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Hormonal treatments modulate pulsatile plasma growth hormone, gonadotrophin and osteocalcin levels in postmenopausal women
Maturitas,
51
17 (1993) 51-62
Elsevier Scientific Publishers Ireland Ltd.
MAT 00728
Hormonal treatments modulate pulsatile plasma growth hormone, gonadotrophin and osteocalcin levels in postmenopausal women N. Mercuri”, F. Petraglia”, A.D. Genazzania, F. Amato”,
M.R. Sgherzi, A. Maietta-Latessaa, V. De Leob, C. NappiC and A.R. Genazzani” Department
of Obstetrics and Gynaecology.
“University of Modena. of Turin (Italy)
bUniversity
of Siena and cUniversity
(Received April 1, 1992; revision received June 22, 1992; accepted June 26, 1992)
Gestrogen plays a role in modulating growth hormone (GH), luteinizing hormone (LH), folliclestimulating hormone (FSH) and osteocalcin secretion in women. Indeed, the postmenopausal period is characterized by changes in plasma GH, LH, FSH and osteocalcin levels. The aim of the present study was to investigate the changes in the secretory patterns of these hormones in postmenopausal women under different therapeutic regimens. A total of 20 subjects took part in the study. They were subdivided into four groups comprising 5 untreated postmenopausal women (time since menopause 1-5 years), 5 postmenopausal women receiving steroid hormone replacement therapy, 5 postmenopausal women receiving salmon calcitonin and 5 fertile women with regular menstrual cycles. Blood samples were collected every 15min for 4 h and hormone levels were measured by radioimmunoassay. Plasma GH levels fell in the postmenopausal women, but were restored under oestrogen-progestogen treatment. The pulsatile patterns of plasma LH and FSH did not show significant differences in the treated and untreated women. Plasma osteocalcin levels showed episodic fluctuations and mean levels decreased under steroid hormone or calcitonin treatment. No significant correlation was observed between plasma GH and osteocalcin or gonadotrophin levels or body mass index.
Key wordr: menopause; hormone replacement therapy; growth hormone; gonadotrophins; treatment; osteocalcin
calcitonin
Introdoetion
The postmenopause is characterized by increased plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels and by decreased steroid hormone levels associated with the cessation of ovulatory function [l]. Because of the failure of the ovary to respond to gonadotrophins the activity of the hypothalamusCorrespondence to: A.R. Genazaani, Department of Obstetrics and Gynaecology, University of Modena, via Del Pozzo 71, 41100 Modena, Italy. 0378-5122/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
52
pituitary-ovarian axis is unchecked [2,3] so that the progressive rise in circulating gonadotrophin levels is related to reduced feedback by ovarian steroids and inhibin [4-81. Some recent reports [9- 1l] have indicated that growth hormone (GH) secretion is also modified by the changes in ovarian activity at the menopause. Postmenopausal women have lower 24-h integrated GH [12,13] and circulating insulin-like growth factor-I (IGF-I) levels than premenopausal women [ 13,141, these differences being related to the gonadal steroid hormone deficiency. Oestrogens in fact play a role in modulating plasma GH levels [ 15-171. A question that remains open is whether oestrogens act on the hypothalamus, the pituitary or on peripheral tissues. The implications of these findings are increased by the observations that IGF-I stimulates the activity of osteoblasts in vitro and increases the skeletal muscle mass [ 181and by the positive correlation found in acromegalic patients between serum osteocalcin (OT) and IGF-I levels [19]. On the basis of this evidence it has been suggested that postmenopausal osteoporosis may result partly from GH deficiency 1201. The aim of the present study was to compare the pulsatile patterns of plasma gonadotrophins (LH and FSH), GH and OT in untreated postmenopausal women and postmenopausal women receiving hormone replacement therapy (HRT) or calcitonin therapy. Subjectsand Methods
The study involved 20 subjects subdivided into 4 groups as follows: 5 untreated postmenopausal women (mean age 55 years, time since menopause l-5 years), 5 postmenopausal women (mean age 60 years) treated for at least 6 months with oestrogen/progestogen replacement therapy comprising transdermal 17/3-oestradiol for 21 days via adhesive patches delivering 50 &day (Estroclim 50, Sigma Tau, Pomezia, Italy) and 10 mg dydrogesterone on days lo-21 (Dufaston Duphar, UCMDifme, Rome, Italy), 5 postmenopausal women (mean age 68 years) treated for 4 months with salmon calcitonin (100 IU) by nasal spray (Calcitonina Armour, Rhone-Poulenc Rorer, Milan, Italy) and 5 fertile women (mean age 24 years) with regular menstrual cycles. The postmenopausal women had no history of amenorrhoea during the premenopausal years and were in good health, as determined by physical examination and urine and blood tests. Their renal function was normal as assessed by serum creatinine (<0.12 mmol/l) and/or creatinine clearance (> 120 ml/l.73 m2). Postmenopausal status was confirmed by low plasma oestradiol levels. Fasting blood samples were collected every 15 min for 4 h, from 08:OOh to 12:OO h. Body mass index (BMI kg/m2) was calculated for each subject, the normal range being 19-25. Bone density (BMD, g/cm2) of the lumbar spine (L2--L4) was determined in all subjects using a 153-Gd source (1 Ci) with photopeaks at 44 and 100 keV (DP3 scanner, Lunar Radiation Corporation, Madison, WI, USA). The Iongterm in vivo precision for spine BMD was 4.9% and the reproducibility coefficient of variation was about 1.5% on the phantom [21]. GH, FSH, LH and OT levels were measured in each plasma sample by doubleantibody radioimmunoassay (RIA) using commercially available reagents (Serono Diagnostics, Rome, Italy, for plasma FSH, LH and GH; Sorin Biomedica, Saluggia,
53
Italy, for OT). The LH RIA sensitivity was 0.5 mIU and the interassay and intraassay coefficients of variation were 5% and 3% respectively. The FSH RIA sensitivity was 0.15 mIU and the interassay and intra-assay coefficients of variation were 5% and 2.5%, respectively. The GH RIA sensitivity was 0.1 @ml and the interassay and intra-assay coefficients of variation were 5% and 3.5%, respectively. The OT RIA sensitivity was 0.2 ng/ml and the interassay and intra-assay coefficients of variation were 6% and 3.5%, respectively. The statistical analysis of the results was performed using analysis of variance (ANOVA) and the Dunnet test for multiple comparison. Pulse characteristics were evaluated by means of the DETECT program [22], which looks for significant secretory episodes using two types of logic, these being (a) first derivatives and (b) linear fitting and plasma concentration segments. The analysis was performed at a nominal P value of 0.01 for false-positive peak detection. The integrated mean value of the 4-h plasma levels was evaluated for each subject and used to express the mean hormone levels.
Changes in plasma GH
Mean f S.E.M. plasma GH levels were lower in the postmenopausal women than in the controls (P < 0.01). However, plasma GH levels were significantly higher (P < 0.01) in the women receiving oestrogen/progestogen replacement therapy than in the untreated postmenopausal women, whereas no differences were found with calcitonin therapy (Fig. 1A). There were no significant differences among the four groups as regards pulsatile GH frequency and duration (Fig. 1B and C). Changes in plasma LH
While mean f S.E.M. plasma LH levels in the eumenorrhoeic controls and the postmenopausal oestrogen/progestogen-treated women were significantly lower than those in the untreated women (P < O.Ol), the calcitonin-treated women had LH plasma levels in the same range as the untreated postmenopausal women (Fig. 2A). The characteristics of the LH pulsatile pattern (frequency and duration) were not significantly different in the treated and untreated postmenopausal women and were in the same range as in the fertile controls (Fig. 2B and C). Changes in plasma FSH
Plasma FSH levels in the postmenopausal women receiving oestrogetiprogestogen replacement therapy were significantly lower than in the untreated and calcitonintreated postmenopausal women (P < 0.01) (Fig. 3A). The FSH pulsatile pattern frequency was higher in the women receiving oestrogen/progestogen therapy than in those receiving calcitonin treatment, the untreated postmenopausal women or the fertile controls (Fig. 3B). There were no significant differences in FSH pulse duration among the four groups (Fig. 3C). Changes in plasma osteocalcin
Mean f S.E.M. plasma OT levels were significantly higher in the untreated postmenopausal women than in the fertile controls and the postmenopausal women
54
A
nplml
0 6
0
5 4 3 2
1 n Y
postmen
controls
?? E2
*CT B
Freauencv (oeaksI4hrr)
controls duration
postmen
+EZ
*CT C
(mln/paak)
100
60
controls
postmen
+E2
Fig. 1. Mean * S.E.M. plasma growth hormone (GH) levels (A), plasma GH pulse frequency (B) and duration (C) in fertile (control), untreated postmenopausal (postm), oestrogen/progestogen treated (Ed or calcitonin (cr) treated postmenopausal women (0, significant difference (P c 0.01) vs. untreated postmenopausal women).
55
conlrols Frenuencv
‘E2
dursllon
B
(oeeka/4hra)
oonlrols
lOOr
+CT
postmen
?? Ez
*CT
(mlnlpedc)
60
oonlrols
‘CT
Fig. 2. Mean f S.E.M. plasma luteinizing hormone (LH) levels (A), plasma LH pulse frequency (B) and duration (C) in fertile (control), untreated postmenopausal (postm), oestrogetiprogestogen treated (E2) or cakitonin (CT) treated postmenopausal women (0, significant difference (P < 0.01) vs. untreated postmenopausal women).
56
7n
mlU/ml
controls
4
A
--
Froquenoy
poolmen
?? E2
*CT
B
~peakel4hrel P ----
3.5
.-_---.-.._
.---
3 2.5 2 1.5
1 0.5 0 controls
postmen
+E2
+GT
60
0 controls
poslmen
?? E2
*CT
Fig. 3. Mean * S.E.M. plasma follicle-stimulating hormone (FSH) levels (A), plasma FSH pulse frequency (B) and duration (C) in fertile (control), untreated postmenopausal (postm), oestrogen/progestogen treated (Ed or calcitonin (CT) treated postmenopausal women (0, significant difference (P < 0.01) vs. untreated postmenopausal women).
----
------
_-____
controls
2.6,
Frequency
._____I._.
poetmen
?? E2
+CT
poetmsn
?? E2
*CT
(peakalrlhrr)
oontrols
C
durdlon (mlnfoeak)
controls
po8tmsn
+E2
*CT
Fig. 4. Mean f S.E.M. plasma osteodcin (OT) levels (A), plasma OT pulse frequency (B) and duration (C) in fertile (control), untreated postmenopausal (postm), oestrogen/progestogen treated (E2) or calcitonin (CT) treated postmenopausal women (0, significant difference (P < 0.01) vs. untreated postmenopausal women).
58
receiving oestrogen/progestogen or calcitonin treatment (P c 0.01) (Fig. 4A). Episodic changes in plasma OT levels were shown in all the subjects and significant pulses were found by DETECT analysis (P < 0.01). The frequency and duration of OT pulsatility was highest in the untreated postmenopausal subjects and it decreased following both types of treatment (P < 0.01) (Fig. 4B and C). To confirm the validity of the results all samples were reassayed (data not shown) and in three subjects plasma OT levels were measured for 8 h (Fig. 5).
osteocalcin A
ng/ml 5
* _
_ _....- _,._.._ _
,_
,,_,
_.. _......... . .
. ..
.
..
.
.-.
.._ _ ,,_. .__..._.__. _.._ __,,,_- _ ,,^.. _..,.,.....,.,__....._.,.......,,..
0
I
I
1 60
0
1
1
I
1 120
.
-.
_.
I
. -- . . .
__
. .._....
I 180
^
,. .
I
I
240
time (minutes) ng/ml
5
2 -
B
“’-. -’“..-.-‘- ...--’ ‘- -’“_” ‘.”“-“-... “- .‘-.- ” ---‘------’ -“-‘--‘- ‘--.“-.--‘--‘ ---‘ - ---.‘-‘-“-----‘----.--’
1 - - ‘- .. .- -
0
I 0
I
I 60
-- --
I
I,,
,
120
I
130
I
I
I
I
,I
I,
240
300
I
330
,
,
420
I,
430
time (minutes) Fig. 5. Plasma osteocalcin level changes after 4 and 8 h in untreated postmenopausal women show a significant pulsatile pattern (P < 0.01, arrows).
59
Discussion The present study confirmed that pulsatile GH, LH and FSH secretory patterns in postmenopausal women differ from those in fertile women and that the changes are oestrogen/progestogen dependent. This is the first time that pulsatile changes in plasma OT have been shown either in fertile or in treated or untreated postmenopausal women. No significant correlation was found between plasma GH, OT, BMI or gonadotrophin levels. The observed gonadal steroid hormone modulation of GH secretion is consistent with the findings of previous studies [23-251. In women of different ages the serum levels of free oestradiol were correlated with 24-h integrated GH concentrations and with GH pulse amplitude [10,23,24,26]. In agreement with previous results, the present data showed augmented resting GH levels after oestrogen/progestogen therapy. The steroid hormone effect may be due to direct oestrogen action on the pituitary or the hypothalamus. The significant percentage of growth hormone releasing factor (GRF) neurons in rat hypothalamus concentrate [ 3H]oestrogen suggesting that these neurons are oestrogen target cells [27]. Other hypothalamic neurons are also targets for circulating oestrogen. In fact, oestrogen modulation of central cathecholamine turnover [28] may have an impact on GH secretion [29,30]. Further possible areas of oestrogen action are the pituitary gland [31] and/or IGF-I secretion [32]. The present results also showed that the LH pulsatile pattern (frequency and duration) was not significantly different in treated and untreated postmenopausal women and fertile controls. In other studies [33,34] the amplitude of the LH pulse increased significantly during oestrogen/progestogen administration from postmenopausal values to values similar to those seen in the luteal phase of the human menstrual cycle (one pulse every 120 min). The variations between our present data and the results of previous studies are probably connected with the different types of HRT progestogen administered or the different experimental protocols and/or statistical methods used for pulse detection. The fact that the pulse frequency of the FSH levels was increased by oestrogen/progestogen therapy suggests that gonadal steroids have a different action on LH and FSH secretion [35]. Our findings showing that plasma OT levels exhibited a pulsatile pattern in all patients are of particular interest. The peak frequency was highest in the untreated postmenopausal women and lowest in those receiving HRT or calcitonin. Plasma OT levels in old men and women have been shown to follow a circadian rhythm; the concentrations are low in the morning, increase in the afternoon and peak during the night [36]. The plasma OT pulses may be the result of secretory and/or metabolic events. OT catabolism is reported to be dependent on renal function [37], while its synthesis is known to be regulated by 1,25dihydroxyvitamin D3 [38]. Recent studies have demonstrated an increase in biochemical indices of bone formation after shortterm administration of recombinant human GH to selected older patients [39,40]. These results, in addition to the experimental findings showing a GI-I/IGF-I effect on OT production [18,19], support the hypothesis that age-related alterations in bone metabolism may in part be related to impaired GH and OT secretion. In the present study, however, no significant correlations were found between plasma GH and changes in OT levels. Further extensive study is necessary to elucidate the correlation between renal
60
function or bone metabolism and OT levels. The fall in mean OT levels in women treated with HRT and calcitonin confirms the data from previous studies [41-431. It would seem that these two types of therapy do not modify the frequency or duration of pulsatile OT levels. In conclusion, the present study showed that the pulsatile pattern of certain pituitary hormone plasma levels (GH, LH and FSH) and of OT, the marker of osteoblast acitivity, is modulated by hormone treatment in postmenopausal women. References
10
11 12
13
14
15
16 17
18 19
Brenner PF. The menopause. Western J Med 1982; 136: 211-219. Lauritzen C. The hypothalamic anterior pituitary system in the climacteric age period. In: Van Keep PA, Lauritzen C, eds. Frontiers in hormone research. Basel: Karger, 1975; 20-29. Yen SSC. The biology of menopause. J Reprod Med 1977; 18: 287-296. Chakravarti S, Collins WP, Forecast JD, Newton JR, Oram DH, Studd JWW. Hormonal profiles after the menopause. Br Med J 1976; ii: 784-786. Studd JWW, Collins WP, Chakravarti S. Plasma hormone profiles after the menopause and bilateral oophorectomy. Postgrad Med J 1978; 54: 25-30. Vale W, Rivier C, Hsueh A, Campen C, Meunier H, Bicsak T, Vaughan J, Corrigan A, Bardin W, Sawchenko P, Petraglia F, Yu J, Plotsky P, Spiess J, Rivier J. Chemical and biological characterization of the inhibin family of protein hormones. Ret Prog Horm Res 1988; 44: I-32. Crilly RG, Francis RM, Nordin BEC. Steroid hormones, ageing and bone. Clin Endocrinol Metab 1981; 10: 115-139. Chang RJ, Judd HL. The ovary after the menopause. Clin Obstet Gynecol 1981; 24: 181- 191. Bellantoni MF, Harman MS, Cho DE, Blackman MR. Effects of progestin-opposed transdermal estrogen administration on growth hormone and insulin-like growth factor-I in postmenopausal women of different ages. J Clin Endocrinol Metab 1991; 72: 172-178. Dawson-Hughes B, Stern D, Goldman J, Reichhn S. Regulation of growth hormone and somatomedin-C secretion in postmenopausal women: effect of physiological estrogen replacement. J Clin Endocrinol Metab 1986; 63: 424-432. Copeland KC, Johnson DM, Kuehl TJ, Castracane VD. Estrogen stimulates growth hormone and somatomedin-C in castrate and intact female baboons. J Clin Endocrinol Metab 1984; 58: 698-703. Thompson RG, Rodriguez A, Kowarski A, Blizzard RM. Growth hormone: metabolic clearance rates, integrated concentrations and production rates in normal adults and the effect of prednisone. J Clin Invest 1972; 51: 3193-3199. Ho KY, Evans WS, Blizzard RM. Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 1987; 64: 51-58. Franchimont P, Urbain-Choffray D, Lambelin P, Fontaine MA, Frangin G, Reginster JY. Effect of repetitive administration of growth hormone-releasing hormone on growth hormone secretion, insulin-like growth factor I, and bone metabolism in postmenopausal women. Acta Endocrinol 1989; 120: 121-128. Weissberger AJ, Ho KKY, Lazarus L. Contrasting effects of oral and transdermal routes of estrogen replacement therapy on 24-hour growth hormone secretion, insulin-like growth factor I and GHbinding protein in postmenopausal women. J Clin Endocrinol Metab 1991; 72: 374-381. Duursma SA, Bijlsma JWJ, van Paassen HC, Slootweg MC. Oestrogens and bone metabolism: a hypothesis. Maturitas 1986; 8: l-6. Wiedemann E, Schwarts E, Frantz AG. Acute and chronic estrogen effects upon serum somatomedin activity, growth hormone and prolactin in man. J Clin Endocrinol Metab 1976; 42: 942-952. Hock JM, Centrella M, Canahs E. Insulin-like growth factor I has independent effects on bone matrix formation and cell replication. Endocrinology 1988; 122: 254-260. Sidenius Johansen J, Giwercman A, Hartwell D, Thoger Nielsen C, Price PA, Christiansen C, Skak-
61
20
21 22
23 24
25 26
21
28 29 30
31 32
33
34
35 36 31 38 39
40 41
kebaek NE: Serum bone gla-protein as marker of bone growth in children and adolescents: correlation with age, height, serum insulin-like growth factor I and serum testosterone. J Clin Endocrinol Metab 1988; 67: 273-278. Nelson ME, Meredith CN, Dawson-Hughes B, Evans WJ. Hormone and bone mineral status in endurance-trained and sedentary postmenopausal women. J Clin Endocrinol Metab 1988; 66: 921-933. Mazess RB, Barden HS, Hanson JA. Precision of DPA with ts3Gd. Abstract 225, presented at the tenth ammal meeting of the American Society of Bone Mineral Research. New Orleans, 1988. Getter KE, Guardobasso V, Robard D. Detection and characterization of peaks and estimation of instantaneous secretory rate for episodic pulsatile hormone secretion. Comp Biomed Res 1986; 19: 170-191. Rudman D, Kutner MH, Rogers CM, Lubin MF, Fleming GA, Bain RP. Impaired growth hormone secretion in the adult population: relation to age and adiposity. J Clin Invest 1981; 67: 1361-1369. Zadik Z, Chalew SA, MacCarter RJ, Meistas M, Kowarski AA. The influence of age on the 24hour integrated concentration of growth hormone in normal individuals. J Clin Endocrinol Metab 1985; 60: 513-516. Vermetden A. Nyctohemeral growth hormone profiles in young and aged man: correlation with somatomedin-C levels. J Clin Endocrinol Metab 1987; 64: 51-58. Frohlander N, von Schouhz B. Growth hormone and somatomedin C during postmenopausal replacement therapy with oestrogen alone and in combination with an anti-oestrogen. Maturitas 1988; 9: 297-302. Shirasu K, Stumpf WE, Sar M. Evidence for direct action of estradiol on growth hormone-releasing factor (GRF) in rat hypothalamus: localization of [3H] estradiol in GRF neurons. Endocrinology 1990, 127: 344-349. Stumpf WE, Sar M, Reisert I, Pilgrim C. Estrogen receptor sites in the developing central nervous system and their relationship to catecholamine systems. Monogr Neural Sci 1983; 9: 205. Martin JB. Neural regulation of growth hormone secretion. Medical progress report. N Engl J Med 1973; 228: 1384. Locatelli V. Neuroregulation of growth hormone secretion. In: Genazzani AR, Petraglia F, Volpe A, eds. Progress in gynecology and obstetrics. Park Ridge: Parthenon Publishing Group, 1990, 449-454. Bethea CL. Estrogen action on growth hormone in pituitary cell cultures from adult and juvenile macaques. Endocrinology 1991; 129: 2110-2118. Berelowitz M, Szabo M, Frohman LA, Firestone S, Chu L, Hintz RL. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science 1981; 221: 1279. Cagnacci A, Melis GB, Paoletti AM, Gambacciani M, Soldani R, Spinetti A, Fioretti P. Influence of oestradiol and progesterone on pulsatile LH secretion in postmenopausal women. Clin Endocrinol 1989; 31: 541-550. Veldhuis JD, Evans WS, Rogol AD, Thomer MO, Stumpf P. Actions of estradiol on discrete attributes of the luteinizing hormone pulse signal in man. Studies in postmenopausal women treated with pure estradiol. J Clin Invest 1987; 79: 769-776. McNeilly AS. The control of FSH secretion. Acta Endocrinol 1988; 288: 31-40. Gundberg CM, Markowitz ME, Mizruchi M, Rosen JF. Osteocalcin in human serum: a circadian rhythm. J Clin Endocrinol Metab 1985; 60: 736-739. Price PA, Williamson MK, Lothringer JW. Origin of the vitamin K-dependent bone protein found in plasma and its clearance by kidney and bone. J Biol Chem 1981; 256: 12760. Price PA, Baukol SA. 1,25 Dihydroxyvitamin D3 increases synthesis of the vitamin K-dependent bone protein by osteosarcoma cells. J Biol Chem 1980; 255: 11660. Salomon F, Cuneo RC, Hesp R, Sonksen PH. The effects of treatment with recombinant human growth hormone on body composition and metabolism in adults with growth hormone deficiency. N EngJ J Med 1989; 321: 1797-1803. Aloia JF, Vaswani A, Meunier PJ. Coherence treatment of postmenopausal osteoporosis with growth hormone and calcitonin. Calcif Tissue Int 1987; 40: 253-259. Johansen JS, Riis BJ, Delmas PD, Christiansen C. Plasma BGP: an indicator of spontaneous bone
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42
43
loss and of the effect of oestrogen treatment in postmenopausal women. Eur J Clin Invest 1988; 18: 191-195. Podenphant J, Christiansen C, Catherwood BD, Deftos LJ. Serum bone Gla protein variations during estrogen and calcium prophylaxis of postmenopausal women. Calcif Tissue Int 1984; 36: 536-540. Christiansen C, Overgaard K, Riis BJ. The optimum regimens for usage of intranasal salmon calcitonin and treatment of postmenopausal osteoporosis. In: Christiansen C, Overgaards K, eds. Osteoporosis 1990, 3: 1888-1891.
51
17 (1993) 51-62
Elsevier Scientific Publishers Ireland Ltd.
MAT 00728
Hormonal treatments modulate pulsatile plasma growth hormone, gonadotrophin and osteocalcin levels in postmenopausal women N. Mercuri”, F. Petraglia”, A.D. Genazzania, F. Amato”,
M.R. Sgherzi, A. Maietta-Latessaa, V. De Leob, C. NappiC and A.R. Genazzani” Department
of Obstetrics and Gynaecology.
“University of Modena. of Turin (Italy)
bUniversity
of Siena and cUniversity
(Received April 1, 1992; revision received June 22, 1992; accepted June 26, 1992)
Gestrogen plays a role in modulating growth hormone (GH), luteinizing hormone (LH), folliclestimulating hormone (FSH) and osteocalcin secretion in women. Indeed, the postmenopausal period is characterized by changes in plasma GH, LH, FSH and osteocalcin levels. The aim of the present study was to investigate the changes in the secretory patterns of these hormones in postmenopausal women under different therapeutic regimens. A total of 20 subjects took part in the study. They were subdivided into four groups comprising 5 untreated postmenopausal women (time since menopause 1-5 years), 5 postmenopausal women receiving steroid hormone replacement therapy, 5 postmenopausal women receiving salmon calcitonin and 5 fertile women with regular menstrual cycles. Blood samples were collected every 15min for 4 h and hormone levels were measured by radioimmunoassay. Plasma GH levels fell in the postmenopausal women, but were restored under oestrogen-progestogen treatment. The pulsatile patterns of plasma LH and FSH did not show significant differences in the treated and untreated women. Plasma osteocalcin levels showed episodic fluctuations and mean levels decreased under steroid hormone or calcitonin treatment. No significant correlation was observed between plasma GH and osteocalcin or gonadotrophin levels or body mass index.
Key wordr: menopause; hormone replacement therapy; growth hormone; gonadotrophins; treatment; osteocalcin
calcitonin
Introdoetion
The postmenopause is characterized by increased plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels and by decreased steroid hormone levels associated with the cessation of ovulatory function [l]. Because of the failure of the ovary to respond to gonadotrophins the activity of the hypothalamusCorrespondence to: A.R. Genazaani, Department of Obstetrics and Gynaecology, University of Modena, via Del Pozzo 71, 41100 Modena, Italy. 0378-5122/93/$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
52
pituitary-ovarian axis is unchecked [2,3] so that the progressive rise in circulating gonadotrophin levels is related to reduced feedback by ovarian steroids and inhibin [4-81. Some recent reports [9- 1l] have indicated that growth hormone (GH) secretion is also modified by the changes in ovarian activity at the menopause. Postmenopausal women have lower 24-h integrated GH [12,13] and circulating insulin-like growth factor-I (IGF-I) levels than premenopausal women [ 13,141, these differences being related to the gonadal steroid hormone deficiency. Oestrogens in fact play a role in modulating plasma GH levels [ 15-171. A question that remains open is whether oestrogens act on the hypothalamus, the pituitary or on peripheral tissues. The implications of these findings are increased by the observations that IGF-I stimulates the activity of osteoblasts in vitro and increases the skeletal muscle mass [ 181and by the positive correlation found in acromegalic patients between serum osteocalcin (OT) and IGF-I levels [19]. On the basis of this evidence it has been suggested that postmenopausal osteoporosis may result partly from GH deficiency 1201. The aim of the present study was to compare the pulsatile patterns of plasma gonadotrophins (LH and FSH), GH and OT in untreated postmenopausal women and postmenopausal women receiving hormone replacement therapy (HRT) or calcitonin therapy. Subjectsand Methods
The study involved 20 subjects subdivided into 4 groups as follows: 5 untreated postmenopausal women (mean age 55 years, time since menopause l-5 years), 5 postmenopausal women (mean age 60 years) treated for at least 6 months with oestrogen/progestogen replacement therapy comprising transdermal 17/3-oestradiol for 21 days via adhesive patches delivering 50 &day (Estroclim 50, Sigma Tau, Pomezia, Italy) and 10 mg dydrogesterone on days lo-21 (Dufaston Duphar, UCMDifme, Rome, Italy), 5 postmenopausal women (mean age 68 years) treated for 4 months with salmon calcitonin (100 IU) by nasal spray (Calcitonina Armour, Rhone-Poulenc Rorer, Milan, Italy) and 5 fertile women (mean age 24 years) with regular menstrual cycles. The postmenopausal women had no history of amenorrhoea during the premenopausal years and were in good health, as determined by physical examination and urine and blood tests. Their renal function was normal as assessed by serum creatinine (<0.12 mmol/l) and/or creatinine clearance (> 120 ml/l.73 m2). Postmenopausal status was confirmed by low plasma oestradiol levels. Fasting blood samples were collected every 15 min for 4 h, from 08:OOh to 12:OO h. Body mass index (BMI kg/m2) was calculated for each subject, the normal range being 19-25. Bone density (BMD, g/cm2) of the lumbar spine (L2--L4) was determined in all subjects using a 153-Gd source (1 Ci) with photopeaks at 44 and 100 keV (DP3 scanner, Lunar Radiation Corporation, Madison, WI, USA). The Iongterm in vivo precision for spine BMD was 4.9% and the reproducibility coefficient of variation was about 1.5% on the phantom [21]. GH, FSH, LH and OT levels were measured in each plasma sample by doubleantibody radioimmunoassay (RIA) using commercially available reagents (Serono Diagnostics, Rome, Italy, for plasma FSH, LH and GH; Sorin Biomedica, Saluggia,
53
Italy, for OT). The LH RIA sensitivity was 0.5 mIU and the interassay and intraassay coefficients of variation were 5% and 3% respectively. The FSH RIA sensitivity was 0.15 mIU and the interassay and intra-assay coefficients of variation were 5% and 2.5%, respectively. The GH RIA sensitivity was 0.1 @ml and the interassay and intra-assay coefficients of variation were 5% and 3.5%, respectively. The OT RIA sensitivity was 0.2 ng/ml and the interassay and intra-assay coefficients of variation were 6% and 3.5%, respectively. The statistical analysis of the results was performed using analysis of variance (ANOVA) and the Dunnet test for multiple comparison. Pulse characteristics were evaluated by means of the DETECT program [22], which looks for significant secretory episodes using two types of logic, these being (a) first derivatives and (b) linear fitting and plasma concentration segments. The analysis was performed at a nominal P value of 0.01 for false-positive peak detection. The integrated mean value of the 4-h plasma levels was evaluated for each subject and used to express the mean hormone levels.
Changes in plasma GH
Mean f S.E.M. plasma GH levels were lower in the postmenopausal women than in the controls (P < 0.01). However, plasma GH levels were significantly higher (P < 0.01) in the women receiving oestrogen/progestogen replacement therapy than in the untreated postmenopausal women, whereas no differences were found with calcitonin therapy (Fig. 1A). There were no significant differences among the four groups as regards pulsatile GH frequency and duration (Fig. 1B and C). Changes in plasma LH
While mean f S.E.M. plasma LH levels in the eumenorrhoeic controls and the postmenopausal oestrogen/progestogen-treated women were significantly lower than those in the untreated women (P < O.Ol), the calcitonin-treated women had LH plasma levels in the same range as the untreated postmenopausal women (Fig. 2A). The characteristics of the LH pulsatile pattern (frequency and duration) were not significantly different in the treated and untreated postmenopausal women and were in the same range as in the fertile controls (Fig. 2B and C). Changes in plasma FSH
Plasma FSH levels in the postmenopausal women receiving oestrogetiprogestogen replacement therapy were significantly lower than in the untreated and calcitonintreated postmenopausal women (P < 0.01) (Fig. 3A). The FSH pulsatile pattern frequency was higher in the women receiving oestrogen/progestogen therapy than in those receiving calcitonin treatment, the untreated postmenopausal women or the fertile controls (Fig. 3B). There were no significant differences in FSH pulse duration among the four groups (Fig. 3C). Changes in plasma osteocalcin
Mean f S.E.M. plasma OT levels were significantly higher in the untreated postmenopausal women than in the fertile controls and the postmenopausal women
54
A
nplml
0 6
0
5 4 3 2
1 n Y
postmen
controls
?? E2
*CT B
Freauencv (oeaksI4hrr)
controls duration
postmen
+EZ
*CT C
(mln/paak)
100
60
controls
postmen
+E2
Fig. 1. Mean * S.E.M. plasma growth hormone (GH) levels (A), plasma GH pulse frequency (B) and duration (C) in fertile (control), untreated postmenopausal (postm), oestrogen/progestogen treated (Ed or calcitonin (cr) treated postmenopausal women (0, significant difference (P c 0.01) vs. untreated postmenopausal women).
55
conlrols Frenuencv
‘E2
dursllon
B
(oeeka/4hra)
oonlrols
lOOr
+CT
postmen
?? Ez
*CT
(mlnlpedc)
60
oonlrols
‘CT
Fig. 2. Mean f S.E.M. plasma luteinizing hormone (LH) levels (A), plasma LH pulse frequency (B) and duration (C) in fertile (control), untreated postmenopausal (postm), oestrogetiprogestogen treated (E2) or cakitonin (CT) treated postmenopausal women (0, significant difference (P < 0.01) vs. untreated postmenopausal women).
56
7n
mlU/ml
controls
4
A
--
Froquenoy
poolmen
?? E2
*CT
B
~peakel4hrel P ----
3.5
.-_---.-.._
.---
3 2.5 2 1.5
1 0.5 0 controls
postmen
+E2
+GT
60
0 controls
poslmen
?? E2
*CT
Fig. 3. Mean * S.E.M. plasma follicle-stimulating hormone (FSH) levels (A), plasma FSH pulse frequency (B) and duration (C) in fertile (control), untreated postmenopausal (postm), oestrogen/progestogen treated (Ed or calcitonin (CT) treated postmenopausal women (0, significant difference (P < 0.01) vs. untreated postmenopausal women).
----
------
_-____
controls
2.6,
Frequency
._____I._.
poetmen
?? E2
+CT
poetmsn
?? E2
*CT
(peakalrlhrr)
oontrols
C
durdlon (mlnfoeak)
controls
po8tmsn
+E2
*CT
Fig. 4. Mean f S.E.M. plasma osteodcin (OT) levels (A), plasma OT pulse frequency (B) and duration (C) in fertile (control), untreated postmenopausal (postm), oestrogen/progestogen treated (E2) or calcitonin (CT) treated postmenopausal women (0, significant difference (P < 0.01) vs. untreated postmenopausal women).
58
receiving oestrogen/progestogen or calcitonin treatment (P c 0.01) (Fig. 4A). Episodic changes in plasma OT levels were shown in all the subjects and significant pulses were found by DETECT analysis (P < 0.01). The frequency and duration of OT pulsatility was highest in the untreated postmenopausal subjects and it decreased following both types of treatment (P < 0.01) (Fig. 4B and C). To confirm the validity of the results all samples were reassayed (data not shown) and in three subjects plasma OT levels were measured for 8 h (Fig. 5).
osteocalcin A
ng/ml 5
* _
_ _....- _,._.._ _
,_
,,_,
_.. _......... . .
. ..
.
..
.
.-.
.._ _ ,,_. .__..._.__. _.._ __,,,_- _ ,,^.. _..,.,.....,.,__....._.,.......,,..
0
I
I
1 60
0
1
1
I
1 120
.
-.
_.
I
. -- . . .
__
. .._....
I 180
^
,. .
I
I
240
time (minutes) ng/ml
5
2 -
B
“’-. -’“..-.-‘- ...--’ ‘- -’“_” ‘.”“-“-... “- .‘-.- ” ---‘------’ -“-‘--‘- ‘--.“-.--‘--‘ ---‘ - ---.‘-‘-“-----‘----.--’
1 - - ‘- .. .- -
0
I 0
I
I 60
-- --
I
I,,
,
120
I
130
I
I
I
I
,I
I,
240
300
I
330
,
,
420
I,
430
time (minutes) Fig. 5. Plasma osteocalcin level changes after 4 and 8 h in untreated postmenopausal women show a significant pulsatile pattern (P < 0.01, arrows).
59
Discussion The present study confirmed that pulsatile GH, LH and FSH secretory patterns in postmenopausal women differ from those in fertile women and that the changes are oestrogen/progestogen dependent. This is the first time that pulsatile changes in plasma OT have been shown either in fertile or in treated or untreated postmenopausal women. No significant correlation was found between plasma GH, OT, BMI or gonadotrophin levels. The observed gonadal steroid hormone modulation of GH secretion is consistent with the findings of previous studies [23-251. In women of different ages the serum levels of free oestradiol were correlated with 24-h integrated GH concentrations and with GH pulse amplitude [10,23,24,26]. In agreement with previous results, the present data showed augmented resting GH levels after oestrogen/progestogen therapy. The steroid hormone effect may be due to direct oestrogen action on the pituitary or the hypothalamus. The significant percentage of growth hormone releasing factor (GRF) neurons in rat hypothalamus concentrate [ 3H]oestrogen suggesting that these neurons are oestrogen target cells [27]. Other hypothalamic neurons are also targets for circulating oestrogen. In fact, oestrogen modulation of central cathecholamine turnover [28] may have an impact on GH secretion [29,30]. Further possible areas of oestrogen action are the pituitary gland [31] and/or IGF-I secretion [32]. The present results also showed that the LH pulsatile pattern (frequency and duration) was not significantly different in treated and untreated postmenopausal women and fertile controls. In other studies [33,34] the amplitude of the LH pulse increased significantly during oestrogen/progestogen administration from postmenopausal values to values similar to those seen in the luteal phase of the human menstrual cycle (one pulse every 120 min). The variations between our present data and the results of previous studies are probably connected with the different types of HRT progestogen administered or the different experimental protocols and/or statistical methods used for pulse detection. The fact that the pulse frequency of the FSH levels was increased by oestrogen/progestogen therapy suggests that gonadal steroids have a different action on LH and FSH secretion [35]. Our findings showing that plasma OT levels exhibited a pulsatile pattern in all patients are of particular interest. The peak frequency was highest in the untreated postmenopausal women and lowest in those receiving HRT or calcitonin. Plasma OT levels in old men and women have been shown to follow a circadian rhythm; the concentrations are low in the morning, increase in the afternoon and peak during the night [36]. The plasma OT pulses may be the result of secretory and/or metabolic events. OT catabolism is reported to be dependent on renal function [37], while its synthesis is known to be regulated by 1,25dihydroxyvitamin D3 [38]. Recent studies have demonstrated an increase in biochemical indices of bone formation after shortterm administration of recombinant human GH to selected older patients [39,40]. These results, in addition to the experimental findings showing a GI-I/IGF-I effect on OT production [18,19], support the hypothesis that age-related alterations in bone metabolism may in part be related to impaired GH and OT secretion. In the present study, however, no significant correlations were found between plasma GH and changes in OT levels. Further extensive study is necessary to elucidate the correlation between renal
60
function or bone metabolism and OT levels. The fall in mean OT levels in women treated with HRT and calcitonin confirms the data from previous studies [41-431. It would seem that these two types of therapy do not modify the frequency or duration of pulsatile OT levels. In conclusion, the present study showed that the pulsatile pattern of certain pituitary hormone plasma levels (GH, LH and FSH) and of OT, the marker of osteoblast acitivity, is modulated by hormone treatment in postmenopausal women. References
10
11 12
13
14
15
16 17
18 19
Brenner PF. The menopause. Western J Med 1982; 136: 211-219. Lauritzen C. The hypothalamic anterior pituitary system in the climacteric age period. In: Van Keep PA, Lauritzen C, eds. Frontiers in hormone research. Basel: Karger, 1975; 20-29. Yen SSC. The biology of menopause. J Reprod Med 1977; 18: 287-296. Chakravarti S, Collins WP, Forecast JD, Newton JR, Oram DH, Studd JWW. Hormonal profiles after the menopause. Br Med J 1976; ii: 784-786. Studd JWW, Collins WP, Chakravarti S. Plasma hormone profiles after the menopause and bilateral oophorectomy. Postgrad Med J 1978; 54: 25-30. Vale W, Rivier C, Hsueh A, Campen C, Meunier H, Bicsak T, Vaughan J, Corrigan A, Bardin W, Sawchenko P, Petraglia F, Yu J, Plotsky P, Spiess J, Rivier J. Chemical and biological characterization of the inhibin family of protein hormones. Ret Prog Horm Res 1988; 44: I-32. Crilly RG, Francis RM, Nordin BEC. Steroid hormones, ageing and bone. Clin Endocrinol Metab 1981; 10: 115-139. Chang RJ, Judd HL. The ovary after the menopause. Clin Obstet Gynecol 1981; 24: 181- 191. Bellantoni MF, Harman MS, Cho DE, Blackman MR. Effects of progestin-opposed transdermal estrogen administration on growth hormone and insulin-like growth factor-I in postmenopausal women of different ages. J Clin Endocrinol Metab 1991; 72: 172-178. Dawson-Hughes B, Stern D, Goldman J, Reichhn S. Regulation of growth hormone and somatomedin-C secretion in postmenopausal women: effect of physiological estrogen replacement. J Clin Endocrinol Metab 1986; 63: 424-432. Copeland KC, Johnson DM, Kuehl TJ, Castracane VD. Estrogen stimulates growth hormone and somatomedin-C in castrate and intact female baboons. J Clin Endocrinol Metab 1984; 58: 698-703. Thompson RG, Rodriguez A, Kowarski A, Blizzard RM. Growth hormone: metabolic clearance rates, integrated concentrations and production rates in normal adults and the effect of prednisone. J Clin Invest 1972; 51: 3193-3199. Ho KY, Evans WS, Blizzard RM. Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 1987; 64: 51-58. Franchimont P, Urbain-Choffray D, Lambelin P, Fontaine MA, Frangin G, Reginster JY. Effect of repetitive administration of growth hormone-releasing hormone on growth hormone secretion, insulin-like growth factor I, and bone metabolism in postmenopausal women. Acta Endocrinol 1989; 120: 121-128. Weissberger AJ, Ho KKY, Lazarus L. Contrasting effects of oral and transdermal routes of estrogen replacement therapy on 24-hour growth hormone secretion, insulin-like growth factor I and GHbinding protein in postmenopausal women. J Clin Endocrinol Metab 1991; 72: 374-381. Duursma SA, Bijlsma JWJ, van Paassen HC, Slootweg MC. Oestrogens and bone metabolism: a hypothesis. Maturitas 1986; 8: l-6. Wiedemann E, Schwarts E, Frantz AG. Acute and chronic estrogen effects upon serum somatomedin activity, growth hormone and prolactin in man. J Clin Endocrinol Metab 1976; 42: 942-952. Hock JM, Centrella M, Canahs E. Insulin-like growth factor I has independent effects on bone matrix formation and cell replication. Endocrinology 1988; 122: 254-260. Sidenius Johansen J, Giwercman A, Hartwell D, Thoger Nielsen C, Price PA, Christiansen C, Skak-
61
20
21 22
23 24
25 26
21
28 29 30
31 32
33
34
35 36 31 38 39
40 41
kebaek NE: Serum bone gla-protein as marker of bone growth in children and adolescents: correlation with age, height, serum insulin-like growth factor I and serum testosterone. J Clin Endocrinol Metab 1988; 67: 273-278. Nelson ME, Meredith CN, Dawson-Hughes B, Evans WJ. Hormone and bone mineral status in endurance-trained and sedentary postmenopausal women. J Clin Endocrinol Metab 1988; 66: 921-933. Mazess RB, Barden HS, Hanson JA. Precision of DPA with ts3Gd. Abstract 225, presented at the tenth ammal meeting of the American Society of Bone Mineral Research. New Orleans, 1988. Getter KE, Guardobasso V, Robard D. Detection and characterization of peaks and estimation of instantaneous secretory rate for episodic pulsatile hormone secretion. Comp Biomed Res 1986; 19: 170-191. Rudman D, Kutner MH, Rogers CM, Lubin MF, Fleming GA, Bain RP. Impaired growth hormone secretion in the adult population: relation to age and adiposity. J Clin Invest 1981; 67: 1361-1369. Zadik Z, Chalew SA, MacCarter RJ, Meistas M, Kowarski AA. The influence of age on the 24hour integrated concentration of growth hormone in normal individuals. J Clin Endocrinol Metab 1985; 60: 513-516. Vermetden A. Nyctohemeral growth hormone profiles in young and aged man: correlation with somatomedin-C levels. J Clin Endocrinol Metab 1987; 64: 51-58. Frohlander N, von Schouhz B. Growth hormone and somatomedin C during postmenopausal replacement therapy with oestrogen alone and in combination with an anti-oestrogen. Maturitas 1988; 9: 297-302. Shirasu K, Stumpf WE, Sar M. Evidence for direct action of estradiol on growth hormone-releasing factor (GRF) in rat hypothalamus: localization of [3H] estradiol in GRF neurons. Endocrinology 1990, 127: 344-349. Stumpf WE, Sar M, Reisert I, Pilgrim C. Estrogen receptor sites in the developing central nervous system and their relationship to catecholamine systems. Monogr Neural Sci 1983; 9: 205. Martin JB. Neural regulation of growth hormone secretion. Medical progress report. N Engl J Med 1973; 228: 1384. Locatelli V. Neuroregulation of growth hormone secretion. In: Genazzani AR, Petraglia F, Volpe A, eds. Progress in gynecology and obstetrics. Park Ridge: Parthenon Publishing Group, 1990, 449-454. Bethea CL. Estrogen action on growth hormone in pituitary cell cultures from adult and juvenile macaques. Endocrinology 1991; 129: 2110-2118. Berelowitz M, Szabo M, Frohman LA, Firestone S, Chu L, Hintz RL. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science 1981; 221: 1279. Cagnacci A, Melis GB, Paoletti AM, Gambacciani M, Soldani R, Spinetti A, Fioretti P. Influence of oestradiol and progesterone on pulsatile LH secretion in postmenopausal women. Clin Endocrinol 1989; 31: 541-550. Veldhuis JD, Evans WS, Rogol AD, Thomer MO, Stumpf P. Actions of estradiol on discrete attributes of the luteinizing hormone pulse signal in man. Studies in postmenopausal women treated with pure estradiol. J Clin Invest 1987; 79: 769-776. McNeilly AS. The control of FSH secretion. Acta Endocrinol 1988; 288: 31-40. Gundberg CM, Markowitz ME, Mizruchi M, Rosen JF. Osteocalcin in human serum: a circadian rhythm. J Clin Endocrinol Metab 1985; 60: 736-739. Price PA, Williamson MK, Lothringer JW. Origin of the vitamin K-dependent bone protein found in plasma and its clearance by kidney and bone. J Biol Chem 1981; 256: 12760. Price PA, Baukol SA. 1,25 Dihydroxyvitamin D3 increases synthesis of the vitamin K-dependent bone protein by osteosarcoma cells. J Biol Chem 1980; 255: 11660. Salomon F, Cuneo RC, Hesp R, Sonksen PH. The effects of treatment with recombinant human growth hormone on body composition and metabolism in adults with growth hormone deficiency. N EngJ J Med 1989; 321: 1797-1803. Aloia JF, Vaswani A, Meunier PJ. Coherence treatment of postmenopausal osteoporosis with growth hormone and calcitonin. Calcif Tissue Int 1987; 40: 253-259. Johansen JS, Riis BJ, Delmas PD, Christiansen C. Plasma BGP: an indicator of spontaneous bone
62
42
43
loss and of the effect of oestrogen treatment in postmenopausal women. Eur J Clin Invest 1988; 18: 191-195. Podenphant J, Christiansen C, Catherwood BD, Deftos LJ. Serum bone Gla protein variations during estrogen and calcium prophylaxis of postmenopausal women. Calcif Tissue Int 1984; 36: 536-540. Christiansen C, Overgaard K, Riis BJ. The optimum regimens for usage of intranasal salmon calcitonin and treatment of postmenopausal osteoporosis. In: Christiansen C, Overgaards K, eds. Osteoporosis 1990, 3: 1888-1891.