in an E2-clamped milieu unmasks tripartite deficits in peptide-signaling pathways in healthy POST, compared with PRE, women. Post hoc analyses indicate that both greater visceral adiposity and lower IGF-I concentrations mark this triple regulatory defect.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GH AND ESTRADIOL (E2) concentrations decrease in parallel in aging individuals (1, 2). In cross-sectional analyses, GH concentrations in premenopausal (PRE) women decline at 50% the rate inferred in men of similar age (3, 4). Gonadal estrogen secretion may contribute to preservation of GH production in young women because GH concentrations fall rapidly after pharmacological ovarian suppression, surgical ovariectomy, or natural menopause (5, 6, 7, 8, 9). Conversely, oral, parenteral, high-dose transdermal and intranasal administration of estrogens and ovarian sex-steroid secretion augment GH production (10, 11, 12, 13, 14, 15, 16, 17, 18, 19).

Because of the strong correlation between E2 availability and GH secretion throughout puberty and adulthood, the basis for hyposomatotropism in the estrogen-deficient menopause remains difficult to parse precisely (7, 10, 20). In particular, one cannot readily distinguish the roles of age vis-à-vis estrogen deprivation in mediating low GH output. The issue is significant clinically because age, hypogonadism, and organic GH deficiency share a phenotype of relative adiposity, sarcopenia, and osteopenia reduced aerobic capacity, peripheral insulin resistance, hyperlipidemia, and low IGF-I concentrations (7, 21, 22, 23, 24). In relation to increased adiposity in aging people, a vicious cycle may ensue, given that abdominal visceral fat (AVF) is a negative predictor of GH secretion (24, 25, 26). On the other hand, lower IGF-I concentrations would be expected to unleash and thereby restore GH secretion via feedback withdrawal (27).

From an experimental perspective, distinguishing the effects of age, gonadal sex steroids, visceral adiposity, and low IGF-I concentrations on GH secretion remains challenging for several reasons. First, most studies have assessed the effects of estrogen administration in postmenopausal (POST) individuals without including PRE controls (7). Second, virtually all investigations to date have used maximally stimulatory doses of GH secretagogues (28, 29), thereby restricting inferences to pharmacological rather than physiological effects. Third, the few direct comparisons made between POST and PRE subjects have evaluated only a single secretagogue type or fixed secretagogue pairs (5). This design precludes an evaluation of relative secretagogue potencies. Fourth, although ghrelin is secreted nearly continuously, clinical protocols usually inject the peptide or an analog as a single nonphysiological bolus (10). How this mode of administration alters age-related responses is unclear. Fifth, only three studies to our knowledge have attempted to reduce confounding by experimentally imposing an identical gonadal sex-steroid milieu in both POST and PRE women (30, 31, 32). And sixth, no analyses have addressed the foregoing issues together, whereas relating GH responses to concomitant AVF mass and IGF-I concentrations.

An investigative paradigm introduced recently by Erickson et al. (30) used a GnRH agonist to down-regulate the pituitary-ovarian axis and transdermal delivery of E2 to sustain comparable estrogen milieus in POST and PRE volunteers. The present study implements this paradigm and submaximal (rather than pharmacological) peptide stimuli to test the hypotheses that age impairs multipathway control of GH secretion, whereas AVF burden and IGF-I concentrations determine pathway-selective drive of GH secretion.


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

Volunteers provided written informed consent and were compensated for time spent in the study according to Mayo institutional review board-approved guidelines. Enrollment comprised 10 healthy PRE subjects of mean ± SEM age 21 ± 1.1 yr (range 19–29) and 10 POST volunteers ages 64 ± 1.4 yr (range 57–70). No woman had received birth control pills or hormone replacement for at least 3 wk before the first leuprolide injection. Pregnancy was excluded by blood human chorionic gonadotropin measurement. Body mass index in PRE averaged 23 ± 0.97 kg/m2 (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) and in POST individuals 26 ± 1.2 (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) kg/m2 (P = NS). Exclusion criteria were any history, symptoms, or signs of ischemic or occlusive arteriovenous events; hepatic, renal, cardiac, pulmonary, malignant, or infectious disease; untreated triglyceride-predominant hyperlipidemia; untreated cholelithiasis; known or suspected breast neoplasm; acute illness; anemia (hemoglobin < 11.8 gm/dl); ongoing psychiatric treatment; drug or alcohol abuse; use of neuroactive drugs, such as antidepressant, antihypertensive, or anticonvulsant agents; more than 3 kg weight change in 6 wk; nightshift work; and unwillingness to provide informed consent. Inclusion criteria were community-living, consenting, informed healthy women aged 18–35 or 50–80 yr. PRE status was defined by cyclic menses with a history of puberty. POST status was defined by amenorrhea for at least 2 yr, FSH more than 45 IU/liter, LH more than 25 IU/liter, and E2 less than 35 pg/ml.

Computerized axial tomography at L4–5 was used to estimate abdominal visceral fat mass, as described earlier (31).

Clinical protocol

The trial was a parallel-cohort comparison of the effects of age on secretagogue actions during controlled estrogen repletion. To achieve age-independent estrogen depletion, the GnRH agonist, leuprolide acetate (3.75 mg im), was administered twice 3 wk apart (30). Leuprolide was given to both POST and PRE subjects to obviate any potential confounding by the down-regulation regimen. The first injection was given in young volunteers within 8 d of menstrual bleeding and 48 h of a negative blood pregnancy test and in older women 3 or more wk after withdrawal of any estrogen supplements. Graded transdermal estradiol repletion was accomplished on an outpatient basis, starting on the day of the second leuprolide injection. The transdermal E2 dose was increased every 4 d beginning at 0.05 mg/d followed by 0.10, 0.15, and 0.20 mg/d [Estraderm (Novartis, Basel, Switzerland)]. The highest E2 dose (0.2 mg/d) was administered for 10 d (d 14–23 inclusive). Infusion studies were performed during the last week of this 10-d window (Fig. 1Go). The transdermal paradigm was designed to elevate serum E2 concentrations into the preovulatory range of 100–150 pg/ml (30, 31). Starting on the last day of the study, oral micronized progesterone (100 mg nightly) was administered for 12 d, according to standards of good medical practice for women with an intact uterus.


Figure 1
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  		FIG. 1. Schema of infusion paradigms used. After a standardized overnight fast, women undertook the indicated five infusion regimens on separate days in randomly assigned order. Saline was infused for 120 min (0800–1000 h), and selective secretagogues were infused beginning at 1000 or 1030 h either as a bolus (GHRP-2 and GHRH, 0.33 µg/kg) or continuously (GHRP-2 and GHRH, 0.33 µg/kg·h x 4 h and L-arginine 30 g over 30 min). Subjects were studied during the last week of a leuprolide-E2 clamp (see Subjects and Methods).

 
Infusion schedule

Five distinct, randomly ordered, separate-day infusion sessions were undertaken in each woman. At 1800 h the night before each study, volunteers received a standardized outpatient meal of 8 kcal/kg distributed as 20% protein, 50% carbohydrate, and 30% fat. Subjects then remained fasting overnight and until the end of sampling. At 0700 h the next morning, two iv catheters were placed in (contralateral) forearm veins to allow simultaneous secretagogue infusion and blood sampling (1 ml) every 10 min for 6 h from 0800 to 1400 h. The five infusion sessions were as follows: 1) GHRH delivered continuously from 1000 to 1400 h at a constant rate of 0.33 µg/kg·h; 2) GH-releasing peptide (GHRP)-2 delivered continuously from 1000 to 1400 h at a constant rate of 0.33 µg/kg·h; 3) L-arginine 30 g delivered continuously from 1000 to 1030 h; 4) GHRP-2 (0.33 µg/kg) iv bolus at 1030 h; and; 5) GHRH (0.33 µg/kg) iv bolus at 1030 h.

The foregoing peptide doses approximate 50% of maximal stimulation (33, 34).

Hormone assays

Plasma GH concentrations were measured in duplicate by automated double-monoclonal immunoenzymatic chemiluminescence assay using 22-kDa recombinant human GH as assay standard (Sanofi Diagnostics Pasteur Access, Chaska, MN) (30). All samples (n = 148) from any given subject were analyzed together. Sensitivity was 0.010 µg/liter (defined as 3 SD above the zero-dose tube). Interassay coefficients of variation (CVs) were 7.9 and 6.3%, respectively, at GH concentrations of 3.4 and 12.1 µg/liter. Intraassay CVs were 4.9% at 1.12 µg/liter and 4.5% at 20 µg/liter. No values fell less than 0.020 µg/liter. Cross-reactivity with 20 kDa GH is less than 5%. Serum LH and FSH concentrations were quantitated by automated chemiluminescence assay (ACS 180; Bayer, Norwood, MA), using as standards the First and Second International Reference Preparations, respectively. Procedural sensitivities for LH and FSH are 0.2 and 0.4 IU/liter. Intraassay CVs for LH were 4.7, 3.5, and 3.8%, and interassay CVs were 8, 3.7, and 4.7% at 4.4, 18.2, and 38.8 IU/liter, respectively. For FSH measurements, intraassay CVs were 5.6, 4.3, and 3.5% and interassay CVs 6, 4, and 2.8% at 4.6, 25.4, and 61.7 IU/liter, respectively. E2 and testosterone were quantified by automated competitive chemiluminescent immunoassay (ACS Corning, Bayer, Tarrytown, NY). For E2, intraassay CVs were 4.1% at 173 pg/ml and 3.9% at 371 pg/ml. Interassay CVs were 7% at 71.2 pg/ml and 4% at 261 pg/ml (multiply by 3.67 for pmol/liter). For testosterone, mean intra- and interassay CVs were 6.8 and 8.3%, with an assay sensitivity of 8 ng/dl (multiply by 0.0347 for nanomoles per liter). Free and bioavailable testosterone concentrations were measured as described (35). Total IGF-I, IGF binding protein (IGFBP)-1 and IGFBP-3 concentrations were assayed by immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX), as described (30, 31, 32).

Analysis

Baseline GH concentrations were averaged over the 2-h preinfusion intervals (0800–1000 h) for all 5 d of study in each subject. As a model-free end point, the peak GH response was defined as the highest GH concentration attained in a three-point moving average of the time series.

Statistical methods

The design was a prospectively randomized, double-blind, parallel-cohort comparison of the effects of age on secretagogue action. There were two age groups and five secretagogues. All subjects completed all sessions. Planning power analyses predicted greater than 85% statistical power to detect a 30% difference due to age if 20 subjects each completed five studies in the combined cohorts (10 PRE and 10 POST women). The effects of age stratum (POST and PRE) and secretagogue type (five infusions) and their interaction were evaluated using a repeated-measures design with age stratum as the between-subject factor and secretagogue type as the within-subject (repeated) factor. A two-way least-squares general-linear ANOVA model (2 x 5) with an interaction term was formulated (36). Departure of the variance-covariance matrix from compound symmetry was adjusted for using the Huynh-Feldt statistic. Wilk’s lambda was applied to evaluate the significance of the interaction between age and secretagogue type. The response variable was the 3-point peak GH concentration. According to the null hypothesis, age stratum and secretagogue type do not individually or jointly (interactively) determine GH secretion. Results were considered significant for experiment-wise 0.05. Post hoc contrasts were made for overall P < 0.001 using Tukey’s honestly significantly different (HSD) test. Trivariate linear regression analyses was used to relate peak GH concentration, AVF mass, and IGF-I concentrations in the combined cohorts, which had similar sex-steroid concentrations at the time of the infusions. Computations were made using SYSTAT (version 11; Point Richmond, CA).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Table 1Go summarizes outpatient screening endocrine data in POST and PRE cohorts. Before any intervention, POST women had higher LH and FSH; similar IGFBP-1, SHBG, free, and bioavailable and total testosterone and TSH; and lower E2, IGF-I, IGFBP-3, prolactin, and albumin concentrations than PRE women.


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  		TABLE 1. Baseline hormone concentrations

 
Graded E2 administration after leuprolide down-regulation stimulated a 2.5-fold increase in IGFBP-1 concentrations in PRE subjects only (P < 0.01) (Table 2Go). IGF-I, IGFBP-3, and SHBG concentrations did not change significantly between d 4 (E2 dose 0.05 mg) and the last wk of the clamp (E2 dose 0.2 mg) (range of decrements or increments 1–19%). FSH and LH concentrations declined over the same interval (P < 0.001 and P < 0.05, respectively). During the peptide infusion phase (d 17–23, E2 dose 0.2 mg/d), POST subjects maintained higher FSH but similar E2, IGFBP-1, SHBG, testosterone, and LH and lower GH, IGF-I, and IGFBP-3 concentrations than PRE women (all P < 0.015) (Table 3Go).


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  		TABLE 2. Hormonal measures during secretagogue infusions

 

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  		TABLE 3. Significance table for secretagogue effects in the combined cohorts

 
GH response to the five secretagogue paradigms are illustrated in Fig. 2Go in one POST and one PRE subject (Fig. 2AGo) and summarized for the two cohorts as the mean (± SEM) (Fig. 2BGo). Visual inspection revealed substantially lower responses to each stimulus type in POST than PRE individuals.


Figure 2
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  		FIG. 2. Individual (A) and combined (B) profiles of GH concentrations sampled every 10 min for 6 h to yield a 2-h baseline and 4-h poststimulus interval. Cohort data are the mean ± SEM (n = 10 PRE and n = 10 POST women).

 
Statistical comparisons were made with respect to peak (3-point moving average) GH concentrations. Two-way ANOVA disclosed significant primary effects of both age (P < 0.001) and secretagogue protocol (P < 0.001) and a weak interaction between age and secretagogue type (P = 0.035) (Fig. 3AGo). In the combined cohorts, the GH response to bolus GHRP-2 exceeded that of all other infusions except for continuous GHRP-2 stimulation (P = 0.08) (P < 0.001 vs. both bolus and continuous GHRH, P = 0.003 vs. L-arginine] (Table 4Go). In PRE women, the descending rank order of GH responses was: GHRP-2 bolus (50 ± 12 µg/liter) = continuous GHRP-2 (32 ± 4.4 µg/liter) > L-arginine (23 ± 3.3 µg/liter) = continuous GHRH (15 ± 2.3 µg/liter) = bolus GHRH (19 ± 4 µg/liter) (overall P < 0.001). In POST women, there was no significant rank order of responses to the five infusion protocols. Thus, the statistical interaction reflected significantly GHRP-2 predominance in PRE but not POST women.


Figure 3
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  		FIG. 3. A, Unstimulated mean (left) and stimulated peak GH concentrations (right, 3-point moving-average estimates) in 10 PRE and 10 POST women studied as described in Fig. 1Go. The overall effects of age, secretagogue type, and their interaction are stated as P values. Means with no shared alphabetic letters differ significantly by Tukey’s HSD test with respect to the combined effects of age and type of peptide infusion. Data are the mean ± SEM. B, Normalized peak GH responses to the indicated secretagogues in PRE and POST volunteers. Data are percentage maximal responses, assuming a value of 100% for bolus GHRP-2 stimulation in PRE women.

 

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  		TABLE 4. Post hoc significance tests

 
For ease of visualization, POST GH responses were evaluated additionally as percentages of PRE responses (POST/PRE x 100%). According to this assessment, mean peak GH concentrations in POST subjects were 26% those of PRE for bolus GHRP-2, 34% for continuous GHRP-2, 33% for L-arginine, 27% for bolus GHRH, and 32% for continuous GHRH (each P ≤ 0.002) (Table 5Go). Expressed alternatively, percentage GH responses to the five secretagogue infusions in POST subjects were only 9.5–27% that of bolus GHRP-2 stimulation in PRE women (Fig. 3BGo). By comparison, the GH response range in PRE volunteers was 29–64% that of the same maximum.


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  		TABLE 5. Trivariate linear regression analyses

 
Computed axial tomography estimates of AVF averaged 20 ± 2.5 cm2 in PRE and 59 ± 18 cm2 in POST women (P < 0.01). Exploratory univariate regression analysis revealed a prominent inverse correlation between the peak GH response to L-arginine and AVF mass, which accounted for 47% of the response variability under the E2 clamp (P = 0.0008) (Fig. 4AGo). AVF also explained 31 and 30% of the variability in GH responses to constant GHRP-2 and constant GHRH stimulation (respectively, P = 0.011 and P = 0.013). Analogous exploratory univariate regressions of peak GH responses on IGF-I concentrations disclosed strongly positive correlations for continuous GHRH and GHRP-2 stimulation (R2 = 0.51 and 0.47, both P < 0.001). Similar inferences applied to L-arginine (R2 = 0.41, P = 0.0023) and bolus GHRP-2 (R2 = 0.36, P = 0.0052) but not bolus GHRH stimulation (Fig. 4BGo).


Figure 4
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  		FIG. 4. A, Linear regression of the natural logarithm of GH responses on cycle threshold estimates of AVF in PRE (closed circles) and POST (open circles) women. The five panels include responses to L-arginine, continuous GHRP-2, continuous GHRH, bolus GHRP-2, and bolus GHRH (top to bottom). Numerical values are the square of the correlation coefficient (R2) and corresponding P value. B, Corresponding regressions on IGF-I concentrations.

 
To corroborate these exploratory relationships, trivariate regression was applied using both AVF and IGF-I concentrations as independent variables. In this analysis, AVF negatively predicted GH responses to L-arginine (partial R2 = 0.51, P = 0.007) but not to other secretagogues. In contrast, IGF-I concentrations positively predicted GH responses to continuous GHRP and continuous GHRH stimulation (both R2 ≥ 0.52 and P ≤ 0.013) but not to any other infusion type. Note that these outcomes combine data in both age cohorts. Thus, one could not necessarily extrapolate inferences to values outside the range of the age-delimited independent variables.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
A paradigm of gonadal axis down-regulation and transdermal E2 addback in young (age 21 ± 1.1 yr) and older (age 64 ± 1.4 yr) healthy women revealed that POST individuals maintain only 48, 43, and 74% of the GH, IGF-I, and IGFBP-3 concentrations observed in PRE subjects. This set of contrasts defines relative biochemical hyposomatotropism in POST volunteers independently of short-term estrogen sufficiency. Near-physiological stimulation with pathway-selective secretagogues further disclosed that POST women achieve peak GH concentrations that are only 26–34% of those attained in PRE individuals independently of the stimulus used, viz., GHRP, GHRH, and putative somatostatin withdrawal. Trivariate regression analysis unveiled that regardless of age AVF is a major (51%) negative determinant of GH responses to L-arginine, whereas the concomitant total IGF-I concentration is a prominent positive correlate of GH responses to continuous GHRH (46%) and GHRP-2 (51%) stimulation. Thus, in the face of comparable short-term E2 availability, POST women exhibit impaired hypothalamo-pituitary responses to near-physiological drive by each major secretagogue pathway. In the same setting, greater AVF predicts excessive somatostatin outflow, and higher IGF-I concentrations forecast enhanced GHRP and GHRH signaling.

The estrogen-clamp paradigm achieved comparable concentrations of E2, SHBG, and total, free, and bioavailable testosterone in POST and PRE women (Table 2Go). Endocrine effects of the graded E2-repletion phase differed by age only with respect to lesser inhibition of FSH concentrations in POST volunteers and greater stimulation of IGFBP-1 concentrations in PRE subjects, resulting in similar final IGFBP-1 values. Lower baseline IGF-I and IGFBP-3 concentrations in POST volunteers persisted during the E2 clamp, consistent with earlier inferences (30, 31, 32).

The mechanisms of putatively impaired hypothalamo-pituitary GH drive in POST women were assessed by submaximal stimulation with a ghrelin analog (GHRP-2) and GHRH, using L-arginine as a positive control. L-Arginine presumptively reduces hypothalamic somatostatin outflow (37, 38, 39). Submaximally effective peptide stimuli were chosen a priori to approximate physiological regulation of GH secretion, given that maximal secretagogue action is rarely if ever realized endogenously. Accordingly, relative hyposomatotropism in POST women cannot be attributed solely to decreased pharmacological secretagogue efficacy, although most protocols have tested agonist efficacy (5, 7). The present paradigm obviated these limitations by administering near-physiological stimuli, thereby unmasking marked reductions (67–74%) in older women.

A fundamental unresolved question had been whether age impairs GH secretion mediated by multiple peptidergic pathways. The query was addressed by inducing GH secretion with each of GHRH, GHRP, and putative somatostatin withdrawal. Reduced GH response to each of these secretagogues in POST, compared with PRE, women provides strong evidence for tripartite failure of regulatory peptide pathways. Moreover, seven of 10 POST women had GH responses to four of the five secretagogue types that were less than those in nine of 10 PRE women, indicating that most often all pathway responses are reduced in any given POST individual. Moreover, age-associated contrasts were independent of the time mode of peptide exposure, viz., bolus or continuous delivery. The comparison is important because GHRH secretion is normally pulsatile, whereas ghrelin concentrations are relatively stable (7, 10, 40). Thus, age-related response deficits are not artifacts of how the stimulus is delivered.

Regression analyses indicated that increased AVF burden strongly predicts low GH secretory responses to L-arginine. The outcome would be consistent with a postulate that unknown factors associated with visceral adiposity mediate excessive somatostatin outflow (37, 38, 39). Candidate factors include low IGFBP-1 and high insulin, free fatty acid, or leptin concentrations and/or altered central nervous system actions signaling (7, 10). In addition, trivariate regression analyses indicated that higher total IGF-I concentrations forecast enhanced effects of continuous GHRP and GHRH stimulation. This positive association could signify greater central activation of GH-regulating pathways, which promote increased GH secretion and thereby higher IGF-I concentrations.

Models of ensemble interactions among GHRP/ghrelin, GHRH and somatostatin may assist objective interpretation of complex experiments (41, 42, 43). In the present study, volunteers received all three of GHRP, GHRH and L-arginine, which exert mechanistically interdependent effects (7, 10). Thus, the question arises whether globally impaired responses could be mediated by a deficit in any one peptide-signaling pathway. Model simulations suggested that age-related attenuation of GHRP/ghrelin’s central inhibition of somatostatin action could explain most of the findings reported here. The mechanistic bases for this inference are that GHRP: (1) acts directly on somatotrope cells to induce GH secretion and oppose pituitary somatostatin restraint (44), and that the latter may be increased in aged animals (2, 7, 45, 46) stimulates hypothalamic GHRH secretion (47), which is putatively reduced in aging (3, 5, 48) drives maximal GH secretion through brain GHRP receptors (49), which are reportedly decreased in older humans (4, 50) antagonizes central nervous system actions of somatostatin, which restrain GHRH secretion (51, 52), which may be elevated in aging (5, 30, 41, 53); and (5) synergizes with GHRH (54, 55), albeit to a reduced extent in older adults (28). According to the model presented in (43), POST women would respond to each of the secretagogue formats applied here significantly less than PRE women (Fig. 5Go).


Figure 5
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  		FIG. 5. Simulated failure of pituitary ghrelin to stimulate GH and oppose somatostatin (SS) in POST women. The model assumes that at the pituitary, ghrelin induces GH secretion and antagonizes somatostatin inhibition less potently in POST than PRE women.

 
A possibility raised by the present data are that POST individuals have reduced hypothalamo-pituitary responses to estrogen per se. Earlier investigations have shown that E2 administration in POST women potentiates submaximal stimulation by injected ghrelin and GHRH and attenuates submaximal inhibition by infused somatostatin (34, 56, 57). However, no studies exist that compare the potency of estrogen in mediating these effects in POST and PRE individuals. In addition, although E2 can induce GH secretion directly by somatotropes in vitro and ectopic pituitary tissue in vivo in the rat (58, 59), whether such direct actions occur in the human or are modified by age is not known.

Caveats include the relatively small number of subjects (n = 20) studied, thus requiring corroboration in larger cohorts. The current design does not test whether testosterone repletion or the duration of estrogen supplementation influences the age dependence of hypothalamo-pituitary responses (5, 7). Given lower GH and IGF-I concentrations and thus putatively less negative feedback in POST than PRE women, the absolute degree of age-related impairment in hypothalamo-pituitary responses to near-physiological agonists may be underestimated. For example, a 37% decrement in IGF-I concentrations induced by administering a GH-receptor antagonist doubled pulsatile GH secretion within 3 d in young adults (60).

In conclusion, in an experimentally estrogen-enriched milieu, POST, compared with PRE, women maintain lower GH, IGF-I, and IGFBP-3 concentrations and secrete one third the amount of GH in response to near-physiological stimulation by both GHRH and GHRP as well as putative somatostatin withdrawal. Pathway-response deficits are evident after bolus and continuous peptide delivery, indicating robustness of outcomes. Mechanistically, AVF is a significant negative correlate of the GH response to somatostatin withdrawal, and the IGF-I concentration is a prominent positive predictor of GH responses to both continuous GHRP-2 and GHRH drive. The collective outcomes indicate that POST women manifest not only multiple GH response-pathway deficits but also pathway-selective impairments associated with higher AVF mass and lower IGF-I concentrations.


   Acknowledgments
 
We thank Heidi Doe for support of manuscript preparation; Ashley Bryant for excellent data analysis and graphics; the Mayo Immunochemical Laboratory for assay assistance; and the Mayo research nursing staff for implementing the protocol.


   Footnotes
 
This work was supported in part by the General Clinical Research Center Grants MO1 RR00585 from the National Center for Research Resources (Rockville, MD) (to the Mayo Clinic and Foundation) and R01 NIA AG019695, R21 DK072095, DK 063609, and RR019991 from the National Institutes of Health (Bethesda, MD).

Disclosure: J.D.V. accepts responsibility for the conduct of this study and has seen and approved the final manuscript. No portion of this article will be published or submitted elsewhere before appearing in JCEM. All authors have nothing to declare.

First Published Online April 3, 2007

Abbreviations: AVF, Abdominal visceral fat mass; CV, coefficient of variation; E2, estradiol; GHRP, GH-releasing peptide; HSD, honestly significantly different; IGFBP, IGF binding protein; POST, postmenopausal; PRE, premenopausal.

Received January 9, 2007.

Accepted March 28, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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