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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 12 6185-6192
Copyright © 2004 by The Endocrine Society

Divergent Effect of Endogenous and Exogenous Sex Steroids on the Insulin-Like Growth Factor I Response to Growth Hormone in Short Normal Adolescents

Régis Coutant, Florence Boux de Casson, Stéphanie Rouleau, Olivier Douay, Elisabeth Mathieu, Frédérique Gatelais, Natacha Bouhours-Nouet, Christelle Voinot, Maurice Audran and Jean Marie Limal

Departments of Pediatrics (R.C., S.R., F.G., N.B.-N., C.V., J.M.L.), Nuclear Medicine (F.B.d.C.), Biochemistry (O.D., E.M.), and Rheumatology (M.A.), University Hospital, and Institut National de la Santé et de la Recherche Médicale, Unité 0335 (M.A.), 49000 Angers, France

Address all correspondence and requests for reprints to: Dr. Régis Coutant, Department of Pediatrics, University Hospital, 4 rue Larrey, 49000 Angers, France. E-mail: recoutant{at}chu-angers.fr.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The lower responsiveness to GH in women than in men is probably due to a divergent effect of gonadal steroids. It is unknown, however, how the progressive increase in sex steroid production that occurs during puberty affects this responsiveness. To compare the effects of puberty and sex steroid administration on responsiveness to GH, we used the IGF-I generation test, in which the peak IGF-I level 24 h after a single injection of GH (2 mg/m2) was studied in 117 healthy short subjects (56 females and 61 males). The subjects, aged 8–16 yr, were divided into four groups: prepuberty, early puberty, midpuberty, or pubertal delay. In the latter group, the IGF-I response was determined before and after priming with oral 17ß-estradiol in girls and im testosterone in boys. We also tested for an association between body composition (by dual energy x-ray absorptiometry) and the IGF-I response to GH. The IGF-I increment in response to GH (change in IGF-I from baseline) was correlated with the growth velocity SD score (P < 0.05). Progression throughout puberty was associated with an increase in both baseline IGF-I (P < 0.05) and the IGF-I increment in response to GH (P < 0.05), with no gender difference. Pubertal category (pre-, early, and midpuberty; P < 0.05) and fat percentage (P < 0.05) were the main positive predictors of the IGF-I increment in response to GH, expressed as micrograms per liter as well as SD score, independently of baseline IGF-I. After sex steroid priming, both the GH peak in response to insulin-induced hypoglycemia and baseline IGF-I were increased (P < 0.05, after vs. before sex steroid). However, the IGF-I increment in response to GH decreased after oral 17ß-estradiol (P < 0.05), whereas it was unchanged after testosterone administration. Endogenous gonadal steroid secretion appears to result in increased responsiveness to GH in peripubertal girls and boys. By contrast, exogenous estrogen and testosterone, respectively, produce a relative decrease and no change in responsiveness to GH in similar populations, possibly through the achievement of sex steroid concentrations exceeding physiological ranges for age. Fat percentage was a positive determinant of the responsiveness to GH, suggesting a link between the energy stores and the anabolic action of GH.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
NUMEROUS STUDIES IN adults with normal, deficient, or excessive GH secretion have found a gender difference in responsiveness to GH. The observation that GH production in healthy women is about 3-fold that in men, whereas IGF-I levels are similar, suggests a relative GH resistance in women (1, 2). In healthy adults as well as in those with GH deficiency (GHD), GH administration led to lower serum IGF-I levels in women than in men (3, 4). Accordingly, women with GHD required higher GH doses than men to similarly normalize IGF-I levels (5). At the other end of GH secretion, a gender difference in serum IGF-I levels for a given serum GH value was also shown in adults with active acromegaly (6). A number of studies have examined whether differences in sex steroid action could account for this gender difference, mainly by studying the effect of exogenous sex steroid administration on responsiveness to GH.

In women, exogenous estrogen administration has been found to influence IGF-I concentration or recombinant human GH (rhGH) requirement. It has been suggested that the negative effect of estrogen on responsiveness to GH is limited to that administered by the oral route (5, 7, 8, 9). However, a dose effect of transdermal estrogen on responsiveness to GH has also been indicated from several studies (7, 10, 11, 12, 13), and this was recently demonstrated in postmenopausal women (14). This suggests that the main factor influencing responsiveness to GH may be the dose of estrogens, more than the route (14, 15). Oral estrogen replacement, when it contained estradiol, generally led to an estrogen concentration above that obtained with transdermal estrogens and above the normal range for the follicular phase of the menstrual cycle (14). Finally, this raises the question of how the progressive increase in estrogen production that occurs during puberty affects responsiveness to GH.

In men, few data are available regarding the effect of androgens on responsiveness to GH. Men with GHD receiving parenteral androgen substitution because of hypogonadotropic hypogonadism had a responsiveness to GH identical to that in eugonadal subjects with GHD for the first 18 months of GH treatment, but the responsiveness increased thereafter (5). Testosterone administration in normal men increased serum IGF-I levels with no change in GH levels (16), suggesting that androgen substitution could increase GH sensitivity. However, it is unknown how low to moderate physiological testosterone production, as is seen throughout puberty, affects responsiveness to GH.

To study the impact of physiological gonadal steroid production on responsiveness to GH, we measured the IGF-I response to a single administration of GH (2 mg/m2) in 117 peripubertal healthy short subjects, 56 girls and 61 boys, aged 8–16 yr, divided into four groups according to their pubertal stage: prepuberty, early puberty, midpuberty, and pubertal delay. Responsiveness to GH in the latter group was studied before and after short-term administration of exogenous sex steroid (17ß-estradiol in girls and testosterone heptylate in boys). We also measured several factors that are known to vary during puberty (17) and that may be related to the changes in responsiveness to GH: body composition assessed by dual energy x-ray absorptiometry, GH secretion assessed by the GH peak to an insulin tolerance test (ITT), and insulin secretion and sensitivity assessed by the measurement of fasting C peptide, as well as by the quantitative insulin sensitivity check index (QUICKI) (18).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

We studied 117 children, aged 8–16 yr (56 girls and 61 boys). These children were primarily referred for assessment of GH secretion using an ITT because of short stature and/or decreasing growth velocity. Puberty in the girls was classified into three stages according to Tanner breast development (19): pre- (breast stage 1), early (breast stage 2), and mid- (breast stage 3–4) puberty. Puberty in the boys was also classified into three stages according to Tanner testicular volume (20): pre- (testis, 1–4 ml), early (testis, 4–8 ml), and mid- (testis, 8–15 ml) puberty. No children in late puberty were studied.

All were assigned to one of four groups according to age and pubertal stage: prepuberty (8–12.5 yr; pubertal stage 1; n = 31; 15 males and 16 females), early puberty (10–14 yr; pubertal stage 2; n = 30; 17 males and 13 females), midpuberty (pubertal stage 3–4; 12–16 yr; n = 28; 15 males and 13 females), or pubertal delay (age, >13 yr in girls and 14 yr in boys; pubertal stage 1; n = 28; 14 males and 14 females). SD scores (SDS) for birth length, birth weight, height, growth velocity (for the last year before evaluation), and body mass index were calculated according to the French standards for age and gender (21, 22, 23). GHD was ruled out (GH peak to the ITT or the arginine-insulin test >10 µg/liter). When a child had a GH response to ITT less than 10 µg/liter, a second test, using arginine insulin, was then performed and ruled out the GHD. Only the results of the ITT are presented. The children were all in good health, with none presenting with gonadotropin deficiency, hypothyroidism, chromosomal abnormalities, dysmorphic syndromes, skeletal dysplasia, chronic illness, or any endocrine or metabolic disease. Gonadotropin deficiency was ruled out by clinical history (no undescended testes, micropenis, or olfactory disorder), sex steroid and gonadotropin measurements, and appropriate follow-up showing that all the children finally underwent pubertal development as well as pubertal acceleration of the growth velocity. None were taking medication. The protocol was approved by our institutional review board. All subjects and families gave their informed consent.

Study design

On the first day the children underwent a standardized ITT after a physiological overnight fast. After placement of a catheter in a peripheral vein, regular insulin was injected at a dose of 0.1 U/kg, iv, at 0800 h. Blood samples were collected at 0, 15, 30, 45, 60, and 120 min for GH measurements. The second day, each subject received recombinant human GH (Saizen, Serono, Inc., Geneva, Switzerland; or Maxomat, Sanofi-Synthelabo, Inc., Paris, France) at a dose of 2 mg/m2 at 0800 h after a physiological overnight fast (14). Treatment was administered by sc abdominal injection by a registered nurse. Blood was sampled 0 and 24 h after injection for measurement of IGF-I and IGF-binding protein-3 (IGFBP-3). Blood was also obtained for baseline (fasting) measurements of C peptide and free fatty acid (FFA) concentrations. The subjects were permitted a normal oral diet; they had three major meals and one snack.

Twenty subjects (13 boys and seven girls; 13 prepubertal and seven during puberty) who had had a GH peak to ITT less than 10 µg/liter underwent a second evaluation, on the average 1 month later, including an arginine-insulin test and a second generation test (with a procedure similar to the first one). Their results were used to assess the precision and reliability of the procedure. The arginine-insulin test was performed as follows. After a physiological overnight fast, a catheter was placed in a peripheral vein; arginine (0.5 g/kg) was injected iv at 0800 h over 30 min, followed by regular insulin (0.1 U/kg) iv at 60 min. Blood samples were collected at 0, 15, 30, 45, 60, 75, 90, 105, and 120 min for GH measurements.

The GH response to ITT and the IGF-I response to GH were also examined after sex steroid administration in the 28 children (14 boys and 14 girls) who had pubertal delay. The boys received a single im injection of testosterone (100 mg) and were reassessed 7 d later. The girls received oral 17ß-estradiol (2 mg once daily) for 3 d before retesting. The rationale for the use of these sex steroid-priming protocols was that they are known to strongly stimulate GH secretion (17, 24).

Body composition

Body composition was investigated by dual energy x-ray absorptiometry using a QDR 4500A densitometer (Hologic, Inc., Waltham, MA). Whole body scans were performed, and body compartments were analyzed using Hologic software, version V8.24a:3. Total and regional body compositions were assessed. Fat mass was expressed as fat percentage.

Insulin sensitivity

The QUICKI was calculated from the fasting concentrations of glucose (expressed in milligrams per deciliter) and insulin (expressed in microunits per milliliter) as an estimate of insulin sensitivity (18): QUICKI = 1/[log(fasting insulin) + log(fasting blood glucose)].

Hormone assays

Serum total IGF-I measurements were performed by immunoradiometric assay (IRMA) after acid-ethanol extraction; serum IGFBP-3 was also measured by IRMA (Immunotech/Beckman Coulter, Villepinte, France). The intra- and interassay coefficients of variation were 5.7% and 8.6% for IGF-I and 4.8% and 6.4% for IGFBP-3, respectively. Serum GH was measured by IRMA (Immunotech/Beckman Coulter). The sensitivity was 0.05 µg/liter, and the intra- and interassay coefficients of variation were 1.5% and 14.03%, respectively. The GH results are expressed in International Reference Preparation 66/217 U, for which 2 mU = 1 µg. Serum C peptide was measured by IRMA (Schering Cis Bio International, Gif sur Yvette, France). The intra- and interassay coefficients of variation were 6.5% and 11.2%, respectively. Testosterone and estradiol were measured by RIA after serum extraction (Immunotech/Beckman Coulter and Schering Cis Bio International). The intra- and interassay coefficients of variation were, respectively, 8.6% and 10.6% for testosterone and 5.4% and 12.8% for estradiol. Plasma glucose was measured with a Hitachi 917 analyzer (Roche, Meylan, France), and plasma FFAs were measured using enzymatic methods (WAKO, Richmond, VA) on a Cobas Mira analyzer (Roche).

Statistical methods

The variables are expressed as the mean ± SD.

In the subset of 20 children who underwent the acute generation test twice (see Study design), the precision of the test was analyzed by the coefficient of variation calculated as the SD of the replicated measurements divided by their mean (25). Its reliability was analyzed by the intraclass correlation coefficient, which expresses the relative magnitude of the two components of total variability, i.e. biological variability (between-subject variability) and random error (method error) (26).

In the three groups of children with normal pubertal development (prepuberty, early puberty, and midpuberty), ANOVAs were used to compare variables. If a statistically significant difference was found, post hoc analysis by Tukey’s procedure was used. For the children with pubertal delay, comparisons before and after sex steroid administration were performed by paired t test. Comparisons between boys and girls were performed by unpaired t test.

To study the effects of puberty, gender, and other variables that could influence responsiveness to GH independently of baseline IGF-I, regression analyses were performed. The IGF-I increment in response to GH (change in IGF-I from baseline = difference between peak and baseline IGF-I) was used as the dependent variable. A model for predicting the IGF-I increment was constructed in several stages. First, clinical parameters (birth weight SDS, birth length SDS, gestational age, midparental height SDS, gender, body composition, and pubertal stage) and biological parameters (serum fasting glucose, C peptide, FFA, QUICKI, and GH peak in response to ITT) were tested in simple regression analyses. Then those associated with a significant regression coefficient were used in a final model for predicting the IGF-I increment, adjusted for baseline IGF-I and gender. To construct this final model, IGF-I was expressed as micrograms per liter as well as SDS. GH peak was defined as the maximal GH value in response to ITT. Normal age- and pubertal stage-related IGF-I values in our laboratory are 200 ± 70 µg/liter (8–12 yr, prepuberty), 300 ± 110 µg/liter (pubertal stage 2), and 470 ± 130 µg/liter (pubertal stage 3–4) in boys, and 230 ± 80 µg/liter (8–12 yr, prepuberty), 350 ± 110 µg/liter (pubertal stage 2), and 460 ± 125 µg/liter (pubertal stage 3–4) in girls. Significance was defined as P < 0.05. All analyses were two-tailed and performed with the SPSS 11.5 statistical package (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical and biological characteristics of the children

The characteristics of the boys and girls in each puberty group are presented in Tables 1Go and 2Go. There was no difference in height SDS or percent fat mass among the puberty groups. As expected, the GH peak in response to ITT and baseline IGF-I increased from pre- to midpuberty in boys as well as girls. Baseline values of IGF-I and IGFBP-3 were not altered by the GH stimulation test, because they were similar on the days of the ITT (d 1) and the acute generation test (d 2). Only the values on d 2 are indicated.


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  		TABLE 1. Baseline characteristics of the male subjects

 

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  		TABLE 2. Baseline characteristics of the female subjects

 
The IGF-I increment 24 h after a single GH administration (difference between peak and baseline IGF-I) was 129 ± 82 µg/liter, 1.16 ± 0.59 SD score, or 45 ± 29% over the baseline value. The IGFBP-3 increment was 200 ± 408 µg/liter, or 8.4 ± 16.6% over the baseline value.

In the whole group of children, the growth velocity SDS correlated with the serum baseline IGF-I (r = 0.33; P < 0.0001), the peak IGF-I (serum IGF-I 24 h after GH administration; r = 0.36; P < 0.0001), the IGF-I increment in response to GH (difference between peak and baseline IGF-I: r = 0.29; P < 0.01), and the GH peak in response to ITT (r = 0.19; P < 0.05), even after adjustment for pubertal stage.

From the subset of 20 children who underwent the acute generation test twice (see Study design), the coefficient of variation, which assessed the precision of the test, was 31 ± 24%, and the intraclass correlation coefficient, which assessed the reliability of the test, was 0.72 (95% confidence interval, 0.29–0.89; P = 0.004).

Responsiveness to GH throughout puberty in the boys

In the boys, progression from pre- to midpuberty was associated with a significant increase in baseline and peak IGF-I (P < 0.001 for both variables, by ANOVA among pre-, early, and midpuberty groups; Fig. 1Go). However, because peak IGF-I increased more than baseline IGF-I throughout puberty, there was a significant rise in the IGF-I increment in response to GH (pre-, early, and midpuberty groups, 60 ± 44, 125 ± 72, and 206 ± 93 µg/liter; P < 0.0001, by ANOVA), suggesting a pubertal increase in the responsiveness to GH.



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  		FIG. 1. Baseline and peak serum IGF-I in boys (upper panel) and girls (lower panel) according to the pubertal category (mean ± SD). The lower whiskers show the SD for baseline IGF-I. The upper whiskers show the SD for peak IGF-I. The IGF-I increment in response to GH is defined by the difference between peak and baseline IGF-I. It is depicted by the white box area, which is not superimposed by the gray box area. Baseline IGF-I, peak IGF-I, and the IGF-I increment in response to GH were compared in the children with normal pubertal development among pre-, early, and midpuberty groups, and in the children with pubertal delay before and after sex steroid administration. Significant comparisons for baseline IGF-I, peak IGF-I, and the IGF-I increment in response to GH are respectively indicated below the lower SDwhiskers, over the upper SDwhiskers, and in the white box areas between the mean baseline and peak IGF-I. IGF-I, To convert values to nanomoles per liter, divide by 7.65. 1, P < 0.05 vs. prepuberty; 2, P < 0.05 vs. midpuberty; a, P < 0.05 vs. before sex steroid administration.

 
Responsiveness to GH throughout puberty in the girls

In the girls, progression from pre- to midpuberty was associated with a significant increase in baseline and peak IGF-I (P < 0.001 for both variables, by ANOVA among pre-, early, and midpuberty groups; Fig. 1Go). As in the boys, peak IGF-I increased more than baseline IGF-I throughout puberty, which caused a significant rise in the IGF-I increment in response to GH (pre-, early, and midpuberty groups, 86 ± 49, 139 ± 83, and 167 ± 89 µg/liter; P < 0.0001, by ANOVA), thus suggesting a pubertal increase in the responsiveness to GH.

Effect of gender on pubertal responsiveness to GH

No difference in the IGF-I increment in response to GH was seen between the boys and girls in pre-, early, or midpuberty (Fig. 1Go).

Effect of sex steroid administration on responsiveness to GH

The significant changes in the somatotropic axis after sex steroid administration in the subjects with pubertal delay are shown in Table 3Go. In the boys, testosterone administration was associated with a significant increase in the GH peak in response to ITT, baseline IGF-I, and peak IGF-I. Because of the parallel increases in baseline and peak IGF-I, the IGF-I increment in response to GH was unchanged (Table 3Go and Fig. 1Go), thus suggesting that testosterone administration did not change responsiveness to GH. Therefore, the large increase in baseline IGF-I after testosterone administration probably resulted from an increase in endogenous GH production, rather than from a change in responsiveness to endogenous GH.


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  		TABLE 3. Significant changes in the somatotropic axis after sex steroid administration in adolescents with pubertal delay

 
In the girls, estrogen administration was associated with a significant increase in the GH peak in response to ITT, a small increase in baseline IGF-I, and no change in peak IGF-I. As a result, the IGF-I increment in response to GH was significantly decreased (P < 0.05; Table 3Go and Fig. 1Go), thus suggesting that oral estrogen administration decreased responsiveness to GH. Therefore, the small increase in baseline IGF-I after estradiol administration probably resulted from an increase in endogenous GH production that was partly counteracted by a decrease in responsiveness to endogenous GH.

Predictors of the IGF-I increment in regression analyses

To study the factors influencing the IGF-I increment in response to GH (in micrograms per liter as well as SDS), regression analyses were performed in the group of subjects with normal pubertal development. The IGF-I increment (micrograms per liter) was significantly correlated with fat percentage, fasting C peptide, baseline IGF-I, QUICKI, and fasting FFA (simple regressions). These variables were subsequently entered into a multiple regression model for predicting the IGF-I increment, adjusted for baseline IGF-I and gender (Table 4Go); the best combination of independent predictors of the IGF-I increment was pubertal stage, fat percentage, fasting C peptide, and fasting FFA (multiple r = 0.73; P < 0.0001), whereas there was no significant influence of gender. The regression coefficient associated with the pubertal stage indicated that early puberty and midpuberty were associated with additional increases in the IGF-I increment in response to GH of 32 and 64 µg/liter, respectively, above that in prepuberty (reference category) and independently of baseline IGF-I.


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  		TABLE 4. Multiple regression analyses for the IGF-I increment in response to GH (expressed as micrograms per liter as well as SDS), adjusted on baseline IGF-I and gender

 
A similar procedure using the IGF-I SDS increment as the dependent variable showed that it was correlated with pubertal stage, fat percentage, GH peak in response to ITT, and baseline IGF-I SDS (simple regressions). In multiple regression analyses with baseline IGF-I SDS and gender as adjusting variables, the best combination of independent predictors of the IGF-I SDS increment was pubertal stage and fat percentage, (multiple r = 0.53; P < 0.0001; Table 4Go). Again, gender was not a significant determinant of the IGF-I SDS increment. The regression coefficient associated with the pubertal stage indicated that early puberty and midpuberty were associated with additional increases in the IGF-I SDS increment in response to GH of 0.16 and 0.32 SDS, respectively, above that in prepuberty (reference category) and independently of baseline IGF-I SDS.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study in healthy short children showed that the IGF-I increment in response to GH increased from pre- to midpuberty in both boys and girls, without gender difference, thus suggesting an increase in responsiveness to GH with puberty. In contrast, testosterone priming in boys with pubertal delay did not change the IGF-I increment in response to GH, whereas estrogen priming in girls decreased this response. Because other factors that vary during puberty may have mediated the apparent effect of puberty on responsiveness to GH, multiple regression analyses were performed. These regression models (adjusted for baseline IGF-I and gender) showed that pubertal stage, fat percentage, and metabolic-hormonal factors related to fat mass (fasting C peptide and FFA) were significant independent predictors of the IGF-I increment in response to GH.

The GH peak in response to ITT, baseline IGF-I, and the amount of IGF-I released into the circulation in response to rhGH (estimated by the change in IGF-I from baseline) increased from pre- to midpuberty. It is well known that circulating IGF-I increases throughout puberty as a result of the production of gonadal steroids (17, 24, 27, 28). Theoretically, this increase could result from an increase in GH production, an increase in responsiveness to GH, or an increased production of IGF-I independent of GH, induced by gonadal steroids or other factors. The major effect of gonadal steroids on circulating IGF-I has been shown to be mediated indirectly through augmented release of GH (17, 24). Subjects with isolated profound GHD due to GHRH receptor mutation failed to increase their serum IGF-I levels during puberty, thus showing that the pubertal rise in serum IGF-I is mostly dependent on GH (29, 30). In apparent contrast with these studies, observations of subjects with GH receptor (GHR)-inactivating mutations have shown higher IGF-I levels in adolescents and adults than in children, thus suggesting a GH-independent sex steroid action on IGF-I (31, 32). The GHR gene alterations in these studies were mutations that created an abnormal splice site (31) or mutations thought to retain the receptor in endoplasmic reticulum (32), associated with detectable levels of GH-binding protein in most cases; a small number of intact receptors thus might still reach the cell surface. Consistent with our results, a unifying hypothesis would be that sex steroids could stimulate some GHR signal transduction (32), thus increasing serum IGF-I in response to endogenous GH secretion.

The IGF-I response to GH increased in a similar manner in boys and girls up to midpuberty, although the mechanisms driving this increase are not well understood. The implication of low concentrations of gonadal steroids, as seen throughout puberty, is possible. In ovariectomized hypophysectomized rats, a single injection of 17ß-estradiol had no effect on hepatic IGF-I expression, whereas combined acute administration of estradiol and GH resulted in significantly greater accumulation of hepatic IGF-I mRNA than GH alone (33). Conversely, in castrated hypophysectomized rats, short-term testosterone administration had no effect on hepatic IGF-I expression, and combined administration of testosterone and GH did not change the hepatic IGF-I mRNA level compared with GH alone (34). These animal studies indicated that estrogens, rather than androgens, may stimulate responsiveness to GH. In humans, suppression of a low male estrogen concentration with the administration of an aromatase inhibitor decreased serum IGF-I, with no change in GH secretion (35), whereas dihydrotestosterone administration had no effect on 24-h GH concentration or serum IGF-I levels (36). Overall, these studies support the hypothesis that exposure to low estrogen levels could increase hepatic responsiveness to GH, whereas androgens would have no effect.

In contrast with these findings, oral 17ß-estradiol in girls decreased the IGF-I increment in response to GH by approximately half, whereas a single im testosterone administration in boys had no effect on the IGF-I increment. Although both exogenous sex steroids similarly stimulated the GH peak in response to ITT, they had a divergent effect on responsiveness to GH. These data suggest that the stimulation of GH production by testosterone was directly responsible for the large increase in baseline IGF-I, with no change in GH responsiveness, whereas the stimulating effect of oral 17ß-estradiol on GH production was partly counteracted by its inhibiting effect on GH responsiveness, thus causing a small increase in baseline IGF-I. The discrepancies between the effects of puberty and sex steroid administration on responsiveness to GH may be due to differences in the origin (endogenous vs. exogenous) or the concentration (low vs. adult) of circulating sex steroids. In our study we used an oral 17ß-estradiol dosage that is usual for an adult female replacement dose and a testosterone dosage that led to an adult male concentration. Notably, the same divergent effect, a lower IGF-I response to GH in females than in males, has already been described in adults whether the sex steroids were of endogenous or exogenous origin, and this divergence has been ascribed to a negative impact of estrogens on responsiveness to GH (3, 4, 5, 6, 37). In women, transdermal estrogens have led to lower estradiol concentrations than oral estrogens (14) and greater IGF-I response to GH (7, 8, 9). Low transdermal estrogen doses caused an increase or no change in serum IGF-I levels (7, 8, 13, 14), whereas high doses decreased these levels (11, 14). Overall, these studies hold with the hypothesis that low estrogen concentrations, as seen in females during the first stages of puberty or in males, could stimulate responsiveness to GH, whereas high concentrations, as seen in adult females, whether eugonadal or requiring replacement, could inhibit responsiveness to GH. In ovariectomized hypophysectomized rats, chronic exposure to 17ß-estradiol reduced GH-induced hepatic IGF-I gene expression and serum IGF-I concentrations, as opposed to the stimulating effect of acute exposure (33).

Responsiveness to GH was related to body fat. This relationship has been suggested from studies of the serum IGF-I concentrations after GH treatment in children and adults with GHD; higher levels were found in obese than in lean patients (38, 39). Insulin secretion (as estimated by fasting C peptide) was also an independent positive predictor of responsiveness to GH in this work. In humans, intraportal insulin delivery efficiently corrected the low serum IGF-I found in diabetic subjects (40). In the hepatoma cell line, insulin up-regulated total and intracellular GHRs (41). Overall, these observations provide a link among the energy stores, insulin secretion, and the IGF-I generation in response to GH, which is thought to mediate most of the anabolic actions of GH (42). However, because the liver is the principal source of IGF-I in the circulation, and because hepatic production of IGF-I is influenced by nutritional factors, this relationship may not be true for tissue IGF-I production, which is more directly related to growth (42, 43, 44).

The IGF-I increment in response to GH was correlated with the growth velocity SDS of the previous year. Accordingly, changes in serum IGF-I in response to GH were correlated with the growth rate in GH-treated children with GHD and idiopathic short stature (ISS), thus linking a biological measurement to a clinical outcome (45, 46). We used the IGF-I response 24 h after a single injection of GH as an estimate of responsiveness to GH for several reasons. IGF-I concentrations have been found to reach a plateau within approximately 18–24 h after a single sc administration of rhGH (14, 47). The GH dose used led to a mean 45% increase in serum IGF-I over the baseline value, which was clearly more than the intraassay variation. However, our study protocol has several limitations. The individual pharmacodynamics of GH and IGF-I may vary, and frequent sampling would have provided a more accurate estimate of responsiveness to GH. Repeated administration would have led to higher IGF-I levels; the administration of rhGH (0.05 mg/kg·d) led to a 71% increase in IGF-I after 4 d in children with ISS (48). Conversely, the use of a lower dose of rhGH (0.011 mg/kg for 4 d) has been suggested to be more sensitive for detecting mild GH insensitivity (49). Finally, IGFBP-3 measurement after 4–7 d of rhGH administration has been shown to provide an additional 10% gain in sensitivity over that of the IGF-I measurement for diagnosing GH insensitivity (50). In our study the IGFBP-3 increment over the baseline value was only 8.4 ± 16.6%. This mean value was close to the intraassay coefficient of variation, thus showing that the IGFBP-3 change after a single administration of GH could not be considered an adequate estimate of the responsiveness to GH.

The coefficient of variation of the acute generation test was 31 ± 24%, indicating that it was moderately precise, whereas the intraclass correlation coefficient was 0.72, indicating fair reliability (a coefficient of 1.00 would have indicated perfect reliability). By comparison, in one study of the reliability of the GH stimulation tests for diagnosing GH deficiency, the highest intraclass correlation coefficient was 0.72 for the ITT (51). However, in another study of 12 children with ISS, the reproducibility of the generation test was found to be poor (52). Several factors could influence the variability in the acute generation test and explain the discrepancies between this study and ours. In our work, the children were hospitalized, their diet was easily controlled, they were not investigated in cases of infectious disease, the GH administration was performed by a registered nurse, and the two tests were performed, on the average, at a 1-month interval. In the study by Jorge et al. (52), diet, infectious disease, and GH administration might have been more difficult to control because GH was administered at home by the parents, whereas the two tests were performed at a 6-month interval. The estimation of reliability is an approach to assessing the impact of random measurement error and provides a measure of the ability of a measurement technique to discriminate between the different members of a sample population. It defines the proportion of variance in the repeated measurements that is attributable to differences between patients and has been considered in some ways a more useful statistic than the assessment of precision (26). As the aims of the acute generation test in this study were to discriminate between subjects and identify trends, its reliability may be more important to consider than its precision. It is likely that the IGF-I generation test, as used in our work, has limited sensitivity and specificity for diagnosing GH insensitivity, just as serum IGF-I and GH provocative tests are known to have limitations for diagnosing GHD. However, we believe that it is a valid tool to show trends in the changes of acute response to GH in a large group of short children.

The children with ISS studied here reached a specific pubertal stage at an appropriate age (19, 20) and were therefore believed to have normally timed puberty (except for those with pubertal delay). These findings are in accord with those of previous studies of pubertal development in short normal subjects, in which the maturational tempo has been shown to usually be normal, although sometimes delayed (24). However, partial insensitivity to GH action has been hypothesized in such children (24, 48, 49, 50). Therefore, the effect of puberty on responsiveness to GH, namely an increase in responsiveness to GH throughout puberty, might be different between these short children and normally growing children. Additional work will be needed to clarify this point.

In conclusion, we showed that endogenous gonadal steroid secretion appears to result in increased GH sensitivity in peripubertal short normal girls and boys. By contrast, exogenous estrogen and testosterone, respectively, produce a relative decrease and no change in GH sensitivity in similar populations, possibly through the achievement of sex steroid concentrations exceeding physiological ranges for age. Besides the putative genetic determinants controlling responsiveness to GH (53), we showed that physiological determinants, namely pubertal stage, fat percentage, and circulating insulin, also influence responsiveness to GH, and they should be taken into account when investigating GH insensitivity in short children.


   Acknowledgments
 
We thank Yannick Simon, who performed the dual energy x-ray absorptiometry, and Anne Liger, Claire Baril, Sophie Dublé, and Martine Chiffoleau, who provided care to the subjects participating in the study.


   Footnotes
 
This work was supported in part by a grant from Programe Hospitalier de Recherche Clinique 1998 (PHRC 1998, Ministère de la Santé).

Abbreviations: FFA, Free fatty acid; GHD, GH deficiency; GHR, GH receptor; IGFBP-3, IGF-binding protein-3; IRMA, immunoradiometric assay; ISS, idiopathic short stature; ITT, insulin tolerance test; QUICKI, quantitative insulin sensitivity check index; rh, recombinant human; SDS, SD score.

Received April 30, 2004.

Accepted September 8, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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