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Original Studies |
Pituitary Research Unit, Garvan Institute of Medical Research, St. Vincent’s Hospital, and Kolling Institute of Medical Research, Royal North Shore Hospital (R.C.B.), Sydney, New South Wales 2010, Australia
Address all correspondence and requests for reprints to: Dr. Ken K. Y. Ho, Pituitary Research Unit, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia. E-mail: k.ho{at}garvan.unsw.edu.au
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Administration of oral, but not transdermal, estrogen in normal
postmenopausal women significantly decreased serum levels of
IGFBP-3 and ALS (P 
0.005). The suppressive
effects were similar with different oral estrogen formulations, and the
degree of suppression increased with estrogen dosage. In hypogonadal
GH-deficient women, oral estrogen treatment also significantly reduced
IGFBP-3 and ALS (P = 0.02). The changes in IGF-I in
each of the four studies paralleled the changes in both IGFBP-3 and
ALS.
In conclusion, exogenous estrogens suppress serum IGFBP-3 and ALS in a route- and dose-dependent manner, which are in parallel with the effects on serum IGF-I. These actions of oral estrogen are independent of endogenous GH status.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Exogenous estrogens exert route-dependent effects on IGF-I, with oral, but not transdermal, estrogen reducing serum levels of IGF-I (5). Little is known about the effects of estrogen on IGFBP-3 and ALS, and in particular whether estrogens exert similar route-dependent effects on both of these proteins. We investigated the effects on serum IGFBP-3 and ALS of 1) oral and transdermal estrogens, 2) different formulations of oral estrogen, and 3) different dosages of oral estrogen in healthy postmenopausal women. We previously observed that the estrogen-induced fall in IGF-I was accompanied by a corresponding rise in circulating levels of GH and GH-binding protein (5), with the latter change considerably reducing the fraction of free GH. To examine whether estrogen affects components of the ternary complex indirectly via changes in GH availability or via direct hepatic effects, we also examined the effects of oral estrogen in a group of hypogonadal GH-deficient women.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Four separate studies were performed, with studies 1, 2, and 3 involving postmenopausal women and study 4 conducted in hypogonadal GH-deficient women. Study 1 compared the effects of oral and transdermal estrogens in an open label, randomized, cross-over study involving nine healthy postmenopausal women, aged 50–69 yr. This study was originally designed to investigate the effects of estrogen administration on glucose tolerance and insulin sensitivity. The metabolic data from this study have been previously published (6). The subjects were at least 1 yr postmenopausal and had not received estrogen replacement therapy for at least 2 months before the study. They were randomized to receive either daily oral conjugated estrogen (Premarin, Wyeth-Ayerst, Philadelphia, PA; 1.25 mg) or twice weekly transdermal 17ß-estradiol (Estraderm-TTS 100, Ciba-Geigy, Sydney, NSW; delivering 100 µg/day) for 12 weeks, before crossing over to the other treatment for another 12 weeks. Blood samples were collected before and at the 10th week of each estrogen treatment period. Samples obtained during the glucose tolerance test were used for the present study. IGF-I, IGFBP-3 and ALS were measured in the fasting samples, whereas GH was measured in all samples (0, 30, 60, 120, and 120 min) obtained during the glucose tolerance test.
Study 2 examined the effects of different oral estrogen formulations, details of which have previously been published (7). Six healthy postmenopausal women , aged 54–71 yr and at least 2 yr postmenopause, were withdrawn from estrogen replacement therapy for at least 2 months before the study. The subjects were randomized to receive 1 month each of three different oral estrogen formulations in a cross-over design. The estrogen formulations were Premarin (1.25 mg/day), ethinyl estradiol (20 µg/day), and estradiol valerate (2 mg/day). All subjects were studied before and at the third week of each treatment period. They were admitted to the Clinical Investigation Unit, and blood samples were taken every hour for 24 h, from which equal aliquots were collected to form a pool for subsequent hormone measurements (7).
Study 3 evaluated the effects of oral estrogen dosage in a double blind, placebo-controlled, cross-over study. Five healthy postmenopausal women, aged 43–65 yr, were withdrawn from estrogen replacement for at least 2 months before randomization to receive placebo or 20 or 30 µg ethinyl estradiol, orally, for 12 weeks. Blood samples were obtained after an overnight fast at the 10th week of each treatment phase.
Study 4 examined the effects of oral estrogen in the absence of GH. This open label study involved nine hypogonadal women, aged 27–68 yr, with organic GH deficiency. Eight of them had previously undergone surgery with or without radiotherapy for pituitary tumors (three nonfunctioning macroadenomas, two prolactinomas, and three craniopharyngiomas). One patient had idiopathic childhood-onset panhypopituitarism. GH deficiency was confirmed by the failure of peak GH to exceed 3 ng/mL during an insulin tolerance test (8). All subjects were receiving stable substitutive therapies for hypoadrenalism and hypothyroidism before and during the study. Six patients who had previously been receiving estrogen replacement were withdrawn from estrogen for at least 2 months before the study. Blood samples were taken before and after 4 weeks of oral estrogen (estradiol valerate, 2 mg/day) administration after an overnight fast.
In all of the above studies, 10 mg medroxyprogesterone acetate (MPA) were coadministered with estrogen during the last 12 days of each monthly cycle. Blood was taken in the estrogen-only phase immediately before the addition of MPA, and was stored at -20 C until assay. All subjects gave informed written consent, and the study protocols were approved by the research ethics committee of St. Vincent’s Hospital.
RIAs
Serum IGF-I was measured by RIA after acid-ethanol extraction as previously described (9), with intraassay coefficients of variation (CVs) of 9.4%, 8.3%, and 10.3% at 48, 254, and 1510 µg/L, respectively. Competitive RIAs for serum IGFBP-3 and ALS were performed in duplicate samples as described previously (2, 10). The intraassay CVs for the IGFBP-3 RIA were 4.6%, 3.3%, and 4.7% at 0.65, 1.52, and 4.17 µg/mL, respectively, and those for interassay CVs were 7.3%, 10.6%, and 10.6%, respectively. The intra- and interassay CVs for the ALS RIA at 35, 140, and 536 nmol/L were 6.6%, 3.9%, and 1.9%, and 13.4%, 6.7%, and 7.9%, respectively. All samples from each patient for each study were run in one assay.
Statistical analysis
The results are expressed as the mean ± SEM. Differences between groups were analyzed by Student’s t tests and ANOVA (StatView 4.02, Abacus Concepts, Berkeley, CA) with repeated measures where appropriate.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Mean LH and FSH concentrations in the postmenopausal women fell
significantly to comparable levels during oral and transdermal estrogen
administration as previously described (6). During the oral estrogen
phase, the mean GH level (2.9 ± 0.5 µg/L) was significantly
higher (P < 0.05) than that observed during the
transdermal phase (1.7 ± 0.4 µg/L) or that obtained before
treatment (0.9 ± 0.3 µg/L) (6). The mean GH concentration
during transdermal estrogen treatment was not significantly different
from that obtained before treatment. The mean IGF-I level fell
significantly to 67.5 ± 11.6% of baseline during oral estrogen
treatment (P < 0.005), but was not significantly
affected by transdermal estrogen treatment (Fig. 1A
). Oral estrogen replacement also
significantly reduced the levels of IGFBP-3 to 86.0 ± 5.5%
(P < 0.01; Fig. 1B) and ALS to 83.5 ± 9.9%
(P = 0.03; Fig. 1C) of baseline. The levels of IGFBP-3
and ALS during transdermal estrogen treatment were not different from
baseline and remained significantly higher (P = 0.03)
than those during oral estrogen treatment.
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As previously reported, all three oral estrogen formulations
significantly reduced mean 24-h LH and FSH and elevated GH levels to
comparable levels (7). Mean IGF-I levels fell significantly by 15–30%
of baseline (P < 0.05) during treatment with each oral
estrogen formulation to mean levels that were not significantly
different between each treatment (Fig. 2A). The mean IGFBP-3 level was 54.0
± 4.0 nmol/L before treatment (Fig. 2B) and fell significantly
(P < 0.001) during treatment with each estrogen
formulation. Mean IGFBP-3 levels obtained during treatments with
ethinyl estradiol (39.5 ± 3.1 nmol/L), conjugated
estrogen (42.4 ± 2.8 nmol/L), and estradiol valerate (38.9
± 3.9 nmol/L) were not significantly different. Similarly, the mean
ALS level was 254.1 ± 16.4 nmol/L before treatment and fell
significantly (P = 0.001) during oral estrogen
treatments (Fig. 2C). As with IGF-I and IGFBP-3, no significant
difference was observed in mean ALS levels during treatments with
ethinyl estradiol (189.3 ± 13.7 nmol/L), conjugated
estrogen (198.3 ± 16.1 nmol/L), and estradiol valerate
(192.1 ± 16.9 nmol/L).
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Estrogen induced a significant dose-related reduction in the mean
circulating level of IGF-I. The mean IGF-I level obtained during
treatment with 30 µg ethinyl estradiol (11.3 ± 1.5
nmol/L) was significantly (P < 0.05) lower than that
during treatment with 20 µg ethinyl estradiol (12.5
± 2.5 nmol/L; Fig. 3A). Mean IGFBP-3
levels also fell in a dose-dependent manner, with the mean level during
treatment with 30 µg (37.4 ± 4.7 nmol/L) significantly lower
(P < 0.05) than that during treatment with 20 µg
(46.0 ± 5.1 nmol/L) ethinyl estradiol; both levels
were lower than that obtained with placebo (59.3 ± 5.7 nmol/L;
Fig. 3B). A similar dose-dependent reduction was found with ALS (Fig. 3C). The reduction in ALS was significant (P < 0.05)
only with the higher dose of estrogen.
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Baseline GH concentrations were below the limit of detection of
the assay in all subjects. The mean IGF-I level in these patients fell
uniformly and significantly with oral estrogen (P <
0.01; Fig. 4A). Both IGFBP-3 and ALS also
fell significantly (P = 0.02) during oral estrogen
treatment (Fig. 4, B and C). The mean IGF-I level fell significantly to
73 ± 11.2%, IGFBP-3 fell to 82.4 ± 5.8%, and ALS fell to
90.2 ± 3.1% of baseline. These changes are comparable to those
in normal women given the same estrogen formulation (study 2), which
resulted in IGF-I level falling to 84.6 ± 10.5%, IGFBP-3 to
76.6 ± 6.1%, and ALS to 79.6 ± 8.0% of baseline (Fig. 2
).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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This is the first paper reporting changes induced by estrogen in all components of the IGF-I ternary complex. ALS, which circulates in vast molar excess to IGF-I, fell in parallel with IGF-I. Our findings on IGFBP-3 are in contradistinction to those previously reported by Bellantoni et al. (11), who observed that transdermal, but not oral, estrogen reduced IGFBP-3 levels in postmenopausal women. The reason for the discrepancy is not obvious. However, oral estrogen consistently suppressed IGFBP-3 in all four of the present studies, indicating that the inhibitory effects are highly reproducible.
GH circulating in blood is partly bound to GH-binding protein (12, 13). We previously reported that oral estrogen administration increases the serum level of this binding protein (14), which can complex free GH, thereby attenuating GH action. This may be one explanation for the decreases in IGF-I, IGFBP-3, and ALS with oral estrogen, as all three proteins are GH regulated. Alternatively, estrogen may suppress the stimulatory action of GH on the three proteins, as it has been shown to inhibit IGF-I expression in response to GH in ovariectomized hypophysectomized rats (15). However, the finding of a uniform reduction in IGF-I, IGFBP-3, and ALS in GH-deficient subjects mitigates against this possibility, but strongly suggests a direct hepatic effect of oral estrogen as a more likely mechanism. Consistent with the present findings is the report that estrogen inhibits IGFBP-3 production by human breast cancer cells (16). As circulating IGF-I is stabilized by IGFBP-3, it is possible that a decrease in IGFBP-3 synthesis could contribute to the fall in serum IGF-I. As ALS and IGF-I are colocalized in hepatocytes (1, 17, 18), and ALS is synthesized primarily in the liver (19), the route dependency of estrogen action thus suggests a first pass hepatic effect similar to that on IGF-I.
In contrast to IGF-I and ALS, IGFBP-3 is synthesized mainly by Kupffer cells in the liver (19, 20). Our data suggest that these sinusoidal cells may also be estrogen sensitive. However, it has been shown that IGFBP-3 may serve as a passenger protein in the ternary complex, with any unbound IGFBP-3 rapidly cleared from the circulation (21). The reduction in the IGFBP-3 level observed with oral estrogen could therefore arise secondarily from a primary reduction in IGF-I and ALS levels.
We conclude that exogenous oral estrogen exerts inhibitory effects on all three components of the IGF-I ternary complex. These effects are route and dose dependent, but independent of endogenous GH status. These findings indicate that IGFBP-3 and ALS are directly or indirectly estrogen-sensitive hepatic proteins.
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Acknowledgments |
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Footnotes |
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2 Recipient of an overseas medical training fellowship from the
Hospital Authority of Hong Kong.
Received September 24, 1999.
Revised December 14, 1999.
Accepted December 29, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) subunit of the high molecular
weight insulin-like growth factor-binding protein complex. J Clin
Endocrinol Metab. 70:1347–1353.[Abstract]
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) and during oral (
) and transdermal (TD; 
) estrogen
treatment. *, P < 0.02 vs.
baseline; #, P < 0.05 vs. TD.
). *, P < 0.05 vs. baseline.
