Metformin and oral contraceptive treatments reduced circulating asymmetric dimethylarginine (ADMA) levels in patients with polycystic ovary syndrome (PCOS)

Atherosclerosis 200 (2008) 336–344

Metformin and oral contraceptive treatments reduced circulating asymmetric dimethylarginine (ADMA) levels in patients with polycystic ovary syndrome (PCOS) Taner Ozgurtas a , Cagatay Oktenli b,∗ , Murat Dede c , Serkan Tapan a , Levent Kenar a , S. Yavuz Sanisoglu d , Zeki Yesilova e , Mufit C. Yenen c , M. Kemal Erbil a , Iskender Baser c a

Department of Biochemistry and Clinical Biochemistry, G¨ulhane Military Medical Academy, Ankara, Turkey Division of Internal Medicine, GATA Haydarpasa Training Hospital, Kadikoy, TR-34668 Istanbul, Turkey c Department of Obstetrics & Gynecology, G¨ ulhane Military Medical Academy, Ankara, Turkey d Department of Monitoring and Evaluation, Turkish Ministry of Health, Ankara, Turkey e Department of Internal Medicine, G¨ ulhane Military Medical Academy, Ankara, Turkey

b

Received 29 July 2007; received in revised form 15 December 2007; accepted 21 December 2007 Available online 20 February 2008

Abstract There is a little information in literature about circulating asymmetric dimethylarginine (ADMA) concentrations in polycystic ovary syndrome (PCOS) and the results reported are discrepant. In this study, therefore, we aimed (1) to determine the circulating ADMA concentrations in 44 women with PCOS and 22 age- and BMI-matched healthy controls, (2) to evaluate its correlations with insulin resistance, gonadotrophins, and androgen secretion, and (3) to compare effects of metformin and ethinyl estradiol–cyproterone acetate (EE/CPA) treatments on circulating ADMA concentrations. In conclusion, our data indicate that circulating ADMA concentrations in non-obese, non-hypertensive and young women with PCOS are significantly higher than healthy controls and they improved by a 3-month course of metformin and oral contraceptive treatments. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Asymmetric dimethylarginine; Polycystic ovary syndrome; Metformin; Ethinyl estradiol–cyproterone acetate

1. Introduction The endothelium plays a key role in the maintenance of vascular homeostasis; in turn, impaired endothelial function has been regarded as an early feature of atherosclerosis and plays an important role in the development of atherosclerotic diseases [1,2]. Although endothelial dysfunction is quite complex, it is characterized by impaired endothelium-dependent vasorelaxation most often accompanied by abnormalities in the production or metabolism of endothelium derived from nitric oxide (NO). Classically, NO inhibits several proatherogenic processes, including smooth ∗

Corresponding author. Tel.: +90 216 346 2600; fax: +90 216 348 7880. E-mail addresses: [email protected], [email protected] (C. Oktenli). 0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.12.054

muscle proliferation, monocyte and platelet adhesion, synthesis of inflammatory cytokines and platelet aggregation, thus exhibiting important antiatherogenic effects. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS) [3,4]. Moreover, since vascular cells are thought to be a major source of ADMA, the doubling of plasma concentrations may reflect an even greater change within endothelial cells [5]. ADMA stimulates many processes involved in atherogenesis such as monocyte adhesiveness [6], expression of proinflammatory and chemotactic factors [7], accumulation of oxidatively modified LDL in macrophages [8]. ADMA is metabolized by the enzyme dimethylarginine-dimethylaminohydrolase (DDAH) [9], which degrades them to citruline and dimethylamine or monomethylamine, respectively. Two isoforms exist (DDAH1 and DDAH2), with distinct tissue distribution

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[10]. Pharmacological inhibition of DDAH increases ADMA concentrations and reduces NO production [5], whereas transgenic DDAH overexpression has the opposite effect [11]. Transgenic mice that overexpress the human isoform of DDAH1 exhibit greater tissue DDAH activity, reduced plasma and tissue ADMA levels, increased tissue NOS activity, reduced systemic vascular resistance [12], and enhanced angioadaptation in response to ischemia or inflammation [13]. ADMA is considered an indicator for endothelial dysfunction [14] and a sensitive marker for cardiovascular risk, which was proved in an end-point study that linked serum concentrations of ADMA to acute coronary events [15]. A number of studies have been published in which strong correlation between ADMA concentrations and increased cardiovascular morbidity and mortality was documented [16–18]. Although there is no threshold for ADMA leading to endothelial impairment established so far, it can be speculated that elevated ADMA concentrations may cause endothelial hyporeactivity. An inverse correlation between endothelium-dependent vasodilatation and ADMA has also been demonstrated after fat intake and experimental hyperhomocysteinemia in humans [19]. The importance of increased circulating ADMA has recently been demonstrated in healthy volunteers who developed hypertension and cardiac dysfunction after exogenous administration of ADMA [20]. In this context, there is compelling evidence that even small modifications of ADMA levels significantly change vascular NO production, vascular tone, and systemic vascular resistance [21,22]. Data from experimental studies document that biologically relevant ADMA blood levels significantly inhibit NOS and reduce NO generation in cultured endothelial cells and in isolated human blood vessels [23,24]. After balloon injury, the regenerating endothelial cells manifest higher intracellular levels of ADMA and impaired endothelium-dependent vasodilatation [25]. The severity of the endothelial dysfunction and the intracellular levels of ADMA are directly related to the intimal thickness of the injured vessel [25]. Continuous ADMA infusion for 4 weeks induces severe microvascular lesions in the myocardium in wild-type as well as nitric oxide synthase knockout mice [26], and these effects depend both on NO-dependent and independent mechanism(s). Exposure of endothelial cells in culture to pathophysiologically relevant concentrations of ADMA reduces NO synthesis, increases superoxide generation and increases the adhesiveness of endothelial cells for monocytes [7]. Systemic administration of ADMA to healthy subjects causes a dosedependent and sustained reduction of NO production and cardiac output and an increase in peripheral vascular resistance accompanied by a rise in mean arterial blood pressure [21]. In hypercholesterolemic adults, elevated ADMA levels are inversely correlated with endothelium-dependent vasodilatation in the forearm [27]. ADMA is a competitive inhibitor, in hypercholesterolemic adults, an intravenous infusion of larginine restores endothelial function and increases urinary nitrate excretion (a surrogate parameter of NO production).

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Moreover, local infusion of ADMA into the brachial artery significantly attenuated endothelial-dependent vasodilatation in healthy volunteers [3]. Consequently, NO inhibition by ADMA may be a relevant proatherogenic mechanism and endogenous ADMA levels may be predictive of vascular lesion formation in both animal models and humans. Polycystic ovary syndrome (PCOS), characterized by infertility, oligo/amenorrhoea and hyperandrogenism, affects 5–10% of women of reproductive age. Women with PCOS have a clustering of cardiovascular risk factors such as obesity, dyslipidemia, impaired glucose tolerance, insulin resistance, and hypertension [28,29]. Cardiovascular risk factors and precocious cardiovascular abnormalities are often evident at an early age, suggesting that the chronically abnormal hormonal and metabolic milieu found in women with PCOS, starting from adolescence, may predispose these women to premature atherosclerosis, making them candidates for early cardiovascular disease. In this context, cardiovascular disease is the leading cause of death in women; particularly those with PCOS are at a seven-fold or greater risk for myocardial infarction [29]. In 2001, Paradisi et al. [30] firstly reported that PCOS is characterized by endothelial dysfunction and resistance to the vasodilating action of insulin. Then, a large number of studies have been focused on the endothelial dysfunction in PCOS [31–34]. Lakhani et al. [35] recently demonstrated that the acetylcholine-induced increase in microvascular perfusion, which is dependent on endothelial NO release, was blunted in women with PCOS. Consequently, in subjects with PCOS, androgens [36], dyslipidemia and insulin resistance [37] all may affect endothelial function, but causative significance of these factors in development of endothelial dysfunction remains uncertain. There is a little information in literature about circulating ADMA concentrations in PCOS and the results reported are discrepant. In a more recent report, Charitidou et al. [38] demonstrated that ADMA concentrations were higher in women with PCOS than controls. However, Demirel et al. [39] reported that ADMA levels in adolescent PCOS subjects were not different than those in controls. In this study, therefore, we aimed (1) to determine circulating ADMA concentrations in 44 women with PCOS and 22 age- and BMI-matched healthy controls, (2) to evaluate its correlations with insulin resistance, gonadotrophins, and androgen secretion, and (3) to compare effects of metformin and ethinyl estradiol–cyproterone acetate (EE/CPA) treatments on circulating ADMA concentrations.

2. Methods A total of 44 non-obese (BMI < 25 kg/m2 ), aged >18 years, women with PCOS were included in our study. They registered for care in our obstetrics and gynecology outpatient clinic with a chief complaint of irregular menstrual cycles and/or clinical hyperandrogenism. All women with PCOS

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had polycystic-appearing ovaries on ultrasound. The control group consisted of aged- and BMI-matched 22 healthy volunteer from students of our nursing school with regular menses (28 ± 2 days) and ultrasonographically normal ovaries. Their clinical, biochemical, and hormonal profiles were within normal limits. All subjects were non-smokers with regular daily activity, normotensive (<120/80 mmHg in two measurements) and were not regular consumers of alcoholic beverages. For at least 3 months before the study, all subjects refrained from using steroid hormones or any other medications likely to interfere with ovarian function, insulin sensitivity, or lipid metabolism. All subjects in the study were off any regular medication, like aspirin, statins, etc. that could affect the outcome of the study. None of the subjects contemplated pregnancy during the study period. Each subject gave her informed consent to the study, which was previously approved by our local ethics committee and institutional review board. The diagnosis of PCOS was made according to the criteria proposed at the Rotterdam revised consensus meeting (The Rotterdam European Society for Human Reproduction and Embryology (ESHRE)/American Society for Reproductive Medicine (ASRM)-Sponsored PCOS Consensus Workshop Group, 2004) [40]. All eligible patients presented with at least two of the three following criteria: (1) oligomenorrhea or amenorrhea, (2) clinical (hirsutism, acne) and/or biochemical signs of hyperandrogenism, and (3) polycystic ovaries. All of the women had normal thyroid-stimulating hormone and prolactin levels, and subjects with possible ovarian tumors, congenital adrenal hyperplasia, BMI greater than 25 kg/m2 , any chronic disease that could interfere with the absorption, distribution, metabolism or excretion of metformin or EE/CPA, renal or liver disease, were excluded from the study. The subjects were randomised to receive either metformin (Glucophage 850 mg, Merck Sant´e S.A.S., France; n = 22) or a monophasic oral contraceptive (n = 22) containing 35 ␮g of ethinyl estradiol and 2 mg of cyproterone acetate (Diane 35, Schering AG, Berlin, Germany). EE/CPA pills were taken daily 21 out of 28 days for a period of 3 months, while one tablet metformin (850 mg) was prescribed to be taken every 12 h. Measurements were taken on recruitment and repeated after 3 months of treatment. Three subjects were lost to follow up (two in metformin arm and one in EE/CPA arm). A full physical examination was performed including assessment of weight, height and waist and hip circumferences. Waist and hip circumferences were measured to the nearest centimetres with a soft tape at the narrowest part of the torso and at the widest part of the gluteal region. Waistto-hip ratio (WHR) was calculated as waist circumference in centimetres divided by hip circumference in centimetres. Body weight was measured using analogue scales in light clothing; height was measured barefoot using a stadiometer. BMI was calculated as weight/(height)2 in kilograms per square meter, and women were considered non-obese at BMI < 25 kg/m2 .

Venous blood samples were collected from an antecubital vein, between 08:00 and 09:00 a.m., after an overnight fast. The samples were centrifuged, aliquoted and immediately frozen at −80 ◦ C for analyses of lipoproteins and hormones. Serum ADMA concentrations were determined by ELISA method (Immundiagnostik AG, Bensheim, Germany). Detection limit of ADMA assay was 0.05 ␮mol/L. Plasma total cholesterol, TG, high-density lipoprotein cholesterol (HDL-C), and glucose levels were measured on Olympus AU-600 (Japan) auto-analyser by using its own commercial kits with enzymatic method. Very low-density lipoprotein cholesterol (VLDL-C) was isolated by ultracentrifugation, and VLDL-C was determined enzymatically. Serum insulin, LH, FSH, total testosterone (TT), estradiol, dehydroepiandrosterone sulphate (DHEA-S) and SHBG tests were determined by electrochemiluminescence immunoassay method, using an automated immunoassay analyser (E170, Roche, Hitachi Co., Osaka, Japan). Serum free testosterone (FT) and androstenedione were measured by radioimmunoassay method (Diagnostic Systems Laboratories, TX, USA). Low-density lipoprotein cholesterol (LDL-C) was calculated by Friedewald’s formula as LDL-C = (TC, mmol/l) − (TG, mmol/l)/(2.2 − (HDL-C, mmol/l)). Homeostasis model assessment for insulin resistance (HOMA-IR) was calculated from fasting plasma insulin and glucose levels as (insulin × glucose)/22.5, where the insulin concentration is in microunits per milliter, and glucose is in millimoles per liter [41]. 2.1. Statistical analysis Data were analyzed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA) software. Descriptives were shown as the mean ± S.D. Differences among the control, metformin and EE/CPA groups were investigated with the ANOVA (analysis of variance) test. Bonferroni test was used as the post hoc test. Pre- and post-treatment values of the parameters were compared with “The Paired samples t test”. Stepwise linear regression analysis was used to show the effects of the other parameters on ADMA. P-Values less than or equal to 0.05 were evaluated as statistically significant.

3. Results There was no difference between baseline characteristics of patient groups and controls with respect to age (data not shown), BMI, TC, HDL-C, LDL-C, LH, FSH and estradiol (Tables 1 and 2). The WHR, ADMA, HOMA-IR, TG, VLDL-C, TT, FT, androstenedione and DHEA-S levels were significantly higher in patients with PCOS than in healthy controls (Tables 1 and 2). As compared with the controls, levels of SHBG were significantly lower in both treatment groups with PCOS (Tables 1 and 2). Clinical characteristics

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Table 1 Comparisons of healthy controls, pre- and post-treatment clinical and laboratory characteristics in metformin group Parameters

Healthy controls (kg/m2 )

Body mass index WHR ADMA (␮mol/l) HOMA-IR (%) Total cholesterol (mmol/l) Triglyceride (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) VLDL-C (mmol/l) LH (IU/l) FSH (IU/l) Estradiol (pmol/l) Total testosterone (nmol/l) Free testosterone (pmol/l) Androstenedione (nmol/l) DHEA-S (␮mol/l) SHBG (nmol/l)

21.44 0.71 0.09 1.11 3.93 0.88 1.42 2.11 0.40 6.07 5.90 119.48 1.29 5.04 5.75 4.79 53.77

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.54 0.01 0.02 0.31 0.62 0.23 0.29 0.40 0.11 3.52 2.01 25.47 0.50 0.82 1.27 0.94 6.75

Pre-treatment 21.81 0.79 0.17 1.68 4.04 1.28 1.25 2.21 0.58 9.19 5.43 127.11 3.44 15.53 10.13 6.61 30.59

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.27 0.01 0.02 0.37 0.56 0.37 0.16 0.49 0.17 1.79 1.77 31.56 0.26 1.54 1.04 1.53 7.21

Post-treatment 21.12 0.78 0.12 1.54 3.76 0.95 1.26 2.12 0.38 7.51 5.52 125.05 3.24 14.17 9.81 6.30 31.48

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.06 0.01 0.02 0.31 0.56 0.31 0.20 0.49 0.14 3.44 1.95 31.53 1.08 3.28 1.27 1.36 6.79

Controls vs. pre-treatment

Pre- vs. post-treatment

NS <0.001 <0.001 <0.001 NS <0.001 NS NS <0.001 NS NS NS <0.001 <0.001 <0.001 <0.001 <0.001

0.001 <0.001 0.004 <0.001 <0.001 <0.001 NS NS <0.001 NS NS NS NS 0.002 NS NS NS

WHR, waist:hip ratio; HOMA-IR, homeostasis model assessment for insulin resistance; ADMA, asymmetric dimethylarginine; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol; DHEA-S, dehydroepiandrosterone sulphate; NS, non-significant.

and biochemical parameters did not differ between treatment groups at baseline (data not shown). In the metformin treated group, pre- and post-treatment clinical and laboratory characteristics and the results of comparisons are shown in Table 1. BMI (P = 0.001), WHR (P < 0.001), ADMA (P = 0.004), HOMA-IR (P < 0.001), TC (P < 0.001), TG (P < 0.001), VLDL-C (P < 0.001) and FT (P = 0.002) decreased significantly after the metformin treatment. The levels of HDL-C, LDL-C, LH, FSH, estradiol, TT, androstenedione, DHEA-S and SHBG did not differ significantly after the metformin therapy. In the EE/CPA treated group, pre- and post-treatment clinical and laboratory characteristics and the results of

comparisons are given in Table 2. The levels of ADMA (P = 0.006), LH (P = 0.001), FSH (P < 0.001), TT (P < 0.001), FT (P < 0.001) and androstenedione (P < 0.001) decreased significantly after the EE/CPA treatment, whereas SHBG (P < 0.001), VLDL-C (P < 0.001), TG (P < 0.001), HDL-C (P = 0.045) and HOMA-IR (P = 0.002) increased significantly. The levels of BMI, WHR, TC, LDL-C, estradiol and DHEA-S did not differ significantly after the EE/CPA therapy. There was a strong positive correlation between ADMA and WHR (ρ = 0.880, P < 0.001), FT (ρ = 0.821, P < 0.001), TT (ρ = 0.665, P < 0.001), TG (ρ = 0.631, P < 0.001), and VLDL-C (ρ = 0.560, P < 0.001) in all patients with PCOS.

Table 2 Comparisons of healthy controls, pre- and post-treatment clinical and laboratory characteristics in ethinyl estradiol–cyproterone acetate group Parameters

Healthy controls

Body mass index (kg/m2 ) WHR ADMA (␮mol/l) HOMA-IR (%) Total cholesterol (mmol/l) Triglyceride (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) VLDL-C (mmol/l) LH (IU/l) FSH (IU/L) Estradiol (pmol/l) Total testosterone (nmol/l) Free testosterone (pmol/l) Androstenedione (nmol/l) DHEA-S (␮mol/l) SHBG (nmol/l)

21.44 0.71 0.09 1.11 3.93 0.88 1.42 2.11 0.40 6.07 5.90 119.48 1.29 5.04 5.75 4.79 53.77

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.54 0.01 0.02 0.31 0.62 0.23 0.29 0.40 0.11 3.52 2.01 25.47 0.50 0.82 1.27 0.94 6.75

Pre-treatment 21.72 0.79 0.17 1.68 4.12 1.27 1.32 2.23 0.57 9.23 5.95 125.18 3.43 15.43 10.75 6.40 29.53

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.24 0.01 0.02 0.37 0.53 0.30 0.17 0.50 0.14 1.51 0.84 29.85 0.20 1.52 1.05 1.28 9.97

Post-treatment 22.09 0.79 0.12 1.74 4.32 1.57 1.37 2.24 0.71 4.42 3.04 127.59 2.06 9.46 6.53 6.30 148.75

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.06 0.01 0.02 0.35 0.46 0.37 0.11 0.43 0.17 1.13 0.52 28.23 0.30 1.18 1.19 1.31 38.63

Controls vs. pre-treatment

Pre- vs. post-treatment

NS <0.001 <0.001 <0.001 NS <0.001 NS NS 0.001 NS NS NS <0.001 <0.001 <0.001 <0.001 <0.001

NS NS 0.006 0.002 NS <0.001 0.045 NS <0.001 0.001 <0.001 NS <0.001 <0.001 <0.001 NS <0.001

WHR, waist:hip ratio; HOMA-IR, homeostasis model assessment for insulin resistance; ADMA, asymmetric dimethylarginine; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol; DHEA-S, dehydroepiandrosterone sulphate; NS, non-significant.

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HOMA-IR correlated positively with WHR (ρ = 0.506, P = 0.001). Stepwise linear regression analysis in patients with PCOS revealed that changes in FT (β = 0.367, t = 5.343, P < 0.001), VLDL (β = 0.144, t = 2.420, P = 0.018), TG (β = 0.185, t = 2.437, P = 0.019) and WHR (β = 0.579, t = 4.472, P < 0.001) levels were independently associated with changes in ADMA (adjusted R2 = 0.67, F = 21.636, P < 0.001).

4. Discussion The present study demonstrated that circulating ADMA levels were higher in young normal-weight women with PCOS than healthy controls and both metformin and oral contraceptive treatments significantly reduced circulating ADMA concentrations after 3 months of therapy. Similar to our results, in a more recent report, Charitidou et al. [38] demonstrated that ADMA levels were significantly decreased after treatment with natural or synthetic estrogens, combined with antiandrogens in women with PCOS. In the one hand, estrogens have been shown to have, among other cardioprotective effects such as vasodilatation, inhibition of smooth muscle cell proliferation and decrease in vascular endothelial permeability, antioxidant properties [42]. H¨ockerstedt et al. [43] speculated that E2 administration to women with metabolic syndrome could result in more effective E2 esterification and antioxidative protection of HDL particles compared to normotriglyceridemic women. In this way, to act as antioxidants in HDL, estrogens need first to be converted to lipophilic fatty acid derivatives, estrogen esters, in a reaction catalyzed by lecithin/cholesterol acyltransferase (LCAT) [44]. Importantly, these fatty acid ester derivatives of E2 are preferential structural forms for association with HDL since unesterified estradiol displays only weak association with HDL [44]. After esterification, estrogens are able to incorporate in HDL, after which they can be transferred to LDL in a process that is at least partly mediated by cholesterol ester transfer protein [45]. Consequently, estrogens incorporated in the lipoprotein particles provide antioxidant protection in the arterial intima [46]. Thus, estrogen esters contained in HDL could enter the arterial subendothelial compartment [46] and could modulate several mechanisms crucial in the development of atherosclerosis. On the other hand, several recent studies have also demonstrated that HDL can stimulate production of NO, which may contribute to the positive cardiovascular effects of HDL [47]. In this context, it has been recently suggested that HDL binds to its receptor, a class B, type I scavenger protein called SR-BI [48], which is also expressed in vascular endothelial cells [49] and delivers estrogen to eNOS, thereby stimulating the enzyme [50], with the resultant generation of NO. However, the authors [50] did not determine esterified E2, and it is not clear how the E2 activated eNOS [46]. It is also not clear the effects of both EE/CPA and metformin on these mechanisms in

our study, since we did not measure esterified E2 like Gong and co-workers [50] did not. However, all of these proposed mechanisms may indicate a possible relationship between estrogens and ADMA metabolism. In this way, direct effects of estrogens on endothelial function and vascular reactivity because of enhanced production or activity of several vasoactive compounds including NO has been previously reported [51]. Estradiol stimulates the expression of both endothelial NO synthase (eNOS) and inducible NOS (iNOS) in vascular cells [51] and stimulates NO-dependent vasodilatation in vivo [52]. There is also evidence that estrogen stimulates eNOS activity directly via the activation of membrane-associated steroid hormone receptors [53]. Furthermore, Holden et al. [54] demonstrated that estrogens can alter the catabolism and release of ADMA in vitro and reduce the circulating concentration in vivo. The authors proposed that increased dimethylarginine dimethylaminohydrolase activity and the subsequent fall in ADMA could contribute to the positive effect of estrogens on NO synthesis [54]. Although studies showing a link between endothelial dysfunction and insulin resistance have been reported in non-obese women with PCOS [33,34], we did not observe any relation between circulating ADMA concentrations and HOMA-IR. Moreover, HOMA-IR not only increased after EE/CPA treatment but also decreased metformin therapy, but ADMA levels decreased significantly after both treatments. Similarly, in a more recent study, ADMA levels were not correlated with HOMA-IR in patients with PCOS [39]. Consistent with a previous report [55], ADMA levels were positively correlated with triglyceride and VLDL-C levels, but not with LDL-C levels. However, Demirel et al. [39] reported that ADMA levels were not correlated with lipid levels in adolescent girls with PCOS. The difference between the present and previous results may derive from differences in the selection of the subjects. Interestingly, in the present study, circulating ADMA concentrations were also positively associated with testosterone levels. On the other hand, the significant reductions of testosterone, free testosterone, androstendione and DHEAS, together with ADMA levels, were also observed after 3 months of treatment by EE/CPA pill. There is little or indirect evidence between ADMA and testosterone. Endothelial dysfunction has also been associated with increased androgen levels in obese women with PCOS [30]. In male patients with idiopathic hypogonadotropic hypogonadism, Cakir et al. [56] reported that the acute testosterone treatment decreased ADMA levels. This is the first study demonstrating the decreased serum ADMA concentrations after metformin therapy in patients with PCOS. Our findings are in line previous studies that reported beneficial effect of metformin on endothelial structure and function [31,32]. Moreover, metformin has been shown to decrease circulating ADMA, both as monotherapy and as add-on therapy to sulphonylurea in poorly controlled type 2 diabetic patients [57]. In the present study, it is unclear if metformin reduced ADMA secondary to improving

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glycemic control or through the its action on adipocyteshormonal axis because the molecular site of metformin action was not evaluated in detail. Experimentally, a cross-talk mechanism between metformin and the NO/NOS system has been described during restoration of microvascular reactivity in diabetic rats [58], in bovine aortic cells [59] and hepatic cells [60]. Elia et al. [61] suggested that metformin regulates the ovarian NO/NOS system during the hyperandrogenized condition. A direct metformin effect in vascular endothelial cells is also possible, and supported by previous findings in rats [62,63]. Mather et al. [64] reported that 3 months of treatment with metformin improved endotheliumdependent vascular responses in patients with IR and type 2 DM, whereas vascular responses of patients treated with placebo remained unchanged. What might be a possible explanation for the lowering effect of metformin on circulating ADMA concentrations? First, one of the physical stigmata of PCOS is a deficit of lean mass and an excess of fat, in particular, abdominal fat, even in the absence of obesity [65]. It has been previously demonstrated that ADMA plasma levels are significantly elevated in morbidly obese subjects and can be reduced by massive weight loss [66]. In this way, metformin exert the potential beneficial action of reducing body weight, perhaps as a result of decreased appetite and reduced caloric intake [67–71]. Second, in the presence of hyperlipidemia, levels of circulating ADMA could be elevated as shown in some studies [27]. Lundman et al. [72] suggested that mild-to-moderate hypertriglyceridemia in young men is associated with endothelial dysfunction and increased plasma concentrations of asymmetric dimethylarginine. In this regard, it has been suggested that metformin improves the dyslipidemia which is commonly associated with PCOS [73–75]. Metformin has been suggested to reduce lipid uptake or synthesis in the intestine and in the hepatocytes [76]. The improvement of obesity and especially abdominal obesity with a subsequent decreased release of FFAs from adipose tissue observed during met-

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formin therapy [71,77] could also be another explanation of the improvement of lipid profile during metformin treatment. In addition, a drop in TC levels after metformin therapy could be due to the decreased androgen levels, as hyperandrogenism has been shown to affect lipoproteins and lipids independently of insulin levels and body weight [78]. Third, it has been suggested that metformin could result in subtle improvements in both hepatic insulin extraction and insulin sensitivity in PCOS subjects, which together could lead to a significant decrease in hyperinsulinemia [67,69,71,75,79,80]. Abbasi et al. [81] reported that plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus. However, the observed effect of metformin on circulating ADMA cannot be attributed solely to the changes of insulin sensitivity in our study. Similarly, Kryzanowska et al. did not find a relationship between parameters of glucose metabolism and ADMA [67]. Finally, consistent with previous reports [67,69,75,80,82], hyperandrogenemia improved after metformin therapy. The mechanism by which hyperandrogenemia might affect vascular reactivity is still unknown. In experimental models, testosterone influences vasocontractile responses, and impairs endothelium-dependent relaxation in hypercholesterolemic rabbits and monkeys [83,84]. Moreover, androgens may act synergistically with insulin resistance [80], inflammatory cytokines on endothelial function [85]. Eventually, while all these characteristics are clearly inter-related, the identification of a cause–effect relationship is exceedingly difficult (Fig. 1). Determining whether or not decreased oxidant status and/or inflammation after metformin contributing of the treatment for observed improvement in circulating ADMA concentrations was not one of the aims of the current study. However, it is necessary to highlight the beneficial effect of metformin on oxidative stress and low-grade inflammation present in PCOS [86,87]. In addition, Elia et al. [61] reported diminished ovarian NOS activity in hyperandro-

Fig. 1. Outline of the possible effects of metformin on circulating ADMA levels (PCOS, polycystic ovary syndrome; ADMA, asymmetric dimethylarginine; DDAH, dimethylarginine-dimethylaminohydrolase; NO, nitric oxide; NOS, nitric oxide synthase).

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genized mice and considering that hyperandrogenization enhanced oxidative stress. In this context, oxidative stress in the vasculature should always stimulate ADMA production and/or inhibit ADMA degradation, in concentrations that significantly inhibit eNOS activity [14] or even uncouple the enzyme which would further increase superoxide production in a positive feedback fashion [88]. Metformin is effective in improving antioxidant defences [89], antioxidant activities in red blood cells from high fructose-fed rats [90], hepatic antioxidant levels in rats [91] and in decreasing xanthine oxidase activity in type 2 diabetic patients [92]. On the other hand, young and non-obese PCOS patients were found to be in a low-grade, chronic inflammation state [93]. Morin-Papunen et al. [94] suggested that metformin treatment was associated with a significant decrease of serum CRP levels, possibly indicating a decrease of the degree of low-grade inflammation. Krzyzanowska et al. [66] proposed that enhanced inflammation might be associated with elevated ADMA levels. As metformin has an antiinflammatory effect, it seems to have some favorable effects on circulating ADMA concentrations. It has been previously demonstrated that ADMA plasma levels are significantly elevated in subjects with hypertension [95]. As our subjects are normotensive, it is currently unclear whether the hypotensive effect of metformin may contribute to the decreased circulating ADMA concentrations in the present study. Consequently, these explanations are obviously speculative and requires experimental validation (Fig. 1). What is the importance of increased circulating ADMA in young women with PCOS? Although there is no threshold for ADMA leading to endothelial impairment established so far, it has been speculated that increased ADMA concentrations may cause endothelial hyporeactivity [15]. Valkonen et al. [15] showed that even slightly increased ADMA concentrations were associated with a strongly elevated risk for acute coronary events. Therefore, in PCOS, it is possible that increased concentrations of ADMA could contribute to the development of endothelial dysfunction in younger age. As a limitation to the study, we did not measure esterified E2 or dehydroepiandrosterone. The lack of assessment of endothelial function by dynamic testing and its correlation with circulating ADMA levels are other limitations of our study. Whether the observed decrease in circulating ADMA levels after the treatments was attributable to improved oxidative stress or whether it was the cause of decreased inflammatory markers cannot be solved in this study. These factors have considerable influences on circulating ADMA concentrations and are potentially affected by metformin. Another limitation of our study is the small sample size, and further studies with a larger study population are needed to demonstrate these findings. In conclusion, our data indicate that circulating ADMA concentrations in non-obese, non-hypertensive and young women with PCOS are significantly higher than age- and BMI-matched healthy controls and they improved by a 3month course of metformin and oral contraceptive treatments.

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