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Determinants of the Growth Hormone-Insulin-Like. Growth Factor Axis ... The objectives of this study were to 1) study the GH-insulin-like growth factor (IGF) axis ...
0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 8 Printed in U.S.A.

Body Composition and Physical Fitness Are Major Determinants of the Growth Hormone-Insulin-Like Growth Factor Axis Aberrations in Adult Turner’s Syndrome, with Important Modulations by Treatment with 17b-Estradiol* CLAUS HØJBJERG GRAVHOLT†, RUNE WEIS NAERAA, SANNE FISKER, JENS SANDAHL CHRISTIANSEN

AND

Medical Department M (Endocrinology and Diabetes) (C.H.G., S.F., J.S.C.) and Pediatric Department A (R.W.N.), Aarhus University Hospital, Kommunehospitalet, DK-8000 Aarhus C, Denmark ABSTRACT The objectives of this study were to 1) study the GH-insulin-like growth factor (IGF) axis in adult untreated Turner’s syndrome compared to that in age-matched controls, 2) examine the effects of sex hormone substitution on this axis, 3) study the effects of route of administration of 17b-estradiol on the measured variables, and 4) examine the effects of sex steroids on hepatic function in Turner patients. Twenty-seven patients with Turner’s syndrome were evaluated before and during sex hormone replacement, and an agematched control group (n 5 24) was evaluated once. Main outcome variables were GH and other measures of the GH-IGF axis, body composition, maximal oxygen uptake, sex hormone-binding globulin, and hepatic enzymes and proteins. The integrated 24-h GH concentration (IC-GH; micrograms per L/24 h) was reduced in women with Turner’s syndrome (T) compared to controls [C; mean 6 SD, 18.3 6 12.0 (T) vs. 37.2 6 29.7 (C); P 5 0.007]. However, multiple regression revealed that fat-free mass (FFM) and maximal oxygen uptake were significant explanatory variables (joint r 5 0.77; P , 0.0005), accounting for 60% of the variance in the 24-h IC-GH. This association was also present in controls. After adjustment for these two variables, any difference in GH concentration between Turner patients and controls disappeared. Serum IGF-I and IGF-II were identical in Turner patients and controls despite the difference in 24-h IC-GH. The level of GH-binding protein (GHBP; nanomoles per L) was higher in Turner women [1.87 6 0.72 (T) vs.

1.22 6 0.33 (C); P 5 0.0005]; after adjustment for FFM, the difference in GHBP levels disappeared between Turner patients and controls. During sex hormone treatment a significant increase was seen in the 24-h IC-GH (P 5 0.02), FFM (percentage of weight; P , 0.0005) and maximal oxygen uptake (milliliters of O2 per kg/min; P 5 0.02). Serum IGF-I was unchanged, whereas serum IGF-II (micrograms per L) decreased significantly [Turner, basal (TB), vs. Turner, treatment (TT), 860 6 135 vs. 823 6 150; P 5 0.04]. Alanine aminotransferase (units per L), g-glutamyl transferase (units per L), and alkaline phosphatase (units per L) were significantly elevated during the basal study period, and all decreased during treatment [alanine aminotransferase, 55 6 55 (TB) vs. 30 6 20 (TT; P 5 0.006); g-glutamyl transferase, 92 6 98 (TB) vs. 43 6 65 (TT; P 5 0.003); alkaline phosphatase, 211 6 113 (TB) vs. 175 6 54 (TT); P 5 0.06]. The route of administration of 17b-estradiol did not affect its actions. In conclusion, we found the GH-IGF axis in Turner’s syndrome to be normal, with body composition and physical fitness exerting the same modifying effects on this axis as seen in the normal population. Sex hormone replacement in Turner’s syndrome is associated with normalizing effects on the GH-IGF axis, body composition, physical fitness, and hepatic function. The lowering of hepatic enzymes is a surprising and hitherto undiscovered action of sex steroids. Finally, the route of administration of 17b-estradiol is of minor importance in Turner’s syndrome. (J Clin Endocrinol Metab 82: 2570 –2577, 1997)

T

stimuli (1, 2), as well as specific alterations in the mode of GH secretion have been reported (3), whereas others have found normal GH secretion (4 –7). The bioactivity of GH produced has been suggested to be lower than that in controls (8), and different isoforms have been found to prevail (9). Body composition, physical fitness, gender, age, and spontaneous GH secretion have been shown to be interrelated. Obesity is associated with low circulating levels of GH (10), a situation that can be reversed by weight loss (11, 12). In nonobese adults a negative association between relative adiposity and GH secretion rate is well established (13–15); intraabdominal fat especially predicts GH secretion (16). Physical fitness is positively associated with spontaneous GH secretion in both women and men (15, 17). Age is a negative determinant for spontaneous GH secretion (14, 15, 18, 19); women seem to produce more GH than men (13), and

URNER’S syndrome is a condition involving total or partial absence of one X chromosome, reduced final height, insufficiency of female sex hormone secretion, and infertility in most cases. Controversy exists concerning the activity of the GH-insulin-like growth factor (IGF) axis in Turner’s syndrome: is it normal or reduced? Diminished GH secretion, both spontaneously and after exogenous Received January 30, 1997. Revision received March 21, 1997. Rerevision received April 23, 1997. Accepted April 25, 1997. Address all correspondence and requests for reprints to: Dr. Claus Højbjerg Gravholt, Medical Department M (Endocrinology and Diabetes), Kommunehospitalet, DK-8000 Aarhus C, Denmark. E-mail: [email protected]. * Presented in part at the Second International Meeting of the Growth Hormone Research Society, London, United Kingdom, November 1996. This work was supported by a grant from the Danish Diabetes Association and an unrestricted research grant from Novo Nordisk. † Supported by a research fellowship from the University of Aarhus.

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circulating levels of estradiol and testosterone explain part of the variations in endogenous secretion of GH (13, 20, 21). After the induction of puberty and after reaching final height, most patients with Turner’s syndrome are faced with the question of continued sex hormone replacement. Sex hormone replacement seems to be beneficial in preventing osteoporosis and for psychological well-being (22, 23), and it is generally recommended to continue sex hormone replacement at least until the age of normal menopause. It is, however, well known that sex steroids, especially orally administered, affect the liver. In Turner’s syndrome this might be of special importance because as many as 80% of adult Turner patients show elevated levels of hepatic enzymes (24). The activity of the GH-IGF axis in adult Turner patients has never been studied in detail, and nothing is known of the effects of sex steroid treatment on GH secretion in these patients. In addition, differential effects of sex steroids on the GH-IGF axis administered either orally or transdermally have been suggested (25). The aims of the present study, therefore, were to 1) study the activity of the GH-IGF axis in adult Turner’s syndrome, taking into consideration the effects of body composition and maximal oxygen uptake and compare them with results from an age-matched control group, 2) study the effect of sex steroid replacement therapy in adult Turner’s syndrome on these variables, 3) examine the effect of oral vs. transdermal administration on these variables, and 4) examine the effects of sex steroids on measures of hepatic function. Subjects and Methods Subjects The study group consisted of 27 patients with Turner’s syndrome and a control group of 24 normal women with presumed normal karyotype. The control group was matched with respect to age, but not body mass index (BMI), as this was found to be impossible because of the characteristic anthropometric pattern in Turner’s syndrome. Age and anthropometric data are presented in Table 1. The karyotypic distribution of the Turner women is shown in Table 2. All subjects received oral and written information concerning the study before giving written informed consent. The protocol was ap-

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proved by the Aarhus County ethical scientific committee and the Danish Authority of Health.

Design All patients were receiving female hormone replacement therapy, but before the initial examination [basal examination (TB)] a 4-month washout period was introduced. None of the Turner patients had experienced spontaneous puberty. After the initial examination, patients were randomized to two regimens of hormone substitution [treatment period (TT)]: oral hormone replacement consisting of 2 mg 17b-estradiol/day from days 1–12, 2 mg 17b-estradiol/day and 1 mg norethisterone acetate/day from days 13–22, and 1 mg 17b-estradiol/day from days 23–28 (Trisekvens, Novo Nordisk, Bagsvaerd, Denmark) or transdermal estrogen replacement consisting of approximately 50 mg 17b-estradiol/55 kgzday for 28 days (Estraderm, Ciba-Geigy, Copenhagen, Denmark) and 1 mg norethisteron (Noretisteron Dak, Nycomed DAK, Copenhagen, Denmark) administered orally from days 13–22. Fifteen subjects were randomly allocated to the group receiving transdermal estrogen, and 12 subjects were allocated to the group receiving oral estrogens. Within the first month of treatment, 3 subjects from the group receiving transdermal estrogen had to be transferred to oral treatment due to irritative dermatitis. These 3 subjects were then transferred to oral treatment and subsequently included as such in the statistical analysis. All patients were studied twice at a 6-month interval, whereas all controls were examined once. Control subjects and Turner patients during sex hormone treatment were studied in the early follicular stage (days 5–10) of the menstrual cycle.

Methods Subjects were admitted at 0800 h after an overnight fast (10 –12 h). After an initial bed rest of at least 45 min, resistance and impedance were measured, and fat mass, fat-free mass (FFM), and total body water (TBW) were determined, employing bioelectrical impedance (Animeter, HTS-Engineering APS, Odense, Denmark) (26). The BMI was calculated TABLE 2. Karyotype distribution of the women with Turner’s syndrome Karyotype

No.

45,X 45,X/46,X,i(Xq) or 46,X,i(Xq) 45,X/46,XY 46,X,del(Xp) 45,X/46,XX

17 6 2 1 1

TABLE 1. Mean 6 SD levels of anthropometric data and hepatic variables in Turner patients at baseline and during sex hormone replacement and in controls TB

Age Ht (cm) Wt (kg) BMI (m2/kg) Fat mass (%) FFM (%) TBW (%) TBW/LBM (%) Resistance (V) W/H (kg/m2) Alanin aminotransferase (U/L) Albumin (g/L) g-Glutamyl transferase (U/L) Bilirubin (mmol/L) Alkalic phosphatase (U/L) a

By paired t test By independent t test. c Wilcoxon test. d Mann-Whitney test. b

33.8 6 8.3 146.8 6 6.9 57.9 6 11.6 26.8 6 4.7 33.4 6 4.5 66.6 6 4.5 51.0 6 5.4 76.3 6 3.7 569 6 66 0.89 6 0.09 55.3 6 54.9 36.7 6 3.1 92.0 6 98.1 9.6 6 5.7 211 6 113

TT

Control

57.9 6 11.8 26.9 6 4.8 31.9 6 4.4 68.1 6 4.4 52.4 6 5.6 76.8 6 3.7 541 6 69 0.88 6 0.09 29.5 6 20.0 35.2 6 3.5 42.6 6 65.2 9.3 6 2.3 174.6 6 54.1

32.7 6 7.8 168.4 6 6.1 67.9 6 14.4 23.8 6 4.2 27.5 6 5.6 72.4 6 5.6 52.4 6 5.8 72.3 6 2.9 565 6 53 0.78 6 0.06 22.0 6 19.6 37.4 6 3.1 13.5 6 9.2 10.0 6 3.5 110 6 28

P (TB vs. TT)a

P (TB vs. C)b

1.0 0.9 0.0005 0.0005 0.0005 0.002 0.002 0.3 0.006 0.08c 0.003 0.8 0.06

0.6 0.0005 0.009 0.02 0.001 0.001 0.4 0.0005 0.9 0.0005 0.006 0.4d 0.0005 0.8 0.0005

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as weight (kilograms) divided by height (meters) squared, and the waist to hip (W/H) ratio was determined in the supine position. A 6-min submaximal exercise test with continuous monitoring of heart rate was performed on a bicycle ergometer (Monark Ergometric 829 E, Monark Exercise, Varberg, Sweden) using a workload of 300-1200 kpm/min, depending on age and reported physical activity by the subject. The mean heart rate during the last 2 min of work (.120 beats/min) was used for calculation of the maximal aerobic capacity (VO2-max) (27), which in our hands previously has been shown to have a day to day intraindividual coefficient of variation of 9% (unpublished observations). This indirect measure of VO2-max correlates well with a direct measure of VO2-max, with a coefficient of variation of less than 10% (28, 29). A cannula was inserted into a cubital vein, and blood sampling was started at 1200 h and continued every 20 min for 24 h. All samples were analyzed for GH; in a subset of subjects (Turner’s syndrome, n 5 15; controls, n 5 11), IGF-binding protein-1 (IGFBP-1) and insulin were analyzed. All other analyses were performed in fasting samples. Serum was separated and stored at 220 C until assayed. The patients received meals served at the hospital at 1230, 1500, 1800, 2100, and 0800 h.

Assays GH was measured with a double monoclonal immunofluometric assay (DELFIA, Wallac Oy, Turku, Finland). The interassay coefficient of variation (CV) in samples varied between 1.7–2.4%, and the intraassay CV varied between 1.9 –3.0% for GH concentrations of 12.08 and 0.27 mg/L. The detection limit was 0.01 mg/L. Serum IGF-I and IGF-II were measured by noncompetitive time-resolved immunofluorometric assays (30). Serum insulin was measured by enzyme-linked immunosorbent assay employing a two-site immunoassay that does not detect proinsulin or split(32–33)- and des(31–32)-proinsulin, whereas split(65– 66)- and des(64 – 64)-proinsulin cross-react 30% and 63%, respectively (31). The intraassay CV was 2.0% (n 5 75) at a serum level of 200 pmol/L, and the interassay CV was 4%. Serum IGFBP-I was measured by a commercial enzyme-linked immunosorbent assay (Medix Biochemica, Kainiainen, Finland). Serum sex hormone-binding globulin (SHBG) was measured by a time-resolved immunofluorometric assay (Wallac Oy). Serum IGFBP-3 was measured by an immunoradiometric assay (Diagnostic System Laboratories, Webster, TX). GHBP was measured by an in-house time-resolved immunofluorometric assay (32). Hepatic enzymes, cholesterol, and other measures of lipid metabolism were determined on a Cobas INTEGRA (Roche, Hvidovre, Denmark).

Statistical analysis Data were examined by Student’s two-tailed unpaired and paired t tests, Mann-Whitney test, or Wilcoxon two-tailed test when appropriate. Multiple linear regression and/or Pearson product moment correlation were used to examine the relations between different variables. Results are expressed as the mean 6 sd. Significance levels under 5% were considered significant. The area under the curve was calculated using the trapezoidal rule.

Results GH-IGF axis in untreated Turner’s syndrome patients vs. controls

24-h integrated concentration of GH (IC-GH) and the GH-IGF axis (Table 3). The 24-h IC-GH (P 5 0.007) and mean GH (P 5 0.004) concentrations were reduced in Turner patients compared with controls. Circulating levels of IGF-I and IGF-II were similar in Turner patients and controls, as were IGFBP-3 and integrated 24-h IGFBP-1 levels, whereas insulin levels were higher in Turner women (P 5 0.04). Serum levels of IGFBP-1 showed an inverse relationship with insulin in women with Turner’s syndrome and controls (Fig. 1, a and c). The level of GHBP was significantly higher in Turner women (P 5 0.0005). Relationship between GH secretion and body composition (Table 1). BMI and W/H were higher in Turner patients than in controls (BMI, P 5 0.009; W/H, P , 0.0005), and FFM was lower in Turner patients than in controls (P 5 0.001). A linear relationship existed between 24-h IC-GH and FFM (TB: r 5 0.57, P 5 0.002; C: r 5 0.45, P 5 0.03; Fig. 2a), BMI (TB: r 5 20.52, P 5 0.005; C: r 5 20.53, P 5 0.008), and W/H (TB: r 5 20.42, P 5 0.03; C: r 5 20.46, P 5 0.02) in Turner patients and controls. There was no significant correlation between age and 24-h IC-GH in Turner’s syndrome patients or controls. Relationship between GH secretion and physical fitness. Maximal oxygen uptake was reduced in Turner subjects compared to controls (P 5 0.003); a significant correlation was present between maximal oxygen uptake and 24-h IC-GH in both groups (Fig. 2c; TB: r 5 0.52, P 5 0.008; C: r 5 0.56, P 5 0.006). However, the relationship was not identical, because in controls, an increase in maximal oxygen uptake was associated with a steeper increase in 24-h IC-GH than that in Turner women (comparison of linear regression slopes, P 5 0.04). Effects of body composition, age, physical fitness, and other variables on spontaneous GH secretion. In addition to indexes of body composition and maximal oxygen uptake, correlation analysis showed the 24-h IC-GH to be negatively associated with GHBP (r 5 20.51, P 5 0.007) in Turner patients and positively associated with SHBG (r 5 0.53, P 5 0.007) in controls. Multiple backward stepwise regression disclosed that FFM, GHBP, and maximal oxygen uptake were signif-

TABLE 3. Mean 6 SD levels of GH, IGFBP-1, IGFBP-3, IGF-I, IGF-II, GHBP, insulin, and maximal oxygen uptake in Turner patients at baseline and during sex hormone treatment and in controls

AUC GH (mg/L z 24 h) Mean GH (mg/L) AUC IGFBP-1 (n 5 15; mg/L) IGFBP-3 (mg/L) Insulin (pmol/L) GHBP (nmol/L) IGF-I (mg/L) IGF-II (mg/L) VO2max (mL O2/kg z min) a b

By paired t test. By independent t test.

TB

TT

Control

P (TB vs. TT)a

P (TB vs. C)b

18.3 6 12.0 0.69 6 0.34 67.4 6 44.3 2884 6 553 43.8 6 38.2 1.87 6 0.72 183.2 6 87.8 859.9 6 134.9 32.9 6 8.4

20.8 6 11.6 0.78 6 0.40 68.9 6 37.4 2752 6 627 38.4 6 29.6 1.78 6 0.77 171.6 6 73.0 822.6 6 150.0 35.9 6 8.2

37.2 6 29.7 1.53 6 1.20 57.7 6 30.7 2794 6 527 27.0 6 11.7 1.22 6 0.33 183.3 6 58.2 792.6 6 131.4 41.4 6 10.0

0.02 0.02 0.8 0.06 0.08 0.2 0.1 0.04 0.02

0.007 0.004 0.5 0.6 0.04 0.0005 1.0 0.08 0.003

GH-IGF AXIS IN TURNER’S SYNDROME

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and IGF-I. However, IGF-I correlated significantly with W/H in patients (r 5 20.49, P 5 0.009), but not with other indexes of body composition. In controls, there was a significant correlation to SHBG (r 5 20.46, P 5 0.02). Multiple linear backward regression disclosed that age was the principal variable related to serum IGF-I concentrations in Turner patients (r 5 0.55, P 5 0.003), whereas in the control group, SHBG and age remained explanatory variables (joint r 5 0.78, P 5 0.0005). Determinants of IGFBP-1 and IGFBP-3. Twenty-four-hour integrated IGFBP-1 levels correlated positively with FFM (r 5 0.57, P 5 0.03) and SHBG (r 5 0.67, P 5 0.006) and negatively with fasting insulin (r 5 20.57, P 5 0.03) and GHBP (r 5 20.51, P 5 0.05) in Turner women; in controls, 24-h integrated IGFBP-1 levels correlated negatively with insulin (r 5 20.67, P 5 0.03) and positively with SHBG (r 5 0.73, P 5 0.01). Multiple backward linear regression disclosed that IGFBP-1 was related principally to SHBG in both Turner patients and controls; this was also illustrated by the strong negative correlation between insulin and SHBG in both groups (TB: r 5 20.43, P 5 0.03; C: r 5 20.71, P 5 0.0005). In women with Turner’s syndrome, there was a positive correlation between IGFBP-3 and fasting serum insulin (TB: R 5 0.55, P 5 0.004). However, this positive correlation was not found in controls. Determinants of GHBP. GHBP was positively related to BMI (TB: r 5 0.53, P 5 0.004; C: r 5 0.61, P 5 0.002) and W/H (TB: r 5 0.41, P 5 0.04; C: r 5 0.63, P 5 0.001) and negatively related to FFM (TB: r 5 20.71, P , 0.0005; C: r 5 20.75, P , 0.0005) in both groups (Fig. 3a). In the untreated group of Turner subjects, GHBP correlated significantly and negatively with 24-h integrated IGFBP-1 concentration (r 5 20.51, P 5 0.05), whereas GHBP correlated significantly and positively with fasting insulin in controls (r 5 0.47, P 5 0.02). In multiple linear regressions, FFM remained the principal discriminative variable for GHBP, and after adjustment for FFM, the initial difference in GHBP disappeared between Turner patients and controls (Turner vs. control, P 5 0.07). There was no significant correlation between either age or IGF-I, and GHBP. FIG. 1. Serum IGFBP-1 (E) and insulin (F) during 24-h sampling. Sampling started at 1200 h. a, Turner subjects in the basal situation. b, Turner subjects during sex hormone replacement. c, Control subjects. Error bars indicate SEM.

icant discriminative variables (r 5 0.81, P , 0.0005), accounting for 65% of the variance in the 24-h IC-GH in Turner patients (Fig. 2, a– c). In controls, SHBG and maximal oxygen uptake remained significant variables (r 5 0.69, P 5 0.002) of 24-h IC-GH. In multiple linear regression, status (Turner or control) was shown to have no impact on 24-h IC-GH when adjusting for other explanatory variables (FFM, GHBP, SHBG, and maximal oxygen uptake). Determinants of IGF-I. Serum IGF-I correlated significantly and negatively to age in both patients and controls (TB: r 5 20.55, P 5 0.003; C: r 5 20.73, P , 0.0005; Fig. 3b); this relationship was identical in both groups, whereas there was no apparent relationship between maximal oxygen uptake

Sex hormone substitution and the GH-IGF axis (Table 4)

During sex hormone substitution, 24-h IC-GH (P 5 0.02) and mean GH (P 5 0.02) increased, IGF-II decreased (P 5 0.04), and IGF-I, IGFBP-3, GHBP, and integrated 24-h IGFBP-1 were unchanged. The mean serum insulin level decreased nonsignificantly (TB vs. TT, P 5 0.08), and the inverse relationship with IGFBP-1 persisted (Fig. 1b). Multiple regression analysis disclosed that 60% of the increase in the 24-h IC-GH was attributable to changes in fasting insulin, IGF-I, maximal oxygen uptake, and FFM (joint r 5 0.78, P 5 0.002). BMI in Turner’s syndrome was unchanged by treatment with sex hormone (P 5 0.9), whereas significant increases in FFM (P , 0.0005) and the TBW/FFM ratio (P 5 0.002) were seen with sex hormone treatment. During sex hormone treatment, a significant increase in maximal oxygen uptake (P 5 0.02) was recorded in Turner’s syndrome.

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The determinants of IGF-I, IGFBP-1, and IGFBP-3 did not change in response to treatment; thus, age was the sole explanatory variable for IGF-I levels in backward multiple regression, SHBG was the sole discriminative variable for 24-h integrated IGFBP-1 concentrations, and fasting insulin showed a positive correlation with IGFBP-3 (r 5 0.48, P 5 0.01). Likewise, FFM remained the only variable significantly related to GHBP in backward multiple linear regression. Route of administration of sex hormone substitution (Table 4)

The route of administration of 17b-estradiol exerted different actions on the integrated 24-h IGFBP-1 levels, as transdermal application caused a significant decrease compared with an increase after oral administration (D AUC IGFBP-1, P 5 0.03). This response to route of administration could not be explained by any of the measured variables, including changes in insulin. The route of administration also affected GHBP; transdermal 17b-estradiol caused a fall in GHBP levels, whereas oral treatment left GHBP levels unchanged (D GHBP, P 5 0.006), and no effect was seen with regard to 24-h IC-GH and IGF-I. Hepatic function and Turner’s syndrome (Table 1)

Alanine aminotransferase, g-glutamyl transferase, alkaline phosphatase were significantly elevated during the basal study period, and all decreased, either significantly or nearly significantly, after sex hormone treatment. In contrast, bilirubin and albumin levels in Turner patients were comparable to those in the control group and were unaffected by sex hormone treatment. Serum albumin, however, decreased significantly in the transdermally treated group compared with the orally treated group (P 5 0.05). Markers of lipid metabolism

There was no difference between the Turner patients and the control group in any of the measured lipid variables (total cholesterol, high density lipoproteins, low density lipoproteins, and triglycerides; data not shown). During treatment, a small, but significant, decrease in high density lipoprotein cholesterol was recorded [1.6 6 0.6 (TB) vs. 1.4 6 0.3 mmol/L (TT); P 5 0.05], whereas all other variables were unchanged. Discussion

Data from the present study indicate that in Turner’s syndrome, physical fitness and body composition are independent predictors of spontaneous GH secretion, a relationship also seen in normal subjects (11, 12, 15, 33), whereas age did not relate to levels of GH, as seen in normal adults (13–15). This implies that the spontaneous GH secretion in Turner’s syndrome is normal in relative terms, with physical fitness and indexes of body composition modulating the 24-h FIG. 2. A, Twenty-four-hour IC-GH vs. FFM. F, Turner subjects in the basal situation. E, Turner subjects in the treatment situation. , Control subjects. Regression lines: full line, Turner subjects in the basal situation; dashed line, Turner subjects in the treatment situation; dotted line, Control subjects. B, Twenty-four-hour IC-GH vs. GHBP. C, 24-h IC-GH vs. maximal oxygen uptake.

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TABLE 4. Mean 6 SD levels of D values (TT 2 TB) of GH, IGFBP1, IGFBP-3, IGF-I, IGF-II, GHBP, insulin, SHBG, albumin, and maximal oxygen uptake in Turner patients

D AUC GH (mg/L) D Mean GH (mg/L) D AUC IGFBP-1 (n 5 15; mg/L) D Fasting serum insulin (pmol/L) D IGFBP-3 (mg/L) D IGF-I (mg/L) D IGF-II (mg/L) D GHBP (nmol/L) D SHBG (nmol/L) D VO2max (mL O2/kg z min) D Albumin (g/L)

Turner (trans.)

Turner (oral)

2.02 6 3.70 0.08 6 0.17 211.7 6 25.1

2.87 6 6.29 0.11 6 0.22 13.0 6 12.7

0.7 0.7 0.03

210.1 6 16.3

22.3 6 15.8

0.2

2207 6 228 272 6 424 211.2 6 36.1 211.9 6 43.9 240.0 6 63.0 235.1 6 106.8 20.35 6 0.26 0.07 6 0.42 21.3 6 6.3 11.9 6 17.2 5.0 6 5.9 1.3 6 5.5 23.6 6 4.5 0.07 6 3.3

P

0.3 1.0 0.9 0.006 0.02 0.1 0.05

Effect of route of administration (transdermal or oral).

FIG. 3. A, GHBP vs. FFM. F, Turner subjects in the basal situation. E, Turner subjects in the treatment situation. , Control subjects. Regression lines: full line, Turner subjects in the basal situation; dashed line, Turner subjects in the treatment situation; dotted line, control subjects. B, IGF-I vs. age.

IC-GH. The 24-h IC-GH increased after treatment with sex hormones, with 17b-estradiol eliciting the same effect, regardless of route of administration. However, only 60% of the variance in the increase in 24-h IC-GH was explained by the measured variables. The remainder of the variance could be due to direct effects of sex hormones on the pituitary gland or the hypothalamus. As serum estradiol increases after sex hormone replacement, this is accordance with previous reports (13, 21). FFM and TBW increased after treatment with sex hormones, but as we also found a significant increase in the TBW/FFM ratio, we conclude that an increase in lean tissue took place. This change was seen without any significant change in W/H or BMI. It appears that female sex hormones per se induce this increase, perhaps via the anabolic and lipolytic effects of estrogens. In postmenopausal women, sex hormone replacement prevents the abdominal fat accumulation normally seen after menopause and increases lipopro-

tein lipase activity in fat depots and lipolysis in abdominal adipocytes, thus modifying adipocyte metabolism toward the situation seen in women before menopause (34 –36), where lipolytic responsiveness and sensitivity are higher than those in postmenopausal women (37). Concurrently, an increase in maximal oxygen uptake was observed during sex hormone treatment. In postmenopausal women, a dramatic decline in muscle force is normally seen, which is preventable by sex hormone replacement therapy (38). Accordingly, female sex hormones alone or perhaps via an increase in endogenous GH secretion seem pivotal in preventing deteriorations in both FFM and maximal oxygen uptake and in Turner patients are actually able to increase these variables. Serum IGF-I and IGF-II were comparable in Turner patients and controls, with IGF-I being the effector hormone of some of the actions of GH. In obesity, IGF-I values are subnormal (39), but despite a marked difference in BMI between Turner patients and controls in this study, we did not find any difference in serum IGF-I. Treatment with sex hormones was associated with a decrease in IGF-II, but not IGF-I. The clinical significance of this decline in serum IGF-II is not clear. Treatment with transdermal 17b-estradiol was associated with a decrease, and oral 17b-estradiol with an increase, in the integrated 24-h serum IGFBP-1 concentration. The reason for this difference in action is obscure, as alterations in insulin levels, a major regulator of IGFBP-1 (40), and other variables that influence IGFBP-1, could not explain this despite the fact that in women with Turner’s syndrome the apparently ubiquitous inverse relationship between insulin and IGFBP-1 was present. IGFBP-1 is exclusively produced in the liver, and one possible explanation could be that oral 17b-estradiol exerts a preferential action on the secretion of IGFBP-1 (and SHBG) in the liver. We found SHBG to be the sole explanatory variable for the 24-h integrated IGFBP-1 concentration. Furthermore integrated 24-h GH concentrations were related to SHBG in controls, whereas not in Turner patients. SHBG is mainly synthesized in the liver (41, 42), and a rise in SHBG is usually seen after oral estradiol treatment, whereas a decrease in SHBG is seen after androgen treatment. Sex steroids have been considered to be the main regulators of SHBG (43); however, in normal subjects, strong correlations between

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SHBG and IGFBP-1 (positive), insulin (negative), and IGF-I (negative) have been observed (43– 45). These correlations were also evident in this study in both normal and Turner subjects. Serum IGFBP-3 was very similar in the two groups, and sex hormone substitution did not change the levels in Turner patients. In Turner patients, but not in controls, a positive correlation between IGFBP-3 and fasting serum insulin was found. This is a novel observation and not readily explainable. The GHBP level was more than 50% higher in women with Turner’s syndrome. A decrease was noted after sex hormone treatment, although the level was still significantly higher than that in controls. However, this difference could be explained by differences in body composition, supporting the view that GHBP primarily arises from GH receptors in visceral adipose tissues (46, 47). Previously in postmenopausal women, oral ethinyl estradiol has been found to increase mean 24-h GH, GHBP, and decrease IGF-I, whereas transdermal 17b-estradiol was associated with an increase in IGF-I to premenopausal levels (25). Thus, two different estradiol formulations were used to test the possible differences in the effects of different routes of administration; different oral formulations of estrogen (conjugated equine estrogen and estradiol valerate) have similar effects on the GH-IGF-I axis (48). It appears that oral 17b-estradiol has different actions in Turner women, as we found no difference in GH and IGF-I responses to oral or transdermal treatment. In girls with Turner’s syndrome, low dose estrogen therapy has been shown to increase IGF-I (49, 50), whereas in pharmacological doses, estrogens suppress circulating IGF-I levels in normal subjects (51). As expected, serum levels of alanine aminotransferase, g-glutamyl transferase, and alkaline phosphatase were elevated in Turner patients compared with controls (24). Rather unexpectedly, however, a decrease in most hepatic proteins and enzymes was noted after treatment with sex hormones regardless of the route of administration. SHBG increased only in the orally treated group, whereas GHBP was unaffected, and a decrease was recorded in the transdermally treated group. This partly contrasts with previous comparisons of transdermal vs. oral replacement in postmenopausal women (52). Although sex hormone treatment had a distinct positive effect on measures of hepatic function, several indexes of hepatic function were still significantly elevated compared with those in controls. Thus, women with Turner’s syndrome seem to have rather distinct alterations in liver function, which are partly alleviated by sex hormone replacement. However, SHBG can also be seen as a measure of hepatic function. SHBG in Turner patients was lower than that in controls and increased in the orally treated group; the lower level was probably due to the lack of estrogens. To our knowledge the rather dramatic changes found here, with partial normalization after sex hormone replacement, have not been reported previously. There was no evidence of increased alcohol consumption among the Turner patients. However, in a recent epidemiological study of morbidity in Turner’s syndrome, we found evidence suggesting increased relative risk of cirrhosis of the liver (unpublished observations). Osteoporosis is frequently found in Turner’s syndrome

(53, 54), and the observed increases in GH secretion, physical fitness, and FFM after sex hormone substitution may have important clinical implications for this disease entity. GH is known to stimulate protein synthesis and has direct and indirect effects on bone metabolism (55). Furthermore, it is recognized that GH substitution plays a psychological role in GH-deficient patients (56). In Turner’s syndrome, specific visuo-spatial deficits are seen, which may be partly alleviated by sex hormone treatment (22). We, therefore, believe the registered increase in 24-h IC-GH along with the other effects attributable to sex hormones to be beneficial for patients with Turner’s syndrome. We think that women with Turner’s syndrome should receive sex hormone substitution at least until normal menopause, e.g. 55 yr of age, and possibly beyond. In conclusion we found the GH-IGF axis in Turner’s syndrome to be normal, with body composition and physical fitness exerting the same modifying effects on this axis as in the normal population. Sex hormone substitution in Turner’s syndrome exerts normalizing effects on the GH-IGF axis, body composition, physical fitness, and hepatic function. The lowering of hepatic enzymes is one surprising and hitherto undiscovered action of sex steroids. Finally, the route of administration of 17b-estradiol has only minor effects on the GH-IGF axis in Turner’s syndrome. Acknowledgments Lone Korsgaard and Eva Sejer Christoffersen are thanked for their expert technical help. Ciba-Geigy and Novo Nordisk are thanked for the generous gift of Estraderm and Trisekvens.

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