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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0615
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5605-5612
Copyright © 2005 by The Endocrine Society

Pioglitazone Treatment Increases Spontaneous Growth Hormone (GH) Secretion and Stimulated GH Levels in Polycystic Ovary Syndrome

Dorte Glintborg, René Klinkby Støving, Claus Hagen, Anne Pernille Hermann, Jan Frystyk, Johannes D. Veldhuis, Allan Flyvbjerg and Marianne Andersen

Department of Endocrinology and Metabolism (D.G., R.K.S., C.H., A.P.H., M.A.), Odense University Hospital, DK-5000 Odense C, Denmark; Medical Research Laboratories (J.F., A.F.), Clinical Institute and Medical Department M (Diabetes and Endocrinology), Aarhus University Hospital, DK-8000 Aarhus, Denmark; and Division of Endocrinology and Metabolism (J.D.V.), Department of Internal Medicine, Mayo Clinical Research Center, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Dorte Glintborg, Kløvervænget 6, Third Floor, DK-5000 Odense C, Denmark. E-mail: dorte.glintborg{at}dadlnet.dk.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Low GH levels, probably due to insulin resistance and increased abdominal fat mass, are well described in polycystic ovary syndrome (PCOS). GH acts as an important ovarian cogonadotropin, and GH disturbances may be an additional pathogenic factor in PCOS. Decreased abdominal fat mass and improved insulin sensitivity during pioglitazone treatment may affect GH secretion.

Objective: The objective of the study was to investigate the effect of pioglitazone on GH levels in PCOS.

Design: Thirty insulin-resistant PCOS patients were randomized to either 16 wk pioglitazone (30 mg/d) or placebo treatment. Before and after intervention, levels of fasting insulin, GH, total IGF-I, free IGF-I, IGF binding protein-1, IGF-II, free fatty acids, testosterone, and SHBG were measured. Patients underwent whole-body dual x-ray absorptiometry scans, pyridostigmine-GHRH tests, and 24-h 20-min integrated blood sampling for measurement of GH.

Results: Peak GH and area under the curve for GH in pyridostigmine-GHRH tests and 24-h mean GH concentrations and pulsatile GH secretion significantly increased after pioglitazone treatment. No significant changes were observed in GH pulse frequency, pulse duration, approximate entropy levels, or basal GH release. Levels of IGF binding protein-1 significantly increased, whereas no significant differences were measured in total IGF-I and free IGF-I. Pioglitazone treatment significantly decreased fasting insulin and homeostasis model assessment levels. No significant changes were observed in Ferriman Gallwey score or androgen levels.

Conclusion: Pioglitazone treatment significantly increased GHRH-stimulated GH levels and 24-h pulsatile GH secretion, probably directly or indirectly due to improved insulin sensitivity.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) is an endocrine disorder characterized by oligo/anovulation and hyperandrogenemia (1). The prevalence of PCOS is 5–8%, thus making this syndrome the most common endocrinopathy among reproductive-aged women (2). Studies using continuous blood sampling or GH stimulation tests have showed lower basal and/or stimulated GH levels in PCOS patients, compared with weight-matched controls (3, 4, 5, 6). Ovarian tissue has both IGF-I and GH receptors (7). GH amplifies intraovarian IGF-I production and acts as an important cogonadotropin able to increase the FSH effect on both ovarian granulosa and theca cells (8). Adjuvant GH therapy reduces gonadotropin requirements in GH-deficient women, and GH cotreatment during in vitro fertilization resulted in an improved fertilization in selected patient groups (8). Changes in GH secretion could therefore have important implications for ovarian function in PCOS.

Studies using metformin or peroxisomal proliferator-activated receptor (PPAR){gamma} agonists in PCOS have showed improved ovulatory function and metformin cotreatment is used in clomiphene-resistant patients to improve results of fertility treatment (9). Whether the effect of insulin sensitizers on the ovarian function in PCOS is in part mediated through improvements in GH dysfunction remains to be established. Two studies by Ibanez et al. showed normalization of insulin sensitivity, body composition, GH secretion, IGF-I levels, and ovarian function during metformin/metformin-flutamide treatment in normal-weight adolescents with precocious pubarche (10) or ovarian hyperandrogenemia (11). These studies have not been reproduced in adult or overweight PCOS patients, but they do, however, indicate that insulin sensitivity and body composition are related to GH levels and ovarian function in hyperandrogen patients.

Most PCOS patients are overweight and have a high waist to hip ratio (WHR), indicating increased abdominal fat storage is seen in both normal and overweight PCOS patients (12). Excess abdominal fat is associated with impaired GH secretion, which can be partly normalized by weight loss (13, 14, 15). Hyperinsulinemia, high abdominal visceral fat mass, and increased free fatty acid (FFA) levels are probably essential for the abnormal GH secretion in obesity (15). Previous studies found FFAs to act directly on the pituitary somatotroph cells by disrupting intracellular signaling (16).

High insulin levels may affect GH secretion independently of FFAs by decreasing levels of IGF binding protein (IGFBP)-1, thereby elevating free IGF-I and increasing the IGF-I mediated feedback inhibition of the pituitary (17). In addition, insulin may have a direct, GH-independent, stimulatory effect on the hepatic IGF-I production (17). Insulin resistance and hyperinsulinemia are observed in more than 50% of PCOS patients (18), and the effects of insulin on the IGF-I system could therefore be of importance in PCOS.

In the present study, we examined the effects of pioglitazone treatment on the spontaneous as well as pyridostigmine (PD)-GHRH-stimulated GH secretion in insulin-resistant individuals with PCOS. The PD-GHRH test was applied as a safe, reliable, and potent stimulator of GH secretion in adults (19, 20). Pioglitazone is a PPAR{gamma} agonist known to increase insulin sensitivity in insulin-resistant type 2 diabetic patients. Increased insulin sensitivity during PPAR{gamma} agonist treatment is mediated through a modulation of fat distribution and fatty acid metabolism including increased sc fat mass and decreased intraabdominal fat mass (21). We therefore hypothesized that pioglitazone treatment could increase the GH secretion in PCOS patients.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

Thirty reproductive-aged women with PCOS were recruited from the Department of Endocrinology and the Department of Fertility, Odense University Hospital, and fertility clinics and departments of gynecology in the area of Funen and Jutland. Criteria for PCOS included irregular periods with cycle length longer than 35 d and free testosterone above reference interval (>0.035 nmol/liter)/ hirsutism. Included patients had elevated fasting insulin levels greater than 50 pmol/liter and/or were overweight [body mass index (BMI) ≥ 30 kg/m2].

Screening fasting blood samples were analyzed for liver enzymes, insulin, hemoglobin A1c, plasma glucose, electrolytes, total and free testosterone, prolactin, and TSH.

Patients were excluded if they were pregnant or expressed a wish for conception during the study period. Patients with diabetes (fasting plasma glucose ≥ 7.0 mmol/liter), hypertension, elevated liver enzymes, renal dysfunction, or congestive heart disease were not included in the study. All patients had serum (s)-prolactin and s-TSH within reference interval.

Patients paused oral contraceptives for at least 3 months before evaluation, and no patient took medicine known to affect hormonal or metabolic parameters. All subjects consented to use barrier contraception combined with spermatocidal cream provided by the Department of Endocrinology during participation in the study. The study was approved by the local ethics committee and the Danish Medicines Agency, and all subjects gave written informed consent.

Methods

Patients were admitted to the Department of Endocrinology, Odense University Hospital, at 0800 h. Examinations were performed during the follicular phase in patients with oligomenorrhea. Patients with amenorrhea (period length > 3 months) were examined randomly. All subjects had a physical examination performed including Ferriman-Gallwey score, blood pressure, WHR, heart and lung stethoscopy, height, and weight. Waist circumference was determined as the minimum circumference between the iliac crest and the lower costae, whereas the hip circumference was defined as the maximum circumference over the gluteal region.

Serial blood sampling

During the first 24 h of admittance, continuous blood sampling was performed using an indwelling iv catheter connected to a peristaltic pump (Carmeda, Upplands Väsby, Sweden) with a flow rate of 9 ml/h with vial shifts every 20 min. The automatic blood sampling system allowed subjects to sleep during the night and move freely around the department area during daytime. During daytime, subjects were served meals at 0900, 1200, 1500, and 1800 h and were allowed to snack. Patients fasted from midnight but were allowed to drink tap water. Blood samples were allowed to clot, centrifuged, and serum stored at –80 C until analysis for GH.

GH stimulation test

On the second day, fasting blood samples were analyzed for testosterone, FFAs, SHBG, LH, FSH, insulin, C-peptide, and lipid profile. At 0830 h (time –60 min) 120 mg PD (Mestinon; Hoffmann-La Roche, Basel, Switzerland) was administered orally. At 0930 h (time 0 min) GHRH 1 µg/kg body weight was administered as an iv bolus. GH was measured at times 0, 20, 30, 45, 60, and 90 min after GHRH injection.

Dual-energy x-ray absorptiometry (DXA) scan

DXA scan in whole-body array mode (DXA, QDR-4500; Hologic, Bedford, MA) was used to measure body composition. DXA scan measurements were used to determine total fat mass, truncal fat mass, and lower extremity fat mass. Technical performance was monitored by daily calibration scans using an anthropomorphic Hologic phantom. The coefficient of variation (CV) for replicate scans of the same individual was 0.8% for measuring fat mass and 0.6% for measuring lean body mass. CV was determined by repeated scanning of 10 of the study participants.

Protocol

After initial examination, patients were randomized to pioglitazone 30 mg/d (Actos, Takeda, Lilly A/S, Indianapolis, IN) or placebo. The study was carried out in a double-blind fashion; compliance was checked after 2 months and at final admittance through tablet count.

Safety parameters included liver enzymes, electrolytes, white blood cell count, and pregnancy test. Control parameters were repeated after 1 and 2 months’ treatment period and before final admittance. After a treatment period of 16 wk, patients were admitted for repeated examinations similar to the initial evaluation program.

Two patients were excluded from the study: One patient in the placebo group became pregnant, and one patient on pioglitazone treatment experienced side effects (dizziness, ankle edema, and anxiety) and was excluded after 1 wk of treatment. In this patient the ankle edemas were not objectively visualized, and the patient was not interested in symptomatic furosemide treatment. No other patient in the pioglitazone group experienced side effects that could be related to pioglitazone treatment.

Eighteen patients (PPAR{gamma}, n = 9; placebo, n = 9) had complete 24-h blood samples during the follicular phase/amenorrhea before and after intervention. Twenty-seven patients (PPAR{gamma}, n = 13; placebo, n = 14) had PD-GHRH tests performed before intervention, and 25 patients (PPAR{gamma}, n = 12; placebo, n = 13) had the test performed after the intervention period.

Assays

Serum levels of GH, LH, FSH, insulin, C-peptide, and estradiol were analyzed by time-resolved fluoroimmunoassay using commercial kits (AutoDELFIA, PerkinElmer Life Sciences, Oy, Turku, Finland). Intraassay CVs were: FSH 1.0–1.4%; LH 1.0–9.3%; estradiol 3.8–5.2%; C-peptide 1.1–5.0%; and insulin 2.1–3.7%. Interassay CVs were: FSH 2.7–2.8%; LH 2.3–3.9%; estradiol 3.7–8.5%; C-peptide 1.1–3.4%; and insulin 3.4–4.0%. Serum total and free testosterone and SHBG were analyzed using the specific RIA and extraction methods previously described (22). For this method, the intraassay CV for total testosterone was 8.2% and for SHBG it was 5.2%. The interassay CV for total testosterone was 13.8% and for SHBG it was 7.5%. Plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, and FFAs were analyzed by enzymatic colonometric reactions using commercial kits (Modular P, Roche, Stockholm, Sweden), and low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation. GH levels were determined by an immunofluorometric assay (Delfia, PerkinElmer Life Sciences). The level of detection for the Delfia was 0.03 mU/liter. The intraassay CVs for GH were 7% at 0.5 mU/liter and 3% at 18 mU/liter. All GH samples from all subjects were run in the same assay. Serum total (extractable) IGF-I was determined after acid ethanol extraction by an in-house time-resolved immunofluorometric assay with mean within and in-between assay CVs less than 5 and 10% (23). Serum-free IGF-I was determined after ultrafiltration by centrifugation at conditions approaching those in vivo (17). The lower detection limit of free IGF-I in the ultrafiltrates was 0.020 µg/liter. Mean within- and between-assay CVs of free IGF-I was 15 and 20%. Serum IGFBP-1 was assessed by an in-house RIA with a mean within- and between-assay CVs less than 6 and 12% (24). From each patient all samples were analyzed in the same run.

We calculated homeostasis model assessment of insulin resistance as (HOMA-r) = fasting insulin * fasting blood glucose/22.5. Reference intervals (RIs) in the follicular phase were as follows: s-FSH, 2.2–6.5 U/liter; s-LH, 1.6–9.3 U/liter; s-estradiol, 80–790 pmol/liter; s-total testosterone, 0.55–1.8 nmol/liter; s-free testosterone, 0.006–0.034 nmol/liter; and s-SHBG, 41–170 nmol/liter. The free androgen index was less than 0.043. RI for fasting insulin was less than 55 pmol/liter, fasting plasma glucose less than 7.0 mmol/liter, and HOMA-r less than 17.1 pmol mmol/liter–2. For fasting lipid parameters, RIs were: total cholesterol, 3.6–6.8 mmol/liter; LDL, 1.8–4.5 mmol/liter; HDL, 0.76–1.68 mmol/liter; and triglycerides, 0.47–2.31 mmol/liter.

Deconvolution

We applied multiparameter deconvolution analysis to quantitate pulsatile GH secretion and endogenous half-life, as previously described by Veldhuis et al. (25). In this model the plasma GH concentration was related to the location, amplitude, and duration of GH secretory bursts, acted on continuously by an endogenous subject-specific hormone half-life (25). The disappearance function for GH was modeled as a one-component exponential decay function with a subject-specific rate constant assuming that the half-life and distribution volume of GH were approximately constant in each individual throughout 24 h (25). The analysis outcome measures of the analysis were pulsatile GH release (the summed mass of GH secreted above basal), basal (nonpulsatile) secretion, and total secretion (combined basal and pulsatile). Pulsatile GH secretion was calculated as the product of secretory pulse frequency and the mean GH mass released per pulse (25). Secretory pulse identification required a calculated burst mass exceeding zero by 95% statistical confidence intervals. The use of the deconvolution technique obviates technical confounding of secretion estimates due to the use of only the absolute peak, incremental peak, and/or integrated GH concentration.

Regularity in GH concentration-time series was quantified using approximate entropy (ApEn) as previously described (26). ApEn quantifies the logarithmic likelihood that similar patterns of data length m remain the same within a tolerance r on the next incremental comparison. It assigns a single nonnegative number to a time series, quantifying a notion of the serial orderliness or regularity of the data. Smaller ApEn values indicate a greater likelihood of successive comparisons remaining close and therefore imply greater regularity, and conversely, larger values imply less regularity. ApEn detects differences in underlying episodic behavior not reflected simply in the mean or variance of hormone concentrations or in peak occurrence or amplitudes. ApEn has been demonstrated to be stable during small changes in noise and infrequent data artifacts. Normalized ApEn parameters of m = 1 (length of compared series) and r = 20% (threshold) of the intraseries SD were used (ApEn 1,20%).

Statistical methods

Parameters were log transformed to achieve log normally distributed variables and were described using geometric mean (± 2 SD). Differences in means within or between patient groups were tested using paired and unpaired t tests, respectively. Basal differences between patients randomized to pioglitazone and placebo were taken into account by further comparing delta ({Delta}) values of hormonal and metabolic variables between the placebo and pioglitazone groups using Mann-Whitney U tests. {Delta} Values were calculated as posttreatment level minus pretreatment level of each analyzed variable. GH responses during PD-GHRH testing were evaluated using maximum GH responses and calculating area under the curve (AUC) for GH using the trapezoidal rule.

We used SPSS (version 11.5; SPSS, Inc., Chicago, IL) in all calculations. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
During pretreatment no significant differences were found in clinical or paraclinical parameters in the pioglitazone group, compared with the placebo group (Table 1Go).


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  		TABLE 1. Clinical, hormonal, and metabolic characteristics

 
Fasting lipid variables in the pioglitazone and placebo group were: total cholesterol, 4.9 (3.1–7.7) vs. 5.1 (3.8–7.0) mmol/liter; triglycerides, 1.4 (0.6–3.4) vs. 1.5 (0.5–4.7) mmol/liter; HDL, 1.3 (0.9–1.9) vs. 1.4 (0.9–2.1) mmol/liter; and LDL, 2.8 (1.5–5.4) vs. 2.9 (1.7–4.9) mmol/liter (all P > 0.05).

PD-GHRH test

Pretreatment peak GH and AUC GH responses to the PD-GHRH test were similar in the placebo group, compared with the pioglitazone group (Table 2Go). After pioglitazone treatment peak GH levels and AUC GH levels increased significantly [40.9 (13.8–120.7) vs. 28.7 (9.8–84.4) mU/liter and 2469(950–6414) vs. 1557(432–5684) mU/liter, P < 0.05]. No significant changes in GH levels were seen in the placebo group.


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  		TABLE 2. GH secretion, PD-GHRH tests, and GH deconvolution analysis

 
{Delta}PD-GHRH stimulated GH levels were significantly higher during pioglitazone treatment, compared with placebo (P < 0.05), whereas no significant changes were found in {Delta}AUC GH levels between pioglitazone and placebo (P = 0.20). Figure 1Go shows geometric mean GH levels during PD-GHRH tests in the two study groups.



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  		FIG. 1. Log GH levels during PD-GHRH test during pioglitazone and placebo treatment. *, P < 0.05 vs. pretreatment.

 
Twenty-four-hour GH profile

At baseline, GH secretory pulse number, length of interpulse interval, pulse amplitude, and basal GH secretion showed no significant difference when comparing the two study groups (Table 2Go).

After pioglitazone treatment, 24-h mean GH concentrations and total GH secretion were significantly increased [mean GH from 0.9 (0.3–3.2) to 1.7 (0.6–5.0) mU/liter, P < 0.001 and total GH secretion from 55.6 (14.2–217.5) to 105.3 (38.9–285.2) mU/liter, P < 0.001]. These observations were due to an increased mass of GH pulses (P < 0.05) and pulsatile GH secretion (P < 0.001). No significant changes were observed in pulse frequency, ApEn levels, or interpulse interval during pioglitazone treatment (Table 2Go).

Placebo treatment had no significant effects on GH secretion parameters.

The results were not significantly changed when comparing {Delta} values of 24-h GH secretion between placebo and pioglitazone groups.

Figure 2Go shows representative 24-h GH secretion profiles in two patients on pioglitazone treatment and one patient on placebo treatment.



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  		FIG. 2. Individual GH profiles from two patients treated with pioglitazone (A and B) and one treated with placebo (C).

 
IGF-I

Pretreatment levels of IGF-I, IGF-II, and IGFBP-1 were comparable in both patient groups (Table 2Go). During pioglitazone treatment, no significant changes were observed in total IGF-I and free IGF-I levels, whereas IGFBP-1 increased significantly. IGF-II levels decreased significantly during pioglitazone. No significant changes were observed during placebo. {Delta}IGF-I was significantly higher in the pioglitazone group, compared with the placebo group (P = 0.03), whereas no significant changes were observed in {Delta}IGFBP-1 levels (P = 0.60).

Insulin resistance and body composition

Fasting insulin levels, C-peptide, and HOMA-r were all significantly decreased after pioglitazone treatment, indicating increased insulin sensitivity (Table 1Go). {Delta}24-h mean GH concentrations were significantly negatively correlated to {Delta}insulin levels in included patients (Pearson correlation r = –0.687, P = 0.003). Pioglitazone did not change mean BMI, WHR, or DXA scan measures of body composition (Table 3Go).


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  		TABLE 3. Measures of body composition

 
Sex hormones

No significant changes were observed in Ferriman Gallwey score, total or free testosterone, LH/FSH, prolactin, or estradiol levels (Table 1Go, data for LH/FSH not shown).

No significant changes were observed in {Delta}SHBG between placebo and pioglitazone (P = 0.60) (Table 1Go).

Menstrual cycle history

Twelve of 14 patients reported improvements of menstrual cycles in the pioglitazone group, compared with five of 14 patients in the placebo group (P < 0.02).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In the present study, pioglitazone treatment significantly increased peak GH response to PD-GHRH stimulation as well as 24-h pulsatile GH secretion in insulin-resistant PCOS patients. During pioglitazone treatment, the peak GH responses to the PD-GHRH test were significantly increased from 28.7 to 40.7 mU/liter and the 24-h mean GH concentrations increased from 0.9 to 1.7 mU/liter as a result of increased secretory burst mass without significant changes in interpulse intervals, pulse regularity, pulse duration, or basal GH secretion. Previous studies using similar GH assays in normal-weight healthy individuals measured peak PD-GHRH-stimulated GH levels of 121 mU/liter and mean 24-h GH concentrations of 3.2–3.7 mU/liter (5, 27, 28). Studies in overweight individuals found decreased 24-h mean GH concentrations of 1.0–1.5 mU/liter (mean BMI in study population, 38–39 kg/m2) (5, 14). Stimulated GH responses have been shown to be reduced by one third in overweight healthy individuals, compared with normal-weight healthy individuals (14, 29, 30). Our findings of low GH levels in PCOS are in agreement with previous studies describing significantly lower stimulated and basal GH levels in obese PCOS patients, compared with weight-matched controls (5, 6). However, even in normal-weight PCOS patients, GH levels are subnormal, thus supporting impaired GH secretion to be an additional pathogenic factor in PCOS (3, 4, 31).

The results of the present study indicate an improvement but not a normalization of GH levels during pioglitazone treatment. The effect of PPAR{gamma} agonist treatment on insulin sensitivity is mediated through changed adipocyte differentiation and FFA metabolism (21). Treatment with PPAR{gamma} agonist often causes weight gain and redistributes visceral fat, shown to be a strong predictor of insulin resistance to sc fat areas (32, 33). Previous studies underlined the important relation between high abdominal fat mass and low GH secretion (13). No measures of intraabdominal and sc fat mass were available in the present study, and this may explain why we found no significant differences of body composition measures. Future studies including computed tomography or magnetic resonance scans are needed to determine the relation between GH secretion and fat distribution during PPAR{gamma} agonist treatment in PCOS patients.

We measured no changes in fasting FFA levels during pioglitazone treatment. However, in the same study cohort, pioglitazone treatment caused a significant improvement in the insulin-mediated FFA suppression during euglycemic hyperinsulinemic clamp studies, hereby indicating significant changes in adipose tissue metabolism (Glintborg, D., A.P. Hermann, M. Anderson, C. Hagen, H. Beck-Nielsen, J. Veldhuis, and J. E. Henrikson, data submitted for publication). We are not aware of previous studies evaluating the effect of PPAR{gamma} agonist on GH secretion in patients with PCOS or type 2 diabetes. However, few studies evaluated the effect of weight loss and changed body composition on GH secretion in healthy overweight individuals. In agreement with the findings in our study, Pijl et al. (34) described increased pulse amplitude and no significant changes in pulse regularity after weight loss in obese females with large visceral fat mass (BMI reduced from 35 to 29 kg/m2). Visceral fat mass was reduced by nearly 50%, thus indicating a significant reduction in circulating FFA levels, but levels of fasting insulin remained unchanged (34). Despite reduced fat mass, GH secretion was still lower than in normal-weight controls (34). Another study on even more obese individuals (BMI 40 kg/m2 before weight loss) showed normalized GH levels during stimulation tests and 24-h measurements after weight loss (14). In that study fasting insulin levels decreased after weight loss, indicating that both fat mass and insulin sensitivity are important for GH secretion.

We observed identical changes in GH secretory pattern during pioglitazone treatment as observed in studies using acipimox, which blocks lipolysis and allows investigation of the isolated effects of decreased FFA levels on GH secretion (35, 36, 37). In both obese subjects and PCOS, acipimox treatment significantly increased GHRH-stimulated GH levels (35, 36, 38). Furthermore, Kok et al. (39) found increased pulsatile and total GH secretion during acipimox treatment in obese subjects with no significant changes in burst frequency and basal production. These results are in keeping with in vivo and in vitro studies showing that FFAs to be able to disrupt the signal transduction pathways in the pituitary somatotrope cells (16). The present data therefore suggest that the effect of pioglitazone on GH levels may be mediated by decreased FFA levels and consequently a restoration of the sensitivity of the somatotrope cells to GHRH.

In the present study, fasting insulin levels decreased significantly from 95 to 54 pmol/liter, and HOMA-r levels decreased from 23.0 to 12.9 pmol mmol/liter, thus suggesting that the insulin sensitivity increased during pioglitazone treatment. Previous studies found insulin to be of major importance for GH regulation (40, 41). High insulin levels have been shown to decrease levels of IGFBP-1, and this may elevate serum levels of free IGF-I and subsequently inhibit the secretion of GH through increased IGF-I feedback on the pituitary (17). In agreement with this hypothesis, several studies found significantly higher levels of serum total IGF-I in PCOS patients, compared with weight-matched controls (5, 42, 43, 44); however, other studies were not able to reproduce this. As recently reviewed by Frystyk (17), measurement of free IGF-I may be a better indicator of the GH secretion in conditions with an abnormal insulin sensitivity. No control group was included in the present study, but two previous studies (45, 46) reported higher serum-free IGF-I levels in PCOS, compared with weight-matched controls. In the present study, we found a significant correlation between changes in insulin levels and GH secretion; however, free IGF-I levels remained unchanged during pioglitazone treatment. Our data therefore indicate a more complex relation between insulin sensitivity and GH secretion. A previous study (47) described increased IGFBP-I levels during PPAR{gamma} agonist treatment; however, in the present study, we could not reproduce these data. In a recently published experimental in vivo study (48), IGFBP-1 was found to affect GH secretion independently of circulating IGF-I levels. These findings are to be reproduced.

We observed only minor changes in estradiol, androgen, and SHBG levels when measuring these parameters in the follicular phase or during periods of amenorrhea. A high frequency of the pioglitazone-treated patients reported more regular menses, compared with the placebo group. In agreement with our study, recent studies using pioglitazone treatment in PCOS reported significantly increased insulin sensitivity and restoration of menses regularity in the majority of treated patients despite unchanged serum levels of SHBG and testosterone (49, 50, 51, 52). GH and IGF-I receptors have been found in the ovary, and high IGF-I levels are believed to increase ovarian androgen production in PCOS (4, 45). Changes in the GH/IGF-I system may be involved in the observed improved menstrual regularity in the present study. However, due to the relatively small number of included subjects and the fact that ovulation rates were not directly established, more studies are needed to reproduce these findings. Wu et al. (53) found improved insulin-stimulated glycogen synthesis during pioglitazone treatment in cultured ovarian granulosa cells from PCOS patients, suggesting normalization of ovarian insulin resistance. Furthermore, the authors found evidence for a mitogenic action of high IGF-I levels. This action was reversed during treatment with troglitazone.

Conclusion

In the present study, pioglitazone treatment significantly increased stimulated as well as spontaneous GH secretion in insulin-resistant overweight PCOS patients without significantly affecting BMI and total fat mass.


   Acknowledgments
 
The authors thank Jane Nielsen, Ellen Andersen, Anna Marie Madsen, Jette Thinggård Jørgensen, Else Marie Eckhoff, Lise Møller Madsen, Henny Hansen, Bente Tøt, Kirsten Westermann, Donna Arbuckle, Anette Madsen, Rikke Kiilsholm, Kirsten Nyborg, Inga Madsen, Joan Hansen, and Susanne Sørensen for excellent technical assistance.


   Footnotes
 
This work was supported by Fonden for Lægevidenskabelig forskning ved Fyns Amt, Jacob Madsen’s and Olga Madsen’s Foundation, Institute of Clinical Research, Odense University Hospital, A. P. Møller’s Foundation, Hans and Nora Buchard’s Foundation, K. A. Rohde’s Foundation, Aase and Ejnar Danielsen’s Foundation, Eva and Carl Adolf Holm’s Foundation, Dagmar Marshall’s Foundation, The Danish Medical Association, A. J. Andersen’s Foundation, the Novo Nordisk Foundation, Overlægerådet Odense University Hospital, the Danish Diabetes Association, and the Danish Medical Research Council.

First Published Online August 2, 2005

Abbreviations: ApEn, Approximate entropy; AUC, area under the curve; BMI, body mass index; CV, coefficient of variation; DXA, dual-energy x-ray absorptiometry; FFA, free fatty acid; HDL, high-density lipoprotein; HOMA-r, homeostasis model assessment of insulin resistance; IGFBP, IGF binding protein; LDL, low-density lipoprotein; PCOS, polycystic ovary syndrome; PD, pyridostigmine; PPAR, peroxisomal proliferator-activated receptor; RI, reference interval; WHR, waist to hip ratio.

Received March 21, 2005.

Accepted July 25, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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