Abstract
Background
Oral administration of estradiol (E2) generally increases GH secretion in postmenopausal women. Oral administration of E2 is associated with a decrease in IGF-1, whereas parenteral or transdermally administered E2 may have no effect on GH. The effect of progesterone (P4) on GH secretion has rarely been studied. We hypothesized that moderately increased serum E2 levels stimulate GH and that P4 modulates E2-stimulated GH secretion.
Study Design
Four parallel groups of randomly assigned postmenopausal women (n = 40). Treatments were saline placebo and oral placebo, saline placebo and oral micronized P4 (3 × 200 mg/d IM), E2 (5 mg IM) and oral placebo, and E2 IM and oral micronized P4. Outcome measures were overnight GH secretion (10 hours), stimulated (ghrelin, 0.3 µg/kg IV bolus) GH secretion, and CT-estimated visceral fat.
Results
Intramuscular E2 administration did not alter nocturnal and ghrelin-stimulated GH secretion. Nocturnal GH secretion was not changed by P4 administration. However, P4 diminished ghrelin-stimulated pulsatile GH release with or without E2 (average, 7.20 ± 2.14 and 9.58 ± 1.97 µg/L/2 h, respectively; P = 0.045). Respective outcomes for mean GH concentrations and GH peak amplitudes were 0.97 ± 0.31 and 1.52 μg/L ± 0.29 (P = 0.025) and 2.76 ± 1.04 and 3.95 μg/L ± 0.90 (P = 0.031). Ghrelin-stimulated GH secretion correlated negatively with P4 concentration with or without correction for visceral fat area in the regression equation (R = 0.49, P = 0.04, β = −0.040 ± 0.016).
Conclusions
Low-range physiological E2 concentrations do not affect spontaneous or ghrelin-stimulated pulsatile GH secretion. Conversely, P4 inhibits ghrelin-stimulated GH secretion in a concentration-dependent fashion. The mechanistic aspects and physiological significance of natural P4’s regulation of ghrelin-evoked GH secretion require further study.
Low-range physiological E2 concentrations do not affect spontaneous or ghrelin-stimulated pulsatile GH secretion. P4 inhibits ghrelin-stimulated GH secretion in a concentration-dependent fashion.
GH is regulated by the time-dependent interplay among the stimulating neuropeptide GH-releasing hormone GHRH and the inhibitory neuropeptide somatostatin. Ghrelin, an acylated GH-releasing peptide (GHRP) synthesized in the stomach, anterior pituitary gland, hypothalamus, kidney, gonad, and placenta, induces GH secretion via combined hypothalamo–hypophyseal mechanisms and concomitantly regulates appetite and insulin secretion. In addition, many other factors affect this system, including negative feedback by liver-derived circulating IGF-1, GH itself, and free fatty acids (1). Clinical studies indicate that estradiol (E2) regulates hypothalamo–pituitary responses to each of these major regulatory peptides (2–4). The highest GH concentrations in humans are present at birth and at puberty, after which GH levels decrease steadily, along with testosterone (T), E2, IGF-1, and IGF-binding protein (IGFBP)-3 concentrations. These data together show that relative sex-steroid deprivation accentuates GH and IGF-I depletion in humans. The combined endocrine changes after puberty have substantial clinical implications to aging-related physical frailty, diminished aerobic capacity, sarcopenia, osteopenia, visceral adiposity, glucose intolerance, and reduced psychosocial well-being (5–8). In contrast to T, E2, synthetic estrogens, and selective estrogen receptor modulators, the possible role of progesterone (P4) on the somatotropic axis is largely unknown (9). Women during their fertile lifespan are exposed to elevated luteal-phase P4 concentrations about half the time. Recent studies have shown that P4 stimulates the local production of GH in cultured human breast epithelium. In turn, GH increases proliferation of a subset of cells that express the GH receptor and have functional properties of early progenitor and stem cells (10). Comparable effects on local GH production have been found in dogs (11).
A number of studies in women have suggested that P4 may enhance spontaneous and stimulated GH secretion (3, 4, 12). The present prospectively randomized, placebo-controlled, double-blind, parallel-group study in postmenopausal women examines the hypothesis that P4 modulates spontaneous nocturnal GH secretion, assessed by 10-minute blood sampling for 10 hours and/or the GH response to a submaximal stimulating dose of ghrelin.
Materials and Methods
Subjects
Forty healthy, ambulatory, community-dwelling postmenopausal women, clinically defined by E2 <50 pg/mL and FSH >30 IU/L, within the allowable age range of 50 to 80 years participated in the overnight Clinical Research Unit (CRU)–based study. Volunteers were recruited by newspaper advertisements, local posters, the Mayo Clinical Trials Center web page, and community (general and minority) bulletin boards. The design of the study was placebo-controlled, double-blind, prospectively randomized assignment to four hormone treatment groups: (i) saline placebo IM and oral placebo, (ii) saline placebo IM and oral micronized P4 (iii) E2 valerate IM and oral placebo, and (iv) E2 valerate IM and oral micronized P4. The treatment paradigm comprised: day 1, IM E2 valerate 2.5 mg or saline placebo 0.12 mL administration during an outpatient visit; day 10 (±2 days), IM E2 valerate 5 mg or saline placebo once, followed by oral micronized P4 200 mg or oral placebo three times daily for 14 days (±2 days) thereafter. The dose of E2 was chosen as an estimate made from IM E2 levels achieved from US Pharmacopoeia. The goal, which was achieved, was to increase serum E2 from very low postmenopausal levels (2 to 12 pg/mL) to ∼100 pg/mL by mass spectrometry (a 10-fold increase). The question was whether such an increase during young women’s midfollicular phase drives GH secretion in older women with or without P4 modulation. The dosage of P4 administered closely mimics a physiological P4 milieu in hypogonadal women, given the very rapid P4 half-life reported by us and others in women (13). At day 23 (±2 days), subjects were admitted to the Clinical Research Unit (CRU) at 5:00 pm and received a prescribed metabolic study meal consisting of 8 kcal/kg of 50% carbohydrate, 20% protein, and 30% fat at 7:00 pm. The subjects remained fasting until the end of the study at 10:00 am the following morning. Breakfast was offered after 10:00 am before discharge from the CRU. Volunteers were allowed to sleep during the overnight sampling window, which comprised 10-minute blood sampling for 12 hours overnight starting at 10:00 pm. At 8:00 am (2 h before the end of sampling), a single intravenous injection of ghrelin (0.3 µg/kg) was administered, and blood sampling was continued for 2 hours. The study was designed to quantify the impact of E2 and/or P4 on nocturnal GH secretion and ghrelin-stimulated GH release. Hormones and peptides were measured in the fasting blood sample obtained at 8:00 am. During the stay in the CRU, P4 or placebo administration was continued. A schematic outline of the study is illustrated in Fig. 1. The Mayo Institutional Review Board required a short course of progestin after completion of the blood sampling to shed the endometrium, which is standard of care in the United States to avert unopposed estrogenic stimulation (hyperplasia) of the endometrium, which would otherwise be present in one of the four groups. Therefore, at the end of the CRU study, subjects in the E2-only group received blinded medroxyprogesterone acetate (5 mg capsules) taken orally for 10 days, whereas women enrolled in the other groups received placebo oral capsules for 10 days.
Figure 1.
Schematic outline of the study. Four randomized groups of postmenopausal women were studied; subjects received E2, P4, and placebo in different combinations. After 24 days, a blood sampling study was performed in the CRU while P4 administration was continued. After the end of the blood sampling, subjects with unopposed E2 exposure received medroxyprogesterone 5 mg for 10 days, and the three groups were treated with placebo.
The protocol was approved by Mayo Institutional Review Board. Witnessed voluntary written informed consent was obtained before study enrollment. Complete medical history, physical examination, and screening tests of hematological, renal, hepatic, metabolic, and endocrine function were normal. Subjects underwent a single-slice CT of the abdomen (level L3 to L4) as an exploratory test of the impact of relative visceral obesity on GH. E2 valerate was obtained from PharmaForce (New Albany, OH), and micronized P4 was obtained from Akorn (Lake Forest, IL).
This investigator-initiated pilot study does not qualify as a clinical trial because it is an acute physiological examination without any health-related or behavioral outcomes per se.
Exclusion criteria
Exclusion criteria were acute or chronic systemic diseases, HIV positivity by medical history, anemia, endocrine disorders (except subjects with hypothyroidism who were biochemically euthyroid on replacement), psychiatric illness, alcohol or drug abuse, or deep venous or arterial thromboses; cancer of any type (except localized basal or squamous cell cancer of the skin treated surgically without recurrence); recent use of estrogen, progestin, anabolic steroids, or glucocorticoids; history of stroke, myocardial infarction, or angina; clinically significant ECG abnormality as determined by study team physicians; allergy to medications used in the study; and substantial recent weight change (loss/gain of ≥6 lbs over 6 weeks), transmeridian travel (exceeding three time zones within the preceding 6 weeks), current or recent night shift work, systemic drugs, abnormal renal, hepatic or hematologic function, concomitant sex-hormone replacement, and unwillingness to provide written informed consent.
Assays
The 10-minute serum samples were assayed in a single batch in each subject by chemiluminescence GH assay, performed using Beckman’s robotics Access ultrasensitive human GH assay (Beckman Coulter, Inc., Atlanta, GA). Within-assay precision was 3.8% to 6.5% (range of 100 runs), sensitivity was 0.010 μg/L, and specificity was 97% 22 kDa GH (14). IGF-1, IGFBP-3, and SHBG were quantified by solid-phase chemiluminescent assay on the Siemens Immulite 2000 Automated Immunoassay System (Siemens Health Care Diagnostics, Deerfield, IL). Intra-assay coefficient of variations (CVs) for IGF-I were 4.9% and 5% at 37 and 225 μg/L, respectively; for IGFBP-3 CVs were 4% and 3.9% at 1.0 and 4.3 mg/L, respectively; and for SHBG CVs were 4.0% and 5.9% at 5.4 and 74 nmol/L, respectively (15–17). IGFBP-1 was measured by a two-site immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX). Intra-assay CVs were 10.2% at 0.49 µg/L and 6.7% at 4.5 µg/L (18). E2 and total T were measured using liquid chromatography–tandem mass spectrometry (Agilent Technologies, Inc., Santa Clara, CA). Intra-assay CVs were: E2, 10.8% at 0.29 pg/mL and 5.1% at 32 pg/mL; T, 8.9% at 0.69 ng/dL, 4.0% at 45 ng/dL, and 3.5% at 841 ng/dL. P4 was measured by a two-site immunoenzymatic sandwich assay on the Roche Cobas e411 (Roche Diagnostics, Indianapolis, IN). Intra-assay CVs were 4.8%, 2.2%, and 1.8% at 0.474, 8.26, and 28.5 ng/mL, respectively (19). Insulin was measured by a two-site immunoenzymatic sandwich assay on the Roche e411 (Roche Diagnostics). Intra-assay CVs were 3.3%, 2.8%, and 2.5% at 18, 61, and 172 mU/L, respectively (20). Leptin was measured by specific immunoassay kit (Linco Research, Inc., St. Louis, MO). The interassay CV was 11% at 20.4 ng/mL (21). Prolactin, FSH, and LH were measured by two-site chemiluminescent sandwich immunoassays on a DXL 800 automated immunoassay system (Beckman Instruments, Chaska, MN). For prolactin, the intra-assay CVs were 3.7%, 2.1%, and 4.8% at 6.1, 16.4, and 34.5 µg/L, respectively; for FSH, the interassay CVs were 3.6%, 3.2%, and 4.7% at 6.5, 16.7, and 58.0 IU/L, respectively; and for LH, the interassay CVs were 9.3%, 6.0%, and 6.0% at 1.4, 15.6, and 48.8 IU/L, respectively (22–24).
Calculations
Deconvolution of 10-minute GH concentration profiles
Variable-waveform deconvolution analysis was used to reconstruct secretion into underlying trains of secretory bursts, superimposed upon basal (time-invariant) secretion, allowing biexponential elimination (fixed fast half-life for rapid diffusion and advection and estimated slow half-life for delayed metabolic elimination). The analysis implements a Matlab-based, adaptive-mesh, multiparameter-search algorithm that has been cross validated (25).
Approximate entropy
Secretory regularity of GH secretion was appraised via the approximate entropy (ApEn) statistic. This metric provides a sensitive (>90%) and specific (>90%) model-free and scale-invariant measure of relative randomness due to loss of feedback control within a network (26).
Statistics
Data were analyzed by ANOVA for the four randomly assigned treatment groups. Post hoc testing was mainly restricted to comparisons between E2(+) and E2(−) subjects and between P4(+) and P4(−) subjects. Age and visceral fat mass were covariates. In the case of non-normal data distribution, the Kruskal-Wallis test was used. Linear regression analysis was applied to identify concentration-dependent effects of P4 and/or E2, corrected for age and visceral fat area. Calculations were performed with Systat 13 (Systat Software, Inc., San Jose, CA). A P value < 0.05 was considered significant for the overall study.
Results
By ANOVA, the four groups were strictly comparable, including parameters of body composition, mean age, and serum concentrations of hormones and binding proteins (Table 1).
Table 1.
Demographic Data
| Plac + Plac (n = 11) | Plac + P4 (n = 9) | E2 + Plac (n = 10) | E2 + P4 (n = 10) | ANOVA | |
|---|---|---|---|---|---|
| Age, y | 61.4 ± 1.7 | 62.8 ± 2.4 | 62.6 ± 1.9 | 63.3 ± 1.2 | 0.48 |
| BMI, kg/m2 | 25.6 ± 1.5 | 26.8 ± 1.7 | 25.5 ± 1.0 | 24.9 ± 1.3 | 0.81 |
| E2, pg/mL | 23.6 ± 1.4 | 23.6 ± 1.4 | 23.5 ± 1.5 | 23.6 ± 1.4 | 1.00 |
| P4, ng/mL | 0.32 ± 0.05 | 0.26 ± 0.03 | 0.39 ± 0.06 | 0.27 ± 0.02 | 0.46 |
| SHBG, nmol/L | 54.8 ± 7.1 | 56.7 ± 6.8 | 60.8 ± 7.3 | 72.4 ± 6.8 | 0.29 |
| T, ng/dL | 13.7 ± 1.7 | 16.1 ± 1.6 | 17.5 ± 1.6 | 22.6 ± 7.2 | 0.45 |
| IGF-1, ng/mL | 124 ± 17.8 | 126 ± 6.9 | 133 ± 12 | 108 ± 16.5 | 0.65 |
| IGFBP-1, µg/L | 3.01 ± 0.47 | 2.40 ± 0.32 | 3.05 ± 0.47 | 4.26 ± 0.88 | 0.15 |
| IGFBP-3, mg/L | 4.04 ± 0.24 | 4.04 ± 0.28 | 4.03 ± 0.19 | 3.41 ± 0.23 | 0.18 |
| Visceral fat, cm2 | 89 ± 17.5 | 99 ± 24.9 | 90 ± 15.3 | 85 ± 13.3 | 0.97 |
| Total fat, cm2 | 313 ± 51 | 311 ± 60 | 325 ± 48 | 300 ± 33 | 0.98 |
Data are shown as mean ± SEM. ANOVA was done with the General Linear Model procedure. Hormone levels were measured in the fasting state during the screening examination.
Abbreviation: Plac, placebo.
Serum GH concentration profiles during 10-hour sampling and those after injection with ghrelin are displayed in Fig. 2. Addback with E2 increased serum E2 levels from 3.59 ± 0.49 to 94 ± 10 pg/mL (P < 0.0001), and under P4 treatment P4 levels increased from 0.2 to 15.5 ± 2.0 ng/mL (P < 0.0001). Replacement with E2 increased serum PRL concentration from 10.0 ± 0.69 to 18.3 ± 1.5 ng/mL (P < 0.0001), increased SHBG from 46 ± 4.1 to 89 ± 6.6 nmol/L (P < 0.0001), increased IGFBP-1 from 2.52 ± 0.31 to 3.83 ± 0.51 μg/L (P = 0.037), and decreased IGFBP-3 concentration from 3.67 ± 0.20 to 2.99 ± 0.16 mg/L (P = 0.012). LH concentration was 24.8 ± 0.8 U/L in E2-depleted women and 4.7 ± 0.8 U/L in E2-treated women (P < 0.0001). Respective FSH concentrations were 67.0 ± 4.0 and 24.8 ± 1.7 U/L (P < 0.0001). Serum insulin, leptin, and IGF-I concentrations remained unchanged.
Figure 2.
Serum GH concentration profiles. The upper panel displays the results of nocturnal blood sampling every 10 minutes for 10 hours. The lower panel displays the effect of ghrelin on serum GH. Data are shown as mean ± SEM.
Nocturnal GH secretion estimated by deconvolution analysis and ApEn in the four experimental groups are depicted in Table 2. By ANOVA, no differences were demonstrable between the groups. In addition, the influence of E2 on GH secretion was quantified both in the presence and absence of P4. The results are depicted in Table 3. In the absence of P4, GH secretion and ApEn were slightly higher in subjects receiving E2, but the differences were not significant before or after correcting for age, visceral fat, and total fat area. When all subjects receiving E2 were compared with those who did not (i.e., irrespective of P4 administration), basal (nonpulsatile) GH secretion was higher in the E2-treated group (P = 0.04) also when corrected for age and total fat surface (P = 0.04). However, the effects on pulsatile and total GH secretion, although higher in the E2-treated groups, did not reach statistical significance. Serum IGF-1 concentration in E2-treated subjects was 104 ± 8.2 µg/L and in E2-depleted women was 117 ± 8.8 µg/L (P = 0.27). ApEn was higher in E2-treated women when corrected for age and visceral fat area (P = 0.04). In a multistep regression analysis using basal, pulsatile, or total GH secretion (both on the original and log-transformed data) as dependent variables and age and visceral fat area and E2 levels as independent variables, only visceral fat area was a significant (negative) predictor of GH secretion (Fig. 3). Comparable negative regressions were found for total fat area. Fat area remained a highly significant negative regressor when the analysis was restricted to estrogen-treated and estrogen-depleted women. Finally, the mean value of the GH concentrations during the 10-hour sampling was negatively correlated with visceral and total fat area but not with age and serum E2 concentration (Fig. 4).
Table 2.
GH Secretion Estimated by Deconvolution Analysis in 40 Healthy Postmenopausal Women
| Plac + Plac | Plac + P4 | E2 + Plac | E2 + P4 | ANOVA P Value | |
|---|---|---|---|---|---|
| Basal GH secretion, µg/L/10 h | 4.24 ± 0.82 | 4.37 ± 1.08 | 5.60 ± 1.42 | 9.25 ± 3.05 | 0.29 |
| Pulsatile GH secretion, µg/L/10 h | 17.69 ± 2.78 | 14.48 ± 3.63 | 18.08 ± 3.16 | 18.38 ± 3.58 | 0.81 |
| Total GH secretion, µg/L/10 h | 21.94 ± 3.16 | 18.85 ± 4.22 | 23.68 ± 3.89 | 27.63 ± 6.21 | 0.63 |
| Mean pulse mass, µg/L | 3.18 ± 0.48 | 2.67 ± 0.56 | 3.00 ± 0.48 | 3.63 ± 2.38 | 0.65 |
| Mean GH concentration, µg/L | 0.61 ± 0.09 | 0.50 ± 0.11 | 0.63 ± 0.10 | 0.73 ± 0.15 | 0.50 |
| ApEn, dimensionless | 0.684 ± 0.07 | 0.734 ± 0.075 | 0.810 ± 0.075 | 0.798 ± 0.01 | 0.48 |
Data are mean ± SEM. ANOVA was performed on logarithmically transformed data. Age was not a statistically significant covariate, in contrast to visceral fat area. Results are based on blood samples drawn at 10-min intervals over 10 h.
Abbreviation: Plac, placebo.
Table 3.
Nocturnal GH Secretion, Mean Serum GH, and ApEn in Healthy Postmenopausal Women
| Influence of E2 on GH Secretion |
P Value | ||
|---|---|---|---|
| No P4 | E2(+) (n = 10) | E2(−) (n = 11) | |
| Basal GH secretion, µg/L/10 h | 5.60 ± 1.42 | 4.24 ± 0.82 | 0.21 |
| Pulsatile GH secretion, µg/L/10 h | 18.08 ± 3.16 | 17.70 ± 2.78 | 0.47 |
| Total GH secretion, µg/L/10 h | 23.68 ± 3.89 | 21.94 ± 3.16 | 0.37 |
| Mean pulse mass, µg/L | 3.00 ± 0.48 | 3.18 ± 0.48 | 0.40 |
| Mean serum GH concentration, µg/L | 0.63 ± 0.10 | 0.61 ± 0.09 | 0.43 |
| ApEn (dimensionless) | 0.810 ± 0.075 | 0.684 ± 0.070 | 0.12 |
| Both with and without P4 | E2(+) (n = 20) | E2(−) (n = 20) | |
| Basal GH secretion, µg/L/10 h | 7.43 ± 1.69 | 4.30 ± 0.65 | 0.04 |
| Pulsatile GH secretion, µg/L/10 h | 18.23 ± 2.33 | 16.25 ± 2.20 | 0.27 |
| Total GH secretion, µg/L/10 h | 25.65 ± 3.59 | 20.55 ± 2.52 | 0.12 |
| Mean pulse mass, µg/L | 3.32 ± 0.44 | 2.95 ± 0.36 | 0.26 |
| Mean serum GH concentration, µg/L | 0.68 ± 0.09 | 0.56 ± 0.07 | 0.15 |
| ApEn (dimensionless) | 0.804 ± 0.050 | 0.707 ± 0.050 | 0.08 |
| Both with and without E2 | P4(+) (n = 19) | P4(−) (n = 21) | |
| Basal GH secretion, µg/L/10 h | 6.94 ± 1.74 | 4.89 ± 0.80 | 0.29 |
| Pulsatile GH secretion, µg/L/10 h | 16.53 ± 2.52 | 17.87 ± 2.04 | 0.68 |
| Total GH secretion, µg/L/10 h | 23.47 ± 3.86 | 22.77 ± 2.42 | 0.88 |
| Mean pulse mass, µg/L | 3.18 ± 0.48 | 3.09 ± 0.33 | 0.89 |
| Mean serum GH concentration, µg/L | 0.62 ± 0.09 | 0.62 ± 0.08 | 0.99 |
| ApEn, dimensionless | 0.768 ± 0.050 | 0.744 ± 0.050 | 0.75 |
Data are shown as mean ± SEM. The main purpose of the analyses was to compare E2 treatment with placebo and P4 with placebo. Statistical comparisons were done with the one- or two-sided Student t tests. GH secretion was estimated by deconvolution analysis of 10-h GH concentration series created by 10-min sampling overnight. The same period was used to calculate mean GH concentrations and ApEn. The test hypothesis was that E2 and/or P4 amplify GH secretion and ApEn.
Figure 3.
Linear regressions between 10-hour nocturnal GH secretion during overnight sampling and abdominal visceral fat in 40 postmenopausal women.
Figure 4.
Regressions between log-transformed basal and pulsatile nocturnal GH secretion and visceral fat area and serum P4 concentrations.
P4 administration did not change overnight 10-hour spontaneous GH secretion estimated by deconvolution analysis or mean serum GH concentration in any of the comparisons with placebo-only–treated women, with E2-only–treated women, and with non-P4–treated women (Table 3). Likewise, after applying covariate corrections for age and for visceral and total fat area, there were no P4 treatment effects on nocturnal GH secretion. However, a negative relation of GH secretion with fat area, especially pulsatile GH secretion, remained significant when P4 was the covariate (Fig. 3). No P4-related differences were found for ApEn.
After ghrelin injection, the pulsatile response of GH in women with E2 addback was similar to E2-deprived women in the absence of P4 (10.69 ± 2.66 and 8.48 ± 3.0 µg/L, respectively; P = 0.58). Comparable results were obtained for the GH-peak amplitude and the mean serum GH concentration in response to ghrelin (data not shown). However, when all data were pooled, P4 diminished GH responses to ghrelin (Fig. 5). GH secretory-pulse mass in women without P4 was 9.6 ± 1.9 µg/L and in women with P4 was 7.2 ± 2.1 µg/L (P = 0.04). Mean ghrelin-stimulated serum GH values were 1.52 ± 0.29 and 0.97 ± 0.31 µg/L (P = 0.029), respectively, and GH-peak amplitude values were 3.9 ± 0.9 and 2.8 ± 1.0 µg/L (P = 0.022), respectively. In addition, GH secretory-burst mass after ghrelin administration was negatively related to serum P4 concentration (Fig. 6).
Figure 5.
GH response after IV ghrelin administration in women with and without P4 treatment. The height of the bars represents the mean, and the error bars represent the SEM. Pulse mass was calculated with deconvolution analysis. Mean concentration was derived from the 2-hour mean serum GH concentration. The amplitude was calculated from the GH concentration just before ghrelin administration and the highest concentration attained.
Figure 6.
Linear regression between log-transformed GH secretory-burst mass after administration of ghrelin (0.33 µg/kg IV) and serum P4 concentrations.
Discussion
This study was designed to quantify the impact of E2 and/or P4, as present in premenopausal women, on nocturnal GH secretion and ghrelin-stimulated GH release in postmenopausal women by sex hormone addback. During short-term sex-steroid hormone addback in healthy postmenopausal women, midfollicular-phase levels of E2 increased basal (nonpulsatile) nocturnal GH secretion but not pulsatile and total secretion. P4 did not affect spontaneous nocturnal GH secretion but diminished ghrelin-stimulated GH release in a concentration-dependent fashion.
Early investigations of the effects of E2 on GH secretion in women demonstrated a stimulating effect of 50 mg oral diethylstilbestrol (27). Later results are conflicting. Orally administered E2 (or other estrogens), usually in a dose of 2 mg/d either alone in short-term studies or combined with progestogens in long-term studies, augmented spontaneous GH secretion, and this effect on GH was associated with decreased serum IGF-1 levels (3, 28–30). In contrast, low-dose transdermal E2 administration (0.05 to 0.1 mg/d) did not increase GH secretion or alter serum IGF-1 concentration (30–33). However, one study, using a higher transdermal E2 dose (0.2 mg/d), yielded high mean serum E2 levels of 350 pg/mL and reported increased GH secretion associated with decreased serum IGF-1 and IGFBP-3 concentrations (34). Collectively, these reports suggest that serum E2 concentrations imposed on postmenopausal women, as occurring in the (early) follicular phase of the menstrual cycle in women, do not increase GH secretion provided that the liver is not exposed to high estrogen levels. The current findings in our postmenopausal women fit this view because E2 levels during addback were in the midfollicular range of 94 pg/mL (35). Notwithstanding the lack of a clear effect on GH secretion, estrogenic effects were clearly present systematically, as evidenced by increased SHBG, IGFBP-3, and prolactin and decreased IGFBP-1 concentrations. In addition, the increased ApEn in E2-treated women (although not statistically significant when tested two-sided) suggests IGF-1/GH feedback disruption and/or increased positive (peptide) signaling on the somatotrope cell population.
The estrogen-deficient state in menopause is accompanied by diminished spontaneous and peptide-stimulated (GHRH, GHRP, and ghrelin) GH secretion (34, 36–38). In one study, treatment with E2 in women with premature ovarian failure resulted in higher GH concentrations than in a comparable group of women with later-life onset of menopause (33). In another study in premenopausal and postmenopausal women, with a leuprolide-downregulated gonadotropic system and addback transdermal E2 resulting in similar serum E2 concentrations, premenopausal women had higher basal and peptide-stimulated GH secretion than postmenopausal women. Both studies underscore the impact of aging per se on the magnitude of estrogen-induced GH secretion (39). Therefore, age as well as lower E2 dose might be a plausible explanation for the absence of estrogen effect on spontaneous GH secretion in our cohort. Whether IM E2 administration has unique effects on GH distinct from the effects of oral or transdermal E2 is not known.
Few studies have addressed the question of whether changing sex steroid concentrations during the menstrual cycle alter GH secretion. Available studies have reported increased GH secretion in the late follicular phase compared with the early phase and that midluteal-phase GH secretion was comparable with that of the early follicular phase. Such data suggest that serum E2 levels during the E2 surge could be responsible for the increased pulsatile GH secretion (12, 27, 40, 41). Increased late-follicular GH pulse frequency and mass were accompanied by raised IGF-1 levels (41). This setting is thus uniquely different from that of exogenous estrogen delivery by any clinical route. Summarizing, the impact of exogenous estrogens (E2) on spontaneous GH secretion depends on both age and body composition (present data), hormone dose, and route of administration. Other explanations for a lack of a lower-dose estrogen effect on GH secretion in our subjects include the absence of circadian and ultradian E2 rhythms.
The effects of ghrelin (GHRP) or GHRH have been studied during the menstrual cycle. Detailed analyses of different ghrelin doses (submaximal to maximal) did not find a phase-dependent change in GH response (41–43). Other studies on the modulating effect of E2 on stimulated GH secretion were carried out in postmenopausal women (4, 34, 36, 38, 44). These investigations revealed that E2 enhances ghrelin (GHRP-2) and GHRH-stimulated GH secretion. Common to these studies was a higher dose of E2 (either 2 mg/d orally or 0.15 to 0.2 mg/d transdermally). Where reported, serum E2 concentrations were 2- to 3.5-fold higher than in the current study. Therefore, a major determinant of the effect of GH-stimulating peptides is likely the mean serum E2 concentration per se. Unfortunately, no E2 dose-response studies have been published.
A salient feature of this study was the inhibitory effect of P4 on ghrelin-stimulated GH secretion but not on spontaneous GH secretion. Few studies have addressed the role of P4 on GH secretion, and no study has done so with respect to ghrelin. P4 administered as a single 300-mg oral dose before bedtime increased nocturnal GH secretion in postmenopausal women, but this was associated with much better sleep quality than placebo-treated control subjects, allowing the consideration that the stimulatory effect on GH secretion was indirect via sleep enhancement (13). In contrast, another study reported that P4 diminished GH responses to insulin-induced hypoglycemia and arginine infusion (45). The mechanistic details of P4 action on GH secretion are not known. However, recent work has indicated that the prolactin-inhibiting effect of P4 is mediated via membrane P4 receptor-α by decreasing cAMP levels and by activating TGF-β1 in the lactotropic population (46).
The physiological role of P4 in somatotropic regulation is not well understood. One might speculate that the E2 surge temporarily increases GH production at ovulation, before the luteal phase when P4 levels rise. Additionally, high P4 levels during gestation might contribute to decreased pituitary GH secretion in concert with rising placental-derived GH (1).
Limitations of this study include the lack of different E2 doses to determine possible interactions between the two sex hormones.
In summary, the present work demonstrates that P4 administered to postmenopausal women does not affect spontaneous GH secretion but inhibits ghrelin-stimulated secretion in a concentration-dependent fashion. Low-dose E2 addback does not consistently increase nocturnal GH secretion or augment ghrelin-stimulated GH release.
Acknowledgments
We thank Jill Smith for support with manuscript preparation, the Mayo Immunochemical Laboratory for assay assistance, and the Mayo research nursing staff for implementing the protocol. Matlab versions of ApEn and deconvolution methodology are available from veldhuis.johannes@mayo.edu.
Financial Support: This work was supported in part by National Institutes of Health Grants R01 AG019695 and R01 AG029362 (to J.D.V.), Metabolic Studies Core of the Minnesota Obesity Center Grant P30 DK050456, National Center for Advancing Translational Sciences Grant UL1 TR000135, and National Institute of Standards and Technology Grant 60NANB10D005Z. Contents are solely the responsibility of the authors and do not necessarily represent the official views of any federal institution.
Disclosure Summary: The authors have nothing to disclose.
Glossary
Abbreviations:
- ApEn
approximate entropy
- CRU
Clinical Research Unit
- CV
coefficient of variation
- E2
estradiol
- GHRP
GH–releasing peptide
- IGFBP
IGF-binding protein
- P4
progesterone
- T
testosterone
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