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Endocrinology Service, Garrahan Pediatric Hospital, Buenos Aires C1245AAM, Argentina
Address all correspondence and requests for reprints to: Dr. Alicia Belgorosky, Laboratorio de Investigación, Hospital de Pediatria Garrahan, C. de los Pozos 1881, Buenos Aires C1245AAM, Argentina. E-mail: . abelgo{at}elsitio.net
Abstract
In girls, but not in boys, pronounced adrenarche and precocious pubarche along with ovarian hyperandrogenism have been related to insulin resistance and reduced fetal growth. However, insulin secretion is increased during puberty in normal boys. The aim of this study was to analyze the possible implication of changes in the GH/IGF-I axis and in insulin sensitivity for the regulation of adrenal androgen secretion of normal prepubertal and adolescent boys. Fifty-six normal boys were divided into the following groups (Gr): Gr1, prepuberty (testicular volume, <4 cc; n = 33); and Gr3, puberty (testicular volume, 4–25 cc; n = 23). Gr1 was subdivided according to age into: Gr1A, early prepuberty (boys younger than 5.9 yr old; n = 16); and Gr1B, late prepuberty (prepubertal boys, 5.9 yr old or older; n = 17). Gr3 was subdivided according to testicular volume into: Gr3A, early puberty (testicular volume, 4–8 cc; n = 13); and Gr3B, late puberty (testicular volume, 10–25 cc; n = 10). To study hormonal changes during the transition between prepuberty and puberty, an additional group, Gr2 (n = 30), was defined by mixing Gr1B and Gr3A. Serum dehydroepiandrosterone sulfate (DHEAS), androstenedione (
4A), insulin, IGF-I, and glucose were determined after overnight fasting. Insulin sensitivity was estimated by the fasting glucose/insulin (G/I) ratio. There was a close correlation between fasting G/I ratio and QUICKI, a quantitative insulin sensitivity check index.
Mean values for Gr1 and Gr3 as well as their subgroups were compared using t test. In Gr1, the mean fasting G/I ratio was significantly higher, and the mean serum IGF-I, serum DHEAS, and serum 4A levels were significantly lower than in Gr3 (P < 0.001). Mean fasting G/I ratios in Gr1A and Gr3A were not significantly different from those in Gr1B and Gr3B, respectively, but the fasting G/I ratio in Gr3A was significantly lower than that in Gr1B (P < 0006). Moreover, body mass index (BMI) in Gr3A was significantly higher than that in Gr1B (P < 0.01). On the other hand, mean serum IGF-I levels in Gr1A and Gr3A were significantly lower than those in Gr1B and Gr3B, respectively (P < 0.0001). The mean serum DHEAS level in Gr1A was significantly lower than that in Gr1B (P < 0.01), but no difference was found between Gr3A and Gr3B. The mean serum 4A in Gr1A was similar to that in Gr1B, but the mean serum 4A in Gr3A was significantly lower than that in Gr3B (P = 0.0001).
Correlation studies within Gr1, Gr2, and Gr3 were also carried out. There was a significant positive correlation between serum DHEAS and age in Gr1 and Gr2, but not in Gr3. In Gr1, no significant correlation was found between serum DHEAS and fasting G/I ratio or between serum DHEAS and serum IGF-I, suggesting that adrenal steroidogenesis in male prepuberty is independent of insulin sensitivity or peripheral IGF-I. In Gr2, a significant negative correlation (P = 0.01) between serum DHEAS and the fasting G/I ratio was found, but not between serum DHEAS and serum IGF-I. Furthermore, a significant negative correlation between BMI and the fasting G/I ratio was also found. Therefore, changes in insulin sensitivity might be involved in adrenal androgen synthesis during the transition from prepuberty to puberty. Finally, in Gr3, DHEAS was not significantly correlated with the fasting G/I ratio or serum IGF-I. A significant negative correlation between serum 4A and the fasting G/I ratio was found in Gr2. In Gr2, but not in Gr3, there was a significant negative correlation between the fasting G/I ratio and age (P = 0.03) and between the fasting G/I ratio and serum IGF-I (P = 0.03).
In conclusion, our data support the hypothesis that the GH/IGF-I axis and insulin sensitivity are not involved in the mechanism of adrenarche in boys. Insulin sensitivity and BMI, however, decrease at early puberty rather than at late puberty, and this change could be involved in modulating adrenal androgen steroidogenesis during the transition between late prepuberty and early puberty.
ADRENARCHE IS the increase in adrenal androgen secretion, principally dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), not accompanied by an increase in cortisol secretion that occurs during prepuberty in higher primates. This event takes place at about 6–8 yr of age in humans (1, 2, 3). The mechanism of this phenomenon is not known. It has been suggested that adrenarche might be secondary to an increment in 17,20-lyase activity by a regulatory effect independent of 17
-hydroxylase activity (4, 5, 6). We had hypothesized that human adrenarche could be a maturation event secondary to a decrease in 3ß-hydroxysteroid dehydrogenase (3ßHSD) mRNA, and we have suggested that 3ßHSD enzyme activity could modulate adrenal androgen steroidogenesis (7, 8). It has been proposed that hyperinsulinemia causes ovarian hyperandrogenism. Insulin can be shown experimentally to have a variety action on steroidogenesis in humans. This action appears to be directly mediated by insulin receptor. However the occupancy of the IGF-I receptor cannot be ruled out (9).
In girls, pronounced adrenarche and precocious pubarche have been related to insulin resistance with subsequent functional ovarian hyperandrogenism and to reduced fetal growth (10, 11). In boys, information about insulin resistance and precocious pubarche is scarce. A single report concludes that precocious pubarche is not associated with this cluster of endocrine-metabolic abnormalities (12). In contrast to adult women, in adult men, serum DHEAS concentrations correlate positively with insulin sensitivity (13), suggesting a physiological sexual dimorphism.
Consequently, the purpose of this study was to analyze the implication of the GH/IGF-I axis and insulin sensitivity in the regulation of adrenal androgen secretion in normal prepubertal and adolescent boys. We have evaluated the relationship among serum IGF-I, body mass index (BMI), fasting serum glucose/insulin ratio (fasting G/I ratio) as an indirect marker of insulin sensitivity, and adrenal androgens in 56 normal boys during early and late prepuberty as well as during pubertal development.
Subjects and Methods
Subjects
Serum samples were collected from patients attending a general pediatric hospital for ambulatory nonendocrine disorders such as strabismus, inguinal hernia, skeletal congenital defects, etc. Serum samples were taken from blood drawn for routine surgical check up. A pediatric endocrinologist was present during blood sampling to obtain oral consent from the parents. Clinical examination was carried out by the same trained pediatric endocrinologist for evaluation of Tanner’s stage of sexual development and assessment of testicular volume, as well as height, BMI, and general well-being. Subjects were included in the study according to the following criteria. Height equal or higher than -1.5 SD score, BMI between 15–85 percentiles of a population of Hispanic subjects older than 5 yr (14), and weight for height between 80–110% of normal for the Argentinean population in subjects less than 5 yr old. The protocol was approved by the research committee of the Garrahan Pediatric Hospital.
The study included 56 normal boys whose clinical characteristics are listed in Table 1
. Subjects were divided according to testicular volume (TV). Group 1 (Gr1) included 33 prepubertal subjects with TV less than 4 cc. To analyze early and late prepubertal periods, Gr1 was subdivided into two subgroups according to median chronological age (5.9 yr): subjects below the median, Gr1A (n = 16) and subjects at the median or above, Gr1B (n = 17). Gr3 included 23 subjects with TV between 4–25 cc. They were subdivided into 2 subgroups according to TV: subjects with TV between 4–8 cc (Gr3A, early puberty; n = 13) and subjects with TV between 10–25 cc (Gr3B, late puberty; n = 10). To study hormonal changes during the transition between prepuberty and puberty, an additional group, Gr2 (n = 30), was defined by mixing the late prepuberty and the early puberty subgroups.
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Serum DHEAS was determined with the Immulite assay (Diagnostic Products, Los Angeles, CA); the sensitivity was 0.05 µmol/liter, and the interassay coefficient of variation ranged from 8.1–15%. Serum androstenedione (4A) was determined by RIA (Diagnostic Systems Laboratories, Inc., Webster, TX); the assay sensitivity was 0.10 nmol/liter, and the interassay coefficient of variation ranged from 7–9.8%. Serum IGF-I was determined by RIA after acid-ethanol extraction of serum, as previously reported (15). Insulin was determined by the Imx sys lens (Abbott Laboratories, Chicago, IL); the assay sensitivity was 1.0 µU/ml, and the interassay coefficient of variation ranged from 3.8–4.5%.
Serum glucose was determined by a Hitachi 911 autoanalyzer. As previously reported (16, 17), the fasting G/I ratio is a useful measure of insulin sensitivity. Furthermore, as a correlation between the quantitative insulin sensitivity check index (QUICKI = 1/log I + log G) and the euglycemic glucose clamp has been reported, QUICKI has been proposed as a simple and accurate method to assess insulin sensitivity (18). To validate the fasting G/I ratio (milligrams per 10-4 U) as an indirect marker of insulin sensitivity in our normal subjects, we studied the correlation of this marker with QUICKI. We found a correlation coefficient of 0.91 (P < 0.0001; Fig. 1) Therefore, in this study insulin sensitivity is estimated by the fasting G/I ratio.
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All statistical analyses were performed using Statistix 7 (Analytical software, Tallahassee, FL). Unpaired t test was employed to assess mean value differences. Simple linear regression analysis between several pairs of variables was carried out. Furthermore, Pearson’s correlation was calculated to compute a correlation matrix for several variables.
Results
The mean (±SD) fasting G/I ratio, serum IGF-I, DHEAS, and 4A in Gr1, Gr2, and Gr3 are shown in Table 2. In Gr1, the fasting G/I ratio was significantly higher (P < 0.001), and serum IGF-I, serum DHEAS and serum 4A were significantly lower than in Gr3 (P < 0.001). There was no difference between early (Gr1A) and late prepuberty (Gr1B) fasting G/I ratios or between early puberty (Gr3A) and late puberty (Gr3B) fasting G/I ratios. However, the fasting G/I ratio decreased between late prepuberty and early puberty. We also found that BMI increased significantly in Gr3A compared with Gr1B (Table 1
) at a time when a significant decrement in the fasting G/I ratio was found.
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The mean serum DHEAS level in Gr1B was significantly higher than in Gr1A (P < 0.02). However, no difference between Gr3A and Gr3B was found. These data indicated that serum DHEAS levels increase during the transition from late prepuberty (Gr1B) to early puberty (Gr3A). Serum 4A levels were similar in Gr1A and Gr1B. During puberty, in contrast to DHEAS, serum 4A increased significantly from Gr3A to Gr3B (P = 0.0001).
The relationships between serum DHEAS and chronological age, fasting G/I ratio, and serum IGF-I in Gr1, Gr2, and Gr3 are shown in Fig. 2. The serum DHEAS level increased significantly with age in Gr1 and Gr2. However, no significant increase with age was found in Gr3, suggesting that DHEAS serum levels increase in late prepuberty and early puberty. There was no significant correlation between serum DHEAS and fasting G/I ratios or between serum DHEAS and serum IGF-I in Gr1, suggesting that during prepuberty insulin and the GH/IGF-I axis are not involved in adrenal androgen steroidogenesis. However, a significant negative correlation (P = 0.01) between serum DHEAS and the fasting G/I ratio, but not between serum DHEAS and serum IGF-I, was found in Gr2. This suggests that peripheral insulin sensitivity, but not extraadrenal IGF-I, might influence adrenal androgen steroidogenesis during the transition between late prepuberty and early puberty. Finally, no correlation was found between serum DHEAS and fasting G/I or between serum DHEAS and IGF-I in Gr3.
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4A and chronological age, fasting G/I ratio, and serum IGF-I in Gr1, Gr2, and Gr3 are shown in Fig. 3. In Gr1, no significant correlation between serum 4A levels and chronological age, fasting G/I ratio or serum IGF-I was observed. However, serum 4A increased significantly with age in Gr2 and Gr3, suggesting that serum 4A increases during transition from prepuberty to puberty as well as during the rest of pubertal development. A significant negative correlation between serum 4A and fasting G/I was only found in Gr2, suggesting again that there is an influence of insulin sensitivity on adrenal androgen steroidogenesis in early puberty. No significant correlation between serum 4A and serum IGF-I was found in any of the groups, suggesting that peripheral IGF-I is not involved in adrenal androgen steroidogenesis during prepubertal and pubertal development.
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. No significant correlation between the fasting G/I ratio and chronological age was found in Gr1, suggesting that insulin sensitivity does not change during prepuberty. On the other hand, a significant negative correlation between serum IGF-I and the fasting G/I ratio was found in Gr1 (P = 0.05). In Gr2, the fasting G/I ratio correlated negatively and significantly with chronological age (P = 0.03), serum IGF-I (P = 0.03), and BMI (r = -0.37; P = 0.03; data not shown in figures), suggesting that BMI and insulin sensitivity are modified in early puberty, and that the GH/IGF-I axis is involved in this change. Finally, no significant correlation between the fasting G/I ratio and chronological age or BMI (data not shown) was observed in Gr3. Furthermore, no significant correlation between the fasting G/I ratio and serum IGF-I was found in Gr3.
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shows the correlation matrix of this analysis. It confirmed that there was absence of correlation either between serum DHEAS and the fasting G/I ratio or between serum DHEAS and serum IGF-I during prepuberty (Gr1). Table 3 also shows that there was a significant negative correlation between serum DHEAS and the fasting G/I ratio, but not between serum DHEAS and serum IGF-I, during the transition from prepuberty to puberty (Gr2). Furthermore, there was a significant negative correlation between BMI and the fasting G/I ratio and a significant positive correlation between BMI and DHEAS in Gr2. On the other hand, no significant correlations were found during puberty (Gr3), except for serum IGF-I and chronological age.
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In the present study we have examined the implications of serum insulin and the GH-IGF-I axis on adrenal androgen steroidogenesis in normal prepubertal and pubertal boys. We have used the fasting G/I ratio as an indirect marker of insulin sensitivity. A single determination of glucose and insulin levels is not the best method for assessing insulin sensitivity. However, more accurate techniques such as the hyperinsulinemic euglycemic glucose clamp technique and the minimal model analysis of a frequently sampled iv glucose tolerance test are either very labor intensive or require large blood sampling. Therefore, they are not feasible for screening large populations of normal children and adolescents (19, 20). That’s why the fasting G/I ratio has been validated as a clinically useful parameter for measuring insulin sensitivity in women with polycystic ovary syndrome (16). Furthermore, it has been reported that the fasting G/I ratio is a useful measure of insulin resistance in girls with premature adrenarche (17). In the present study we checked the usefulness of the measurement of serum glucose and insulin in the fasting state by calculating QUICKI in every sample. QUICKI has been shown to be an accurate method for assessing insulin sensitivity in humans (18). We found a very high correlation between the fasting G/I ratio and QUICKI.
Insulin can be shown experimentally to have a variety of direct actions on steroidogenesis in humans (21). Hyperinsulinism and insulin resistance were present in small for gestational age girls with exaggerated adrenarche, and it had been speculated that insulin resistance might be responsible for the initially reduced growth and subsequently exaggerated adrenarche in these girls (11). In boys, pronounced adrenarche and precocious pubarche were not found to be associated to this cluster of endocrine and metabolic abnormalities in the only study available (12). However, several reports have described an increment in insulin secretion in early puberty as a compensatory response to reductions in insulin sensitivity in normal adolescents of the two sexes (22, 23, 24). In our study we found that, as previously described (25, 26), serum DHEAS levels increased during prepuberty, but we also showed that there was an increment during the transition from late prepuberty to early puberty. However, the fasting G/I ratio did not increase during prepuberty. These results suggest that insulin is not involved in the development of normal human adrenarche during the prepubertal years in boys. As we previously described, adrenarche might be an adrenal maturation event secondary to a decrement in 3ßHSD activity (7, 8) along with an increment in 17,20-lyase activity by a regulatory effect independent of 17-hydroxylase activity (5, 6).
The fasting G/I ratio increased from late prepuberty to early puberty, and it remained unchanged during late puberty. We also found a significant negative correlation between the fasting G/I ratio and serum DHEAS when late prepubertal and early pubertal subjects were analyzed together. The fact that the decrement in insulin sensitivity is associated with an increment in serum DHEAS during the transition to early puberty suggests that peripheral insulin is involved in adrenal androgen steroidogenesis after adrenarche in normal adolescent boys. Even though this negative correlation does not necessarily imply a cause-effect relationship, published evidence suggests than insulin amplifies 17-hydroxycorticosteroid intermediates response to ACTH (27). On the other hand, in polycystic ovary syndrome, a condition marked by insulin insensitivity, adrenal hyperandrogenism is often associated with ovarian hyperandrogenism (28). However, our data differ from the report of Potau et al. (12). In that study insulin sensitivity increased with advanced puberty. However, Potau’s study was performed in short normal children. In our study the mean height SD scores were near 0 in all groups, and ranges varied from -1.5 to 2.
Even though serum IGF-I levels increased from early prepuberty to late prepuberty and also during the rest of pubertal development, no correlation between serum DHEAS and serum IGF-I levels was found. Therefore, serum IGF-I is probably not involved in the regulation of adrenal androgen steroidogenesis. According to a previous report (29), there is a significant negative correlation between serum IGF-I and insulin levels in prepuberty and early puberty. The pattern of IGF-I and insulin levels may be related to changes in GH release. However, the fact that the main change in insulin sensitivity occurs in early puberty, and it remains unchanged in late puberty, when the most significant increment in serum IGF-I takes place, suggests that other factors, which are relevant in late puberty, might affect insulin sensitivity. Indeed, it was previously reported that BMI was the best predictor of insulin sensitivity in normal pubertal boys (30, 31). In line with these reports, we found that BMI increased significantly in early puberty when the fasting G/I ratio decreased. The increment in BMI in early puberty could be secondary to an increment in IGF-I (32) along with moderate increases in testicular T. In late puberty, however, at a time when serum IGF-I and T levels reach their maximum, there was no further increase in BMI or in insulin resistance. It is possible that T-induced lipolysis (33, 34, 35) prevents further increments in body adiposity.
Differences in the pattern of changes in serum 4A and DHEAS levels were observed during prepuberty and puberty. Serum 4A did not change throughout prepuberty, but increased from late prepuberty to early puberty and during puberty. These increments might be secondary to an increase in testicular secretion of 4A. Similarly to what was observed with serum DHEAS, we found a significant negative correlation between the fasting G/I ratio and serum 4A when late prepubertal and early pubertal boys were analyzed together, suggesting again that insulin regulates adrenal androgen steroidogenesis in early puberty. Our results also suggest that peripheral IGF-I is not involved in 4A synthesis.
In summary, the findings of this study indicate that the GH/IGF-I axis and insulin resistance are not involved in the mechanism of adrenarche during prepuberty in boys. We also found that in normal boys, insulin sensitivity decreases at early puberty rather than at late puberty, following changes in BMI. It is suggested that the increment in T secretion characteristics of late puberty offsets possible effects of the GH/IGF-I axis on body adiposity. These data suggest that peripheral insulin could be involved in adrenal androgen steroidogenesis, particularly during early puberty. As this evidence is partially based on correlations, additional studies are necessary to confirm these findings.
Acknowledgments
The contributions of D. Chirico and L. Del Rio are acknowledged. We thank the National Pituitary Agency for the generous supply of IGF-I RIA reagents.
Footnotes
This work was supported by grants from CONICET, FONCYT, and Ministerio de Salud (Beca Carrillo-Oñativia) of Argentina.
Abbreviations: 4A, Androstenedione; BMI, body mass index; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; G/I, glucose/insulin; Gr, group; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; QUICKI, quantitative insulin sensitivity check index.
Received January 19, 2001.
Accepted December 5, 2001.
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