1 Department of Obstetrics and Gynecology and 2 Department of Genetics, Mersin University, School of Medicine, 33079, Mersin, Turkey
3 To whom correspondence should be addressed. Email: devrimertunc{at}hotmail.com
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Abstract |
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Key words: IRS-1 polymorphism/metformin/polycystic ovary syndrome
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Introduction |
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There is still no consensus about which patients benefit from and should use the insulin-sensitizing drugs. Moghetti and colleagues (2000) found that higher body mass index (BMI) and plasma insulin concentration, lower serum androstenedione concentration and less severe menstrual abnormalities were baseline predictors of clinical efficacy measured by improved menstrual cyclicity. In contrast, Fleming et al. (2002)
suggested that high sex hormone-binding globulin (SHBG) concentration and lower free androgen index, but not BMI and insulin measures, predicted the ability to establish normal ovarian function. At present, therefore, there are insufficient data to limit the use of metformin treatment to a specific subgroup of women with PCOS.
The insulin receptor is a tetrameric membrane glycoprotein composed of two - and two
-subunits, linked together by disulphide bonds. Upon binding of its ligand, conformational changes occur that results in autophosphorylation of the
-subunit (Hubbard et al., 1994
). The first step after activation of the insulin receptor is phosphorylation of a cytoplasmic protein, insulin receptor substrate-1 (IRS-1) (White, 1997
). IRS-1 is a docking protein that needs to become phosphorylated in order to activate the enzyme phosphatidylinositol 3-kinase (PI3K), a necessary step for the initiation of several effects of insulin such as glucose transport. Molecular scanning of the IRS-1 gene has revealed several amino acid substitutions (Ura et al., 1996
). The most common IRS-1 variant, a glycine to arginine change at codon 972 (G972A), is more prevalent among subjects who have features of the insulin resistance syndrome associated, or not, with type 2 diabetes (Clausen et al., 1995
; Jellema et al., 2003
). Some of the phenotypic features of G972A, i.e. obesity (Clausen et al., 1995
) and hyperandrogenism (Witchel et al., 2001
), show similarities to those of PCOS. There are conflicting reports about G972A in PCOS. It has been suggested that G972A was more prevalent in patients with PCOS who are insulin-resistant (El Mkadem et al., 2001
). In contrast, Ehrmann et al. (2002)
found no association of G972A with the phenotypic features of PCOS.
Not all the mechanisms of action of metformin in the improvement of insulin resistance have been brought into the light. Recently, it has been suggested that metformin can induce phosphorylation of insulin receptor -subunit and IRS proteins (Wiernsperger and Bailey, 1999)
. Depending on this feature of metformin, we supposed that the G972A variant of IRS-1 may modulate the response to metformin, and aimed to investigate if the response to metformin varies according to the presence of this variant in women with PCOS.
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Materials and methods |
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BMI was calculated as weight (kg) divided by the height squared (m). According to the recommendation of the World Health Organization, waist circumference was obtained as the minimum value between the iliac crest and the lateral costal margin, whereas hip circumference was determined as the maximum value over the buttocks. The waist-to-hip ratio (WHR) was calculated by dividing the waist measurements by those of the hips. All patients received metformin (Metformin, Glukofen®, Ilsan-Hexal, Istanbul) at a dose of 500 mg three times a day for 6 months. All patients received individual, written and verbal diet and lifestyle counselling at inclusion. Basal and 6 month hormone levels were measured. IRS-1 genotype was determined at the end of the 6 months by one of the authors (E.M.E.).
Outcome measures
Primary outcome measures were sex steroid levels including 17-estradiol (E2), total testosterone and androstenedione (AS), SHBG, free testosterone index (FTI) and dehydroepiandrosterone sulphate (DHEAS). FTI was calculated by the equation: total testosterone (nmol/l)x100 divided by SHBG (nmol/l). This value correlates quite well with the free testosterone level measured by equilibrium dialysis and appears to be useful to assess the clinical androgen status in women, except in pregnancy (Vermeulen et al., 1999
).
Secondary outcome measures were BMI, WHR and metabolic parameters including fasting insulin, fasting C-peptide, fasting glucose and glucose levels during a 75 g oral glucose tolerance test (OGTT). The degree of insulin resistance was estimated using homeostasis model assessment (HOMA) analysis as follows: fasting serum insulin (µU/ml) x fasting plasma glucose (mmol/l) divided by 22.5 (Matthews et al., 1985). The HOMA method has been validated recently to be a good index of insulin resistance in subjects with a broad range of insulin sensitivity and has a good correlation with the insulin-mediated glucose uptake calculated by the euglycaemic hyperinsulinaemic glucose clamp (Bonora et al., 2000
).
Biochemical and hormonal measures
After an overnight fast, blood samples were collected for fasting glucose, insulin and C-peptide levels. All subjects then underwent a 75 g OGTT. For sex steroids, blood was drawn within 7 days of menstruation in spontaneously menstruating women. In women with long-lasting oligo/amenorrhoea, blood was drawn on the 7th day of progesterone withdrawal bleeding to avoid the effect of progesterone on measured parameters. We used 10 mg of medroxyprogesterone acetate (Farlutal®, Deva, Istanbul) for 10 days for this purpose. Serum was obtained from each subject for measurement of total testoserone, E2, DHEAS, AS, 17-hydroxyprogesterone (17
-OHP) and SHBG. The same procedures were repeated at 6 months.
Assay methods
Plasma glucose levels were determined by the glucose oxidase method immediately after blood samples were obtained. Blood samples for hormones were centrifuged immediately, and serum was stored at 20 °C until assayed. E2, total testosterone, DHEAS and insulin levels were determined by a competitive electro-chemoluminescent immunoassay method using an auto-analyser (Elecys 2010 RDM, Roche Diagnostics GmbH, Mannheim, Germany). FSH and LH levels were determined by a sandwich electro-chemoluminescent immunoassay method using the same auto-analyser. AS, C-peptide, IGF-1 and SHBG levels were determined by competitive enzyme-linked chemoluminescent immunoassay (Immulite 1000 Biodpc, CA).
Detection of the Gly972Arg polymorphism in the IRS-1 gene
Genomic DNA was obtained from human leukocyte nuclei isolated from whole blood by standard techniques using phenol/chloroform. The genotyping was performed as described elsewhere (van Dam et al., 2004). Briefly, the G972A genotype was obtained by PCR using the primers 5'-CTTCTGTCAGGTGTCCATCC-3' (forward) and 5'-TGGCGAGGTGTCCACGTAGC-3' (reverse). The product of amplification (236 bp) was subsequently digested by the restriction enzyme, and the fragments were run in the agarose gel stained with ethidium bromide, and analysed under ultraviolet light.
Power analysis and randomization
Because no data have been published previously on the efficacy of metformin treatment according to IRS-1 genotype, the study power was based upon predicted changes in the testosterone levels, using data derived from the literature (Harborne et al., 2003a). When considering a change of 0.2 ng/ml in testosterone levels before and after treatment, eight patients in each group would be sufficient to detect a difference at a significance level of 5% with a power of 80% (G*Power v2.0). The data concerning the prevalence of the G972A variant of IRS-1 in PCOS are inconsistent (el Mkadem et al., 2001
; Sir-Petermann et al., 2001
; Ehrmann et al., 2002
). Assuming an approximate incidence of 13% G972A variance (the mean percentage of three studies),
60 patients would be required to ascertain at least eight patients in a group, i.e. in the G972A variant group. We performed randomization at initial recruitment of the patients from a cohort of women with PCOS admitted between 1996 and 2003 (computer-generated randomization in blocks of four, ClinStat v08.05.96). IRS-1 genotype was determined at the end of the sixth month by one of the authors, so the clinicians who assessed the clinical and biochemical features were unaware of the IRS-1 genotype of the patients.
Statistical analysis
All statistical analyses were performed using SPSS v.10 (SPSS Inc., Chicago, IL) for Windows (Microsoft, Redmon, Washington). Changes in outcomes over time in both groups were examined using two-way analysis of variance for repeated measures in a general linear model. This method has the advantage of excluding the variability between subjects and is the best method to compare the outcomes in unbalanced groups (Vickers, 2001). The time x IRS genotype interaction tested whether the mean change after treatment differed between G972A-negative and positive patients. To control for mediating variables, we added potential confounders as continuous covariates in the model. We used age as covariate for BMI and WHR; age and BMI as covariates for FSH, LH, 17
-OHP, SHBG, FTI and metabolic variables; and age, BMI and SHBG as covariates for sex steroids. Changes in outcomes after treatment within each group were assessed by paired t-test in normally distributed data. Data that showed skewed distribution according to the KolmogorovSmirnov test were analysed by Wilcoxon matched pairs test. A P-value
0.05 was considered statistically significant.
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Results |
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Anthropometric variables and glucose and insulin metabolism
Our primary aim was to detect a differential effect of metformin on the variables on the basis of IRS-1 genotype in women with PCOS. Although the BMI in G972A-negative women decreased compared with baseline, the effect of metformin was not dependent on the IRS-1 genotype. Marked differences at the end of follow-up were noted between groups when glucose and insulin metabolism were analysed. In general, metformin had no effect on glucose and insulin metabolism in patients with the G972A variant, except fasting glucose levels (Table I). However, we found that metformin had differential effects on fasting insulin levels and on insulin resistance (Table I). Metformin had lowered the fasting insulin levels and insulin resistance more effectively and significantly in G972A-negative women (P<0.001 for both). Although significant decreases were observed in the levels of fasting glucose, C-peptide and 2 h glucose in G972A-negative women, the response to metformin in these parameters did not change according to IRS genotype (Table I).
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Discussion |
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Knowledge about the role of IRS-1 protein variants in the development of insulin resistance usually originates from studies on subjects with non-insulin-dependent diabetes mellitus (NIDDM). Although the role of variant IRS-1 proteins, especially the most abundant type Gly972Arg variant, in the development of NIDDM is controversial, it is generally accepted that this variant is responsible at least for obesity-related insulin resistance (Clausen et al., 1995). Almind et al. (1996)
observed a diminution in insulin action in transfection studies of the G972A variant. It has been suggested that this mutation may affect the tertiary structure of IRS-1 that results in defective binding to PI3K, and impairs glucose metabolism by this means, without disturbing the mitogenic functions of insulin.
The reason for the insulin resistance that is seen in women with PCOS remains to be elucidated, but it is generally believed that it originates from post-receptor defects (Venkatesan et al., 2001). The nature of post-receptor defect(s) is poorly understood, but it seems to be multifactorial. The theory of increased serine/threonine phosphorylation of the insulin receptor that is unique to PCOS and prevents tyrosine phosphorylation of insulin receptor
-subunit has gained popularity in the last decade (Dunaif et al., 1995
). There are controversial reports about the prevalence and the role of the G972A variant in insulin resistance in women with PCOS. While Ehrmann et al. (2002)
found no association between the G972A variant and the metabolic features of patients with PCOS, el Mkadem et al. (2001)
suggested that this variant is more prevalent in insulin-resistant PCOS patients. These last authors found that women who are carriers for the G972A variant were more obese, had higher fasting insulin levels and were more insulin resistant as demonstrated by HOMA. In another study, 2 h glucose values were found to be higher in patients with the G972A variant, whereas fasting insulin levels and HOMA were comparable with those of controls (Sir-Petermann et al., 2001
).
Although metformin has been used for 40 years, its mechanisms of action are not fully understood. The postulated mechanisms are reduced absorption from the gastrointestinal system, decreased endogenous gluconeogenesis from liver, and increased peripheral glucose uptake. At the cellular level, there is growing evidence that metformin may augment tyrosine phosphorylation of insulin receptor
-subunit and IRS proteins. It may also augment insulin-dependent and non-dependent cellular glucose uptake through the family of glucose transporter proteins (Wiernsperger and Bailey, 1999
).
We found in our study that metformin had differential effects on fasting insulin levels and HOMA indices on the basis of IRS genotype. However, there were also remarkable improvements in other metabolic variables within the IRS-1 intact group after 6 months metformin therapy. The decrease in insulin resistance in the intact IRS-1 group and no change in the variant group made us think that the IRS-1 genotype may play an important, but probably secondary role in this differential effect. In a study concerning the mechanism of action of metformin, metformin provoked tyrosine phosphorylation of insulin receptor -subunit and IRS-2 proteins, but not IRS-1 (Gunton et al., 2003
). As IRS-1 is essentially distributed across the muscle and adipose cells, variant IRS-1 protein may not be able to propagate the signals that are transmitted from the tyrosine-phosphorylated insulin receptor
-subunit, and thus may not be able to increase the glucose uptake into the muscle and adipose cells. However, this hypothesis must be confirmed with appropriate in vitro studies. Fasting blood glucose levels significantly decreased in both groups compared with baseline. The decrease in fasting glucose levels in both groups may be attributable to the dietary therapy or, more importantly, to the effects of metformin on basal endogenous glucose production from liver through phosphorylation of insulin receptor
-subunit and IRS-2 proteins (Gunton et al., 2003
). While C-peptide and 2 h glucose levels were significantly different compared with baseline in IRS-1 intact women, no change was observed in these parameters in PCOS women with the G972A variant.
An interaction has been found between hyperinsulinism and hyperandrogenism in most studies. Furthermore, the decline in the insulin level with insulin-sensitizing agents is usually accompanied by a decrease in androgen levels, although the exact mechanism(s) remains to be proven (Harborne et al., 2003a). Hyperandrogenism in PCOS is predominantly of ovarian origin; patients with PCOS exhibit an ovarian overproduction of 17
-OHP and AS after GnRH agonist challenge (Ehrmann et al., 1995
). However, 5070% of these patients also exhibit adrenal hyperandrogenism, because hypersecretion of AS and DHEA after adrenocorticotrophic hormone (ACTH) is frequently found in PCOS (Carmina et al., 1992
).
17-OHP and AS levels were found to be decreased indifferently with metformin therapy when comparing both groups, in our study. The decline in these potent androgen precursors in both groups made us think that not only is the decrease in insulin levels operational, but additional actions of metformin should be required. Indeed, several studies have suggested that metformin also has direct ovarian effects. It has been suggested that metformin could decrease AS production by at least 40% and 17
-OHP production by 10% from thecal cells (Attia et al., 2001
). A subsequent study confirmed that insulin could augment AS and 17
-OHP production from thecal cells, and metformin could inhibit their production in the presence or absence of insulin (Mansfield et al., 2003
). Furthermore, the decrease of production of these products was more pronounced in the presence of insulin in the latter report. Ovaries have been shown to contain all components of insulin signalling molecules. Several disorders in IRS proteins have been reported in both ovarian granulosa and theca cells. IRS-1 has been found to be increased in granulosa cells, whereas IRS-2 is decreased (Wu et al., 2003
). A more recent study has reported that there is no difference in distribution of IRS molecules in granulosa cells, instead both IRS-1 and IRS-2 are increased in theca cells (Yen et al., 2004
). However, the common suggestion of these studies is that a disequilibrium in distribution of IRS proteins may lead to the disturbances of ovarian function in women with PCOS. Notably, although it is known that thiazolidinediones act through peroxisome-proliferator activator receptor molecules (Yki-Jarvinen, 2004
), Wu et al. (2003)
observed that troglitazone was able to reverse the distribution of IRS proteins in PCOS ovary cells in a similar way to ovarian cells from normal women.
Although there are studies reporting a decreased secretion of DHEAS in response to metformin therapy (Nestler and Jacubowicz, 1997; Jacubowicz et al., 2001
), many studies established an increased secretion of this adrenal product after metformin (Crave et al., 1995
; Fleming et al., 2002
; Harborne et al., 2003b
). The reports on the evaluation of adrenal androgen production after metformin therapy are more confusing. After 1 month of metformin treatment, the ACTH stimulation test was performed in patients with PCOS, and the enzymatic activities of C17-hydroxylase and C17,20 lyase were found to be decreased (la Marca et al., 2000
). Whereas Unluhizarci et al. (1999)
found no difference in 17
-OHP or AS response after ACTH stimulation, after a 3-month course of metformin, Vrbikova et al. (2001)
found increased enzymatic activities of 3
-hydroxysteroid dehydrogenase and C17,20-lyase and a significant decrease in the activity of 17
-hydroxysteroid dehydrogenase.
While there was no significant change in the level of DHEAS in women with intact IRS-1 after 6 months of metformin treatment, we observed an increase in DHEAS in women with the G972A variant. As DHEAS is a good marker of adrenal function, we can suppose that metformin acted differently on adrenal glands on the basis of IRS genotype. Although the levels of 17-OHP and AS, precursors of more potent androgens, decreased in both groups, the levels of total testosterone and FTI did not change after metformin therapy in PCOS women with the G972A variant. For this reason, we speculated that the increase in the adrenal function after metformin therapy was not counteracted by the suppression of ovarian androgen production in these women. This finding may involve other as yet not studied factors such as the role of IRS proteins on adrenal gland function and on the conversion of weak androgens to more potent androgens. Further research may help to clarify this issue.
In conclusion, despite the small sample size in our study, we found a differential effect of metformin therapy in PCOS women on the basis of IRS genotype. Complex subcellular mechanisms warrant further investigations to explain this differential effect. There is still a continuing debate about the selection of patients that would benefit from metformin therapy. This study may call attention to the importance of molecular markers in the management of women with PCOS and may bring an alternative viewpoint.
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Submitted on November 17, 2004; resubmitted on December 13, 2004; accepted on December 15, 2004.
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