N-Acetyl-cysteine treatment improves insulin sensitivity in women with polycystic ovary syndrome

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1 FERTILITY AND STERILITY VOL. 77, NO. 6, JUNE 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. N-Acetyl-cysteine treatment improves insulin sensitivity in women with polycystic ovary syndrome Anna Maria Fulghesu, M.D., a Mario Ciampelli, M.D., a Giuseppe Muzj, M.D., a Chiara Belosi, M.D., a Luigi Selvaggi, M.D., a Gian Franco Ayala, M.D., b and Antonio Lanzone, M.D. b Università Cattolica del Sacro Cuore, Rome, Italy Objective: To evaluate the effect of N-acetyl-cysteine (NAC) on insulin secretion and peripheral insulin resistance in subjects with polycystic ovary syndrome (PCOS). Design: Prospective data analysis. Setting: Volunteer women in an academic research environment. Patient(s): Six lean and 31 obese subjects, aged years. Intervention(s): Patients were treated for 5 6 weeks with NAC at a dose of 1.8 g/day orally. A dose of 3 g/day was arbitrarily chosen for massively obese subjects. Six of 31 obese patients with PCOS were treated with placebo and served as controls. Main Outcome Measure(s): Before and after the treatment period, the hormonal and lipid blood profile and insulin sensitivity, assessed by an hyperinsulinemic euglycemic clamp, were evaluated and an oral glucose tolerance test (OGTT) was performed. Result(s): Fasting glucose, fasting insulin, and glucose area under curve (AUC) were unchanged after treatment. Insulin AUC after OGTT was significantly reduced, and the peripheral insulin sensitivity increased after NAC administration, whereas the hepatic insulin extraction was unaffected. The NAC treatment induced a significant fall in T levels and in free androgen index values (P.05). In analyzing patients according to their insulinemic response to OGTT, normoinsulinemic subjects and placebo-treated patients did not show any modification of the above parameters, whereas a significant improvement was observed in hyperinsulinemic subjects. Conclusion(s): NAC may be a new treatment for the improvement of insulin circulating levels and insulin sensitivity in hyperinsulinemic patients with polycystic ovary syndrome. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: Polycystic ovary syndrome, insulin, insulin resistance, N-acetyl-cysteine, obesity Received July 11, 2001; revised and accepted February 26, Reprint requests: Antonio Lanzone, M.D., Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore, Rome, Italy (FAX: ; a Department of Obstetrics and Gynecology. b OASI Institute for Research, Troina (Enna), Italy /02/$22.00 PII S (02) Polycystic ovary syndrome (PCOS) is a common disease that affects up to 10% of women of reproductive age, in which hyperandrogenism, enlarged cystic ovaries, and chronic anovulation often coexist with obesity, hyperinsulinemia, and insulin resistance (1). Furthermore, recent data provide evidence that the metabolic disturbances associated with the syndrome may have important consequences for long-term health, such as an increased risk of developing cardiovascular disease and diabetes (2). Obesity in women with PCOS is rather high, ranging from 30% to 60% (3), whereas hyperinsulinemia is present in more than 50% of patients with PCOS (4). Furthermore, although about 70% of obese women with PCOS exhibit an exaggerated insulin secretion, this feature is also present in 20% 40% of lean PCOS subjects. Recently, some papers have been published concerning the activity of N-acetyl-cysteine (NAC) on insulin secretion in pancreatic -cells, as well as on the regulation of the insulin receptor in human erythrocytes (5). NAC is commonly used as a safe mucolytic drug, but at higher doses it increases the cellular levels of reduced glutathione (GSH), an antioxidant (6), which has been shown to influence insulin receptor activity in vivo. 1128

2 In vitro (7) and in vivo (8) data show that NAC is able to improve insulin secretion in response to glucose. In humans, oral NAC administration was also proposed for the prevention of endothelial damage due to oxidant agents in non insulin-dependent adult diabetic subjects (9). The above reported data seem to suggest a possible role of NAC in ameliorating the insulin resistance associated with PCOS. The present study was performed to evaluate the effects of long-term NAC administration on insulin secretion, metabolism, and peripheral insulin sensitivity in patients with PCOS. MATERIALS AND METHODS Subjects We studied 37 women affected by PCOS, aged years. All women were in good health and had good thyroid function and a normal glomerular filtration rate, as demonstrated by normal creatinine clearance levels. All patients had spontaneous onset of puberty and normal sexual development, and all had been affected by oligomenorrhea with chronic anovulation since puberty. As described elsewhere (10), PCOS was diagnosed by a finding of bilaterally normal or enlarged ovaries (maximum ovarian volume 12 cm 3 ) with the presence of at least 7 10 microcysts per ovary ( 5 mm in diameter) at the time of ultrasound and the presence of the following clinical and endocrine factors: amenorrhea or oligomenorrhea, hirsutism, and plasma androgen levels at the upper limit or above the normal range (androstenedione, ng/ml; T, ng/ml). The presence of impaired glucose tolerance or diabetes was considered an exclusion criterion. No patient showed hyperprolactinemia (normal range of prolactin ng/ml) or clinical evidence of hypercorticism or thyroid dysfunction. Late-onset congenital hyperplasia was excluded by an adrenocortocotropic hormone stimulation test (11). No patient had taken any medication known to affect carbohydrate metabolism for at least 3 months before the study. The body mass index (BMI) was calculated according to the following formula: body weight in kilograms/height in meters squared. A patient with a BMI 25 kg/m 2 was considered obese. Informed consent was obtained from each patient before the entry into the study. The protocol was previously approved by our Institutional Review Board. Clinical Protocol Patients were hospitalized at day 7 8 after spontaneous menstrual bleeding or randomly during amenorrhea. The presence of a dominant follicle, recent ovulation, or luteal phase was excluded by ultrasound and serum P evaluations at the time of the entry in the study. After 3 days of a standard 300-g carbohydrate diet (prescribed before hospitalization) and after a 12-hour overnight fast, baseline lipid, hormone, and sex hormone binding globulin (SHBG) plasma determinations were performed; patients then underwent a 75-g oral glucose tolerance test (OGTT). Insulin sensitivity was assessed by the use of the hyperinsulinemic euglycemic clamp technique (8), which was performed the day after the OGTT. As described elsewhere (10), a retrograde IV catheter was inserted for blood sampling and kept in a warming device at 60 C to arterialize the venous blood samples. Another indwelling catheter was inserted in the contralateral forearm vein (Cavafix; B Braun, Melsungen, Germany) for the glucose and insulin infusions. A two-step primed constant infusion of human insulin (Actrapid HM; Novo Nordisk, Denmark) was administered at a rate of 40 miu m 2 /min. Having reached insulin circulatory concentrations of about 100 IU/mL within 10 minutes, the steady state velocity of insulin infusion was fixed to maintain these levels during the clamp. Blood samples were taken every 5 minutes for 4 hours from the arterialized line, and blood glucose concentrations were immediately measured with a glucose analyzer. The exogenous glucose infusion was adjusted according to a standard algorithm to maintain the blood glucose concentration between 80 and 88 mg/dl. Patients left the hospital 3 days after hospitalization and had 5 6 weeks of treatment by NAC (Fluimucil; Zambon, Italy) 1.8 g/day orally and 3 g/day for massively obese subjects (BMI 30 kg/m 2 ). This dose was chosen for its effectiveness in increasing synthesis of GSH as recommended for urotoxicity prophylaxis under cyclophosphamide treatment (6). In the present study, we arbitrarily chose a NAC dose of 1.8 g/day for all patients. We observed that obese patients with PCOS with a BMI 30 kg/m 2 did not respond to the treatment (six patients). To test the hypothesis that the NAC effective dose is weight dependent, we increased the dose to 3 g/day in these patients; this dose was effective in ameliorating insulin sensitivity and decreasing insulin levels. Since we were not able to specify if the metabolic improvements found in these patients were due to the increased NAC dose alone or also to time-dependent effects (10 instead 5 weeks of treatment), we decided to exclude these subjects from the study. Six obese BMI-matched patients were selected without randomization for treatment with placebo. Patients were instructed not to modify their usual diet during the study. The presence of an ovulatory cycle was monitored during therapy by weekly assays of P serum levels. Anovulatory patients were rehospitalized irrespective of the cycle phase, whereas ovulatory subjects were rehospitalized in the early follicular phase of the subsequent menstrual cycle. After the treatment with NAC, patients repeated the OGTT, the clamp, the lipid blood profile, and the hormonal assay within 3 days. FERTILITY & STERILITY 1129

3 Samples for hormone assay were promptly centrifuged, and plasma was stored at 20 C until assayed; samples for biochemical assay were assayed immediately. Data Analysis Plasma baseline concentrations of gonadotropins, E 2, SHBG, androstenedione, T, 17-hydroxyprogesterone, and dehydroepiandrosterone sulfate were measured before and after NAC administration. Total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, very low-density lipoprotein (VLDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and non-esterified fatty acid levels were also determined during both hospitalizations. The free androgen index (FAI), which measures the biologically active amount of androgens, was calculated according to the formula T)([ ] log 10 SHBG)] as proposed by Rajokhowa et al. (12). Insulin, C-peptide, and glucose serum concentrations were analyzed in all samples after oral glucose load. A normal glycemic response to OGTT was defined according to the criteria of the American Diabetes Association (13). Insulin, C-peptide, and glucose responses to glucose load were expressed as area under the curve (AUC), which was calculated by the trapezoidal rule. The insulinemic response to the OGTT test was considered normal when the insulin levels were under 100 IU/mL at 30 and 60 minutes and the AUC was lower than 15,000 IU/mL 240 minutes, as established by the standard procedures of our laboratory (14). During the clamp study, total body glucose utilization (M) was determined between 120 and 240 minutes and expressed as mg/kg/min. This index represents the ability of the subject to metabolize the administered glucose at predetermined insulin plasma concentrations, that is, the insulin-mediated glucose disposal. We preferred this index for the measure of the insulin sensitivity because the M/I ratio fails to narrow the range of the individual sensitivity values (15). Hepatic insulin extraction was estimated from the difference between the incremental areas of C-peptide and insulin divided by the incremental area of C-peptide, as proposed by other investigators (10, 12). This calculation was based on the concept that insulin and C-peptide are secreted in equimolar concentrations by the pancreatic -cells; furthermore, in contrast to insulin, very small amounts of C-peptide are metabolized by the liver, rather, the kidney is the major site of degradation. Therefore, the ratio between insulin and C-peptide permits the analysis of hepatic insulin removal (16). The incremental area was calculated by the difference between AUC and basal AUC (basal AUC area of the curve due to unstimulated secretion and calculated assuming a constant value during a 4-hour period). Assays Glucose serum levels were determined by the glucose oxidase technique. Plasma samples for hormone determinations were maintained at 20 C until assayed. All hormones were measured by radioimmunoassay methods using commercial kits. For LH and FSH assays, a monoclonal doubleantibody technique was used. Steroids were assayed by the dextran-charcoal technique. Intra-assay and interassay coefficients of variation were below 8% and 15%, respectively, for all hormones. Total cholesterol and triglycerides concentrations were determined by an enzymatic assay (Bristol, Paris, France); HDL levels were determined after precipitation of chylomicrons, VLDL and LDL (Boehringher, Mannheim, Germany). VLDL was separated as supernatant from LDL and HDL by lipoprotein centrifugation. A magnesium chloride/phosphotungstic acid technique was used to precipitate LDL from the bottom fraction after ultracentrifugation. NEFAs were determined by an acylcoenzime A-oxidase-based colorimetric method. Statistical Analysis Data are presented as mean SD. Data were stored and analyzed using the Statistical Package for Social Sciences, TABLE 1 Endocrine and metabolic features in all studied subjects with PCOS before and after the NAC and placebo treatment. Pre-NAC Post-NAC FSH (IU/L) LH (IU/L) Prolactin ( g/l) E 2 (pmol/l) Androstenedione (nmol/l) T (nmol/l) a 17 OH-progesterone (nmol/l) DHEAS ( mol/l) SHBG (nmol/l) a FAI a Fasting glucose (mmol/l) Glucose AUC (mmol/l 240 ) 1, , Fasting insulin (pmol/l) AUC insulin (pmol/l 24 ) 114,664 52,177 97,078 55,771 a AUC C-peptide (pmol/l 240 ) 480, , , ,686 M (mg/kg/min) a Cholesterol (mg/dl) b Triglycerides (mg/dl) a HDL (mg/dl) LDL (mg/dl) c VLDL (mg/dl) NEFA (mg/dl) Note: Data are expressed as mean SD. a P.05. b P.001. c P Fulghesu et al. NAC and insulin in polycystic ovary syndrome Vol. 77, No. 6, June 2002

4 FIGURE 1 Metabolic parameters in the normoinsulinemic women with PCOS (14 patients) before and after NAC treatment. Data are expressed as mean SD. A, AUC of insulin expressed as IU/mL 240 minutes. B, AUC of C-peptide expressed as ng/ml 240 minutes. C, Total body glucose utilization (M) expressed as mg/kg/min. *P.05 Before (pre) vs. after (post) NAC treatment. version 5.0, on an IBM-compatible computer. Abnormally distributed variables were logarithmically transformed. Student s two-tailed t-test for paired data was used to compare the effects of NAC or placebo within each group of subjects. Comparisons between the groups were made by one-way analysis of variance, and any significant difference was identified by using the Bonferroni correction for multiple comparisons. P.05 was considered statistically significant. RESULTS Clinical Results The PCOS study group consisted of six lean and 31 obese subjects. No patient showed evidence of acanthosis nigricans. The BMI in the obese patients ranged from 25 to 45; 20 patients were massively obese (BMI 30 kg/m 2 ). The BMI after treatment did not show statistically significant changes in the entire study group ( vs ; P NS) or in the separate analysis of the three study groups (hyperinsulinemic pre-nac kg/m 2 vs. hyperinsulinemic post-nac kg/m 2, P NS; hyperinsulinemic pre-placebo kg/m 2 vs. hyperinsulinemic post-placebo kg/m 2, P NS; normoinsulinemic pre-nca kg/m 2 vs. normoinsulinemic post-nca kg/m 2, P NS). Three patients ovulated during NAC treatment. No differences in the glomerular filtration rate were observed in relation to treatment (creatinine clearance: pre- NAC vs. post-nac ml/minute). No side effects were observed during treatment. Metabolic and Hormonal Results Based on the insulinemic response to OGTT of the NACtreated patients (six lean and 25 obese women with PCOS), 14 patients were classified as normoinsulinemic (four lean and 10 obese), and 17 patients as hyperinsulinemic (two lean and 15 obese). All the placebo-treated patients with PCOS were obese and hyperinsulinemic. Table 1 shows the endocrine and metabolic parameters at baseline and at the end of the NAC treatment. After therapy, a significant decrease in total circulating T levels (P.05) and FAI (P.05), as well as a significant increase in SHBG levels (P.05), was found. Fasting glucose, insulin, and glucose AUC were similar before and after treatment. The insulin-auc after glucose load was significantly reduced, and the peripheral insulin sensitivity was increased after NAC administration, while the C-peptide AUC was not significantly modified. Total cholesterol (P.001), plasma triglycerides (P.05), and LDL (P.005) levels significantly decreased after NAC treatment. In a separate analysis of the patients divided into hyper- and normoinsulinemic subjects, these results were confirmed only in the hyperinsulinemic subgroup, whereas no significant changes were found in normoinsulinemic subjects or in the placebo group. Figures 1, 2, and 3 show the metabolic changes in the studied patients when divided into normo- and hyperinsulinemic groups; data were also compared with the hyperinsulinemic placebo-treated group. In the normoinsulinemic group (Fig. 1) as well as in the placebo group (Fig. 2), no change occurred in circulating levels of insulin, pancreatic C-peptide secretion, and insulin sensitivity, whereas a significant improvement for all these parameters was found in the hyperinsulinemic NAC-treated subjects (Fig 3). FERTILITY & STERILITY 1131

5 FIGURE 2 Metabolic parameters in the hyperinsulinemic women with PCOS (17 patients) before and after NAC treatment. Data are expressed as mean SD. A, AUC of insulin expressed as IU/mL 240 minutes. B, AUC of C-peptide expressed as ng/ml 240 minutes. C, Total body glucose utilization (M) expressed as mg/kg/min. *P.05 Before (pre) vs. after (post) NAC treatment. Furthermore, the insulin hepatic extraction was unchanged after NAC treatment both in the entire group (pre- NAC vs. post-nac , P NS) and in the separate analysis of hyper- and normoinsulinemic subjects (normoinsulinemic: pre-nac vs. post- NAC , P NS; hyperinsulinemic: pre-nac vs. post-nac , P NS). Table 2 shows some NAC effects on hormonal values in the two treated groups and in the placebo group. In the hyperinsulinemic group, the circulating T levels and FAI significantly decreased, while SHBG significantly increased, after NAC administration. No significant modifications were found in the normoinsulinemic group or in the placebotreated patients. DISCUSSION Insulin resistance and hyperinsulinemia are now recognized as common features in women affected by PCOS (17, 18); interestingly, almost all obese subjects, but also several lean patients with PCOS (19 21), present these features. Chronic hyperinsulinemia may be a risk factor for several FIGURE 3 Metabolic parameters in the hyperinsulinemic women with PCOS (six patients) before and after placebo treatment. Data are expressed as mean SD. A, AUC of insulin expressed as IU/mL 240 minutes. B, AUC of C-peptide expressed as ng/ml 240 minutes. C, Total body glucose utilization (M) expressed as mg/kg/min Fulghesu et al. NAC and insulin in polycystic ovary syndrome Vol. 77, No. 6, June 2002

6 TABLE 2 Hormonal values before and after the NAC treatment in the normo- and hyperinsulinemic groups or the placebo treatment in hyperinsulinemic women. Normoinsulinemic (n 14) NAC-hyperinsulinemic (n 17) Placebo-hyperinsulinemic (n 6) Pre-NAC Post-NAC Pre-NAC Post-NAC Pre-NAC Post-NAC E 2 (pg/ml) H-P (ng/ml) A (ng/ml) T (ng/ml) a DHEAS (ng/ml) 1, , , ,025 1,020 1, , SHBG (nmol/l) a FSH (IU/L) LH (IU/L) FAI a Note: Data are expressed as mean SD. a P.05. clinical pathologies such as gestational and non insulindependent diabetes, hypertension, thrombosis, and cardiovascular disease (1, 22). Lipid alterations induced by hyperinsulinemia (23, 24) may also promote atherosclerosis with arterial stiffening and narrowing. Insulin excess may also exert an endocrine impact on the ovary; in vitro, insulin has been shown to directly stimulate androgen secretion and to enhance LH-mediated responses in isolated thecal tissue to a greater extent than in normal ovaries (25). Furthermore, in vivo data seem to confirm that insulin might influence ovarian as well as adrenal steroidogenesis (26, 27). On the basis of the above considerations, it seems very important to test the efficacy of insulin-lowering drugs in patients with PCOS (1); thus, we wanted to analyze the potential insulin-sensitizing properties of NAC in patients with PCOS. To our knowledge, this is the first study focusing on this specific end point. NAC is recognized by the Department of Health as a medicine that fluidizes bronchial mucous secretions. In vivo and in vitro studies have demonstrated that NAC represents a precursor in GSH biosynthesis and is deacetylated into cystine (6, 28). In a recent paper, it was demonstrated that oral treatment with the antioxidant NAC in non insulin-dependent diabetic patients (1.2 g for 1 month) significantly reduced intraerythrocyte GSH disulfide (GSSG) concentrations and increased reduced GSH levels and the GSH:GSSG ratio (29) and was able to prevent endothelial damage after oxidative stress in these patients. Human erythrocytes have also been shown to possess insulin receptors, which are internalized in response to the binding of insulin (30, 31); it has been demonstrated that NAC administration is able to protect these receptors from oxidant agents. In an in vivo human study about the specific effects of NAC on insulin metabolism, healthy volunteers were submitted to a NAC infusion during a hyperglycemic clamp (8); the study showed an increase of glucose consumption under NAC administration, that is, an improvement of insulin sensitivity. In our study population, NAC administration was well tolerated by all the patients and no adverse effect was observed. The results of our study are encouraging; indeed, we obtained a significant (paired comparison) reduction of both circulating insulin levels and peripheral insulin resistance, as well as a significant reduction of T levels and FAI values in the whole study group. Furthermore, when data were analyzed in relation to pretreatment insulin response to glucose load, hyperinsulinemic subjects showed a significant increase in insulin sensitivity, as well as a concomitant reduction in circulating insulin levels and secretion after OGTT. In normoinsulinemic subjects, we did not observe any change in C-peptide and insulin levels or in peripheral insulin sensitivity after NAC treatment, which suggests that the treatment is effective only in those patients who were compromised from a metabolic point of view. The lack of any modifications in the obese hyperinsulinemic placebo-treated patients further confirmed the effectiveness of NAC administration. It could be argued that the magnitude of the observed changes is not quite significant from a clinical point of view, especially when compared with previously reported data about the use of metformin or troglitazone (32 35). However, it should be considered that our trial was 5 6 weeks FERTILITY & STERILITY 1133

7 long, whereas the above reported data are the result of weeks of treatment. It could be hypothesized that a longer NAC treatment would lead to superimposable results. Furthermore, it should also be kept in mind that NAC administration caused no side effects in our study population. The NAC effect in the hyperinsulinemic group is probably due to an improvement in insulin receptor activity leading to a secondary decrease in the -cell responsiveness to OGTT. Any change in the hepatic insulin clearance was found after NAC treatment, thus arguing against the possibility of a drug action on the liver insulin metabolism. The decrease in circulating insulin levels was followed by a significant reduction in T levels and FAI in patients responding to treatment. No changes in these levels were observed when insulin levels were unmodified by NAC. We assume, therefore, that the improvement in insulin resistance contributes to the decrease of androgen levels. Many studies have reported about the presence of higher serum levels of triglycerides, VLDL, and LDL and lower levels of HDL in patients with PCOS (1). In evaluating lean and obese PCOS subjects compared with weight-matched women with normal ovaries, it has been demonstrated that dyslipidemia in PCOS is associated with insulin resistance (36). In accordance with other recent studies about the effects of insulin-sensitizing agents on lipidic patterns, we found a significant decrease of plasma lipids, particularly cholesterol, triglycerides, and LDL (17, 37), after NAC-mediated insulin reduction, which suggests a possible additional benefit of this drug on long-term health in patients with PCOS. In conclusion, the preliminary results on the effectiveness of the antioxidant drug NAC suggest that it may be a new treatment for hyperinsulinemia in patients with PCOS, which is of note also because of the absence of side effects. 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