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The Journal of Clinical Endocrinology & Metabolism 91(9):3559 –3565 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2006-0948

Estradiol Potentiates Ghrelin-Stimulated Pulsatile Growth Hormone Secretion in Postmenopausal Women Johannes D. Veldhuis, Daniel M. Keenan, Ali Iranmanesh, Kristi Mielke, John M. Miles, and Cyril Y. Bowers Endocrine Research Unit (J.D.V., K.M., J.M.M.), Department of Internal Medicine, Mayo School of Graduate Medical Education, Rochester, Minnesota 55905; Department of Statistics (D.M.K.), University of Virginia, Charlottesville, Virginia 22904; Research and Development Office (A.I.), Veterans Affairs Medical Center, Salem, Virginia 24153; and Department of Medicine (C.Y.B.), Tulane University Health Sciences Center, New Orleans, Louisiana 70112 Context: Ghrelin and an estrogen-rich milieu individually amplify pulsatile GH secretion by increasing the amount of hormone released per burst. However, how these distinct agonists interact in controlling pulsatile GH output is not known. Objective: The objective of the study was to test the hypothesis that elevated estradiol (E2) concentrations potentiate hypothalamo-pituitary responses to a near-physiological ghrelin stimulus. Design: This was a double-blind, placebo-controlled, prospectively randomized, parallel-cohort study. Setting: The study was conducted at an academic medical center. Subjects: Twenty-one postmenopausal women participated in the study. Interventions: Eleven subjects received placebo (Pl) and 10 others E2 transdermally in escalating doses over 3 wk to mimic late follicular-phase E2 concentrations. Saline or a submaximally stimulatory amount of ghrelin (0.3 ␮g/kg) was infused iv on separate

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H AND IGF-I production increase by 3- to 10-fold in puberty and decrease progressively thereafter beginning in young adulthood (1). The parallelism between pulsatile GH secretion and sex-steroid concentrations across the human lifetime suggests that waning of gonadal steroidogenesis in older individuals contributes to relative hyposomatotropism in aging (2). This notion is supported by the capability of short-term supplementation with estradiol (E2) or testosterone to double GH concentrations in young and older hypogonadal patients and elderly healthy adults (3–10). Estrogen stimulates GH secretion by augmenting the amount of hormone released per burst (11–13). The size of GH secretory bursts in turn is determined by at least three peptides, viz., GHRH, ghrelin [a GH-releasing peptide (GHRP)], and somatostatin (an antagonist of both GHRH and GHRP) (14 –26). Puberty as well as the administration of estrogen or an aromatizable androgen in prepubertal children enhances responsiveness to synthetic analogs of ghrelin First Published Online June 27, 2006 Abbreviations: E2, Estradiol; GHRP, GH-releasing peptide; IGFBP, IGF binding protein; Pl, placebo. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

randomly ordered mornings fasting after 17–21 d of Pl or E2 administration. Outcomes: Outcomes included serum concentrations of E2, ghrelin, GH, IGF-I, IGF binding protein (IGFBP)-1 and IGFBP-3, and the estimated mass and waveform of stimulated GH secretory bursts. Results: Administration of E2 yielded late follicular-phase E2 concentrations. Compared with Pl, E2 did not alter ghrelin concentrations but reduced IGF-I and IGFBP-3 and elevated IGFBP-1 concentrations. Compared with saline, ghrelin infusion amplified pulsatile GH secretion by 7.1-fold (P ⬍ 0.01). The effect of E2 alone was 2.0-fold placebo and that of combined ghrelin/E2 10.4-fold (P ⬍ 0.01). Ghrelin and E2 accelerated initial GH release individually but nonadditively by more than 2-fold (P ⬍ 0.01). Conclusions: Estrogen augments ghrelin’s near-physiological stimulation of pulsatile GH secretion and mimics ghrelin’s acceleration of initial GH release. Thus, we hypothesize that estrogen and a GH secretagogue act via independent as well as convergent mechanisms. (J Clin Endocrinol Metab 91: 3559 –3565, 2006)

(27, 28). Studies in adults are conflicting, in that E2 supplementation may or may not potentiate stimulation of GH secretion by near-maximally effective doses of ghrelin analogs (reviewed in Ref. 29). Possible explanations for disparities are confounding effects of endogenous ghrelin and/or the use of pharmacological doses of agonists, which would test secretagogue efficacy but not potency (30). Addressing these issues could help clarify why the actions of GHRP are increased more in girls than boys during puberty, reduced in aging individuals with lower sex-steroid concentrations, and gender independent in other settings (18, 21, 27, 28, 31). The present study tests the hypothesis that a young adultlike estrogen-enriched compared with estrogen-deficient milieu in postmenopausal women will amplify hypothalamosomatotrope responses to ghrelin, a naturally occurring GHRP. To mimic a physiological secretagogue signal, a submaximal rather than pharmacological dose of ghrelin was used (25). Analyses were then designed to quantify both the size and shape of induced GH secretory bursts under the hypothesis that E2 and ghrelin may regulate both end points (32–34). Subjects and Methods Subjects Twenty-one healthy postmenopausal women were enrolled in and completed the two study sessions (below). Participants provided written

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informed consent approved by the Mayo Institutional Review Board. The protocol was first reviewed by the U.S. Food and Drug Administration under an investigator-initiated new drug number. Exclusion criteria were recent transmeridian travel or night-shift work (within 2 wk); significant weight change (⬎2 kg in 1 month); body mass index less than 20 kg/m2 or greater than 30 kg/m2; acute or chronic systemic illness; use of neuroactive agents; psychiatric treatment; substance abuse; known or suspected cardiac, cerebral, or peripheral arterial or venous thromboembolic disease; poorly controlled hypertension or hypertriglyceridemia; any history or suspicion of endometrial or breast cancer; and untreated gallstones. Some enrollees continued to take a thiazide diuretic, an angiotensin-converting enzyme inhibitor, multivitamins, ferrous sulfate, calcium carbonate, aspirin, or ibuprofen on nonstudy days. Each subject had an unremarkable medical history and physical examination and normal screening laboratory tests of hepatic, renal, endocrine, metabolic, and hematologic function, as described (12, 13). The median (range) age was 64 (50 – 69) yr in the placebo (Pl) and 62 (52– 68) yr in the E2-treated volunteers. Corresponding body mass indices (kilograms per square meter) were 25 (20 –29) and 26 (21–29), respectively. Postmenopausal status was confirmed by concentrations of FSH greater than 50 IU/liter, LH greater than 20 IU/liter, and estradiol less than 20 pg/ml (⬍74 pmol/liter). Volunteers stopped any postmenopausal hormone replacement at least 6 wk before study.

Statistical design The study was a parallel-cohort design in which the order of the two infusions was prospectively randomized and double blind. The Food and Drug Administration restricted the infusion of ghrelin to once per subject but approved a parallel design (Pl vs. E2).

Estrogen clamp Placebo or E2 was applied each night in matching transdermal patches. In the case of E2 administration, the schedule achieves stepwise elevation of E2 concentrations into the young-adult late follicular-phase range (12). Doses (milligrams per day) were escalated once every 4 nights as follows: 0.05, 0.10, 0.15, and 0.20. The last dose was continued for 7 d (d 15–21). Infusion sessions were scheduled during the last 5 d (17–21, inclusive). Thereafter, micronized progesterone was administered (100 mg orally for 12 d) to women with an intact uterus according to good standards of clinical practice (13).

Sampling paradigm Volunteers were admitted to the Mayo General Clinical Research Center on the evening before study to allow overnight adaptation. To obviate food-related confounds, subjects received a constant meal (turkey sandwich or vegetarian alternative) of 500 kcal containing 55% carbohydrate, 15% protein, and 30% fat at 1800 h. Participants then remained fasting overnight until 1200 h the next day. On the day of combined sampling and infusion, catheters were inserted in contralateral forearm veins at 0700 h. Blood was withdrawn at 0800 h for later assay of serum estradiol, LH, FSH, IGF-I, IGF binding protein (IGFBP)-1, and IGFBP-3 concentrations. Further samples (1.5 ml) were collected in chilled plastic tubes containing EGTA every 10 min for 4 h between 0800 and 1200 h. At 0900 h, a single bolus was injected of saline or ghrelin (0.3 ␮g/kg, delivered in 3.7% mannitol). Plasma was separated on ice and frozen at ⫺70 C within 30 min. Lunch was provided before discharge.

Dosimetry The ghrelin stimulus is approximately half maximal according to dose-response estimates in young adults (25, 35, 36).

Hormone assays Plasma GH concentrations were measured in duplicate by automated ultrasensitive double-monoclonal immunoenzymatic chemiluminescence assay using 22-kDa recombinant human GH as assay standard (Sanofi Diagnostics Pasteur Access, Chaska, MN) (12). All samples (n ⫽ 50) from any given subject were analyzed together. Sensitivity is 0.010 ␮g/liter (defined as 3 sd above the zero-dose tube). Interassay coeffi-

Veldhuis et al. • Estradiol Enhances Ghrelin Action

cients of variation were 7.9 and 6.3% at GH concentrations of 3.4 and 12 ␮g/liter, respectively. Intraassay coefficients of variation were 4.9 and 4.5% at 1.1 and 20 ␮g/liter GH. No values fell less than 0.020 ␮g/liter. Cross-reactivity with GHBP or 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 (13). E2 was quantitated using the same platform, as described (37). Prolactin, total IGF-I, IGFBP-1, and IGFBP-3 concentrations were measured by immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX), as reported (12, 38). Total ghrelin concentrations were assayed on a serum pool collected 10, 20, and 30 min after bolus ghrelin injection using RIA kits purchased from Linco Research, Inc. (St. Charles, MO) (catalog no. GHRA-88HK). Inter- and intraassay coefficients of variation were, respectively, 5.2 and 4.7%, and the detection threshold was 280 pg/ml.

Deconvolution analyses of pulsatile and basal (nonpulsatile) GH secretion Earlier deconvolution methods in some cases yield nonunique estimates of hormone secretion and elimination rates (39). To address this technical impasse, pulsatile and basal GH secretions were estimated simultaneously using a new variable-waveform secretory-burst model statistically conditioned on biexponential kinetics and a priori estimated pulse times, as recently validated (40 – 43). The mass of GH secreted per burst was defined as the product of secretory-burst amplitude, a unitarea normalized waveform (burst shape) and a weak linear function of the preceding interpulse interval. The secretory-burst waveform (threeparameter generalized gamma density) was considered common to any given intervention and either cohort, as stated algebraically in the supplemental data published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org. GH pulse-onset times were created for each 4-h GH time series by a selective smoothing (anisotropic diffusion) algorithm, as described (42). All secretion parameters were then estimated simultaneously, conditioned on the selected pulse-time sets. The principal analytical outcomes are pulsatile GH secretion (micrograms per liter per 3 h), defined as the summed mass of GH secreted in bursts after saline or secretagogue infusion, and the modal time latency (minutes) to achieve maximal secretion within any given burst (12, 13, 44).

Other statistical comparisons The rank-sum test was used to contrast the intraindividual incremental effect of ghrelin stimulation over that of saline in the two study cohorts. In view of the unbalanced statistical design, exact P values were estimated by random permutations on ranks. Fifty thousand permutations were performed for each comparison by random reassignment of interventional ranks in the combined cohorts (n ⫽ 21 subjects). Purely chance assortment would predict a median normalized rank of 10.5. The null hypothesis of no E2 effect over Pl further forecasts a difference (E2 minus Pl) in ranks of zero. The critical P value of the observed difference was one sided, given the a priori postulate that E2 potentiates the ghrelininduced response. The se of the mode of the GH secretory-burst waveform was calculated analytically, as described and verified earlier by bootstrap estimates (12, 13). Hormone concentrations were compared in the E2 and Pl cohorts via an unpaired Student’s t test. Data are presented as the mean ⫾ sem (45).

Results

E2 administration caused a sense of abdominal bloating, breast tenderness, headache, or mild pedal edema in three volunteers. None discontinued participation. Saline and ghrelin infusions were occasionally (four subjects each) associated with facial warmth. Figure 1A gives mean fasting hormone concentrations in the two cohorts. E2 concentrations (picograms per milliliter) averaged 7.5 ⫾ 0.51 (Pl) and 352 ⫾ 9.1 (E2) (P ⬍ 0.001, multiply by 3.67 for units of picomoles per liter). Total IGF-I



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TABLE 1. Impact of E2 vs. Pl administration on GH and ghrelin concentrations Placebo (n ⫽ 11)

Interventions

GH (␮g/liter) Saline infusion Ghrelin infusion Peak GH to ghrelin ratio Saline infusion Ghrelin infusion

1.2 ⫾ 0.33 8.1 ⫾ 1.9a 0.80 ⫾ 0.21 3.4 ⫾ 0.26a

E2 (n ⫽ 10)

2.0 ⫾ 0.94 15 ⫾ 1.5a 1.7 ⫾ 0.57 5.8 ⫾ 0.51a,b

Data are the mean ⫾ SEM for indicated n. a P ⬍ 0.05 vs. saline/placebo. b P ⬍ 0.05 vs. placebo.

FIG. 1. A, Impact of Pl (O) and transdermal E2 administration on fasting morning concentrations of (left to right) E2, IGF-I, IGFBP-1, and IGFBP-3 in postmenopausal women. B, Total serum ghrelin concentrations. Data are the mean ⫾ SEM (n ⫽ 11, Pl; and n ⫽ 10, E2). Multiply E2 concentrations in picograms per milliliter by 3.67 to convert to picomoles per liter.

concentrations were 25% lower (P ⬍ 0.05), IGFBP-1 62% higher (P ⬍ 0.01), and IGFBP-3 27% lower (P ⬍ 0.05) in the E2 than Pl cohort. FSH concentrations (international units per liter) were reduced in the presence of E2 (47 ⫾ 2.0) vs. Pl (75 ⫾ 2.4) (P ⬍ 0.01), but LH concentrations (international units per liter) did not differ (29 ⫾ 1.0 vs. 25 ⫾ 0.74, respectively). In response to ghrelin vis-a-vis saline injection, serum total ghrelin concentrations (30 min mean) rose significantly by 1.65fold (P ⬍ 0.05) in the Pl group and 2.2-fold (P ⬍ 0.01) in the E2-treated cohort (Fig. 1B). Administration of E2 did not affect total ghrelin concentrations after either saline or ghrelin infusions. Table 1 summarizes peak GH concentrations achieved after saline or ghrelin injection. Ghrelin increased peak GH concentrations by 6.8-fold (P ⬍ 0.01), and E2 did so by 1.6-fold (P ⬍ 0.05) over saline. The combined effect of ghrelin and E2 was 12.5-fold that of saline (P ⬍ 0.005). Peak GH to ghrelin ratios

rose with ghrelin infusion, consistent with the properties of a nonlinear dose-responsive curve (30). E2 vs. Pl significantly increased peak GH to ghrelin ratios during ghrelin infusion (P ⬍ 0.05), pointing to increased potency of ghrelin in the presence of E2. Deconvolution analysis was applied to assess GH secretion. Figure 2A illustrates GH concentration time series in three subjects from each cohort, whose responses are typical (the median and flanking two ranks of responses in the Pl and E2 groups). Deconvolution-based estimates revealed that injection of ghrelin in the Pl setting stimulated pulsatile burst-like GH secretion 7.1-fold more than saline/Pl (P ⬍ 0.01); E2 alone amplified GH secretion 2-fold more than saline/Pl (P ⬍ 0.10); and combined ghrelin/E2 did so by 10.4-fold (P ⬍ 0.01) (Fig. 2B). Formal statistical comparison of responses to E2 to ghrelin and Pl to ghrelin was made using rank permutations. The null hypothesis assumes chance assortment of the strength (rank) of incremental GH responses to ghrelin over saline among the 21 women studied independently of estrogen status. Thus, for individual Pl and E2 interventions, the median random rank would be 10.5, and the (E2 minus Pl) difference in random ranks would be zero. However, the actual difference in ranks was 4.6 (P ⬍ 0.05), which rejects the null hypothesis of no treatment effect (Fig. 3). Thus, E2 increases (nearly doubles) the amount of GH secreted in the presence of ghrelin (P ⬍ 0.05). GH secretory-burst waveform (shape), defined as the time evolution of GH release within bursts, was quantified by the modal time delay (minutes) to attain maximal GH secretion (Fig. 4). The mode denotes maximal secretion in the burst. The time (minutes) when the mode occurs reflects how rapidly GH is released. Statistical comparisons of the modes disclosed that, in the Pl group, infusion of ghrelin, compared with saline, reduced the time latency to achieve peak GH secretion by 2.1fold (P ⬍ 0.01). Exposure to E2 vs. Pl shortened the time delay to maximal GH secretion by 2.3-fold after saline (P ⬍ 0.01) but did not further abbreviate the delay after ghrelin stimulation (P ⬎ 0.80). Discussion

The present investigations reveal that E2 and ghrelin stimulate pulsatile GH secretion via both distinctive (independent) and shared (convergent) mechanisms in postmenopausal women. First, a distinct interaction was identified by the capability of E2 supplementation to amplify the amount of ghrelin-induced GH secretion without altering ghrelin concentrations. In particular, E2 supplementation increased

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FIG. 3. Impact of Pl vs. E2 pretreatment on pulsatile GH secretion (micrograms per liter per 3 h) driven by saline or ghrelin (0.3 ␮g/kg iv bolus). Data are the median, interquartile range, and extrema (n ⫽ 11, Pl; and n ⫽ 10, E2).

FIG. 2. A, Illustrative GH concentration time series in three postmenopausal women (median and flanking two responders) in each cohort. Volunteers underwent blood sampling every 10 min for 4 h on two occasions after receiving either transdermal Pl or E2 for 17–21 d. Saline or ghrelin (0.3 ␮g/kg) was given by bolus iv injection at 70 min (x-axis) (see Subjects and Methods). B, Mean responses for the cohort as a whole. Data are the mean ⫾ SEM.

the ratio of peak GH concentrations to total ghrelin concentrations (Table 1), consistent with an enhancement of ghrelin’s potency. Second, shared effects were inferred by the finding that E2 and ghrelin individually but nonadditively accelerate initial GH release within secretory bursts (Fig. 5). The collective outcomes indicate that E2 and ghrelin act via independent and convergent mechanisms to augment the size of GH secretory bursts and trigger more rapid exocytosis of GH stores, respectively. A novel analytical tool was applied to estimate both the shape and the size of GH secretory bursts. The shape of bursts has been viewed traditionally as symmetric to simplify calculations (46). Direct estimates show that this is not the case (41, 47, 48). In fact, estrogen and ghrelin nonadditively change the shape of secretory bursts to favor rapid initial release of available GH-

containing granules in pituitary cells. In addition, the two hormones additively increase the amount of GH released in each burst, quite independently of changing its shape. By this means, the accompanying analyses suggest that E2 and ghrelin share a common mechanism to facilitate prompt GH release but activate different mechanisms to augment the amount of GH secreted. The paradigm of graded transdermal E2 administration reduced IGF-I and IGFBP-3 and elevated IGFBP-1 concentrations. This tripartite response pattern has been recognized after oral estrogen administration but not typically after conventional transdermal E2 replacement (1, 49). As discussed by Friend et al. (9), the difference may be explicable in part by the fact that higher E2 concentrations are usually attained after oral than transdermal regimens. The current protocol was designed to mimic late-follicular phase E2 concentrations (12, 13), which are sufficient to lower free IGF-I concentrations measured by ultrafiltration dialysis (50). Whether transdermally delivered E2 also lowers free IGF-I levels is not known. Lower IGF-I availability could withdraw negative feedback and thereby contribute to the greater stimulatory action of ghrelin observed here. Confirmation of this postulate would require quantifying ghrelin-stimulated GH secretion during experimental infusion of IGF-I in a low and high estrogenic milieu. One study using GHRH as the secretagogue reported that E2 administration paradoxically augments feedback inhibition by exogenous IGF-I (44). No comparable data exist to clarify how ghrelin, E2, and IGF-I act in concert to regulate pulsatile GH secretion. Estrogen does not appear to influence blood total ghrelin concentrations. In one study, 2 d of orally administered con-



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FIG. 4. Histograms of 50,000 random permutations of normalized rank sums to assess whether E2 (top) vs. Pl (middle) administration affects the relative strength (rank) of GH increments (ghrelin minus saline) in the combined cohorts (n ⫽ 21 women). Under the null hypothesis, the difference in rank sums (E2 minus placebo) would average zero (bottom). The actual (observed) difference in rank sums between E2 and Pl responses was 4.6 (P ⬍ 0.05).

jugated estrogens in peripubertal girls did not alter total ghrelin concentrations (51). The present data in postmenopausal women given transdermal E2 are congruent with such findings. In contrast, in women with anorexia nervosa, combined administration of a synthetic estrogen and progestin elevated total ghrelin concentrations (52). In another analysis, 6 months of oral E2 supplementation in postmenopausal women increased active ghrelin concentrations by 14%, without affecting total ghrelin concentrations (53). Thus, no study using estrogen alone has demonstrated a significant increase in plasma total ghrelin concentrations. Whether acute transdermal E2 supplementation as studied here alters acylated ghrelin levels is not known but could be important by way of influencing ghrelin bioactivity. At present the mechanisms that control ghrelin’s acylation status are not well known (1). Infusion of a submaximally stimulatory dose of ghrelin (0.3 ␮g/kg) (25, 35, 36) induced peak plasma GH concentrations of 8.1 ␮g/liter in the Pl setting and 15 ␮g/liter in the E2 setting. These pulse sizes are attained normally during sleep or shortterm fasting in young adults (1, 2). The purpose of mimicking physiological GH pulses was to obviate the difficulty in interpreting pharmacological responses, which reflect secretagogue efficacy only (30). Therefore, the finding that E2 potentiates the GH response to a submaximal ghrelin stimulus could denote that estrogens increase secretagogue potency or enhance pituitary sensitivity to ghrelin. Other studies indicate that E2 sup-

FIG. 5. A, Analytically reconstructed GH secretory-burst shape (waveform) in postmenopausal volunteers supplemented with Pl (n ⫽ 11) or E2 (n ⫽ 10) (top and bottom panels, respectively). The secretoryburst waveform is the reconstructed time course of GH release. B, Quantification of burst shape by the modal time delay to achieve maximal GH release. Compared with Pl/saline, E2 and ghrelin individually accelerated GH release by 2.1- and 2.3-fold, respectively. Their effects were nonadditive.

plementation increases the stimulatory potency of and somatotrope sensitivity to GHRH and conversely reduces the inhibitory potency of and pituitary sensitivity to somatostatin (54, 55). Ghrelin and E2 individually but nonadditively accelerated the initial release of GH within secretory bursts by 2-fold. Laboratory observations raise the possibilities that E2 may trigger rapid GH release by up-regulating pituitary GHRP receptors, down-regulating somatostatin subtype-5 receptors, or augmenting pituitary GH stores (1, 29, 56 –59). In addition, atomicforce microscopy indicates that GH release by porcine somatotropes requires transient fusion of exocytotic vesicles with membrane porosomes (60). If this cellular model also applies to the human, then ghrelin and E2 may individually recruit secretory vesicles or facilitate their fusion with the membrane, thus driving rapid initial GH release. The fact that the enhancing effects of ghrelin and E2 were not additive suggests that the

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two effectors may regulate similar steps in the release process. In contradistinction, E2 administration accelerated GH release within individual secretory bursts driven by combined infusion of GHRH/GHRP-2 or GHRH/l-arginine (13). Pulses of ghrelin also evoke GH secretory bursts of more rapid onset and offset than those stimulated by GHRH under in vitro conditions (34). These observations suggest that the mechanisms by ghrelin and GHRH, but not ghrelin and E2, trigger initial GH release differ. Certain caveats should be considered. First, the present investigation used a submaximally stimulatory dose of ghrelin to mimic physiological secretagogue action. Serum concentrations of ghrelin confirmed this expectation. When investigationally practicable, detailed dose-response analyses should allow one to quantify the full nonlinear interaction between E2 and ghrelin. In the only dose-response study of this nature, administration of E2 potentiated GHRP-2-driven GH secretion at the highest dose of secretagogue tested (3 ␮g/kg), which was still not maximal (33). Second, the mean concentration of E2 attained experimentally was intended to and did approach that in the late-follicular and preovulatory phase of the normal menstrual cycle when pulsatile GH secretion typically doubles (61– 63). This model differs from clinical replacement doses of estrogen in postmenopausal women. The present data should have relevance to understanding the basis of large GH pulses in healthy pubertal girls and young women studied in the late follicular phase of the menstrual cycle (1, 11). Third, given the growth-related and anabolic significance of constitutive signaling by the human ghrelin receptor (64), an important future question is how constitutive receptor function, E2 concentrations, and ghrelin availability jointly determine GH secretion. In summary, experimental elevation of E2 concentrations in postmenopausal women amplifies ghrelin’s near-physiological stimulation of pulsatile GH secretion, consistent with independent mechanisms. Conversely, elevated E2 concentrations mimic the action of ghrelin by evoking rapid initial GH release, suggesting shared stimulus-response mechanisms. Acknowledgments We thank Sharon Kaufman, Heidi Doe, and Ashley Bryant for excellent support of manuscript preparation and graphical presentation; the Mayo Immunochemical Laboratory for assay assistance; and the Mayo research nursing staff for conduct of the protocol. Received May 3, 2006. Accepted June 16, 2006. Address all correspondence and requests for reprints to: Johannes D. Veldhuis, Endocrine Research Unit, Department of Internal Medicine, Mayo School of Graduate Medical Education, General Clinical Research Center, Mayo Clinic, Rochester, Minnesota 55905. E-mail: veldhuis. [email protected]. This work was supported in part by General Clinical Research Center Grant MO1 RR00585 to the Mayo Clinic and Foundation from the National Center for Research Resources (Rockville, MD) and Grant R01 NIA AG014799 from the National Institutes of Health (Bethesda, MD). Disclosure: J.D.V. accepts responsibility for the conduct of this study and has seen and approves the final manuscript. No portion of this article will be published or submitted elsewhere before appearing in The Journal of Clinical Endocrinology & Metabolism. All authors have nothing to disclose.

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