Pioglitazone reduces the adrenal androgen response to corticotropin-releasing factor without changes in ACTH release in hyperinsulinemic women with polycystic ovary syndrome

Pioglitazone reduces the adrenal androgen response to corticotropin-releasing factor without changes in ACTH release in hyperinsulinemic women with polycystic ovary syndrome Daniela Romualdi, M.D.,a Maddalena Giuliani, M.D.,a Gaetano Draisci, M.D.,b Barbara Costantini, M.D.,a Francesca Cristello, M.D.,a Antonio Lanzone, M.D.,a,c and Maurizio Guido, M.D., Ph.D.a a Department of Obstetrics and Gynaecology and b Department of Anesthesiology and Intensive Care, Università Cattolica del Sacro Cuore, Rome, Italy; and c “Oasi” Institute for Research, Troina, Italy

Objective: The hypothalamic-pituitary-adrenal (HPA) axis seems to hyperfunction at both central and peripheral levels in polycystic ovary syndrome (PCOS). Hyperinsulinemia is involved in the adrenal hyper-responsiveness to ACTH. The present study was performed to investigate the role of insulin in the derangement of the hypothalamic-pituitary compartment of the HPA axis in PCOS. Design: Prospective clinical study. Setting: Academic research center. Patient(s): Fifteen hyperinsulinemic PCOS women. Intervention(s): Hormonal and lipid assays, oral glucose tolerance test, and corticotropin-releasing factor (1 ␮g/kg CRF) test before and after 4 months of treatment with the insulin sensitizer pioglitazone (30 mg/day). Main Outcome Measure(s): Glycemic and insulinemic response to glucose load; pituitary and adrenal response to CRF. Result(s): We observed a significant reduction in insulin secretion after therapy. Pioglitazone administration did not modify ACTH and cortisol response to CRF. A significant reduction in the adrenal CRF-induced secretion of androstenedione (A) (area under the curve [AUC] 202.76 ⫾ 78.68 ng/mL · 90 minutes to 147.05 ⫾ 52.06 ng/mL · 90 minutes) and 17OH-progesterone (AUC 152.92 ⫾ 59.56 ng/mL · 90 minutes to 117.10 ⫾ 63.25 ng/mL · 90 minutes=) occurred after treatment. A trace response to CRF was observed for DHEAS and testosterone both before and after pioglitazone. Conclusion(s): In PCOS subjects, insulin may enhance adrenal steroidogenesis by acting directly on the peripheral gland, with no significant effects on the pituitary response to CRF stimulation. (Fertil Steril威 2007;88: 131– 8. ©2007 by American Society for Reproductive Medicine.) Key Words: Adrenal, CRF, insulin, PCOS, pioglitazone

Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies in female subjects, affecting about 6%–15% of women of reproductive age. Hyperandrogenism, chronic anovulation, and infertility are the main hallmarks of this heterogeneous condition (1–2). The pathogenesis of the syndrome is still unclear, although insulin resistance and the concomitant hyperinsulinemia frequently found in these patients are believed to play a paramount role. Hyperinsulinemia contributes to the clinical and biochemical manifestations of the hyperandrogenism by affecting various levels of the hypothalamic-

Received July 31, 2006; revised and accepted November 17, 2006. Supported by a grant from the Ministry of Public Health “La prevenzione dell’handicap mentale” RC 2004 PREV 1– 4 IRCCS, Troina, and a grant from Ministero dell’Università e della Ricerca (MIUR) 2004. Reprint requests: Maurizio Guido, M.D., Ph.D., Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore, L.go Agostino Gemelli, 8-00168 Rome, Italy (FAX: ⫹39-063057794; E-mail: [email protected]).

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pituitary-ovarian axis as well as the hepatic production of SHBG (3). Although the ovary is considered the principal source of androgen excess in PCOS, several lines of evidence suggest that adrenal steroid production might take part in determining the androgenic milieu of the syndrome. Elevated adrenal androgens and a hyper-responsiveness of the gland to ACTH, in fact, were found in a percentage ranging from 25% to 70% of PCOS patients in different series (4 –5). We previously demonstrated that PCOS patients with high insulin circulating levels are those who exhibit the highest basal and ACTH-stimulated adrenal androgen levels when compared to both normoinsulinemic PCOS women and control subjects (6). Furthermore, it was reported that insulin infusion enhances 17OH-progesterone (P) and 17OH-pregnenolone secretion after ACTH in hyperandrogenic women (7), thus providing evidence for a direct stimulatory effect of insulin on the adrenal gland.

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Concordant with these findings, the pharmacologic lowering of insulin levels by insulin sensitizing agents was proven to decrease the adrenal androgen response to ACTH in studies by us and other investigators (8 –10). However, in PCOS, the hypothalamic-pituitary-adrenal (HPA) axis seems to be up-regulated also at the central level: a higher ACTH response to corticotropin-releasing hormone (CRH) was observed in PCOS compared with normo-ovulatory women (11), and this abnormality appeared to be correlated with the presence of obesity (12), a feature strictly linked to the severity of hyperinsulinism in such patients. This observation led to the hypothesis of an involvement of insulin in the dysregulation of the HPA axis also at the central level in PCOS women. Earlier reports in the literature regarding non-PCOS subjects further support this contention: insulin is able to cross the blood-brain barrier (13); ACTH levels are increased during high-insulin clamp studies (14); and there is a positive correlation between CRH-stimulated ACTH levels and homeostasis insulin resistance indexes (15). Therefore, to improve our understanding of this aspect, the present study was undertaken to investigate the effects of the treatment with the insulin sensitizer pioglitazone on the pituitary-adrenal response to CRH in a cohort of obese hyperinsulinemic PCOS women. MATERIALS AND METHODS We recruited 15 caucasian obese women (body mass index [BMI] ⬎27 kg/m2) with PCOS (age range 18 –33 years) attending our divisional outpatients’ services. All of the women had spontaneous onset of puberty and normal sexual development, and all had oligomenorrhea with chronic anovulation since puberty. None had thyroid diseases nor had taken medications known to affect plasma sex steroid levels for at least 3 months before entering the study. In accordance with the Rotterdam Consensus Criteria (16), polycystic ovary syndrome was diagnosed in the presence of at least two of the three following clinical findings: chronic anovulation, clinical and/or biochemical evidence of hyperandrogenism, and ultrasonographic appearance of polycystic ovaries. In particular, the ovarian morphology was defined as polycystic in the presence of bilateral normal or enlarged ovaries with at least 10 microcysts (2– 8 mm diameter) from the inner margin to the outer margin in longitudinal cross-section associated with an augmented stromal area/total area ratio (17). Pregnancy or possibility of pregnancy and nursing, significant liver (aspartate aminotransferase [AST], alanine aminotransferase [ALT], total bilirubin, or alkaline phosphatase ⬎2 times the upper limit of normal) or renal (serum creatinine ⬎159.12 ␮mol/L) impairment, neoplasm, cardiovascular disease, and unstable mental illness were considered exclusion criteria. 132

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Because of the impact of body fat distribution on androgen levels and glucose metabolism (18, 19), waist-to-hip ratios were measured. Waist circumference was obtained as the minimum value between the iliac crest and the lateral costal margin, and hip circumference was determined as the maximum value over the buttocks. All patients also underwent a transvaginal ultrasonography, and the grade of hirsutism was detected using the Ferriman-Gallwey score (⬍8: no hirsutism; 8 –16: low hirsutism; score value 17–24: moderate hirsutism; ⬎24: severe hirsutism) (20). Informed consent was obtained from each patient, and the study protocol was approved by our Institutional Review Board. Studies were conducted during the early follicular phase of spontaneous or induced (10 mg/day medroxyprogesterone acetate for 7 days) menstrual cycles (day 3–7). On the first visit, the patients were hospitalized and underwent a gynecologic and medical examination. After following a standard carbohydrate diet (300 g/day) for 3 days and fasting overnight for 10 –12 hours, blood samples were collected to perform the following laboratory investigations: basal hormone assessment, ALT, AST, alkaline phosphatase, cholesterol, lipoproteins, total protein, albumin, creatinine, nitrogen urea, Ca⫹⫹, Na⫹⫹, K⫹, partial thromboplastin time, and fibrinogen. Patients then underwent an oral glucose tolerance test (OGTT). The OGTT was performed as follows: at 9:00 AM after overnight fasting, an indwelling catheter was inserted into the antecubital vein of one arm. Blood samples were collected basally and, after ingestion of 75 g glucose in 150 mL water within 5 min, at 30, 60, 90, 120, 180, and 240 minutes. Insulin, glucose, and C-peptide were assayed in all samples. The following day, after another overnight fasting, a CRF test was performed as follows: At 7:00 AM, an indwelling catheter was inserted into the antecubital vein and saline solution was infused slowly throughout the test to keep the vein patent. Blood samples were collected just before (0) and 15, 30, 60, and 90 minutes after the injection of 1 ␮g/kg CRF (Clinalfa, Laufeifingen, Switzerland). Plasma cortisol (F), DHEAS, ACTH, A, T, and P were assayed. The first day of the following menstruation, therapy with pioglitazone (Actos, Takeda, Italy) was started: one pill of 30 mg daily in the morning for 4 months. During the study, chronically stabilized therapies not interfering with the parameters under evaluation were permitted. The use of antidiabetic and estroprogestinic drugs was not allowed. Patients were recommended not to modify their usual diet. Main parameters of liver function were monitored each month during the treatment.

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After the pioglitazone treatment, all patients had a second hospitalization during the early follicular phase of a spontaneous or induced (10 mg/day medroxyprogesterone acetate for seven days) menstrual cycle (day 3–7) and repeated the same protocol study. On each visit, the compliance with treatment was checked with a questionnaire about the side effects and a subjective evaluation of the tolerability of the administered drug; the patients were also asked about incidental missed administrations, but all reported to have correctly followed the scheduled treatment. A count of capsules returned was made on each control visit. Assays Plasma samples for glucose concentration were collected in tubes containing an inhibitor of glycolysis (sodium fluoride) to be analyzed within 5 hours. Plasma samples for insulin and C-peptide concentrations were placed in tubes standing in ice, centrifuged for 10 min at 1,000g using a 4226 ALC centrifuge (ALC, Milan, Italy) and remained frozen at ⫺30°C until assayed. Glucose plasma concentrations were determined by the glucose oxidase technique with a glucose analyzer (Beckam, Fullerton, CA). All hormone assays were performed with commercial radioimmunoassay kits (Radim, Rome, Italy). The intraassay and interassay coefficients of variation for all hormones were ⬍8% and ⬍15% respectively. For each determination, all samples from the same patient were assayed simultaneously. Total cholesterol and triglyceride concentrations were determined by an enzymatic assay (Bristol, Paris, France). High-density lipoprotein (HDL) concentrations were determined after precipitation of chylomicrons, very low-density lipoprotein (VLDL), and low-density lipoprotein (LDL) (Boehringer, Mannheim, Germany). The VLDL was separated (as the supernatant) from LDL and HDL by lipoprotein ultracentrifugations. A magnesium chloride/phosphotungstic acid technique was used to precipitate LDL from the bottom fraction after ultracentrifugation. Nonesterified free fatty acids (NEFA) were determined by an acyl– coenzyme A oxidase– based colorimetric method. All lipid assays were performed according to our standard laboratory procedures. Data Analysis All data are presented as mean ⫾ SD. The OGTT data were analyzed as area under the curve (AUC) after the glucose ingestion, calculated by the trapezoidal rule and expressed as pmol/L · 240 minutes for insulin and C-peptide and nmol/L · 240 minutes for the glucose plasma levels. The glycemic responses were defined in accordance with the criteria of the National Diabetes Data Group (21). A Fertility and Sterility姞

normal insulinemic response to OGTT was considered to be at a threshold AUC value of 15,000 UI/L · 240 minutes, as previously described (22). Descriptive statistics per visit and analysis of variance were used in the evaluation of the clinical features. The significance of differences among the visits’ measures was determined with the use of one-way analysis of variance, and any significant difference was identified by using the Bonferroni correction for multiple comparisons. For all analyses, P⬍.05 was considered to indicate statistical significance. The incidence rates of adverse events were evaluated and calculated by MEDDRA code (23). RESULTS All patients completed the study therapy without major protocol violation as far as inclusion and exclusion criteria were concerned. No adverse events were reported by the treated subjects, and no abnormalities were detected in hepatic or renal chemistries, in complete blood counts, or in electrocardiograms throughout the treatment. Table 1 shows the clinical and metabolic features of studied subjects before and after pioglitazone treatment. As expected, no differences were found in BMI and hirsutism score. Mean tryglicerides and total cholesterol levels were in the normal range at baseline and were not modified by pioglitazone treatment. Nevertheless, we observed a trend toward reduction in plasma LDL and VLDL concentrations and a significant (P⬍.01) rise in HDL levels. Similarly, serum NEFA decreased in response to pioglitazone treatment, although not significantly. Pioglitazone administration was able to significantly reduce insulin secretion as well as C-peptide levels in terms of AUC after OGTT (P⬍.01 and P⬍.05, respectively), with no changes being observed in the glycemic response to the glucose load. Table 2 shows the main hormonal parameters before and after pioglitazone treatment. A trend toward reduction was seen for plasma T levels, although it was not statistically significant. Sex hormone binding globulin plasma values were significantly higher (P⬍.05) than basal when evaluated after pioglitazone administration. The insulin-lowering treatment induced a significant decrease in the basal plasmatic concentrations of A and P (P⬍.05 for both). Gonadotropins, E2, and the remnant basal hormonal plasma levels were comparable before and after the study protocol. CRF Test Figure 1 shows ACTH and F response to CRF stimulus before and after pioglitazone administration. The ACTH secretion showed an increase of 118% as a peak 15 minutes after CRF injection and then declined, recovering values similar to baseline about 1 hour after the stimulus. No differences were found when ACTH response to CRF was 133

TABLE 1 Main changes induced by pioglitazone treatment in clinical and metabolic parameters in hyperinsulinemic PCOS women. Data are presented as mean ⴞ SD.

Age (ys) BMI (kg/m2) Score Total cholesterol (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) LDL (mg/dL) AUC insulin (␮UI/mL/240=) AUC glucose (mg/dL/240=) AUC C-peptide (ng/mL/240=)

Pre-treatment

Post-treatment

26.73 ⫾ 4.98 34.66 ⫾ 2.77 11.33 ⫾ 9.90 160.15 ⫾ 27.03 109.75 ⫾ 13.21 35.61 ⫾ 4.18 103.75 ⫾ 19.72 23,423.04 ⫾ 4,568.27 28,616.14 ⫾ 2,985.21 1,793.70 ⫾ 211.30

— 35.01 ⫾ 2.88 10.31 ⫾ 8.81 163.80 ⫾ 31.43 104.93 ⫾ 30.16 46.66 ⫾ 4.15b 99.00 ⫾ 31.90 12,540.05 ⫾ 3,058.77b 25,991.18 ⫾ 4,590.43 1,234.50 ⫾ 257.50a

Note: AUC ⫽ area under the curve after oral glucose load; BMI ⫽ body mass index; HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein; PCOS ⫽ polycystic ovary syndrome. a P⬍.05; b P⬍.01. Romualdi. HPA-axis in PCOS: effect of pioglitazone. Fertil Steril 2007.

evaluated after pioglitazone treatment in terms of plasma levels at each time point as well as in terms of AUC. Adrenal F secretion showed an increment of 53% as a peak 15 minutes after CRF stimulus, then plateaued and slowly returned to basal level after 90 minutes; pioglitazone administration did not modify adrenal F production after CRF injection when evaluated either as plasma levels or by AUC. Figure 2 shows the androgen response to CRF administration before and after pioglitazone treatment. Androstenedione plasma levels showed an increment 15 minutes

after CRF injection and returned to basal values after 60 minutes. After treatment, the maximum CRF-induced increase in A levels occurred at 30 minutes. Pioglitazone administration was able to markedly reduce the adrenal discharge of A after CRF stimulus, as evidenced by the significant difference (P⬍.01) in AUC values before and after treatment. Furthermore, we observed a statistically significant decrease in plasma A levels at each time point of the curve. The plasma 17-OHP response to CRF showed a peak in the plasma concentrations 15 minutes after CRF injection

TABLE 2 Hormonal changes induced by pioglitazone treatment in hyperinsulinemic PCOS women. Data are presented as mean ⴞ SD.

E2 (pg/mL) P (ng/mL) FSH (mUI/mL) LH (mUI/mL) SHBG (nmol/L) A (ng/mL) T (ng/mL) 17OH-P (ng/mL) DHEAS (ng/mL)

Pre-treatment

Post-treatment

48.24 ⫾ 6.99 0.68 ⫾ 0.28 5.01 ⫾ 1.35 8.15 ⫾ 2.29 16.31 ⫾ 6.64 1.78 ⫾ 0.83 0.71 ⫾ 0.19 1.34 ⫾ 0.32 2,114.8 ⫾ 1079.65

46.52 ⫾ 15.85 0.73 ⫾ 0.44 4.98 ⫾ 0.97 7.14 ⫾ 3.55 29.03 ⫾ 11.58a 1.58 ⫾ 0.67 0.57 ⫾ 0.20 1.19 ⫾ 0.43 2,056.9 ⫾ 996.43

Note: PCOS ⫽ polycystic ovary syndrome. a P⬍.05. Romualdi. HPA-axis in PCOS: effect of pioglitazone. Fertil Steril 2007.

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FIGURE 1 ACTH and cortisol response to the CRF test in PCOS women before (squares/solid bars) and after (circles/ clear bars) 4 months of pioglitazone therapy. Left panel: curves of secretion. Right panel: AUC values.

Romualdi. HPA-axis in PCOS: effect of pioglitazone. Fertil Steril 2007.

and returned to basal values after 90 minutes. After pioglitazone therapy, a significantly lower peak of 17-OHP discharge occurred 30 minutes after the CRF bolus; both the plasma concentrations at each time point and the absolute AUC values were significantly decreased in the post-treatment evaluation. Exogenous CRF administration did not modify either DHEAS or T production (maximum variation in detected plasmatic concentrations during the test 0.6% and 1.7%, respectively). The same pattern was observed after pioglitazone treatment, which seemed to be unable to affect the adrenal production of these steroids (AUC for testosterone: pretreatment 59.23 ⫾ 6.80 ng/mL · 90 minutes, post-treatment 55.14 ⫾ 5.62 ng/mL · 90 minutes; AUC for DHEAS: pretreatment 164,595.8 ⫾ 32,425.4 ng/mL · 90 minutes; posttreatment 170,361.7 ⫾ 31,998.6 ng/mL · 90 minutes). DISCUSSION Several lines of evidence suggest that an alteration in the adrenal steroidogenesis could be present in patients with PCOS, so that some investigators have hypothesized that an adrenal defect could underlie the pathogenesis of the syndrome (4, 6). This abnormality is believed to originate from a hyper-responsiveness of the gland to normal ACTH circulating concentrations (5, 24) more than from a pituitary corticotropin hyperproduction (25). However, several years ago, we found that a stimulation of the HPA axis, both directly by exogenous CRF and indirectly by naloxone, led to higher ACTH and F production in PCOS than in control subjects (11, 26). Because the ACTH/F ratio was near to Fertility and Sterility姞

unity, we concluded that the hyper-responsiveness of the HPA axis in PCOS women could be central in origin. On the other hand, the possibility of an abnormal drive on the adrenals by factors other than ACTH has not been ruled out. In this regard, previous studies suggest that the PCOSrelated hyperinsulinism may bring a substantial contribution to this derangement. In fact, hyperinsulinemic patients with PCOS were demonstrated to respond to an ACTH stimulus more than normoinsulinemic and control women in terms of both A and P (6). These data were supported by the evidence that a decrease in insulin levels, obtained through an amelioration of insulin hepatic clearance by naltrexone treatment (8) as well as through an improvement of insulin secretion and peripheral sensitivity by insulin-lowering drugs (9, 10), is able to reduce the adrenal androgen response to ACTH stimulus in PCOS women with hyperinsulinism. Pioglitazone, a new insulin sensitizing agent belonging to the thiazolidinedione class, is able to enhance insulin activity with a post–insulin receptor mechanism of action (27). The different chemical structure of this compound, which exhibits a higher affinity to the specific receptor peroxisome proliferators–activated receptor ␥, allows a more potent insulin sensitizing effect with a much lower hepatotoxicity compared with troglitazone and rosiglitazone (28). In the current study, to investigate at which level of the HPA axis insulin may exert its steroidogenic effect, we evaluated the adrenal response to CRF stimulus in a selected population of hyperinsulinemic PCOS patients before and after pioglitazone therapy. In accordance with our previous studies (10, 29), a 4-month pioglitazone treatment was ef135

FIGURE 2 Response to the CRF test in terms of A and P in PCOS women before (squares/solid bars) and after (circles/clear bars) treatment with pioglitazone. Left panel: curves of secretion. Right panel: AUC values. *P⬍.05 post-treatment vs. pre-treatment; **P⬍.01 post-treatment vs. pre-treatment.

Romualdi. HPA-axis in PCOS: effect of pioglitazone. Fertil Steril 2007.

fective in markedly reducing insulin pancreatic secretion. This effect was followed by a significant decrease in P and A levels and by an increase in SHBG plasma concentrations, the latter attributable to the decreased insulin inhibitory action on the hepatic synthesis of this protein (3). A trend toward improvement was seen for the T and lipid profiles. The pathogenetic role of insulin in PCOS seems not to be limited to its interference on the peripheral endocrine glands. It is well known, in fact, that insulin receptors are localized in several structures of the central nervous system, where they are implicated in the signaling pathways of the neuroendocrine regulation of the reproductive system (30). The pituitary is one of the most important sites of insulin action. In particular, in women with PCOS, the insulin/insulin-like growth factor (IGF) 1 system seems to be involved in the dysregulation of the LH secretory pattern (31, 32). The present study is the first report in the literature investigating the effect of a decrease in insulin levels on the pituitary response to CRF in PCOS. On the basis of our results, the marked drop in insulinstimulated levels obtained with pioglitazone treatment was not able to affect either basal and CRF-stimulated ACTH levels. This finding indirectly suggests that the insulin influence on the adrenal steroidogenesis does not originate from 136

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an enhancement of ACTH secretion at pituitary levels, thus supporting the finding of comparable plasma corticotropin levels in hyperinsulinemic women with PCOS and control subjects. Similarly, adrenal F production seems not to have been affected by insulin reduction due to pioglitazone treatment. These data are in accordance with several previous studies hypothesizing a possible selective role of hyperinsulinism in sex steroid adrenal production (6, 8). Most authors, indeed, agree that cytochrome (c) P450-17␣, a key enzyme for the synthesis of ovarian and adrenal androgens, could be involved in the hyperproduction of 17-OHP and A in hyperinsulinemic women with PCOS (33). In fact, the insulin/IGF system might represent the trigger factor responsible for the hyperstimulation of cP450 –17␣, leading to the dysregulation of the two sequential enzymatic steps (17-hydroxylase and 17,20-lyase) necessary for adrenal 17-OHP and A synthesis (7, 34). Consequently, in line with our previous study on the effect of pioglitazone on the adrenal response to ACTH stimulus, the administration of this drug for 4 months was able to significantly reduce the secretion of androstenedione and P. Furthermore, it could not be undervalued that thiazolidinediones display a direct inhibitory effect on the enzymatic activities of cP450 –17␣ and 3␤-hydroxysteroid dehy-

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drogenase type 2 (35). Among the members of this drug family, troglitazone is the compound with the most relevant enzymatic inhibitory activity, whereas rosiglitazone and pioglitazone were reported to exert a direct but weaker interference on both cP450 –17␣ and 3␤-hydroxysteroid dehydrogenase type 2. No responses to CRF stimulus were seen in plasma levels of T and DHEAS, neither before nor after pioglitazone treatment. This finding is not easily explained, because little data are available in the literature dealing with the ability of exogenous CRF stimulation to influence the plasma levels of these two steroids (36, 37). It could be speculated that the 90 minutes after CRF stimulation was not an adequate period to observe significant changes in T and DHEAS production. In conclusion, this study, which for the first time analyzed the effect of a decrease in insulin levels on the pituitary response to CRF in PCOS, seems to suggest that the insulin interference on HPA-axis is mainly exerted directly on the adrenal steroidogenesis.

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Vol. 88, No. 1, July 2007