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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-1493
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 11 4471-4478
Copyright © 2008 by The Endocrine Society

Estradiol Supplementation in Postmenopausal Women Attenuates Suppression of Pulsatile Growth Hormone Secretion by Recombinant Human Insulin-like Growth Factor Type I

Johannes D. Veldhuis, Daniel M. Keenan, Joy N. Bailey, Adenborduin Adeniji, John M. Miles, Remberto Paulo, Mihaela Cosma and Cacia Soares-Welch

Departments of Medicine (J.D.V., J.N.B., A.A., J.M.M., M.C., C.S.-W.) and Pediatrics (R.P.) Endocrine Research Unit, Mayo School of Graduate Medical Education, Clinical Translational Science Center, Mayo Clinic, Rochester, Minnesota 55905; and Department of Statistics (D.M.K.), University of Virginia, Charlottesville, Virginia 22904

Address all correspondence and requests for reprints to: Johannes D. Veldhuis, Departments of Medicine, Mayo School of Graduate Medical Education, Clinical Translational Science Center, Mayo Clinic, Rochester, Minnesota 55905. E-mail: veldhuis.johannes{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Why pulsatile GH secretion declines in estrogen-deficient postmenopausal individuals remains unknown. One possibility is that estrogen not only enhances stimulation by secretagogues but also attenuates negative feedback by systemic IGF-I.

Site: The study took place at an academic medical center.

Subjects: Subjects were healthy postmenopausal women (n = 25).

Methods: The study included randomized assignment to estradiol (n = 13) or placebo (n = 12) administration for 16 d and randomly ordered administration of 0, 1.0, 1.5, and 2.0 mg/m2 recombinant human IGF-I sc on separate days fasting.

Analysis: Deconvolution analysis of pulsatile and basal GH secretion and approximate entropy (pattern-regularity) analysis were done to quantify feedback effects of IGF-I.

Outcomes: Recombinant human IGF-I injections increased mean and peak serum IGF-I concentrations dose dependently (P < 0.001) and suppressed mean GH concentrations (P < 0.001), pulsatile GH secretion (P = 0.001), and approximate entropy (P < 0.001). Decreased GH secretion was due to reduced secretory-burst mass (P = 0.005) and frequency (P < 0.001) but not basal GH release (P = 0.52). Estradiol supplementation lowered endogenous, but did not alter infused, IGF-I concentrations while elevating mean GH concentrations (P = 0.012) and stimulating pulsatile (P = 0.008) and basal (P < 0.001) GH secretion. Estrogen attenuated IGF-I’s inhibition of pulsatile GH secretion (P = 0.042) but was unable to restore physiological GH pulse frequency or normalize approximate entropy.

Conclusion: Short-term estrogen replacement in postmenopausal women selectively mutes IGF-I-mediated feedback on pulsatile GH secretion. Disinhibition of negative feedback thus confers a novel mechanism by which estrogen may obviate hyposomatotropism.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GH and IGF-I concentrations decline exponentially with age in adults (1). However, maximally stimulated GH secretion remains unchanged in older adults (2). In addition, exogenous GH stimulates hepatic IGF-I synthesis normally in elderly individuals (3). The rate of fall of GH secretion with age is attenuated by 50% in premenopausal women compared with men of similar age (4), raising the consideration that an estrogenic milieu preserves GH production (1). In support of this hypothesis, estrogen supplementation stimulates pulsatile GH secretion in hypogonadal girls and women (5, 6, 7). The basis of estrogenic stimulation is not fully understood (1). However, oral and higher doses of transdermal estradiol often decrease IGF-I concentrations, which could enhance GH secretion by feedback withdrawal (1). Other candidate mechanisms for estrogenic stimulation include enhancement of the hypothalamic release and stimulatory potency of GHRH (8, 9), potentiation of GH-releasing peptide (GHRP)/ghrelin action (10, 11), reduction of somatostatin’s inhibitory potency at the pituitary level (12), and attenuation of negative feedback by GH itself (13). Nonetheless, whether estrogen influences the negative-feedback effect of any given systemic concentration of IGF-I is not known.

Adequate concentrations of estradiol (E2) stimulate pituitary GH synthesis and secretion directly (14), induce hypothalamic GHRP receptors, elevate pituitary IGF-binding protein (IGFBP)-2, attenuate signal transduction via GH receptors (15), reduce IGF-I concentrations (5), and inhibit expression of the hypothalamic GH receptor and pituitary somatostatin receptor subtype 5 (16, 17). Each of these effects would plausibly augment GH secretion, and several could attenuate IGF-I feedback. In addition, E2 down-regulates pituitary GHRH receptors, blunts GH responses to GHRH, up-regulates hypothalamic somatostatin expression and IGF-I receptor number, and induces pituitary IGF-I receptors and somatostatin receptor subtype 2 (1, 16, 18). These countervailing actions would be expected to reduce GH secretion and accentuate inhibition by IGF-I and GH. Such complex pathway interactions make it difficult to predict whether or how estrogen regulates IGF-I negative feedback.

Exogenous IGF-I represses GH secretion in normal fasting adults, patients with type I diabetes mellitus, and subjects with GH-receptor mutations (1). Given the capability of E2 to augment GH secretion (1), the present investigation tests the hypothesis that estrogen supplementation can relieve feedback inhibition of GH secretion by systemic IGF-I.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
The postulate is that E2 supplementation relieves the negative-feedback effect of increased systemic IGF-I concentrations on GH secretion in healthy postmenopausal women. The hypothesis was tested using a three-step interventional strategy, viz. 1) overnight fasting after a standardized evening meal to lower morning IGF-I concentrations, 2) graded elevation of circulating IGF-I concentrations by sc injection of saline or three doses of recombinant human (rh)IGF-I in randomly assigned order on separate days, and 3) prospectively randomized double-masked administration of placebo vs. estradiol orally to control the sex-steroid milieu.

Subjects

Participants provided written informed consent approved by the Mayo Institutional Review Board. The protocol was reviewed by the National Institutes of Health and U.S. Food and Drug Administration under an investigator-initiated new drug assignment. Exclusion criteria included known or suspected cardiac, cerebrovascular, or peripheral arterial or venous thromboembolic disease; personal history of breast or endometrial cancer; concomitant or recent use of neuroactive medications; anemia; and failure to provide written informed consent. Additionally disallowed were recent transmeridian travel (exceeding three time zones within 10 d), nightshift work, significant weight change (≥2 kg in 3 wk), acute or chronic systemic disease, psychiatric illness requiring treatment, and alcohol or drug abuse. Inclusion criteria comprised an unremarkable medical history and physical examination and normal screening laboratory tests of hepatic, renal, endocrine, metabolic, and hematological function. Individuals were clinically postmenopausal for at least 1 yr, and ovariprival status was confirmed by high concentrations of FSH (>50 IU/liter) and LH (>20 IU/liter) and a low concentration of E2 (<30 pg/ml, <10 pmol/liter). Subjects stopped any hormone replacement at least 6 wk before participation.

Timeline of interventions

Twenty-five postmenopausal women were each studied four times (Fig. 1Go, top). Admissions to the Clinical Translational Unit (CRU) were scheduled during the administration of placebo (oral capsule) or micronized E2 (1 mg orally twice daily) for 16 d. This regimen stimulates GH secretion by approximately 2-fold (7). CRU visits were scheduled at least 48 h apart within the 10-d time window comprising d 7–16 inclusive (Fig. 1Go, bottom).


Figure 1
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  		FIG. 1. Overall protocol design (A) and timeline of interventions (B and C).

 
CRU sampling and injection protocol

Volunteers were asked to report to the CRU in the late afternoon. To obviate nutritional confounds, participants received a standardized meal the night before at 1800 h (8 kcal/kg of 20% protein, 50% carbohydrate, and 30% fat) and remained fasting thereafter until the end of sampling at 1400 h the next day. Blood was withdrawn repetitively (1.0 ml every 10 min) for 8 h beginning at 0800 h. Caffeinated beverages, sleep, and exercise were disallowed during the morning sampling session. Saline or rhIGF-I (1.0, 1.5, and 2.0 mg/m2; maximal single dose, 4 mg) was injected sc at 0800 h after the first blood sample was obtained. The rhIGF-I was obtained from Tercica Inc. (South San Francisco, CA) and used here experimentally after obtaining approval by the U.S. Food and Drug Administration. The doses reflect the estimated daily blood production rate of 3–3.5 mg IGF-I in young adults (1).

Safety considerations

Continuous electrocardiographic monitoring and hourly plasma glucose measurements were performed after rhIGF-I administration.

Hormone assays

Serum GH concentrations were determined in duplicate by automated ultrasensitive two-site immunoenzymatic chemiluminescence assay performed on the DxI automated system (Beckman Instruments, Chaska, MN). Interassay coefficients of variation (CV) were 6.1% at 0.46 µg/liter, 4.3% at 3.0 µg/liter, 5.0% at 7.2 µg/liter, and 4.8% at 13.6 µg/liter. Intraassay CV were 4.7% at 0.37 µg/liter, 3.5% at 2.5 µg/liter, and 3.2% at 14.8 µg/liter. The lowest detectable GH concentration at 95% confidence is 0.008 µg/liter determined by processing a six-point calibration curve, five quality controls, and 10 replicates of zero calibrator in multiple assays.

E2 concentrations were quantified by tandem liquid chromatography ion spray mass spectrometry (ThermoFisher Scientific, Franklin, MA, and Applied Biosystems-MDS Sciex, Foster City, CA). Intraassay CV were 3.1, 5.0, and 3.5% at 29, 109, and 325 pg/ml, respectively (multiply by 3.67 to convert to picomoles per liter). Interassay CV were 8.6, 9.0, 6.6, and 4.8% at 24, 61, 125, and 360 pg/ml, respectively.

IGFBP-1, IGFBP-3, and total IGF-I concentrations were measured by immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX) (8). Interassay CV for IGF-I were 9% at 64 µg/liter and 6.2% at 157 µg/liter. Intraassay CV were 3.4% at 9.4, 3% at 55, and 1.5% at 264 µg/liter.

LH and FSH were assayed using the DxI automated two-site immunoenzymatic system (Beckman Instruments, Chaska, MN). For LH, intraassay CV were 4.3 and 4.0% at 1.2 and 38.5 IU/liter and interassay CV 9.3, 6.0. and 6.0% at 1.4, 15.6, and 48.8 IU/liter, respectively. For FSH, intraassay CV were 3.2 and 2.8% at 8.6 and 47.1 mIU/ml and interassay CV 3.6, 3.2, and 4.7% at 6.5, 16.7, and 58.0 mIU/ml, respectively.

Deconvolution analysis

Each 8-h GH concentration time series was analyzed using a recently validated deconvolution method (19). The automated program first detrends the data and normalizes concentrations to the unit interval [0, 1]. Second, successive potential pulse-time sets, each containing one fewer burst, are created by a smoothing process (a nonlinear adaptation of the heat-diffusion equation). Third, a maximum-likelihood expectation (MLE) deconvolution method using the Matlab7 pattern-search algorithm (The MathWorks, Natick, MA) estimates all secretion and elimination rates simultaneously for each candidate pulse-time set (19). The deconvolution model specifies basal secretion (β0), two half-lives ({alpha}1, {alpha}2), an accumulation process and weak interpulse-length dependency for secretory-burst mass ({eta}0, {eta}1), random effects on burst mass ({sigma}A), procedural and measurement error ({sigma}{epsilon}), and a three-parameter secretory-burst waveform (β1, β2, β3). In the present analysis, the rapid half-life was assumed to be 3.5 min and contribute 37% of total decay (20). Lastly, model selection is performed to distinguish among the candidate pulse-time sets using the Akaike information criterion (21). Observed interpulse intervals are described by a two-parameter Weibull process (more general form of a Poisson renewal process). The parameters (and units) are frequency (number of bursts per unit time, {lambda} of Weibull distribution), regularity of interpulse intervals (unitless {gamma} of Weibull), slow half-life (min), basal and pulsatile secretion rates (concentration per unit time), mass secreted per burst (concentration), and waveform mode (time delay to maximal secretion after burst onset in minutes) (19).

Approximate entropy (ApEn)

ApEn is a scale- and model-independent univariate regularity statistic used to quantitate the orderliness (subpattern consistency) of serial stationary measurements. GH data were subjected to first-differencing to ensure stationarity. Mathematical models and feedback experiments establish that pattern orderliness monitors feedback and/or feedforward interactions within an interlinked axis with high sensitivity and specificity (both > 90%) (22). Reduced pattern regularity typifies hormone secretion in puberty and aging, during diminished negative feedback or fixed exogenous stimulation, and by autonomous neuroendocrine tumors.

Statistical analysis

The primary outcome was the mean GH concentration observed after injection of saline and each dose of rhIGF-I. Hourly mean GH concentrations were evaluated by three-way analysis of covariance (ANCOVA). The model comprised three categorical factors: placebo and E2, three doses of rhIGF-I, and eight time blocks with the GH response to saline serving as the covariate. Post hoc testing was by Fishers least-significantly different criterion (23). In a pilot analysis in eight subjects, a single dose (1.0 mg/m2) of rhIGF-I reduced mean GH concentrations by 45 ± 19% (SD). As an approximation, statistical power to detect 50% attenuation of such inhibition by the estrogen intervention would exceed 90% at P < 0.05 by a priori one-tailed unpaired t test if 19 individuals completed the study.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Subject characteristics and baseline hormonal data are summarized in Table 1Go. Twenty-five women participated, 13 of whom received placebo and 12 E2. Age and body mass index (BMI) were similar in the cohorts. Compared with placebo, administration of E2 lowered FSH, increased E2, SHBG, and GH, and did not affect the single 0800-h fasting IGF-I concentration. On the saline day, the 8-h mean (nine samples) IGF-I concentration was 113 ± 0.83 (placebo) and 94 ± 0.94 (E2) µg/liter (P < 0.01). Two-way ANCOVA using the saline response as a covariate revealed that injection of rhIGF-I dose-dependently elevated peak and mean IGF-I concentrations (both P < 0.001) (Fig. 2Go). Concomitant administration of E2 did not alter IGF-I responses.


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  		TABLE 1. Baseline characteristics and hormonal measurements

 

Figure 2
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  		FIG. 2. Administration of rhIGF-I elevates mean and peak total IGF-I concentrations dose-dependently and comparably in placebo (n = 13) and E2-treated (n = 12) postmenopausal women. P values reflect two-way ANCOVA. Means with unshared superscripts are significantly different by Fisher’s least-significantly different test.

 
Three-way ANCOVA showed that E2 stimulated (P < 0.001 vs. placebo) and rhIGF-I inhibited (P < 0.001 vs. saline) 10-min GH concentrations (Fig. 3AGo). Time after rhIGF-I injection was also a significant factor (P < 0.001), which interacted with IGF-I dose (P < 0.001). E2 treatment interacted with IGF-I dose (P < 0.001) but not time (P = 0.23). There was no three-way interaction. Post hoc comparisons revealed that E2 overcame submaximal but not maximal inhibition by IGF-I; i.e. E2 significantly relieved inhibition by rhIGF-I doses of 1.0 (P = 0.001) and 1.5 (P = 0.008) but not 2.0 mg/m2 (Table 2Go). Three-way ANCOVA of hourly data further confirmed that E2 and IGF-I regulate mean GH concentrations (Fig. 3BGo). E2 elevated hourly mean GH concentrations after saline injection and relieved suppression by exogenous IGF-I when the dose was 1.0 mg/m2 but not higher.


Figure 3
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  		FIG. 3. A, GH concentration-time profiles (mean ± SEM) in 13 placebo-treated and 12 E2-treated postmenopausal women who underwent blood sampling every 10 min for 8 h fasting. Subjects each received 0 (saline), 1.0, 1.5, and 2.0 mg/m2 rhIGF-I by sc injection in randomized order on separate days at 0800 h. B, E2 compared with placebo supplementation reduces the inhibitory effect of rhIGF-I on hourly mean GH concentrations. Data are the mean ± SEM (n = 12 E2, n = 13 placebo). Two-way ANCOVA was applied followed by post hoc Fisher’s least-significantly different test.

 

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  		TABLE 2. Slopes of GH secretion (micrograms per liter per 8 h) regressed on BMI (kilograms per square meter)

 
Deconvolution analysis was used to assess the mechanisms by which IGF-I inhibits and E2 stimulates GH production over 8 h of sampling. In particular, two-way ANCOVA revealed that increasing doses of rhIGF-I suppress 1) pulsatile GH secretion (P = 0.001), 2) GH secretory burst number (P < 0.001), and 3) GH secretory-burst mass (micrograms GH released per unit distribution volume per pulse) (P = 0.005), but not basal GH secretion (P = 0.52) (Fig. 4Go, A–C). By way of comparison, supplementation with E2 stimulated pulsatile GH secretion (P = 0.008), elevated basal GH secretion (P < 0.001), and did not oppose inhibition of GH burst frequency by IGF-I (P = 0.62). Administration of E2 attenuated inhibition of pulsatile GH secretion by the lowest dose of rhIGF-I (1.0 mg/m2) (P = 0.042) and did not overcome inhibition by higher doses of IGF-I. Power for the latter comparison was 0.92 to detect a 30% difference at P < 0.05 in 25 subjects.


Figure 4
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  		FIG. 4. Increasing IGF-I concentrations suppress pulsatile GH secretion (A), E2-stimulated basal GH secretion (B), GH pulse number (C), and the irregularity of GH secretion patterns (ApEn) (D). Data were analyzed as described in Fig. 3Go. Curved solid lines denote a monoexponential fit with the indicated R2 values. Interrupted lines define the IGF-I increment and the IGF-I concentration producing 50% asymptotic inhibition. In the absence of an E2 effect, regression was performed on the combined data.

 
ApEn (irregularity) of 8-h GH concentration time series decreased significantly with increasing rhIGF-I dose (P < 0.001), denoting feedback-imposed orderliness (see Subjects and Methods) (Fig. 4DGo). E2 did not relieve this effect (P = 0.48 by two-way ANCOVA).

Waveform shape, defined by the mode of the GH secretory burst (time from burst onset to maximal secretion), did not differ after treatment with E2 or rhIGF-I (two-way ANCOVA P > 0.35, average mode 20.5 ± 1.8 min). Neither intervention altered the slow-phase GH half-life (average 15.5 ± 0.4 min, P > 0.26). Pulse-renewal variability also seemed to be independent of E2 or rhIGF-I exposure (mean {gamma} of Weibull distribution 2.6 ± 0.48, P > 0.39), recognizing that for 8-h data series, a type II error would be possible.

Quadratic or exponential regression was used to estimate IGF-I concentrations that suppressed GH secretory-burst mass, pulse number, basal secretion, and ApEn by 50% of the difference between baseline and the asymptote. Whether or not E2 was present, an IGF-I concentration increment of 32 µg/liter was sufficient to diminish pulse number by 50% asymptotically. IGF-I increments of 51 µg/liter (placebo) and 107 µg/liter (E2) were required to inhibit GH secretory-burst mass to an analogous degree (P < 0.01). The IGF-I increment needed to reduce ApEn comparably was 6.3 µg/liter with or without E2 supplementation. Even the highest IGF-I level did not decrease basal GH secretion in the placebo group, but an IGF-I concentration increment of 122 µg/liter did so during E2 administration (P < 0.005). These data demonstrate distinguishable sensitivities of individual GH secretory measures to inhibition by IGF-I and selective effects of E2 to limit suppression of GH secretory-burst mass and elevate basal GH secretion.

Electrocardiographic monitoring revealed occasional atrial premature contractions in three patients not requiring intervention. The absolute plasma glucose concentration range was 61–89 mg/dl in the 25 subjects after rhIGF-I injection. This range was no different from that observed in the same subjects on the saline day.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Salient outcomes of this investigation are that 1) oral E2 supplementation does not alter the capability of graded doses of exogenous rhIGF-I to increase total IGF-I concentrations in postmenopausal individuals, suggesting that estrogen does not accelerate IGF-I clearance; 2) higher IGF-I concentrations suppress mean and nadir GH concentrations and inhibit pulsatile GH secretion by decreasing both secretory-burst mass and number; 3) elevated IGF-I concentrations enforce more orderly GH secretion patterns but do not alter GH half-life, secretory-burst shape, or the variability of interpulse intervals; 4) E2 supplementation amplifies both pulsatile and basal GH secretion without increasing pulse number; 5) E2 administration relieves submaximal but not maximal inhibition of pulsatile GH secretion by exogenous IGF-I; and 6) E2 is unable to overcome IGF-I’s suppression of secretory-burst number or ApEn (pattern regularity). Therefore, an estrogen-enriched milieu in postmenopausal women can selectively antagonize the feedback actions of elevated systemic IGF-I concentrations on pulsatile GH secretion.

Mean hormone concentrations are determined jointly by the size and number of secretory bursts, elimination half-life, and underlying basal secretion (19). The present analyses indicate that imposing young adult-like concentrations of IGF-I in postmenopausal women reduces mean GH concentrations by decreasing both the size and number of GH secretory bursts without altering estimated GH half-life or basal secretion. The fact that rhIGF-I attenuates both the size and number of GH secretory bursts would be consistent with suppression of pulsatile GHRH drive to somatotropes (1). Whereas the capability of oral estrogens to lower endogenous IGF-I concentrations could in principle contribute to augmented pulsatile GH secretion in such settings (1, 5, 7), our data establish that E2 administration also mutes feedback by any given (submaximally inhibitory) IGF-I concentration in postmenopausal women. Peak preovulatory serum E2 concentrations reported in three recent studies comprising a total of 346 healthy young women averaged 320 ± 214 (SD) pg/ml. The present experimental mean E2 concentration of 449 pg/ml falls within 0.60 SD (P = 0.27) of the normal mean so estimated (24, 25, 26). Thus, our data could have applicability to understanding the preovulatory rise of GH secretion in young women (1).

In experimental models, IGF-I inhibits hypothalamic GHRH secretion and stimulates somatostatin release in vitro and likewise represses GHRH and induces somatostatin gene expression in vivo (1, 27, 28). In humans, IGF-I administration suppresses fasting GH concentrations and impairs GH responses to exogenous GHRH and GHRP/ghrelin (28, 29, 30). In the only study in postmenopausal women, a single iv dose of rhIGF-I suppressed the GH response to GHRH by 50% (29), and inhibition was not overcome by oral E2 administration. The present rhIGF-I dose-response analysis conforms with this outcome in that E2 diminishes hypothalamo-pituitary sensitivity to submaximal repression by IGF-I but does not overcome maximal suppression by IGF-I (inhibitory efficacy). This inference was confirmed by showing that E2 supplementation doubles the increment in IGF-I concentrations required to reduce the size of GH secretory bursts by 50% of the total decrement. In other investigations, administration of L-arginine, a putative inhibitor of hypothalamic somatostatin release, counteracted exogenous IGF-I’s suppression of fasting and GHRH-stimulated GH secretion (31, 32). These effects argue against clinically significant direct pituitary inhibition in humans in vivo, unlike direct inhibition of GH release in vitro by sustained exposure to IGF-I (27). Thus, a plausible feedback model (Fig. 5Go) is that IGF-I evokes periventricular outflow of somatostatin, which represses both GHRH secretion from the arcuate nucleus and GH release by somatotropes (1, 33). The converse of this scenario could explain potentiated effects of GHRH in volunteers given pegvisomant to lower systemic IGF-I concentrations. The model would also be consistent with the results of transgenic knockout of the somatostatin gene, which elevates both IGF-I concentrations and GH secretion (34). However, somatostatin-gene silencing does not preclude repression of hypothalamic GHRH by elevated GH and IGF-I concentrations, suggesting the existence of somatostatin-independent inhibitory pathways in the mouse. Whether analogous pathways operate in the human is not known.


Figure 5
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  		FIG. 5. Proposed mechanisms of estrogen-induced attenuation of negative feedback by IGF-I. In this model, IGF-I evokes somatostatin outflow, which in turn inhibits hypothalamic GHRH release and pituitary GHRH action. Estradiol is viewed as potentiating GHRH and ghrelin drive and possibly also relieving direct pituitary inhibition by somatostatin or IGF-I. However, E2 is unable to reverse IGF-I’s intrahypothalamic repression of GHRH pulse frequency.

 
Only two clinical investigations have employed variable doses of rhIGF-I to investigate feedback control (35, 36). One study did not use either randomly ordered or separate-day infusions as required for valid scientific interpretation (35), and neither study controlled the sex-steroid milieu experimentally (36). The accompanying paradigm is unique by way of administering randomly ordered doses of rhIGF-I on separate days in an E2-deficient vs. E2-enriched milieu. This paradigm unveiled that estrogen selectively antagonizes negative feedback by IGF-I on GH secretory-burst mass but not number. The basis for this mechanistic distinction has not been established. A plausible speculation would be that IGF-I-induced somatostatin outflow reduces both the size and number, whereas E2 augments the size but not the number, of GHRH pulses. In this regard, estrogen receptor-{alpha} is expressed in 65% of arcuate-nucleus GHRH neurons but in few if any periventricular somatostatin neurons (1). In addition, estrogen reduces brain GH receptor number and GH feedback onto a GHRP stimulus (13, 17), potentiates GH responses to GHRH, ghrelin, and GHRP, and opposes submaximal inhibition by infused somatostatin in women (8, 10, 11, 12). Whether E2 also restricts somatostatin outflow to the pituitary gland or attenuate IGF-I’s putatively direct inhibition of somatotropes is not known (1).

Protein-unbound (free) IGF-I concentrations correlate with negative feedback more strongly than total IGF-I concentrations under some conditions (37). This point is of interest, because oral E2 administration can lower both total and free IGF-I concentrations (38). Although fasting total IGF-I levels averaged over 8 h were reduced here by 17% in the E2 group during saline infusion, they increased linearly with IGF-I dose and indistinguishably in the E2- and placebo-treated cohorts. Whether E2 supplementation might further augment GH secretion by reducing free IGF-I availability to tissue sites mediating negative feedback is not known.

In conclusion, an experimental paradigm of randomly ordered, separate-day, double-blind administration of rhIGF-I in doses of 0, 1.0, 1.5, and 2.0 mg/m2 in a low- vs. high-estrogen milieu demonstrates that systemic IGF-I inhibits GH secretory-burst mass and frequency but not basal GH secretion. E2 supplementation augments basal and pulsatile GH secretion and antagonizes IGF-I’s repression of pulsatile GH secretion without altering its effects on burst number, interburst-interval variability, or the orderliness of GH secretion. Accordingly, estrogen’s disinhibition of the feedback effect of any given systemic total IGF-I concentration (present data) and its attenuation of GH feedback onto a GHRP/ghrelin stimulus (13) together confer dynamic mechanisms by which a sex steroid can amplify pulsatile GH secretion.


   Acknowledgments
 
We thank Kay Nevinger and Donna Scott for support of manuscript preparation, Ashley Bryant for data analysis and graphics, the Mayo Immunochemical Laboratory for assay assistance, and the Mayo research nursing staff for implementing the protocol. Recombinant human IGF-I was provided by Tercica Inc. (South San Francisco, CA).


   Footnotes
 
This work was supported in part via the Center for Translational Science Activities (CTSA) Grant 1 UL 1 RR024150 to the Mayo Clinic and Foundation from the National Center for Research Resources (Rockville, MD) and R01 NIA AG29362 and AG19695 from the National Institutes of Health (Bethesda, MD).

Disclosure Statement: The authors have nothing to declare.

First Published Online August 26, 2008

Abbreviations: ANCOVA, Analysis of covariance; ApEn, approximate entropy; BMI, body mass index; CRU, Clinical Translational Unit; CV, coefficient of variation; E2, estradiol; GHRP, GH-releasing peptide; IGFBP, IGF-binding protein; rh, recombinant human.

Received July 11, 2008.

Accepted August 19, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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