Eur J Endocrinol
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DOI: 10.1530/eje.1.02195
European Journal of Endocrinology, Vol 155, Issue 1, 127-130
Copyright © 2006 by European Society of Endocrinology
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CLINICAL STUDY

The androgen receptor CAG repeat modifies the impact of testosterone on insulin resistance in women with polycystic ovary syndrome

Matthias Möhlig1,2, Annette Jürgens4, Joachim Spranger1,2, Kurt Hoffmann3, Martin O Weickert1,2, Hans W Schlösser6, Thilo Schill6, Georg Brabant7, Andreas Schüring5, Andreas F H Pfeiffer1,2, Jörg Gromoll4 and Christof Schöfl1

1 Department of Endocrinology, Diabetes and Nutrition, Charité-University Medicine Berlin, Berlin, Germany, 2 Departments of Clinical Nutrition and 3 Epidemiology, German Institute of Human Nutrition, Potsdam-Rehbruecke, 14558 Nuthetal, Germany, 4 Institute of Reproductive Medicine, and 5 Department of Obstetrics and Gynecology, University of Münster, 48129 Münster, Germany, and 6 Departments of Gynecological Endocrinology and 7 Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany

(Correspondence should be addressed to C Schöfl; Email: christof.schoefl{at}charite.de)


   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Objective: Hyperandrogenism is a central feature of the polycystic ovary syndrome (PCOS) and might worsen insulin resistance (IR) often seen in PCOS. Androgens act through the androgen receptor (AR). A polymorphic CAG repeat sequence within the AR gene was reported to modulate its transactivation activity. Therefore, we investigated a putative interaction between testosterone and the CAG repeat length polymorphism with respect to IR.

Design: In 63 PCOS women with normal glucose tolerance free testosterone, the biallelic CAG repeat length and a multiplicative interaction term were investigated by multiple linear regression analysis for an association with IR as indicated by the homeostasis model assessment of IR (HOMA-IR).

Results: Free testosterone was correlated with HOMA-IR. The impact of testosterone on HOMA-IR was modified by the AR CAG length as indicated by an interaction term. This interaction remained significant after adjustment for smoking, age and body mass index. While there was a positive association of free testosterone with HOMA-IR, the interaction term was inversely associated. The model, which explained 42.5% of the variation of HOMA-IR predicted that in carriers of short CAG lengths, an increase in testosterone increased IR. This effect attenuated with rising biallelic CAG length until it turns into the opposite at a CAG length longer than 23. The results were confirmed by using CIGMA as another measure of IR.

Conclusions: The association between testosterone and IR is modified by the CAG repeat polymorphism within the AR. Therefore, the evaluation of testosterone effects on IR seems to require consideration of the AR CAG repeat polymorphism in PCOS women.


   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
The polycystic ovary syndrome (PCOS) is one of the most frequent endocrine diseases affecting about 5–10% of reproductive women (1). PCOS is a heterogenous disorder characterized by hyperandrogenism, chronic anovulation and infertility. A large percentage of PCOS women are insulin resistant and suffer from metabolic alterations (2, 3). Insulin resistance (IR) has been shown to correlate with hyperandrogenism (2–4). It is, however, controversial whether hyperandrogenism causes IR or whether, vice versa, IR accounts for hyperandrogenism. Arguments for IR leading to hyperandrogenism come from trials that show an improvement of hyperandrogenism and fertility by lifestyle or pharmacological interventions that improve insulin sensitivity (5–8). On the other hand, antiandrogenic medication improved insulin sensitivity in hyper-androgenic women (9) indicating that androgens could well contribute to IR. Taken together, there is evidence that testosterone could affect insulin sensitivity in women with PCOS.

The effects of testosterone are mediated at the molecular level through activation of the androgen receptor (AR). A CAG repeat polymophism within the AR gene was found to influence transactivation. An inverse relationship exists between AR CAG repeat length and the transcriptional activity of testosterone target genes (10). We, therefore, tested the hypothesis that an interaction between circulating testosterone and the CAG repeat length polymorphism exists and that this interaction has an impact on IR in PCOS women.


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

In 63 PCOS women with normal glucose tolerance (according to the World Health Organization definition) (11), who were referred to our clinic because of hirsutism or oligo/amenorrhea were included after written informed consent was obtained. The cohort has been published in part previously (12, 13). The diagnosis of PCOS was based on: a) the presence of chronic ovulatory dysfunction, i.e. oligomenorrhoea (four or less cycles in the last 6 months)- or amenorrhoea (no cycles in the last 6 months), and b) clinical signs of hyperandrogenism, i.e. hirsutism or acne or c) laboratory findings, i.e. hyperandrogenaemia, defined as serum androgen levels (dehydroepiandrosterone sulphate (DHEAS), 17-OH-progesterone, androstenedione or total testosterone) above the upper limit of normal for the respective assay and d) the exclusion of other disorders such as Cushing’s syndrome, late-onset 21-hydroxylase deficiency, thyroid dysfunction, hyperprolactinaemia or androgen-secreting tumours. These diagnostic criteria for PCOS are consistent with the most commonly used diagnostic criteria for PCOS, often referred to as the National Institutes of Health consensus criteria (14). The clinical and endocrine features of the women are given in Table 1Go. Six patients suffered from Hashimoto’s thyroiditis and were euthyroid under thyroid hormone replacement therapy. Five women were suffering from arterial hypertension and were normotensive under antihypertensive drugs. All other women (n = 52) were free of other diseases and were not taking any medication.


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  		Table 1 Clinical and endocrine features of the PCOS cohort (n = 63). Continuous variables are given as means ± S.E.M. and frequencies as n (%).
 
All women were studied within the first 10 days following menstruation in case of mild oligomenorrhoea or at random if they suffered severe oligo- or amenorrhoea. Blood was sampled in the morning after an overnight fast and the samples were stored at –20 °C until analysis. IR was quantified by calculating homeostatic model assessment of IR (HOMA-IR) (fasting insulin (mU/l) x fasting glucose (mmol/l))/22.5) using the mean of three fasting glucose and insulin values (15). In 61 women, IR was additionally assessed by a 2-h continuous infusion of glucose with model assessment (CIGMA) using a computer program kindly provided by Dr Levy (16). Body mass index (BMI) was calculated as body weight (kg) divided by body height squared (m2).

Assays

All biochemical and endocrine parameters were determined as previously described (12). Free testosterone was calculated from total testosterone and sexual hormone binding globulin as published (17) using a web-based calculator (http://www.issam.ch/freetesto.htm).

The AR CAG repeat length polymorphism was determined by PCR and gel electrophoresis as described elsewhere (18). The AR gene is localized on the X-chromosome. Therefore, the mean length of the two alleles was calculated for each woman. These biallelic means are given throughout the manuscript and were used for further analysis as has been performed by others (19, 20).

Statistical analysis

Statistical analyses were performed with SPSS software (version 8.0, SPSS Inc.; Chicago, IL, USA). Significance was considered as two-tailed {alpha}< 0.05. To describe continuous variables, mean values and S.E.M. are presented. Correlation analysis was performed using the Pearson correlation coefficient. The relation between testosterone, AR CAG length and IR was modelled by multiple linear regression analysis with HOMA-IR or CIGMA as the dependent variable.


   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
The clinical and endocrine characteristics of the cohort are depicted in Table 1Go. The mean biallelic CAG length of the AR was 21.9 ± 0.3 ranging from 16.5 to 26. Nearly, 52.3% of the women had a CAG length between 21 and 23, 22.2% had a CAG length shorter than 21 and in 25.5% the CAG length was longer than 23.

There was a significant correlation between free testosterone and HOMA-IR (r = 0.295, P = 0.019). The biallelic CAG length of the AR neither significantly correlated with HOMA-IR (P = 0.08) nor with free testosterone (P = 0.46). Correlation analysis, however, does not consider a putative interaction between two variables with respect to a third dependent parameter. To address such an interaction between free testosterone and the AR CAG repeat length polymorphism with respect to IR, we performed multiple linear regression analysis including a multiplicative interaction term. The interaction was significantly associated with HOMA-IR (P = 0.009) and the impact of the interaction term remained significant (P = 0.002) even after adjustment for age, BMI and smoking status (smoker vs non-smoker). The fully adjusted model explained 42.5% of the variation of HOMA-IR (r2 = 0.425) (Table 2Go). While there was a positive association of free testosterone with HOMA-IR, the interaction term was inversely associated. The results were confirmed using either the long allele (P = 0.022) or the short allele (P = 0.019) instead of the mean biallelic length for calculation.


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  		Table 2 Multivariate regression model with HOMA-IR as the dependent variable unadjusted and adjusted. The adjusted model was used to calculate Table 3Go.
 
To better illustrate the impact of the interaction, we used this model to calculate HOMA-IR values for different biallelic mean CAG repeat lengths and free testosterone concentrations (Table 3Go). At a biallelic CAG length of 23 free testosterone had no or only minimal impact on IR. At CAG lengths shorter than 23, a rise in free testosterone was associated with increased IR. This effect increased with shorter CAG lengths. On the other hand, at CAG lengths longer than 23 increasing concentrations of free testosterone improved IR. This effect of free testosterone on IR was more pronounced the longer the CAG repeat lengths were. Viewing the data the other way around, at a given free testosterone of 20 pmol/l, the impact of the CAG length on HOMA-IR was minimal. At a lower free testosterone concentration HOMA-IR increased, while at higher testosterone levels HOMA-IR decreased with longer CAG lengths. Together, this demonstrates that the association of circulating free testosterone with IR is modified by the AR gene polymorphism.


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  		Table 3 HOMA-IR depending on free testosterone (f-testo) and the AR CAG repeat length as predicted from a regression model.
 
The results were confirmed using CIGMA, a second measure of IR. Again, the fully adjusted model explained a great proportion (56.6%) of the variation of CIGMA (r2 = 0.566) and the interaction term remained significant (P = 0.007). Furthermore, the mode of interaction was similar as described above for HOMA-IR.


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Hyperandrogenism is a key feature of the PCOS syndrome and might contribute to IR, which is often observed in PCOS women (2, 3, 9). At the molecular level, testosterone effects are mediated through activation of the AR. The ability of the receptor to enhance transcription of testosterone-regulated genes was shown in vitro to depend on a highly polymorphic CAG repeat microsatellite in exon 1 (10). The shorter the CAG repeat length, the stronger the androgenic activity of the AR. In men, this polymorphism has been associated with metabolic parameters such as high density lipoprotein cholesterol, fasting insulin or fat mass (18). Therefore, it appears reasonable to consider the CAG length polymorphism, when investigating the relation between testosterone and IR in PCOS.

The mean biallelic CAG length was similar to that reported from other PCOS cohorts (19, 20). There was no correlation between free testosterone and the CAG length, which is in accordance with a recently published study (19). Others, however, have demonstrated either a positive or negative association (20–22).

In our PCOS women with normal glucose tolerance, insulin sensitivity ranged from very sensitive to highly resistant. IR was calculated as HOMA-IR, a widely used and accepted measure of IR. HOMA-IR has been shown to correlate with euglycaemic clamp data in cohorts of similar size (15). However, HOMA-IR is a measure of basal insulin sensitivity. To obtain additional information about the insulin sensitivity in the stimulated state we used CIGMA (16). Using CIGMA confirmed the results calculated from HOMA-IR.

Insulin sensitivity correlated with testosterone as described (2–4), but not with the biallelic CAG length, which is consistent with a recent report (20). The CAG repeat length, however, was a significant modifier of the testosterone effect on insulin sensitivity as indicated by multiple linear regression analysis. At short CAG lengths, an increase in testosterone impairs insulin sensitivity. This effect attenuates with rising biallelic CAG length until it turns into the opposite effect at a CAG length longer than 23. However, only a minority of the women had a biallelic CAG repeat length longer than 23. We, therefore, cannot exclude the possibility that our model, which in general assumes linear interrelations, overestimates the modifying effect of the CAG polymorphism at longer CAG repeats. The interaction between testosterone and the genetically determined and hence invariable polymorphism within the AR supports the view that hyperandrogenism in PCOS women affects IR.

A limitation of the study, which needs to be addressed is the relatively small cohort size. Nevertheless, it seems to be the homogeneity of the cohort of PCOS women on the one hand and the robust effect of the interaction on the other hand that enabled the detection of its impact on insulin sensitivity in PCOS women. The general importance, however, of its role for the high prevalence of IR in PCOS requires further investigation.

In conclusion, the AR CAG repeat polymorphism was found to modify the impact of testosterone on IR in PCOS women. Therefore, it seems to be necessary to consider this in studies addressing metabolic effects of testosterone in PCOS women.


   Acknowledgements
 
The authors thank L Pekel and K Sprengel for excellent technical assistance.


   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received 12 April 2006
Accepted 28 April 2006




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